Keywords: Cytosolic Ca2+ concentration 35S: Cauliflower mosaic virus 35S typeIII effector, acetyltransferase, bromodomain, Ralstonia solanacearum PopP2-

Figure 32. Targeting of GTE proteins by PopP2.

Materials and Methods

. Primers used in this study. Figure 2. PAMPs and DAMPs recognition by PRRs (adapted from He and Wu, 2016). During plant-microbe interactions, PAMPs are recognised by PRRs triggering PTI responses. Wellknown PAMPs are derived from bacterial flagellin and elongation factor Tu (EF-Tu) and fungal chitin, which are recognised by the PRRs FLS2, EFR and CERK1, respectively. Plant elicitor proteins (Peps) and oligogalacturonides (OGs) are plant-derived molecules resulting from damage caused during pathogen infection or herbivore attack, called DAMPs. Peps and OGs are perceived by the PRRs PEPR and WAK1, respectively. Upon perception of PAMPs/DAMPs, PRRs often recruit BAK1, an adaptator playing a central role in the regulation of PTI. Then, PRRs autophosphorylate and transphosphorylate, triggering PTI responses, mediated by, among others, receptor like cytoplasmic kinases (RLCKs).

PAMP-triggered immunity (PTI)

Figure 4. PTI and ETI downstream signalling events (adapted from Lu and Tsuda, 2021). After microbe perception, cellular responses will be triggered leading to activation of plant immune responses. These cellular responses include calcium influx, ion fluxes, ROS burst, activation of MAPK cascades, activation of phytohormone signalling, transcriptional reprogramming, and sometimes programmed cell death. Solid arrows indicate known mechanisms. Dashed arrows indicate signalling mechanism remaining elusive. Question marks show connections that have not been characterised experimentally. B.4.4.b. Jasmonic acid (JA) and ethylene (ET)

JA controls the production of defence compounds that confer resistance to several pathogens and vertebrate herbivores. Pathogen perception and tissue injury trigger JA synthesis that will promote the expression of several secondary metabolites and proteins involved in plant immunity including alkaloids, terpenoids, anti-nutritional proteins and some PR proteins (Campos et al., 2014).

While JA-mediated defence mechanisms were long thought to be specific for necrotrophs, this assumption is now questioned. Originally, this assumption was Figure 6. Eukaryotic chromatin structure (adapted from Doğan and Liu, 2018). Eukaryotic genomic DNA is wrapped around histone octamers to form nucleosomes. Chromatin loops are the result of the interaction of nucleosomes from distant regions. They are parts of Topologically Associated Domains (TADs), regions of tens of kilobase pairs that represent areas of enhanced chromatin interaction within TADs but suppressed between TADs. The TADs together constitute A/B compartments, areas enriched in active or repressed chromatin. They are themselves sorted into distinct nuclear spaces, called chromosome territories.

Summary

Microbial pathogens have developed sophisticated strategies to defeat host immune responses and promote infection, among which manipulation of host epigenetic-related processes plays a prominent role. Here, we identify GTE9 and GTE11, two Arabidopsis Bromodomaincontaining proteins with an extra-terminal domain (BET), as substrates of the YopJ family PopP2 acetyltransferase from the soil-borne bacterium Ralstonia solanacearum. We show that GTE9 and GTE11 function as epigenetic readers that interact in planta with histone H4 through their bromodomain. PopP2 acetylates several lysine residues flanking the bromodomain of GTE9 and GTE11 and, remarkably, alters the ability of GTE9 to bind histone H4 in vivo.

Moreover, bacterial manipulation of GTE9 and GTE11 transgenically overexpressed in Arabidopsis promotes wilt disease development in a PopP2-dependent manner. Taken together, our study highlights a virulence strategy employed by a bacterial plant pathogen that manipulates host epigenetic readers using a YopJ family acetyltransferase.

 B.3.2.b. Heterodimerisation of NLRs.............................................................................. B.3.2.c. NLRs can associate in higher structure shapes: the resistosomes .................... B.3.2.d. NLRs cooperate with helper NLRs ................................................................... B.4.4.a. Salicylic acid (SA) .............................................................................................. B.4.4.b. Jasmonic acid (JA) and ethylene (ET) ................................................................ B.4.4.c. Abscisic acid (ABA) ............................................................................................ B.4.4.e. Hormonal crosstalk ........................................................................................... B.4.5. Transcriptional PRRs ................................... B.5.2.b. Effectors can interfere with MAPK signalling cascades .................................... B.5.2.c. Effectors manipulate hormonal pathways........................................................ B.5.2.d. Effectors can interfere with plant cytoskeleton ................................................ B.5.2.e. Effectors can alter host gene transcription ...................................................... B.5.2.f. Effectors can counteract ETI responses ............................................................. B.5.2.g. AvrAC,an effector that can disturb both PTI and ETI ....................................... B.5.3. Effectors target in their own way different pathways in a redundant manner ...... B.2.1.a. Histone methylation ......................................................................................... B.2.1.b. Histone acetylation,marker of active transcription ......................................... B.2.2. Histone marks regulators have different roles in plant response to pathogens ..... Les mécanismes épigénétiques contribuent à réguler l'expression des gènes sans en changer leur séquence, en influençant la structure de la chromatine. De plus en plus d'études montrent que les agents pathogènes ont développé des stratégies de virulence pour interférer avec les mécanismes épigénétiques de l'hôte. Bien décrits chez les modèles animaux, de tels mécanismes d'interférence sur l'épigénome de cellules hôtes végétales demeurent encore méconnus, et plus particulièrement en réponse à des bactéries. Ralstonia solanacearum est la bactérie responsable du flétrissement bactérien, qui affecte plus de 250 espèces végétales dont des grandes cultures et des plantes modèles comme Arabidopis thaliana. En tant que facteur de virulence majeur de R. solanacearum, PopP2 est une acetyltransférase de la famille YopJ qui atténue la résistance basale d'Arabidopsis en ciblant des facteurs de transcription WRKY. Pour mieux comprendre les fonctions de virulence de PopP2, des interacteurs ont été recherchés par une approche double hybride. Les protéines GTE9 et GTE11 de la famille GTE (General Transcription factor, group E) ont ainsi été identifiées. Ces protéines possèdent un bromodomaine connu pour interagir avec des résidus lysine acétylés, notamment présents chez des histones, suggérant que ces protéines pourraient être impliquées dans des processus épigénétiques. Précédemment, des travaux réalisés dans l'équipe ont révélé que GTE9 et GTE11 (i) co-localisent et interagissent avec PopP2 dans le noyau de cellules végétales, et (ii) sont acétylées par PopP2. De plus, GTE9 et GTE11 interagissent in planta avec l'Histone H4 via leur bromodomaine, suggérant que ce sont des lecteurs épigénétiques ciblés par PopP2. Dans ce contexte, les principaux objectifs de ma thèse furent de mieux comprendre la fonction de GTE9 et GTE11 en essayant de déterminer la façon dont PopP2 pourrait les manipuler et si ces protéines jouent un rôle dans la réponse d'A. thaliana vis-à-vis de R. solanacearum. Des analyses de spectrométrie de masse nous ont permis de cartographier les résidus lysine de GTE9 et GTE11 modifiés par PopP2. Plusieurs de ces résidus sont conservés entre les deux protéines et situés autour de leur bromodomaine. Par une approche de FRET-FLIM semi-quantitatif in vivo, nous avons montré que l'interaction GTE9-H4 est altérée par l'activité acetyltransferase de PopP2 suggérant que l'acétylation de GTE9 par PopP2 le dissocie de la chromatine. En sus de GTE9 et GTE11, PopP2 acétyle plusieurs autres protéines GTE. Concernant le rôle de GTE9 et GTE11 dans la réponse de la plante à R. solanacearum, des lignées d'A. thaliana sur-exprimant GTE9 et GTE11 sont plus sensibles à R. solanacearum et cela dépend de l'activité enzymatique de PopP2. Collectivement, nos données indiquent que GTE9 et GTE11 s'apparentent à des lecteurs épigénétiques qui sont ciblés par une bactérie phytopathogène à l'aide d'une acétyltransférase de la famille YopJ. Les GTEs pourraient être des cibles clefs de virulence car nous avons également identifié PopP1, une autre acetyltransferase YopJ de R. solanacearum, comme interagissant aussi avec certaines GTEs. Il reste à déterminer comment le ciblage des protéines GTEs par PopP2 facilite l'infection chez Arabidopsis par R. solanacearum. Pour répondre à cette question, une approche ChIP-seq visant à identifier les régions chromatiniennes ciblées par GTE9 et GTE11 a été initiée (approche en cours de réalisation). En parallèle, nous voulions identifier les sites de la chromatine visités par PopP2 chez Arabidopsis. Pour cela, une seconde analyse ChIP-seq a été entreprise en générant divers outils moléculaires, incluant des versions étiquetées de PopP2 délivrées in planta via un système de sécrétion de type III bactérien. De façon générale, ce projet de thèse a permis de progresser sur la compréhension d'une stratégie de virulence développée par une bactérie phytopathogène qui manipule des composantes épigénétiques pour favoriser l'infection.

B.2.2.a. Histone methylation plays an ambiguous role in plant

Epigenetic mechanisms contribute to the regulation of gene expression without changing its sequence by influencing the chromatin structure. Increasing evidence reveals that pathogens display virulence strategies that can interfere with host epigenetic mechanisms. This is particularly well described in animal pathogens but less in plant pathogens. Especially, very little evidence relate such virulence strategies used by plant pathogenic bacteria. Ralstonia solanacearum is the causal agent of the bacterial wilt disease, which can affect more than 250 plant species including major crops and model plants such as Arabidopsis thaliana. As a potent R. solanacearum virulence factor, PopP2 is an acetyltransferase from the YopJ family that dampens basal immune responses by targeting defensive WRKY transcription factors. In order to better understand the virulence activities of PopP2, we searched for PopP2-interacting proteins using a yeast two hybrid assay, and identified the GTE9 and GTE11 proteins from the GTE family (General Transcription factor, group E). GTE proteins possess a bromodomain, a specific protein module allowing interaction with acetylated lysine residues, notably on histones tails suggesting that they could be involved in epigenetic-related processes. GTE9 and GTE11 were previously shown to (i) co-localise and interact with PopP2 in the plant nucleus, and (ii) to be acetylated by PopP2. Also, GTE9 and GTE11 were shown to interact in planta with Histone H4 through their bromodomain, suggesting that they function as epigenetic readers whose manipulation by PopP2 would promote R. solanacearum virulence. In this context, the main objectives of my PhD were to better understand the the function of GTE9 and GTE11, by trying to determine how they can be manipulated by PopP2 and whether these proteins play a role in the plant response to R. solanacearum. Mass-spectrometry-based analysis enabled us to map the lysine residues modified by PopP2 in GTE9 and GTE11. Several of these residues are conserved between the two proteins and localised on either side of their bromodomain. By semi-quantitative FRET-FLIM assay performed in vivo, we demonstrated that GTE9 interaction with Histone H4 is altered by PopP2 acetyltransferase activity suggesting that PopP2 uses acetylation to dissociate GTE9 from chromatin. In addition to GTE9 and GTE11, PopP2 acetylates several other GTE members. Regarding the role of GTE9 and GTE11 in the plant response to R. solanacearum, GTE9 and GTE11 over-expressing lines displayed an enhanced disease response to R. solanacearum that depended on PopP2 enzymatic activity. Overall, these data indicate that GTE9 and GTE11 behave as epigenetic readers that are manipulated by a plant bacterial pathogen through their targeting by a YopJ family acetyltransferase. GTE proteins could represent key virulence targets for R. solanacearum since PopP1, an additional YopJ family acetyltransferase that belongs to its effector repertoire, also interacts with several of these proteins. How the targeting of GTE proteins is mechanistically impacting the overall course of R. solanacearum infection remains elusive. To answer this question, we undertook a ChIP-seq analysis aimed at identifying the chromatin regions targeted by GTE9 and GTE11 (approach in progress). In addition to this approach, we wanted to identify more globally the chromatin sites visited by PopP2 in Arabidopsis. For this, we have initiated a second ChIP-seq analysis using various molecular tools including tagged versions of PopP2 for in planta delivery through a bacterial type III secretion system. Overall, this PhD work allows to progress on the understanding of a virulence strategy used by a plant bacterial pathogen that consist in manipulating host epigenetic components to promote infection.

Je tiens à remercier infiniment Laurent Deslandes. Tu m'a épaulée depuis le stage de master jusqu'à la fin de ma thèse, toujours présent pour répondre à mes questions et m'aiguiller dans ce projet, et m'apprendre les ficelles du métier. Même dans les moments où tu étais moins disponibles je savais que je pouvais compter sur toi. Ma thèse s'est bien passée en grande partie grâce à ton encadrement, tu es un encadrant en or ! Un grand merci à Françoise Jardinaud, pour m'avoir aider à comprendre comment analyser mes données en RT-qPCR.

Merci à toutes les personnes qui ont été gentilles avec moi pendant mon passage au LIPME.

Merci à mes amis, qui m'ont suivie de près ou de loin pendant ma thèse. Merci à Estelle et Aymeric, pour tous les bons moments de détente passés ensemble ; à Beubeu et Erla pour m'avoir sauvé de la dépression lors du 1 er confinement ; à Julie pour notre soutien mutuel de thésardes parfois en souffrance ; et petite dédicace à Amélie, pour m'avoir souvent dit d'être moins dure avec moi-même et ramenée vers la voie de l'optimisme tant de fois.

Papa, merci de m'avoir soutenue toutes ces années, jusqu'à mon dernier diplôme (normalement) le voilà tu l'attendais avec fierté, et m'y voilà ! Je ne le dis pas souvent mais tu es dans mon coeur papa.

Mamounette, sans les journées et les soirées à me forcer à me faire faire mes devoirs en étant petite, je n'en serais probablement pas là. Tu suis peut être de loin mon parcours désormais mais je pense souvent que c'est grâce à ta persévérance à me forcer d'être moins paresseuse que j'y suis arrivée (je suis un caillou mais pas trop finalement hein ?).

Je t'aime très fort Mamounette.

Merci aussi à toute ma famille, pour m'avoir soutenue et toujours compris lorsque je me renfermais et ne voulais pas parler de ma thèse, alors que vous vouliez en savoir plus.

And last but not least, I want to deeply thank you, Bram. We began sharing our lives at the end of both our PhDs and I know the end of mine has been difficult with my rollercoaster mood. Thank your for not holding it against me, never reproaching it to me, and for supporting me through all of it. Thank you for loving me. 5. Quantitative FRET-FLIM measurements showing that catalytically active PopP2 alters the ability ofGTE9 to interact with histone H4 in nuclei of N. benthamiana cells. As one of the most important criteria in crop production is yield, crop diseases are a major concern in one's mind as they can cause yield loss (Savary et al., 2019).

Figures and Tables list

Results and Discussion

Supplementary Table

Supplementary Table 6. Primers used in this study

Complements to the manuscript

However, we should remember that most plants are resistant to most pathogens and that only the most successul pathogens infect plants and cause disease. To summarise the different mechanisms underlying plant-pathogen interactions, several concepts were introduced in the literature and in particular related to plant defence responses to pathogens. Non-host resistance describes the concept in which all members of a plant species are resistant to all variants of a pathogen.

The plant species is then considered as non-host and is, therefore, fully resistant to this pathogen. Otherwise, if a pathogen is able to infect a plant species and only some genotypes of that species are resistant to the pathogen, we consider it to be host-resistance. In addition, if a pathogen fails to infect a plant genotype, the interaction is considered "incompatible" and if the pathogen succeeds infecting the plant genotype and causes symptoms, the interaction is considered "compatible" (Heath, 1981). Nowadays, a plant genotype is said "susceptible" to a pathogen when the interaction is compatible while it is said "resistant" to a pathogen when the interaction is incompatible.

To prevent pathogens from infecting them, plants possess various protection mechanisms. Plants protect themselves from outside threats with reinforced surfaces like tree bark; a wax layer called cuticle that covers leaves, fruits, flowers and non-woody stems; and cell wall that gives both structure and protection against pathogens and pests. Despite these mechanical barriers, pathogens can invade plants through wounds or degrade these barriers, resulting in disease.

Interaction with microbes also triggers the activation of an innate immune system in plants and constitute another level of protection from infection. To summarise Jones and Dangl, 2006). The zig-zag model describes the arms-race that takes place between plants and pathogens upon interaction. Plants can detect conserved molecules from pathogens, PAMPs, through cell surface receptors, PRRs. The PAMP recognition by PRRs triggers PTI responses. During evolution, pathogens have evolved effectors capable of disturbing cellular processes to promote infection, known as ETS. Some effectors can be detected by plants through NLR (R) proteins leading to ETI responses, often associated with a programmed cell death (Hypersentive Response (HR) response). To counteract activation of host immune responses and evade recognition, pathogens evolve new effectors or delete/modify others. In turn, plants can also evolve new recognition mechanisms/capabilities to improve pathogen recognition and immunity activation.

Effector-R protein

Effector-R protein the mechanisms underlying plant-pathogen interactions, the zig-zag model proposed by Jones and Dangl in 2006 integrated a great deal of knowledge about these mechanisms into a simple and elegant model described below. This model has the benefit of including a large number of known mechanisms related to plantpathogen interactions and of taking into account the molecular arms race between plants and pathogens. However, this model suffers from certain limitations that will also be discussed later in this introductory chapter and that takes into account recent advances in the understanding of the mechanisms involved in the activation of plant immune responses.

B. The zig-zag model: a model to summarise the molecular bases of plant-pathogen interactions molecular bases

• B.1.

Presentation of the model

The zig-zag model describes plant-induced immune responses against pathogens in several layers (Figure 1) (Jones and Dangl, 2006). The first layer that corresponds PTI interference by effectors is denominated Effector-Triggered Susceptibility (ETS). On the plant side, plants have evolved specific immune receptors, the R proteins, that are able to specifically recognise effectors directly or indirectly, leading to Effector-Triggered Immunity (ETI). A successful pathogen is a "discrete" pathogen that will not be defeated by the plant's immune responses. Therefore, evolution selects pathogens that evade recognition by the plant immune system, so ones that do not possess a recognised effector anymore or a mutated effector variant that evades recognition, or that possess new effectors that counteracts the recognition of other effectors. This new layer, called the 2 nd ETS, is countered by mechanisms evolved in plants to prevent pathogens from infecting plants. PTI and ETI downstream signalling involves various events such as transcriptional reprogramming in plant cells with, for instance, up-regulation of defence-related genes and genes encoding proteins associated with hormone signalling (Jones and Dangl, 2006).

• B.2. PAMP-Triggered Immunity (PTI) B.2.1. PAMPs are conserved microbial motifs

PAMPs are generally regarded as general elicitors perceived by apoplastic cell surface receptors. One reason for this is that some of them are well conserved among different pathogen genera. For instance, flagellin is one of the most studied PAMPs in plant and animal pathogenic bacteria. In plants, the conserved 22amino-acid (aa) peptide flg22 that corresponds to a domain of the flagellin amino terminus (N-term) is a potent elicitor of immune responses in Arabidopsis and other plant species.

Known PAMPs include a large number of different relatively conserved molecules, such as motifs from bacterial or fungal cell wall proteins, elongation protein factors, proteases, cell-wall degradation enzymes... Some specific exemples include peptidoglycan fragments, lipopolysaccharides, protein elongation factor Tu (EF-Tu) from bacterial pathogens, chitin from fungal pathogens, as well as β-1,3 or β-1,6-glucans from oomycetes (Saijo et al., 2018).

We tend to think that PAMPs are highly conserved patterns and are widely recognised by PRRs, but this may not be so strict, as evidence in the literature show examples of PAMPs recognised by a narrower range of plant species. Indeed, the flgII-28 peptide is a flagellin peptide distinct from flg22 that elicits responses in some Solanaceae species but not in Arabidopsis (Cai et al., 2011). Another example is the peptide elf18 from EF-Tu, which is sufficient to elicit defence responses in Brassicaceae species but not in plants outside this family. This suggests that PAMP diversification facilitates PTI evasion and that, despite the role of PAMPs as general elicitors, evolutionary pressure may increase the host-pathogen specificity of PAMP perception (Saijo et al., 2018;Yu et al., 2017).

B.2.2. PRRs are extracellular receptors that recognise PAMPs

Most PRRs are divided into two categories: Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs). They are transmembrane receptors with an extracellular domain, a transmembrane domain and an intracellular domain. The main difference between RLKs and RLPs resides is the presence of an intracellular kinase domain in RLKs but not in RLPs. RLKs are involved in various plant growth and developmental processes, such as cell expansion, cell division and cell proliferation, but also in disease resistance and responses to abiotic stresses. RLPs are mainly associated with the detection of pathogenic threats. The extracellular domains of PRRs determine the specificity of ligand binding. These domains include leucine-rich repeats (LRRs), lysine motifs (LysMs), lectin motifs and epidermal growth-factor-like domains (Tang et al., 2017;Yu et al., 2017) (Figure 2).

The two more common extracellular domains are LRR and LysM that can be found in both RLKs and RLPs. LRR-RLKs and LRR-RLPs are involved in the perception and signalling of peptide ligands. They include the well-studied Flagellin-sensing 2 (FLS2) and the EF-Tu receptor (EFR). LysM-RLKs and LysM-RLPs function in the perception and signalling of microbial patterns containing acetylated glucosamine. For instance, chitin perception is possible through the LysM-RLK CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) in Arabidopsis (Tang et al., 2017).

One of the first signalling steps after PAMP perception by RLKs is the phosphorylation of downstream proteins by the kinase domain of RLKs. Since RLPs lack a kinase domain, signalling following PAMP perception by RLPs involves the association of RLPs with RLKs. However, RLKs also tend to act as complexes with other RLKs, triggering signalling processes (Saijo et al., 2018). For instance, LRR-RLKs have been shown to recruit BRI1-ASSOCIATED RECEPTOR KINASE (BAK1), a LRR-RLK and other members of the SOMATIC EMBRYOGENESIS RECEPTOR KINASES family (Tang et al., 2017). After ligand perception, many LRR-RLPs form a complex with the adaptor LRR-RLK SOBIR1 and then with BAK1 (Saijo et al., 2018).

B.2.3. PRR/PAMP examples

flg22 recognition by the LRR-RLK FLS2 in Arabidopsis is well described in the literature (Figure 2). This receptor was discovered by screening flg22-insensitive Arabidopsis mutant plants (Boller and Felix, 2009). In the resting state, this receptor does not form a stable complex with BAK1 but interacts with several Receptor-Like Cytoplasmic Kinases (RLCKs) including BOTRYTIS-INDUCED KINASE 1 (BIK1) and BR-SIGNALLING KINASE 1 (BSK1) which will be involved in early signalling (He and Wu, 2016). Upon perception of flg22 by FLS2, FLS2 and BAK1 associate and this association is required to mediate immune responses (Tang et al., 2017). The formation of this RLK-RLK complex induces a rapid phosphorylation of BAK1 and FLS2 and allows initiation of intracellular signalling (Yu et al., 2017). This includes the release of BIK1 and BSK1 which will positively regulate some PTI responses including the production of Reactive Oxygen Species (ROS) (Tang et al., 2017;Yu et al., 2017).

A chitin receptor was also identified by screening Arabidopsis mutants with altered responses to chitin leading to the identification of the LysM-RLK CERK1. It belongs to the LysM receptor-like kinase (LYK) family which comprises five members in Arabidopsis including CERK1, also known as LYK1. Upon chitin perception, CERK1 heterodimerises with LYK5, resulting in homodimerisation and phosphorylation of CERK1 in response to chitin. Although CERK1 was the first PRR identified that perceives chitin, LYK5 has been shown to have a higher affinity for chitin than CERK1, suggesting that LYK5 is the primary chitin-binding protein in Arabidopsis instead of CERK1 (Cao et al., 2014). CERK1 interacts with BIK1 in a similar way than FLS2, and phosphorylated CERK1 phosphorylates BIK1 upon chitin perception leading to the activation of ROS production. CERK1 also interacts with the RLCK PBL27 and phosphorylates it, leading to the activation of the Mitogen Activated Protein Kinases (MAPKs) cascade (Yu et al., 2017). These different interactions and responses following chitin perception provide an insight into the signalling events triggered by chitin perception. Surprisingly, CERK1, in association with the two RLPs LYM1 and LYM3, recognises bacterial peptidoglycans and induce cellular responses, suggesting that a PRR can mediate the recognition of different PAMPs depending on its association with other proteins (He and Wu, 2016;Tang et al., 2017). domain that is supposed to act as a signalling domain, a nucleotide-binding APAF1, R gene products and CED-4 domain (NB-ARC) proposed to act as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms, and the C-term LRR domain that determines recognition specifity (Sun et al., 2020). Most NLRs are structured with these three domains and are commonly classified based on their N-term part, the CC-NLRs or TIR-NLRs. Other NLRs display a different structure such as additional domains or truncated NLRs lacking the LRR domain or both the NB-ARC and the LRR domain (Baggs et al., 2017). NLRs are found in different cellular compartments: they are present in the cytoplasm, the nucleus, or also at the plasma membrane. Furthermore, their location can change after activation (Song et al., 2021b). In resting state, the amount of NLRs is kept at a basal level and/or in an inactive state in order to avoid unnecessary activation of immunity to maintain the trade-off between development and immunity, both The first proposed mechanism of effector recognition was based on the gene-forgene hypothesis (Flor, 1971). According to this hypothesis, the outcome of a plantpathogen interaction depends on two factors: the pathogen's avirulence genes, now known as effector-encoding genes, and the plant's R genes, now known as NLR-encoding genes. The gene-for-gene hypothesis suggested that for every avirulence gene in a pathogen there was a corresponding R gene in plants and that the product of an avirulence gene could correspond to a ligand interacting with its matching plant R protein. The absence or presence of one or the other determined the success or failure of the interaction (Flor, 1971). Direct effector recognition has been documented for some NLRs: AvrL567 from the fungus Melampsora lini is directly recognised by the flax NLRs L5, L6, and L7 (Ellis et al., 2008), and the barley NLR MLA directly recognises the effector AVR A from the fungal pathogen Blumeria graminis f. sp. hordei (Saur et al., 2019) (Figure 3A). Nevertheless, scientists tend to think that direct effector recognition is not the major recognition mechanism. 3A). This is what we call the "guard model" (van Wersch et al., 2020).

In detail, we distinguish the guard model from the decoy model. In the guard model, a NLR guards a plant protein that has a specific biological function, whereas the decoy mimics a host effector target, but has no real biological function. The decoy only serves as a warning signal to trigger immune responses (van Wersch et al., 2020). For instance, the Arabidopsis NLR HOPZ-ACTIVATED RESISTANCE1 (ZAR1) guards several host proteins from different effectors of Pseudomonas syringae and Xanthomonas camprestris that are either guardees or decoys. For example, PBL2 is a RLCK targeted by the X. campestris effector AvrAC. Uridylation of PBL2 by AvrAC triggers ZAR1 activation (Wang et al., 2015b). In that case, PBL2 is a guardee as this RLCK is also required for the perception of PAMPs like flg22 and elf18 (Zhang et al., 2010). Another example is the targeting of the RLCK HOPZ-ETI-DEFICIENT1 (ZED1) by the effector HopZ1a of P. syringae. HopZ1a is an acetyltransferase that can acetylate ZED1 and trigger ZAR1-mediated immune responses. ZAR1 guards ZED1 but in this case ZED1 is considered as a decoy because in absence of ZAR1, ZED1 does not contribute to plant immunity or susceptibility in response to P. syringae (Lewis et al., 2013). It is not always clear whether a guarded protein is a guardee or a decoy but in both cases the recognition mechanism is indirect.

B.3.1.c. A particular type of decoy: the Integrated Decoys (ID) Some NLRs harbour unusual domains called integrated domains (IDs) and are present in many plant species (NLR-IDs) (Grund et al., 2019;Kroj et al., 2016;Sarris et al., 2016). Some IDs from NLR-IDs mimick virulence targets of effectors making these domains integrated decoys but for most of NLR-IDs we do not know whether their IDs serve as decoys (Figure 3A). Well-characterised examples of NLR-IDs include the Arabidopsis RRS1 (RESISTANCE TO RASTONIA SOLANACEARUM1)

and the rice NLRs RGA5 and Pik-1. RRS1 will be detailed later in this introduction in a specific section. In rice, both the RGA5 and Pik-1 NLRs harbour a C-term Heavy-Metal-Associated (HMA) domain, a domain orignally found in a Saccharomyces cerevisiae copper chaperone. The HMA domain of RGA5 interacts directly with the effectors AVR-PiA and AVR1-CO39 from the fungus Magnaporthe oryzae (Césari et al., 2014). Are HMA domain-containing proteins targeted by effectors? This is not yet known, but Pi21, a HMA-containing protein, is an important factor for total susceptibility to M. oryzae (Fukuoka et al., 2009;Zhang et al., 2016b). However, it has been recently documented that the effector AVR-Pik from the same fungus, which is recognised by the NLR-ID Pik-1 which possesses a HMA domain, targets HMA domain-containing proteins. Indeed, AVR-Pik variants have been demonstrated to target proteins from the HMA isoprenylated plant protein family (HIPP) (Oikawa et al., 2020). Various variants of AVR-Pik have been demonstrated to interact with the HMA domains of OsHIPP19 and OsHIPP20, and OsHIPP20 was shown to be important for M. oryzae infection since a Oshipp20 knock-out mutant is less susceptible to the fungus than the WT (Maidment et al., 2021;Oikawa et al., 2020).

B.3.2. NLRs work often in cooperation

NLRs have frequently been observed to act in cooperation with other NLRs (Figure 3B). Interestingly, the first hints of cooperation between NLRs might be found in the location of the genes on chromosomes as in vascular plants, NLR-encoding genes are often arranged in pairs or located in complex multi-gene clusters. An Several NLRs have been reported to form homodimers and for some of them this homodimerisation plays a role in NLR activation and/or signalling function (Li et al., 2015a;Nguyen et al., 2021). For instance, the TIR domain of the flax NLR L6 was found to mediate L6 self-association and immune signalling was abolished when L6 homodimerisation was disabled (Bernoux et al., 2011). Similar results

were obtained on the CC domain of the barley NLR MLA10 (Maekawa et al., 2011).

B.3.2.b. Heterodimerisation of NLRs

Homodimerisation is not the only way NLR can associate. Some NLRs form heterodimers and participate in the regulation of their immune functions (Nguyen et al., 2021). Two well-studied heterodimer-forming pairs of NLRs are the TIR-NLRs RESISTANCE TO PSEUDOMONAS SYRINGAE 4 (RPS4) with RRS1 from Arabidopsis, and the CC-NLRs RGA4/RGA5 from rice. In Arabidopsis, RRS1 and RPS4 heterodimerise due to a strong affinity of their TIR domains that can be compromised if the domains are mutated (Williams et al., 2014). The specific mechanisms of RPS4/RRS1 interaction will be detailed later in this introduction. In rice, RGA4 and RGA5 form a heterodimer via their CC domains that prevents cell death activation by RGA4. RGA4 is released from RGA5 after recognition of the M.

oryzae effector AVR-Pia, leading to activation of cell-death (Césari et al., 2014).

B.3.2.c. NLRs can associate in higher structure shapes: the resistosomes Recent breakthroughs have revealed interesting complex assocation structures of NLRs known as resistosomes that largely ressemble the inflammasomes described in animal immunity activation (Ma et al., 2020;Martin et al., 2020;Wang et al., 2019).

In 2019, Wang and colleagues revealed the switching mechanism of the Arabidopsis CC-NLR ZAR1. In a resting state, ZAR1 interacts with the pseudokinase RKS1. Upon infection with Xanthomonas campestris pv. campestris, the secreted AvrAC effector uridylates the RLCK PBL2 which will activate ZAR1.

Indeed, uridylated-PBS2 interacts with the ZAR1-RKS1 complex and changes its conformation leading to a pentamerisation in a ring-like structure of the ZAR1-RKS1-PBL2 complex, called the ZAR1 resistosome (Wang et al., 2019). This resistosome forms a funnel-shaped structure that can insert into the plasma membrane to form a pore, like a calcium-channel, which triggers plant immune signalling (Bi et al., 2021).

Furthermore, it was shown in 2020 that the TIR-NLR RECOGNITION OF XopQ 1 (ROQ1) of Nicotiana benthamiana and the TIR-NLR RPP1 of Arabidopsis form tetrameric resistosomes after activation by the Xanthomonas euvesicatoria effector XopQ, and the Hyaloperonospora arabidopsidis effector ATR1, respectively (Martin et al., 2020;Ma et al., 2020). These new results hint that cooperation of NLRs through oligomerisation, either in pairs or through higher structures like resistosomes, is more common than previously thought and is important for NLR activation and signalling. It is also quite interesting to notice that these oligomeric structures in plant NLRs are similar to the ones described in animal NLRs, for which this type or higher level of oligomerisation has already been found several years ago (van Wersch et al., 2020).

B.3.2.d. NLRs cooperate with helper NLRs

In addition to homo-or hetero-oligomerisation of NLRs, NLRs can also cooperate with another type of NLR known as helper NLRs (hNLRs) (Figure 3C). The presence of hNLRs is necessary for the activation of NLR-mediated immune responses. The hNLRs do not function in effector recognition but rather in downstream signalling of NLR activation (Jubic et al., 2019). Three important families of helper NLRs have been reported: the ACTIVATED DISEASE RESISTANCE 1 (ADR1) family, the N REQUIRED GENE 1 (NRG1) family and the NLR required for cell death (NRC) family which is specific to Solanaceae (Castel et al., 2019;Wu et al., 2017). Which family is involved depends on the type of NLRs with which it cooperates but they all contribute to the activation of ETI-mediated defence responses (Saile et al., 2020).

For instance, TIR NLR signalling pathways involve the ADR1 and NRG1 family of hNLR and require another protein called Enhanced disease susceptibility 1 (EDS1) (Feehan et al., 2020).

• B.4. How is pathogen perception translated into immune responses?

PAMP recognition induces the formation of a PRR complex that will lead to a series of cellular responses summarised in Figure 4. These events start from transphosphorylation of PRRs after PAMP-induced complex formation to downstream signalling comprising calcium signalling, plasma membrane depolarisation, activation of MAPK-signalling cascades, ROS burst, activation of hormonal pathways, and transcriptional reprogramming. It also involves RLCKs, heteromeric G proteins, plasmodesmata and stomata closure, and callose deposition (Yu et al., 2017).

While the recognition phenomena between PTI and ETI are distinct, the signalling events following PAMP or effector perception overlap for many signalling events.

Some signalling pathways are triggered by both PAMP or effector perception but differ in intensity or duration (Yuan et al., 2021a).

B.4.1. Ca 2+ influx and other ion fluxes

During PTI, one of the earliest responses after PAMP treatment is an increase in cytosolic Ca 2+ concentration ([Ca 2+ ]cyt). This increase starts after 30-40 seconds, peaks at 2-6 minutes and lasts for about 30 minutes before returning to the resting state. The intensity of the increase in [Ca 2+ ]cyt depends on the dose and the type of PAMP perceived but in any case, this [Ca 2+ ]cyt induces the opening of ion channels (H + influx, K + , Cl -and NO -efflux) leading to membrane depolarisation and apoplastic alkanisation (Bigeard et al., 2015;Yu et al., 2017).

Calcium signalling is also triggered in ETI via NLR signalling but the influx of [Ca 2+ ]cyt is slower and more sustained than the one triggered during PTI. Recent discoveries about NLR resistosomes that can act as channels on the plasma membrane lead scientists to wonder whether these resistosomes might be involved in [Ca 2+ ]cyt influx but this requires further investigation (Yuan et al., 2021a). ]cyt and is required for stomatal closure and callose deposition (Tang et al., 2017;Yu et al., 2017).

ROS production is also induced during ETI, with the difference that the ROS burst is biphasic with two peaks and the second peak is usually much stronger and sustained than the first. Interestingly, the ETI-triggered ROS burst requires exposure to PAMPs, suggesting that ETI-associated ROS bursts depends on PRR signalling (Ngou et al., 2020;Yuan et al., 2021b). This shows that in addition to common signalling pathways, PTI and ETI appear to depend on each other for full potentiation of responses. pathogens. This cascade is also required for stomatal immunity (Thulasi Devendrakumar et al., 2018). The MEKK1-MKK1/2-MPK4 cascade was thought to have a negative role on plant defence as disruption of this cascade resulted in autoimmunity (Thulasi Devendrakumar et al., 2018). However, reports have shown that this MAPK is guarded by SUPPRESSOR OF MKK1 AND MKK2 2 (SUMM2), a CC-NLR that monitors phosphorylation of a substrate of MPK4 (Bigeard et al., 2015;Thulasi Devendrakumar et al., 2018). This suggests that autoimmunity in the disrupted mutant of the MEKK1-MKK1/2-MPK4 cascade may be due to SUMM2 monitoring mechanisms. Challenging summ2 mekk1 and summ2 mkk1 mkk2 mutant plants with Pseudomonas syringae DC3000 led to a greater susceptibility than in summ2 mutants, suggesting that MEKK1-MKK1/2-MPK4 cascade promotes plant defence against pathogens. Thus, it seems that MEKK1-MKK1/2-MPK4 cascade also positively regulates immune responses against pathogens and is guarded by SUMM2 that will trigger immunity activation when the cascade is disrupted (Thulasi Devendrakumar et al., 2018).

The activation of MAPK cascade upon treatment with PAMP takes a few minutes.

Indeed, in A. thaliana, treatment with flg22 triggers the activation of four MAPKs (MPK3, MPK4, MPK6 and MPK11) within 1-2 minutes and MAPK activity reaches its peak at 10-15 minutes (Bigeard et al., 2015). During ETI, MAPK activation is slower but more sustained. Although MAPK cascades activation following PAMP perception is known to be mediated by RLCKs, how NLR signalling activates MAPK cascades remains unknown (Yuan et al., 2021a). Surprisingly, MAPK cascades cannot be triggered by the RRS1/RPS4 and RPP4 TIR-NLRs after inducible expression of AvrRps4 or AvrRpp4 in Arabidopsis, revealing that NLR-mediated activation of MAPK cascade cannot be triggered without PRR signalling (Ngou et al., 2020).

B.4.4. Phytohormones

Phytohormones play a central role in plants as they are involved in both development and immunity. The main hormones involved in immunity are salicylic acid, jasmonic acid, ethylene and abscisic acid. We can also include cytokinins, auxin, gibberellins and brassinosteroids to a lesser extent (Shigenaga and Argueso, 2016). While hormonal pathways related to plant immunity promote plant resistance to pathogens, this is not without a fitness cost to the plants. Hence, fine-tuning of strong hormone-mediated immune responses is necessary to maintain the trade-off between growth and immunity (Ballaré and Austin, 2019;van Butselaar and Van den Ackerveken, 2020;Vos et al., 2015).

B.4.4.a. Salicylic acid (SA)

Despite its roles in plant development and resistance to abiotic stresses, the phenolic compound SA is primarily known as a key plant hormone required to promote plant defence responses against many biotrophic and hemibiotrophic pathogens. SA accumulation at pathogen infection sites correlates with plant resistance to pathogens (Shigenaga and Argueso, 2016). SA or related metabolites are involved in regulating HR and System Acquired Resistance, which represents a kind of systemic induced resistance. Several genes are required for SA response including Nonexpresser of PR genes 1 (NPR1), identified by forward genetic screens for mutants insensitive to SA induction. NPR1, together with other NPR proteins, seem to operate as hubs mediating the expression of SA-responsive genes, including the PATHOGENESIS RELATED (PR) genes that encode several proteins with antimicrobial activities promoting plant resistance to a wide range of pathogens (Ding and Ding, 2020;Verma et al., 2016).

based on studies performed on a limited number of dicot plants, including Arabidopsis, tomato and tobacco. However, there is increasing evidence showing the importance of JA-induced defence mechanisms in monocot and gymnosperm species in response to pathogens and herbivores showing that JA is not always associated with resistance against necrotrophs (Campos et al., 2014). Indeed, JA elicits immunity in rice against biotrophic and hemibiotrophic parasites such as the root-knot nematode Meloidogyne graminicola and the bacterial pathogen Xanthomonas oryzae. As such JA is involved in defence mechanisms against different types of pathogens but it depends on the pathosystem (Nahar et al., 2011;De Vleesschauwer et al., 2013).

After pathogen perception, ET biosynthesis is stimulated and is then sensed by its receptors triggering a signalling cascade involving the Ethylene-responsive factor (ERF) transcription factor (TF) family, which plays an important role in defence regulation. In dicots, ET is best known to act synergistically with JA to promote plant defence against necrotrophic pathogens (Broekgaarden et al., 2015;Pieterse et al., 2012).

B.4.4.c. Abscisic acid (ABA)
ABA is a major hormone associated with various stages of plant growth but also with abiotic stresses including drought and salinity. There is also a growing body of research on the role of ABA in plant responses to biotic stresses. One of the best known roles of ABA in plant responses to biotic stresses is the control of stomatal closure in response to pathogen attack to prevent or restrict pathogen entry (Chen et al., 2020). ABA can induce the opening of Ca 2+ channels to close stomatal pores and activate Open Stomata1 (OST1), a key regulator of stomatal closure. PAMPinduced stomatal closure can be triggered in an OST1-dependent or -independent manner (Su et al., 2017;Zheng et al., 2018). However, ABA has a broader role in plant defence against pathogens than the control of stomatal closure to prevent pathogen entry.

In many pathosystems, ABA has been reported to negatively affect plant defences.

In some studies, a host plant species pretreated with ABA could lead to an increased susceptibility to a pathogen. This has been demonstrated in rice infected with the fungus Magnaporthe grisea (Jiang et al., 2010), in wheat in response to the fungus Fusarium graminearum (Buhrow et al., 2016), but also in barley in response to the fungus M. oryzae (Ulferts et al., 2015). Other studies have investigated the response of plants mutated in the ABA pathway to pathogens. For instance, an ABA-hypersensitive mutant showed increased susceptibility to P.

syringae (Goritschnig et al., 2008). Another example is the infection of barley mutants in ABA synthesis with M. oryzae which led to a lower susceptibility implying a negative role of ABA in barley defence responses to M. oryzae (Ulferts et al., 2015).

In other pathosystems ABA seems to play a beneficial role in plant defence. In A.

thaliana, infection by the oomycete Pythium irregular of ABA-insensitive mutants or mutants impaired in ABA biosynthesis revealed increased susceptibility compared to the wild-type (WT), suggesting that ABA has a positive role in plant defence in this pathosystem (Adie et al., 2007). Furthermore, exogenous application of ABA to rice prior to infection by the fungus Cochliobolus muyabeanus increased basal resistance and limited pathogen progression (de Vleesschauwer et al., 2010). Finally, Arabidopsis mutants impaired in ABA biosynthesis were

shown to be more susceptible to Bamboo mosaic virus (BaMV), suggesting that ABA pathway has a positive role in Arabiopsis defences against this virus (Alazem et al., 2014). Thus, ABA seems to be involved in plant response to pathogens and its role is pathosystem-dependent.

B.4.4.e. Hormonal crosstalk

All of the plant hormones described above are involved in mediating immune responses against various biotic stresses. It is clear that there is no single master hormone that governs the activation of immunity in plants. Instead, plant immunity is modulated by a complex hormonal network called hormonal crosstalk.

In dicots, one parameter influencing the type of hormonal response against a given pathogen is the pathogen's lifestyle. SA is generally required for defence against biotrophic or hemi-biotrophic pathogens, whereas JA is mostly required for defence against necrotrophic pathogens, making these two pathways act antagonistically to each other. Other hormones, like cytokinins, auxin, gibberellins, and brassinosteroids play an important role in plant development, but also play a role in plant immune responses and are often involved in SA or JA/ET pathways, adjusting hormone signalling to adapt immune responses to the invading pathogen (Shigenaga and Argueso, 2016). It is also worth mentioning that hormone-mediated immune responses require a lot of energy that cannot be used for growth at the same time. Thus, hormonal crosstalk might be essential to adapt the immune response to invading pathogen, but it might also be cost saving in fitness to combine different pathways to promote defence to pathogens (Shigenaga and Argueso, 2016;Vos et al., 2015;Yang et al., 2012).

B.4.5. Transcriptional reprogramming

Plant-pathogen interactions induce a massive and dynamic reprogramming of plant gene expression, referred to as transcriptional reprogramming. Transcriptional reprogramming is the main link between signal transduction and the activation of defense mechanisms.

At the top of plant immunity transcriptional regulation, the SARD1 (System Acquired Resistance Deficient 1) and CBP60g (Calmodulin binding protein 60g)

TFs act as a convergent point in PTI and ETI signalling (Peng et al., 2018). Originally

shown to be involved in the SA signalling pathway, chromatin-immunoprecipitation sequencing (ChIP-seq) analysis demonstrated that SARD1 and CBP60g directly regulate the expression of a large number of plant immunity-related components.

Since they regulate many positive and negative regulators of immune responses, they are considered as master regulators of plant immunity (Sun et al., 2015).

In addition to these master regulators, several families of TFs are involved in the transcriptional reprogramming associated with plant immunity. TFs of the basic leucine-zipper subfamily TGA (bZIP/TGA) are positive regulators of SA-mediated gene expression and defence against biotrophs. The basic-Helix-Loop-Helix (bHLH) TF family includes members that are key regulators in JA responses and crosstalk with other phytohormones (Tsuda and Somssich, 2015).

Another important family of defensive TFs is the WRKY family. WRKY TFs are characterised by a conserved 60-aa DNA-binding domain called the WRKY domain, containig a highly conserved WRKYGQK heptad motif responsible for DNA binding activity. These TFs bind to the cis-regulatory "W-box" motifs in their DNA target sequences via their WRKY domain(s). W-boxes have been identified in the promoters of many genes associated with PTI or ETI and WRKY TFs have been shown to act as positive and negative regulators of ETI and PTI. For instance, AtWRKY33 is a negative regulator of SA-mediated plant defences but is a positive regulator of plant resistance against the necrotroph Botrytis cinerea; and AtWRKY70, AtWRKY46 and AtWRKY53 are involved in the positive regulation of plant defence against P. syringae (Garner et al., 2016;Pandey and Somssich, 2009;Tsuda and Somssich, 2015).

B.4.6. Hypersensitive Response and Systemic Acquired Resistance

After the perception of a pathogen, one way to stop the progression of the pathogen in plant is to condemn the site of infection. This phenomenon is called the Hypersensitive Response (HR) and is characterised by a rapid and localised cell death at the site of pathogen penetration. HR is often associated with ETI notably (Balint-Kurti, 2019).

While HR is a way of stopping pathogen invasion at the site of infection, plants can also protect distal tissues from present and future infections. This systemic defence is called system acquired resistance (SAR) and relies on the activation of systemic immune responses conferring broad-spectrum resistance at the whole plant level. It involves mobile signals generated at the site of infection, which then translocate to distal tissues to prepare the plant for future infections. Mobile

Extracellular

signals include SA, pipecolic acid (a methylated derivative of SA) and ROS to name a few (Shine et al., 2019).

• B.5. How phytopathogens facilitate infection: focus on Effector-

Triggered Susceptibility

To counteract basal immune responses and promote infection of their host, adpated pathogens have developed specialised proteins called effectors that promote infection by interfering with various plant cellular processes (Figure 5). In with DNA into the host cell (Costa et al., 2015;Hayes et al., 2010). Filamentous pathogens do not have these needle-like secretion systems but use different secretion systems via specialised structures like haustoria (Bozkurt and Kamoun, 2020). Once inside the cell, effectors will target various host components to make the environment beneficial to the pathogen, or to counter immune responses to promote infection. Some effectors can be recognised or detected by the infected plant leading to ETI responses (cf. B.3., Figure 3). There is increasing evidence of plant pathogen effectors targeting MAPK cascades.

For instance, the P. syringae HopAI1 effector targets MPK3 and MPK6 in Arabidopsis. Indeed, in presence of HopAI1, these MAPKs are permanently dephosphorylated on a key threonine residue, indicating that HopAI1 is a phosphothreonine lyase, an enzyme that covalently modifies phosphorylated threonines, leading to permament dephosphorylation of this residue and impairment of PTI responses (Zhang et al., 2007a (Üstün and Börnke, 2015). Degradation of RPT6 decreases proteasome activity and impairs the turnover of NPR1, a major regulator of SA-dependent responses (Üstün and Börnke, 2015;Üstün et al., 2013).

Effectors can also interfere with phytohormone synthesis. The Phythophthora sojae effector PsAvh238 is a virulence factor promoting disease in soybean (Glycine max). PsAvh238 interacts with soybean type 2 1-aminocyclopropane-1carboxylate synthases (Type2 ACSs), and trigger the destabilisation of these enzymes which are involved in ethylene biosynthesis, promoting infection by P.

sojae (Yang et al., 2019).

B.5.2.d. Effectors can interfere with plant cytoskeleton Some effectors have been reported to interfere with the plant cytoskeleton that is essential for many cellular processes including cell division and growth, vesicle trafficking, endocytosis, organelle movement, but is also important in plant defence responses (Li and Day, 2019).

The effector HopZ1a from P. syringae, among all its functions, has also been shown

to target microtubule networks in Arabidopsis. Lee and colleagues demonstrated that HopZ1a acetylates tubulin and causes a strong decrease in microtubule networks, triggering alteration of the secretory pathway and suppression of a cellwall mediated defense, callose deposition (Lee et al., 2012).

Disturbing the localisation of a protein can also contribute to disturbing the organisation of the cytoskeleton. Considering that the plant transcriptional machinery is vast and complex, targeting master regulators could be a potent virulence strategy for blocking immune responses. The effector VdSCP41 from the plant pathogenic fungus Verticillium dahliae contributes to pathogen virulence and has been shown to interact with the Arabidopsis master regulators CBP60g and SARD1 and cotton GhCBP60b. Binding of VdSCP41 to CBP60g compromises its transcriptionnal activity and may subsequently interfere with PTI and other defence pathways (Qin et al., 2018).

Another virulence strategy consists in deploying effectors that can directly affect gene expression. In Arabidopsis, the nematode Heterodera glycines HgGLAND4 effector binds genomic DNA and was shown to repress the expression of a reporter gene in planta. A DNA-binding site is located at defence-related genes encoding lipid transfer proteins (LTPs), and these LTP genes were found to be downregulated by HgGLAND4, suggesting that this effector might act as a transcriptional repressor to dampen LTP-triggered defences (Barnes et al., 2018).

Acting in a similar way, the well-known transcription activator-like effectors (TALEs) from Xanthomonas spp. are known to target specific DNA sequences as well. These effectors contain series of tandem aa repeats between 33 and 35 residues thanks to which they can bind to specific host DNA sequences, called effector binding elements (EBEs), and activate gene transcription (Boch et al., 2014). To activate transcription, TALEs interact with the general plant transcription factor TFIIAγ, and coordinate the transcription of susceptibility genes (Hui et al., 2019;Yuan et al., 2016). TALEs targets include various genes encoding sugar transporters, abscisic acid synthesis hormones, and sulphate transporters (Boch et al., 2014;Cernadas et al., 2014;Peng et al., 2019). (Liu et al., 2011). HopAR1 can prevent this event by cleaving RIPK, preventing phosphorylation of RIN4 and activation of RPM1mediated immunity (Russell et al., 2015).

ETI can also be countered by targeting the regulation of NLR proteins. In Nicotiana benthamiana, the P. syringae effector HopBF1 acts as a host client of the plant chaperone Hsp90 by exhibiting a minimal protein kinase folding, resulting in HopBF1-Hsp90 interaction and Hsp90 phosphorylation by HopBF1.

Phosphorylation of Hsp90 results in a reduction in its ATPase activity used to catalyse the folding of its target proteins, such as NLRs. As Hsp90 phosphorylation delays and reduces HR elicited by an autoactive variant of RPM1, it has been suggested that HopBF1 causes NLR destabilisation by inhibiting Hsp90 chaperon activity, thus attenuating ETI (Lopez et al., 2019).

Finally, another mean to alter ETI-related transcriptional responses can be by interfering with the function of TFs. The P. syringae HopD1 effector contributes to virulence in Arabidopsis and has been shown to target the NAC transcription factor NTL9, a positive regulator of plant immunity, resulting in the dampening of ETI responses (Block et al., 2014).

B.5.2.g. AvrAC, an effector that can disturb both PTI and ETI Increasing evidence suggests the existence of intricate interactions between PTI and ETI (Yuan et al., 2021a). In this context, a potent virulence strategy would consist in interfering with both PTI and ETI using a single effector. The AvrAC (XopAC) effector from Xanthomonas campestris pv. campestris (Xcc) is an uridyltransferase. In Arabidopsis, AvrAC uridylates two kinases, the RLK BIK1, known for its role in PTI activation, and RIPK, involved in RPM1-mediated responses (Feng et al., 2012b). Uridylation of these two kinases by AvrAC compromises PTI and ETI responses. Indeed, transgenic lines expressing avrAC are strongly impacted in PTI responses normally triggered upon flg22 treatment such as MAPK activation and ROS burst. In these lines, increased bacterial growth of the type III secretion mutant strains Xcc ΔhrcV and Pst ΔhrcC was also observed.

In addition, these transgenic lines supported greater growth of Pst delivering AvrB, which normally triggers RPM1-triggered immunity in Arabidopsis (Feng et al., 2012b). However, plants evolved the decoy PBL2, a paralog of BIK1, enabling the detection of AvrAC activity by the ZAR1 immune complex (Wang et al., 2015b).

C. Adjustments and limitations to the zig-zag model

The zig-zag model has the benefit of summarising the evolutionnary arms race between plants and pathogens, but it was developed at a time when knowledge about plant responses to pathogens was mainly obtained from a few pathosystems.

Indeed, as our knowledge of other pathosystems and the underlying molecular mechanisms that govern these interactions increase, the limitations and restrictions of the zig-zag model become even more apparent. The following section will briefly describe the limitations and adjustments of the zig-zag model.

One of the first adjustment made to the zig-zag model was to expand the definition of patterns. Originally, patterns were only motifs from conserved molecules found on pathogens. This definition was too restrictive, as host motifs generated upon damage caused by a pathogen or modified "self" can also be recognised in planta to trigger PTI: Damage Associated Molecular Patterns (Choi and Klessig, 2016;Erb and Reymond, 2019) (Figure 2). Nowadays, the term PAMPs is as commonly used as MAMPs (Microbe Associated Molecular Patterns) and other terms are used depending on the type of patterns referred to, like Herbivore-Associated Molecular Patterns or Nematode-Associated Molecular Patterns (Choi and Klessig, 2016;Erb and Reymond, 2019).

Other definitions and concepts from the original model were challenged by results

indicating that there is a blurred dichotomy between ETI and PTI (Cook et al., 2015;Thomma et al., 2011). For instance, PAMPs were often considered to be highly conserved motifs in pathogens, whereas effectors were more likely to be less conserved as they experience greater evolutionary pressure. Whilst this definition is accurate in many cases, several examples have shown that some effectors are in fact highly conserved within and between different pathogen species and that some PAMPs are instead narrowly distributed (Cook et al., 2015;Thomma et al., 2011).

The blurred dichotomy is also reflected in the distinction between PTI and ETI responses. For a long time, scientists have observed that many of the responses induced during PTI and ETI overlap, and that PTI and ETI differ mainly in intensity and duration of these responses (Lu and Tsuda, 2021;Tsuda and Katagiri, 2010).

This overlap already hinted that ETI and PTI might not be strictly seperate pathways, and recently, evidence has shown a real interplay and interdependence between PTI and ETI (Lu and Tsuda, 2021). Indeed, two recent studies have demonstrated that PTI and ETI potentiate each other and that NLR signalling requires PRR signalling for full activation (Ngou et al., 2021;Yuan et al., 2021b). In detail, mutation of the PTI-related components BAK1 and BKK1, altered RPS2-, RPS4-, and RPS5-mediated resistance in A. thaliana (Ngou et al., 2021;Yuan et al., 2021b). Conversely, ETI-related responses also appear to be PTI-dependent. This is well illustrated with the RPS4/RRS1-dependent cell death response triggered by AvrRps4 which is aborted when this effector is transgenically expressed in A.

thaliana instead of being delivered by a bacterial T3SS (Ngou et al., 2020). Also, AvrRpt2-induced HR is compromised in PTI defective mutants (Yuan et al., 2021b).

These results show that ETI-mediated resistance and induction of cell death are dependent on PTI. Furthermore, Tian and colleagues showed that TIR-NLR signalling seems to be required in PTI, as mutation of the TIR NLR-signalling pathways EDS1/PAD4/ADR1 and EDS1/SAG101/NRG1 resulted in attenuated PTI responses, like SA accumulation or induction of defense-related genes after PAMP treatment (Tian et al., 2020). This could also mean that PTI also relies on signalling mechanisms we thought specific to ETI (Pruitt et al., 2020). In any case, either PTI requires ETI for full activation of responses, and/or these two pathways have much more in common than we thought. Overall, these adjustments and new findings revealed that ETI and PTI are not as distinct as previously thought and have prompted researchers to think of adjusted or alternative models to the zig-zag model. Among the various alternative models imagined by researchers are the invasion model, its variation the spatial invasion model, the danger model and its variation the spatial immunity model (van der Burgh and Joosten, 2019; Cook et al., 2015;Gust et al., 2017;Kanyuka and Rudd, 2019). To keep it simple, this manuscript will mainly consider the zig-zag model, its adjustments and limits keeping in mind that ETI and PTI are not as distinct as previously described. A. Chromatin is a dynamic environment regulated by epigenetic factors

• A.1.

Chromatin structure influences gene expression

In eukaryotes, genomic DNA is tightly condensed in the nucleus at different levels (Figure 6). The first level of DNA compaction is chromatin. Chromatin consists of nucleosomes which are units of 146 base pairs of DNA wrapped around histone octamers composed of the H2A, H2B, H3, H4 histones and the linker histone H1 (Luger et al., 1997). Beyond the nucleosomal scale, chromatin loops allow interactions between a regulatory component and its target gene even if they are distant in the genome (Doğan and Liu, 2018). Chromatin loops are themselves part of larger structures of tens of kilobase pairs called Topologically Associated Domains (TADs). TADs are chromatin regions that exhibit suppressed interactions with neighbouring TADs and enhanced chromatin interactions within the TAD.

They allow distant chromatin areas to interact, but with spatial constraints that may confer target specificity of cis-regulatory elements (Doğan and Liu, 2018).

TADs are part of structures of hundreds to thousands of kilobase-pairs called A/B compartments. A/B compartments are spatially distinct compartments of chromatin that are enriched in active chromatin, known as euchromatin, and repressed chromatin, known as heterochromatin. Finally, the highest level of chromatin organisation is at the level of chromosomes. During cell replication, DNA tightly condense in the chromosomes to protect the genetic information during replication. During interphase, when DNA is not as condensed as during replication, chromosomes still occupy distinct nuclear spaces called chromosome territories (Doğan and Liu, 2018).

Despite the complex organisation of genetic information, chromatin is a highly dynamic structure that varies at different stages of the plant life cycle such as development or in response to stress (Pikaard and Scheid, 2014;Probst and Mittelsten Scheid, 2015). Importantly, chromatin structure influences the accessibility of genes for transcription machinery and is de facto involved in the regulation of gene transcription. Indeed, at the nucleosomal scale, DNA wrapped around histone octamers is less accessible to the transcription machinery than DNA located between nucleosomes (Workman and Kingston, 1998). During plant development and in response to stress, chromatin structure varies and this correlates with differential regulation of gene transcription (Lauria and Rossi, 2011;Li et al., 2007a). Changes in chromatin structure are regulated by a variety of mechanisms involving epigenetic factors.

• A.2. Epigenetics, or how to study the regulation of chromatin condensation status

Historically, "epigenetics" was defined by Waddington as "the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being" (Waddington, 1968). The word "epigenetics" itself refers to everything that is "in addition to changes in the genetic sequence" (Weinhold, 2006). In this manuscript, we will use a derived definition that is somewhat broader: "a phenomenon that changes the final outcome of a locus or chromosome without changing the underlying DNA sequence" (Goldberg et al., 2007). These phenomena largely include all processes that modify chromatin condensation status. DNA methylation is one of the major epigenetic marks associated with gene repression. In plants, this mark is mainly found on the 5 th carbon in the pyrimidine ring of cytosine nucleotides in the CG, CHG and CHH sequence contexts (where H is A, C or T) but this mark also exists on the 6 th carbon of the purine ring of adenine in much lower abundance (Liang et al., 2018;Ouyang et al., 2020). De novo DNA methylation is catalysed by the RNA-dependent DNA methylation pathway (RdDM) that involves small interfering RNAs (siRNAs), and various proteins including ARGONAUTE proteins, RNA polymerases, DICER-LIKE proteins and the methylase Domains rearranged methylase 2 (DRM2) (Deleris et al., 2016;Zhang et al., 2018).

Methylated DNA must be maintained by various DNA methyltransferases such as METHYLTRANSFERASE 1, DRM1, or CHROMOMETHYLASE3 (Zhang et al., 2018).

If not maintained, DNA methylation can be passively erased but there are also glycosylases that are responsible for active DNA demethylation by excising the methylated cytosine nucleotide (Zhang et al., 2018).

Mechanistically, DNA methylation is involved in the regulation of gene expression, transposon silencing and chromosome interactions (Zhang et al., 2018). Regarding gene expression regulation, DNA methylation can occur in the promoter or within the gene. When in the promoter, DNA methylation is usually associated with repression of gene transcription, as it does prevent binding of transcription factors or promote other epigenetic marks associated with gene repression (Domcke et al., 2015;Zhang et al., 2018). However, this is not always the case, and the relationship between DNA methylation and transcription factors may depend on the cellular/biological context (Zhu et al., 2016a).

DNA methylation occurs at different stages of the plant life cycle, including growth, development, fruit ripening, but also in response to biotic and abiotic stress (Zhang et al., 2018).

B.1.2. DNA methylation mainly represses plant immunity

DNA methylation has been shown to be involved in the regulation of plant defences in some pathosystems. Dowen and colleagues showed that A. thaliana mutants impaired in the maintainance of DNA methylation were more resistant to Pst DC3000 compared to the WT line suggesting that DNA methylation is negatively correlated with plant defence against this pathogen (Dowen et al., 2012). They also showed that DNA methylation appears to directly or indirectly modulate some defence-related genes like PR1 since mutants in DNA methylation maintainance showed constitutive and inducible misexpression of this gene (Dowen et al., 2012).

During the interaction between A. thaliana and the fungal pathogen Fusarium oxysporum, the DNA demethylase triple mutant rdd (ros1 dml1 dml3) was found to be more susceptible than wild-type plants (Le et al., 2014). This suggests that DNA hypermethylation is linked to plant defence against F. oxysporum. Moreover, many stress-responsive genes were down-regulated, and that correlated with an enrichment for transposable elements in their promoters (Le et al., 2014).

Together, these studies show that DNA methylation and its regulators are directly or indirectly involved in regulating plant defences against fungal and bacterial pathogens, mainly by repressing stress-responsive genes.

• B.2. Histone marks influence chromatin condensation state leading to differential regulation of defence-related genes in response to pathogens B.2.1. Histone marks: Post-Translational Modifications on histone tails influence chromatin condensation status

Post-Translational Modifications (PTMs) on histone tails, also known as histone marks, are another major epigenetic mark that influences chromatin structure.

Histones consist of a structured core and an unstructured tail at their N-term that can carry various PTMs. The different types of histone marks include acetylation, methylation, phosphorylation, ubiquitylation and sumoylation. Some histone marks are associated with gene activation or repression such as histone methylation, histone acetylation and histone ubiquitylation (Kouzarides, 2007;Roudier et al., 2011).

Histone marks are dynamically regulated by various enzymes and proteins that can directly or indirectly modify histone marks and lead to changes in chromatin condensation state and transcription activity. The addition of histone marks is catalysed by enzymes called "writers", and enzymes that can erase PTMs are called "erasers". In addition, proteins carrying a domain that can detect and bind specific histone marks are called "readers". It is important to keep in mind that although different epigenetic marks are sometimes studied separately, several marks are present at a certain locus and it is the combination of the different histone marks that determines the outcome in terms of chromatin condensation state or gene activity. Thus, whether it be writers, erasers or readers, depending on the chromatin state and histone mark environment, these histone mark regulators can be positively or negatively involved in gene transcription (Jenuwein and Allis, 2001;Roudier et al., 2011). In this introduction, histone methylation and acetylation will be detailed as well as their implications in plants. respectively (Jacob et al., 2009;Zhang et al., 2007c). On the other hand, H3K4me3

is associated with active transcription (Zhang et al., 2009).

B.2.1.b. Histone acetylation, marker of active transcription

In Arabidopsis, histone acetylation occurs on the lysine residues on H2A, H2B, H3 and H4 tails (Zhang et al., 2007b). In general, histone acetylation, catalysed by histone acetyltransferases, is associated with permissive chromatine and active gene transcription, whereas histone deacetylation, catalysed by histone deacetylases, is associated with repression of gene transcription (Eberharter and Becker, 2002). Indeed, the addition of an acetyl group on the amine of a lysine residue neutralises the positive charge of the amine residue, leading to chromatin relaxation as the DNA-histone interaction is weakened (Figure 7A) (Eberharter and Becker, 2002;Kumar et al., 2021). Plant development and response to environmental stimuli/stress is regulated in part epigenetically, notably by histone acetylation. Upon environmental or developmental stimulus, a signalling cascade is activated and leads to the expression of histone acetyltransferases (HATs) and histone deacetylases (HDACs), enzymes responsible for histone acetylation and histone deacetylation respectively. In cooperation with other co-activators or corepressors, HATs and HDACs target the genome, facilitating gene activation or repression respectively, leading to a fine-tuning of cellular processes (Figure 7B) (Kumar et al., 2021).

B.2.2. Histone marks regulators have different roles in plant response to pathogens

B.2.2.a. Histone methylation and plant-pathogen interactions

In Arabidopsis, the histone methyltransferase SDG8 regulates the tri-methylation of H3K36 (Li et al., 2015b). SDG8 has been shown to be involved in the plant response to B. cinerea and Alternaria brassicicola as well as to P. syringae (Berr et al., 2010;Zhang et al., 2020). The sdg8-1 mutant infected with B. cinerea, A.

brassicicola or P. syringae had reduced resistance, suggesting that SDG8 plays a role in plant defence against these pathogens (Berr et al., 2010;Zhang et al., 2020).

SDG8 seems to act via H3K36me3-related activation of various genes in the JA/ET and SA pathways, involved in plant defence against necrotrophs and biotrophs respectively (Berr et al., 2010;Zhang et al., 2020). It has also been shown that SDG8 is required to maintain the NLR-encoding gene LAZ5 in an active transcriptional state through H3K36me3 (Palma et al., 2010).

In addition, the histone demethylase IBM1 has recently been reported to be a positive regulator of Arabidopsis immunity against P. syringae. Infection of ibm1 mutants with P. syringae resulted in greater symptom development compared to the WT and the up-regulation of defense marker genes such as PR1, and PR2, normally induced upon bacterial infection, was abolished. In ibm1 mutants, these gene loci displayed enrichment of the inactive mark H3K9me2 and a reduction of the active mark H3K4me3. Since IBM1 can also bind directly to the gene body of PR1, and PR2 , it has been suggested that IBM1 facilitates the activation of these defence genes by directly or indirectly controlling the level of H3K9me2 and H3K4me3 (Chan and Zimmerli, 2019).

In rice, the histone lysine demethylase JMJ704 acts as a positive regulator of rice immunity against bacterial blight caused by Xanthomonas oryzae pv. oryzae.

Expression of JMJ704 is induced upon infection with X. oryzae pv. oryzae and knock-down of JMJ704 led to reduced resistance of rice to bacterial blight (Hou et al., 2015). In this study, Hou and colleagues also demonstrated that JMJ704 participated in the repression of negative regulators of rice immunity against X.

oryzae pv. oryzae such as NRR, OsWRKY62 and Os-11N3 by reducing active H3K4me2/3 (Hou et al., 2015).

B.2.2.b. Histone acetylation and plant response to pathogens

Since HATs catalyse histone acetylation, which correlates with active transcription, it is tempting to imagine that, in general, HATs are involved in the plant response to pathogens by activating transcription of immunity-related genes.

In Arabidopsis, the Elongator subunit 2 (ELP2) possesses a HAT domain and positively contributes to plant immunity against the necrotrophs B. cinerea and A.

brassicicola (Wang et al., 2015a). Indeed, upon infection by these two necrotrophs, elp2 mutant was found to be more susceptible than WT plants and the induction of some JA/ET signaling pathway marker genes, such as PDF1.2 and WRKY33, was reduced or delayed (Wang et al., 2015a). In addition, the levels of histone H3K9/14ac in the defence-related genes NPR1, PR2, PR5, EDS1 and PAD4 were lower than in WT plants which could partly explain the delay in defence gene induction in the mutant (Wang et al., 2013). Thus, ELP2 participates in the regulation of histone acetylation levels at some defence genes to promote efficient defence induction.

HDACs are also involved in plant responses to pathogens. For instance, in Arabidopsis, HDA19 is involved in both JA-and SA-mediated defence responses.

An early study revealed that HDA19 expression was induced upon infection with A.

brassicicola, JA treatment and wounding (Zhou et al., 2005). Infection of the hda19 knock-down mutant with A. brassicicola led to an increased susceptibility associated with decreased transcription of several JA/ET pathway-related genes.

Also, HDA19 overexpressing line showed increased resistance to the pathogen associated with upregulation of PR genes (Zhou et al., 2005). Another study showed that HDA19 expression was also induced in response to Pst DC3000 and infection of hda19 mutants or HDA19 over-expressing lines with this pathogen led to reduced and increased susceptible response, respectively, associated with altered PR1 and PR2 expression (Choi et al., 2012). By ChIP assays, Choi and colleagues also demonstrated that under unchallenged conditions, HDA19

associates directly with PR1 and PR2 promoter regions and deacetylates histones at these loci. This suggests that HDA19 acts as a negative regulator of SA-related defences by favouring repressive chromatin under unchallenged conditions at loci such as PR1 and PR2, preventing unnecessary defence activation (Choi et al., 2012). Thus, HDA19 functions as a positive and a negative regulator of JA-and SArelated defence responses, respectively.

Histone acetylation also influences the regulation of NLR-encoding gene expression. HDA9, an Arabidopsis HDAC regulating H3 acetylation, including H3K9ac, seems to be a negative regulator of plant immunity against Pst DC3000, as infection of the hda9-1 mutant resulted in enhanced disease resistance to the pathogen (Yang et al., 2020). Upon pathogen infection, SNC1 is strongly upregulated in hda9-1 mutant and more than in WT plants, suggesting that HDA9 has a role in the regulation of SNC1 expression in response to pathogen infection.

Furthermore, levels of H3K9ac at the SNC1 locus were higher in the hda9-1 mutant than in the WT upon infection but not under normal conditions. This suggests that HDA9 regulates negatively SNC1 expression upon infection, likely by reducing H3K9 acetylation levels at this locus (Yang et al., 2020). We have seen that chromatin modifiers can add or remove chemical groups from DNA or histones, but there is another type of epigenetic regulator that can affect chromatin condensation: the chromatin remodelling complexes (CRCs). CRCs are protein complexes comprising enzymes that modify the DNA-histone interaction in a non-covalent manner, since there is no change in chemical groups. CRCs are also referred to as chromatin remodeling ATPases, as they use the energy of ATP to modify the contact between DNA and histone octamers, displace histone octamers along DNA, and exchange or remove nucleosomes (Clapier and Cairns, 2009).

Conserved from yeast to humans and plants, there are four sub-families of chromatin remodelling ATPases : INO80/SWR1, CHD, ISWI and SWI/SNF (Han et al., 2015). They differ in the domains they harbour, and in their biochemical activities. They are involved in various chromatin-related mechanisms including DNA damage repair, homologous recombination, maintainance of other epigenetic marks, control of gene expression, or antagonise with specific chromatin silencing mechanisms (Han et al., 2015).

B.3.2. Chromatin remodelling complexes regulate plant responses to pathogens

Chromatin-Remodelling Factor 5 (CHR5) from A. thaliana is necessary for accurate plant immune responses against bacterial pathogens. chr5 mutant infected with different strains of P. syringae displayed higher bacterial growth than WT plants, notably for the strains P. syringae pv. maculicola, and the avirulent strains Pst DC3000 (avrRpt2) and Pst DC3000 (avrRps4), delivering the effectors AvrRpt2 and AvrRps4 that are recognised by the NLRs RPS2 and RPS4, respectively. This result suggests that CHR5 is a positive regulator of plant immunity in response to various P. syringae strains. Moreover, CHR5 was shown to be required for the up-regulation of the NLR SNC1 and associated autoactivation of immune responses in the bon1 auto-immune mutant (Zou et al., 2017).

The Arabidopsis chromatin-remodelling component SWP73A functions as a negative regulator of NLR expression (Huang et al., 2021). SWP73A is an ortholog of BAF60 from mammals, which is known to be involved in inflammatory responses during infection. The swp73a mutant showed a higher expression of the NLR RPS2

and the defence-related PR1 gene (Huang et al., 2021). Infecting these mutants with Pst DC3000 (AvrRpt2) and Pst DC3000 (AvrRps4) avirulent strains led to a reduction in bacterial growth suggesting that SWP73A negatively regulates ETI.

This was confirmed by the analyses carried on SWP73A over-expressing lines, which had lower expression of RPS2 and PR1 genes and increased bacterial growth compared to WT plants when infected with Pst DC3000 (AvrRpt2) or Pst DC3000 (AvrRps4). In details, SWP73A affects the transcription of several NLRs such as RPS2, RRS1, RPS4 and ZAR1 (Huang et al., 2021). By ChIP-seq, the authors showed that SWP73A can bind directly to the promoters of several NLRs including RPS2 and ZAR1 but not RPS4 and RRS1. This implies that SWP73A directly regulates the expression of some NLRs but for others, its regulatory action might be indirect. In fact, the regulation of RPS4 and RRS1 by SWP73A seems to occur via another component, Cell division cycle 5 (CDC5), a key regulator of alternative splicing (Palma et al., 2007). It appears that SWP73A directly down-regulates CDC5

probably leading to a splicing defect in RPS4, an important factor for RPS4 activity (Huang et al., 2021). Furthermore, binding of SWP73A to promoters is dependent on the repressive mark H3K9me2, which is reduced on RPS2 and CDC5 promoters upon infection with Pst DC3000 (AvrRps4) or Pst DC3000 (AvrRpt2). Thus, this study showed that under normal conditions, the chromatin-remodelling component SWP73A participates in the repression of some NLRs by binding direcly to their promoters thanks to the repressive mark H3K9me2. SWP73A can also negatively and indirectly regulate the expression of NLRs like RRS1 and RPS4 via downregulation of CDC5, a key regulator of alternative splicing, important for NLR activity. However, under pathogen infection, the repression of CDC5 and RPS2 is abolished/reduced correlating with a decrease in H3K9me2 levels which reduces the association of SWP73A with the CDC5 and RPS2 promoters. Interestingly, under infection with avirulent strains Pst DC3000 (AvrRpt2) and Pst DC3000 (AvrRps4), two small-RNAs were induced, mi3440 and siRNA-SWP73A which target SWP73A for down-regulation, presumably to prevent this negative regulator from inhibiting immune responses.

These small-RNAs are regulatory non-coding RNAs, a type of epigenetic regulator derived from RNAs that do not encode proteins but regulate gene expression at various levels in different biological processes, including in response to biotic stresses.

• RNAs (sRNAs) like microRNAs (miRNAs), small interfering RNAs (siRNAs) and long ncRNAs (lncRNAs) (Cech and Steitz, 2014;Morris and Mattick, 2014). Regulatory ncRNAs regulate gene expression at the transcriptional, post-transcriptional and epigenetic levels.

LncRNAs are transcripts of more than 200 nucleotides that modulate many cellular processes. In mammals, lncRNAs regulate gene silencing, protein activity, messenger RNA stability, mRNA processing, transcription; they serve as scaffold for higher-order complexes or as decoys or enhancers of protein-encoding gene promoters (Geisler and Coller, 2013). In plants, lncRNAs have similar function than those in mammals. They are involved notably in transcription regulation, gene silencing via the RdDM pathway, alternative splicing and chromatin structure (Wang and Chekanova, 2017).

miRNAs and siRNAs are small ncRNAs of 20-24 nucleotides. In most eukaryotic organisms, including plants, they are generated by DICER-LIKE proteins or the ribonuclease III-like enzyme DICER and induce gene silencing in a sequencespecific way (Baulcombe, 2004). miRNAs are derived from single-stranded RNA precursors encoded by MIR genes. They can repress gene expression by transcript cleavage, or translation repression (Yu et al., 2019). Most siRNAs are processed from long double-stranded RNAs from repeats and transposable elements in heterochromatin. They are involved in gene silencing by DNA methylation mediated by the RdDM pathway (Yu et al., 2019).

B.4.2. LncRNAs, miRNAs and siRNAs do also their part during plant infection by a pathogen

In different pathosystems, various lncRNAs have been shown to be induced upon infection or PAMP treatment such as in A. thaliana in response to the fungal pathogen Fusarium oxysporum or to Pst DC3000, or in wheat in response to the fungus Blumeria graminis f.sp. tritici (Seo et al., 2017;Xin et al., 2011;Zhu et al., 2014). Functional analyses using knock-down or knock-out lines revealed that some of these lncRNAs have a role in the plant defence responses to pathogens.

For instance, during infection of Arabidopsis by F. oxysporum, knock-out or knockdown mutants of the lncRNAs TAR-191, TAR-197, TAR-212 and TAR-224 showed earlier and stronger symptom development compared to WT plants (Zhu et al., 2014). In the Arabidopsis-Pst DC3000 pathosystem, knock-down of the lncRNA ELENA1 also showed increased susceptibility (Seo et al., 2017). These studies suggested that lncRNAs are positively involved in plant defence against pathogens.

However, much remains to be discovered about the mechanisms underlying their roles in the regulation of plant defence. The most documented example is ELENA1.

Indeed, ELENA1 was shown to participate in the up-regulation of PR1 expression in challenged conditions, likely through an interaction with Mediator Subunit 19a, a component of the transcriptional coactivator complex Mediator that connects TFs to RNA polymerase II (Seo et al., 2017).

There is increasing evidence of a regulatory role of miRNAs in plant immunity in response to pathogens (Song et al., 2021a). Upon pathogen infection, miRNAs expression can be altered, resulting in differential regulation of immune responses.

Since miRNAs function mainly by repressing gene expression, they positively or negatively regulate immune responses by silencing positive or negative regulators of immune responses like hormone receptors, TFs, proteins involved in ROS burst activation or NLRs (Song et al., 2021a). As an example, the Arabidopsis miR472 is a negative regulator of PTI and ETI against Pst DC3000 (Boccara et al., 2014).

Regarding PTI regulation, flg22-induced ROS production and callose deposition was increased in the miR472 mutant suggesting that miR472 has a negative role in PTI regulation. Moreover, in the miR472 over-expressing lines, fewer transcripts of several NLRs, including RPS5, were detected. In addition, RPS5-mediated immunity was dampened in these over-expressing lines, suggesting that miR472 negatively regulates RPS5-mediated resistance (Boccara et al., 2014). miRNAs work also cooperatively with siRNAs, including endogenous siRNAs or phase secondary interfering RNA, a specific type of siRNAs generated by cleavage of a miRNA target (Song et al., 2021a). siRNAs influence immune responses to pathogens by silencing components involved in hormonal pathways, TF signalling, NLRs, or ETI (Huang et al., 2019;Song et al., 2021a).

In pathogens, epigenetic mechanisms also participate in the regulation of gene expression. In the above section, I deliberately focused on the role of epigenetic mechanisms in plant immunity, but the regulation of gene expression by epigenetic mechanisms in pathogens could deserve its own detailed section. Very briefly, epigenetic mechanisms are involved in pathogen development and pathogenesis (Dubey and Jeon, 2017;Sánchez-Romero and Casadesús, 2020). While the main epigenetic mechanism in bacteria is DNA methylation, genes from fungal pathogens can be regulated by DNA methylation, histone modifications, sRNAs and Transposable Elements (Sánchez-Romero and Casadesús, 2020;Sánchez-Vallet et al., 2018). Notably, it has been shown that DNA methylation might play a role in R. Competitively binds to a subunit of the SAGA HAT complex, causing a decrease in acelytation levels at the promoters of various genes including immunity-related genes correlated with their down-regulation upon infection Relocates an acetyltransferase into the nucleus triggering histone acetylation at susceptibility genes favouring their transcription and infection Li et al., 2018 solanacearum pathogenesis (Erill et al., 2017). In addtion, epigenetic mechanisms are important for fugal pathogenesis (Zhu et al., 2016b). For instance, genes encoding recognised effectors can be silenced by sRNAs, leading to the promotion of virulence (Sánchez-Vallet et al., 2018). Thus, plant-pathogen interactions are characterised by an important regulation of gene expression, involving notably epigenetic mechanisms from both sides of the interaction.

C. Parasites and pathogens can manipulate the host epigenome to their benefit Considering that more and more evidence reveals a key role of the plant epigenome in the regulation of immune responses against pathogens and that it acts upstream of the regulation of gene expression, it seems obvious that pathogens have evolved virulence strategies that consist in bypassing or counteracting host epigenetic mechanisms to promote infection. While for plant pathogens few examples are documented, more studies describe the manipulation of the host epigenome by animal pathogens. The following paragraphs provide an overview of the diverse and sophisticated virulence strategies used by animal pathogens to subvert the host epigenome and present the current state of knowledge about the manipulation of host epigenome by plant pathogens. I will focus in this section on the action of nucleomodulins, which are bacterial proteins targeted to the nucleus (Table 2, Figure 8). The effector Rv2966c from M. tuberculosis, the causal agent of tuberculosis, is a functional DNA methyltransferase (Sharma et al., 2015). Once secreted into the Various effectors from plant and animal pathogens and parasites were shown to interefere with the host epigenome, supposedly to favour infection. Some can directly or indirectly modify DNA methylation and histone marks such as acetylation, methylation or phosphorylation. Others target histone marks by manipulating cytoplasmic acetyltransferase of histone mark regulators such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), or epigenetic readers. Orange: effectors from animal pathogens; green: effectors from plant parasites or pathogens. PTMs. Since it binds to specific DNA sequences and the transcription of these sites is altered upon mycobacterial infection, Rv2966c was hypothesised to cause the down-regulation of these loci by methylating their DNA sequence (Sharma et al., 2015). This effector introduces a way of manipulating the host epigenome, presumably to the benefit of the pathogen, although much remains to be elucidated about its mode of action and the consequences of DNA methylation on host transcription and immune responses.

Histone methylation

TrAP

C.1.1.b. Targeting chromatin remodelling to prevent transcription

The LpqH lipoprotein from M. tuberculosis alters host immunity by inhibiting the expression of several immunity-related genes. The expression of one of these immunity-related genes, CIITA, is affected by this lipoprotein, possibly via an alteration of its epigenetic regulation (Pennini et al., 2006). Indeed, in animal cells treated with LpqH, histone acetylation at CIITA promoter region was altered, together with the recruitment to this promoter of Brahma-related gene 1, a component of the SWI/SNF chromatin remodelling complex. This indicated that expression of the immune-related CIITA gene was likely inhibited by manipulating the accessibility of chromatin to the transcription machinery through changes in histone acetylation levels and chromatin remodelling. However, the underlying molecular mechanisms remain elusive (Pennini et al., 2006). The animal pathogenic bacterium Shigella flexneri manipulates a host epigenetic reader by using the effector OspF (Harouz et al., 2014). OspF is a phosphothreonine lyase that catalyses eliminylation on its targets, a reaction that transforms a phosphothreonine residue into a dehydrobutyrine, permanently blocking phosphorylation of that residue. OspF was shown to target MAPKs by causing their permanent dephosphorylation, and thus their permanent inactivation (Li et al., 2007b). This permanent inactivation has various consequences on the proteins that are normally regulated directly or indirectly by MAPK-mediated phosphorylation.

This includes Heterochromatin protein 1 γ (HP1γ), an epigenetic reader that recognises methylated H3K9. HP1γ is normally phosphorylated downstream of the MAPK ERK by the kinase MSK1, but whose phosphorylation is impaired because OspF blocks the ERK phosphorylation cascade. As a result, non-phosphorylated HP1γ accumulates at the promoters of target genes, possibly retained by interaction with OspF, impeding active transcription of pro-inflammatory genes in response to the bacterial pathogen (Harouz et al., 2014).

C.1.2. Pathogen interference with histone marks

C.1.2.a. OspF indirectly alters H3 phosphorylation

In addition to altering the phosphorylation status of an epigenetic reader, OspF also indirectly causes an alteration of H3 phosphorylation (Arbibe et al., 2007). By blocking MAPKs in an inactive conformation via eliminylation, OspF alters the subsequent phoshorylation of H3 at the promoters of immune-related genes and the recruitment of the transcription machinery to these promoters, preventing the transcription of pro-inflammatory chemokines and cytokines, important components of animal immune response to infection (Arbibe et al., 2007).

C.1.2.b. Several effectors can directly methylate host histones

As histone methylation is involved in gene activation and repression, targeting this mark would represent a potent strategy to alter pathogen-induced transcriptional reprogramming. Surprisingly, several effectors from different pathogens are methyltransferases capable of directly modifying histones in vitro and/or in vivo (Rolando et al., 2015). These include BaSET from Bacillus anthracis, NUE from Chlamydia trachomatis, and RomA from Legionella pneumophilia. Remarkably, these effectors all contain a SET domain, a domain found in eukaryotic histone methyltransferases responsible for the methylation of histone H3 and H4 tails on lysine residues. This suggests that pathogens have evolved effectors mimicking host histone methyltransferases, possibly to disrupt the regulation of gene transcription (Rolando et al., 2015). However, for many of these effectors, the link between histone methyltransferase activity and their function in infection remains elusive. RomA is among the effectors for which we know a little more. The RomA type 4 effector from L. pneumophilia methylates mammalian H3K14 in vitro and in vivo, resulting in a genome-wide decreased acetylation at this residue. This reveals a potential switch between an active and a repressive histone mark (Rolando et al., 2013). The authors also identified a large set of H3K14 methylated promoter regions upon L. pneumophilia infection, including immunity-related genes (Rolando et al., 2013). Strikingly, H3K14 methylation has never been observed before in mammalian cells and therefore represents a particularly ingenious strategy used by L. pneumophilia to manipulate the host epigenome.

C.1.2.c. Manipulation of histone acetylation by animal bacterial pathogens Similar to effectors capable of methylating histones, the M. tuberculosis effector Rv3423 acetylates H3K9 and H3K14 in vitro (Jose et al., 2016). Considering that acetylation is involved in the regulation of gene transcription, Rv3423 might disturb host gene transcription to promote the intracellular survival of M. tuberculosis.

However, the role of Rv3423 during infection and its impact on gene expression by triggering histone acetylation remain uncharacterised (Jose et al., 2016).

Instead of directly modifying histone acetylation levels, effectors can also target regulators of histone acetylation such as HATs. For instance, the effector NleC from enteropathogenic and enterohaemorrhagic Escherichia coli is a protease that targets the HAT p300 and likely promotes its degradation in infected cells.

Consequently, p300 degradation correlates with altered secretion of Interleukin-8, a major cytokine involved in immune responses against enteropathogenic and enterohaemorrhagic Escherichia coli (Shames et al., 2011).

• C.2. Plant pathogens can also alter the host epigenome from the HAT Spt-ADA-Gcn5-Acetyltransferase (SAGA) complex (Kong et al., 2017). Interestingly, the competitive binding of ADA2 to PsAvh23 alters the formation of the ADA2-General Control Non-depressive 5 (GCN5) subcomplex that mediates acetylation of nucleosomal histones. Also, PsAvh23 decreases H3K9ac levels upon its interaction with ADA2 in soybean. In addition, in PsAvh23expressing roots, various genes involved in plant defence against P. sojae are down-regulated; and this correlated with decreased levels of H3K9ac marks at their promoters (Kong et al., 2017). (Berger, 2007;Li et al., 2018). In vivo, transient expression in N. benthamiana of a version of GmTAP1 forced into plant nuclei revealed increased acetylation levels on H3, and H2K5 notably. Moreover, PsAvh52 induces increased acetylation levels of H3K9 and H2AK5 in soybean roots and this is dependent on the ability of PsAvh52 to interact with GmTAP1. Finally, in soybean roots expressing PsAvh52, the transcription of several susceptibility genes was increased, like SWEET, lypoxygenase and a polygalacturonase encoding genes.

These genes showed an increase in H3K9 and H2AK5 acetylation levels 6 hours after P. sojae infection in soybean roots expressing PsAvh52. Together, these data highlight how P. sojae affects histone acetylation to control host gene expression and promote infection (Li et al., 2018).

• C.3. From animal to plant parasites, effectors can affect the host epigenome in similar ways and yet differently

This section has provided an overview of animal and plant parasites that can alter the host epigenome, notably via effector activities (Figure 8).

It is quite impressive to note the diversity of strategies used by parasites, including pathogens, to alter the host epigenome. They include the targeting of DNA methylation, histone marks (phosphorylation, methylation and acetylation), but also epigenetic readers. Interestingly, evidence in the literature describe the manipulation of histone methylation and acetylation by animal and plant parasites.

This suggests that there has probably been evolutionary selection of virulence mechanisms in both animal and plant pathogens targeting epigenetic regulatory mechanisms that are common to both kingdoms.

Althought they sometimes target similar epigenetic mechanisms, the means in which pathogens manipulate these mechanisms can be quite diverse, particularly with regard to the manipulation of histone marks, since some effectors can directly or indirectly modify histones, or manipulate epigenetic regulators to alter histone mark levels, resulting in differential gene transcription, which is thought to benefit the pathogen.

Targeting the host epigenome appears to be a powerful and profitable strategy, as some pathogens have evolved several effectors and/or proteins that can target the host epigenome, such as the effectors Rv3423 and Rv2966c and the lipoprotein LpqH from M. tuberculosis and the effectors PsAvh23 and PsAvh52 from P. sojae.

There also appears to be a co-evolution of some pathogens towards the development of effectors with similar functions, such as effectors with a SET domain, domain usually found in eukaryotic histone methyltransferases and found functional in the effectors NUE, BaSET and RomA from C. trachomatis, B. anthracis and L. pneumophilia, respectively.

However, it should be noted that it is not always easy to assess the actual role of an effector on an epigenetic mechanism for two main reasons. On one hand, the link between levels of epigenetic marks, transcription and functional characterisation of targets is often indirect and mainly relies on correlations, more than on causalities. On the other hand, it is quite challenging to determine, from a given experiment, whether the observed effect on an epigenetic mechanism is directly caused by the function/activity of the effector or whether it is a side effect of the considered epigenetic mechanism or its interconnection with other epigenetic mechanisms. For instance, in the Geminiviruses-Arabidopsis interaction, given that KYP proteins are required for the maintenance of DNA methylation, it is difficult to link their manipulation by viral TrAP proteins and its putative consequences on histone and DNA methylation. This is also why these studies propose working hypotheses based on correlations, due to limititations that are inherent to their experimental model. Another limitation is that despite the importance of the epigenome in plant-pathogen interactions, its influence is vast and extremely complex, and far from being fully understood.

Finally, while most of the effectors or proteins described as being capable of 3-The Ralstonia solanacearum species complex (RSSC)

Figure 9. The RSSC consists of three species (adapted from Paudel et al., 2020).

After the 2014 taxonomic revision, the RSSC was defined as comprising three species: Ralstonia pseudosolanacearum which includes phylotypes I and III (strains originating in Asia and Africa respectively), Ralstonia solanacearum which corresponds to phylotype II (strains originating in America), and Ralstonia syzygii which corresponds to phylotype IV (strains originating in Indonesia and Japan), and subdivided into three subspecies R. syzygii subsp. syzigii, R. syzygii subsp. indonesiensis and R. syzygii subsp. celebesensis.

A. Pathogen description, classifications and taxonomy and Kelman, 1964). A complementary classification system was introduced, defining biovars, sorting the RSSC strains by their ability to oxidise different carbohydrate sources (Hayward, 1964). However, the classification race/biovar was not fully satisfying as it did not provide sufficient discrimating features to effectively represent the genetic diversity within the species complex, even though the concept of a species complex was not official yet. Later, Prior and Fegan introduced a classification based on genetic differentiation, distinguishing strains into phylotypes (Prior and Fegan, 2005). Interestingly, these phylotypes correlated with the geographical origins of the strains: phylotype I corresponds to strains originating from Asia, phylotype II from America, phylotype III from Africa and surrounding islands and phylotype IV from Indonesia and Japan (Figure 9). For many years and until today, this phylotype classification system is mostly used, although R. solanacearum has undergone various taxonomic changes, even in recent years before being considered as a species complex.

• A.

A complicated taxonomy

The taxonomy of R. solanacearum is quite complex as this pathogen changed genus several times before entering the genus Ralstonia in 1995. While it was first described in 1986 as Bacillus solanacearum, it was transferred in different genera, from Bacterium to Pseudomonas and Xanthomonas genera and passing through Burkholderia before settling on Ralstonia in 1995. Today, R. solanacearum is now referred to as a species complex due to the high genetic diversity among R.

solanacearum strains (Paudel et al., 2020).

More recently, in 2014, a taxonomic revision was carried out leading to the division of R. solanacearum phylotypes into different species (Prior et al., 2016;Safni et al., 2014). Indeed, phylogenetic analyses based on DNA-DNA hybridisation, DNA base composition, intergenic spacer region sequences, partial endoglucanase gene sequences, and other analyses based on denitrification assays and proteomics allowed to distinguish three different species (Prior et al., 2016;Safni et al., 2014).

Phylotype I and III represent the species Ralstonia pseudosolanacearum, phylotype II remains R. solanacearum and phylotype IV is a distinct species, Ralstonia syzygii, subdivided in three subspecies names R. syzygii subsp. syzygii, R. syzygii subsp.

indonesiensis and R. syzygii subsp. celebesensis (Figure 9).

B. Pathogenicity of the RSSC and its virulence determinants

• B.1. The RSSC is an important threat for crop production due to its wide host range and persistence in the field RSSC strains are the causal agent of bacterial wilt disease, which can infect more than 250 plant species from 54 plant families, including Solanaceae, Cucurbitaceae, Musaceae, Zingiberaceae and Asteraceae (Hayward, 1991). Of The RSSC is a devastating plant bacterial pathogen with a worldwide distribution. Many strains thrive in equatorial regions but other strains are adapted to more temperate climates. While in many areas the RSSC is endemic, its presence in Europe is less pronounced and for this reason it is considered a quarantine pathogen in Europe.

these 250 plant species, many crops can be affected by RSSC bacteria including tomato, tobacco, eggplant, potato, banana, and ginger. It has also been reported that RSSC bacteria can infect or be hosted by weeds and ornamentals such as the weeds Urtica dioica and Solanum dulcamara and the ornamental species Rosa spp., Anthurium spp. and Pelargonium spp. (Bergsma-Vlami et al., 2018;Champoiseau et al., 2009;Poussier et al., 1999;Wenneker et al., 1999;Wicker et al., 2007).

With its genetic diversity and wide host range, the RSSC is considered the second most important pathogenic bacteria in terms of economic impact (Mansfield et al., 2012). While assessing the overall yield loss caused by RSSC bacteria is extremely complicated given the numerous factors that play a role in the calculation, local yield loss and incidence reports have been published at the country level with a focus on one host species (Bragard et al., 2019;Mamphogoro et al., 2020). For instance, yield loss in potato production has been reported in different countries:

with 15% in Bangladesh, 95% in Nepal and 50% in Kenya (Karim et al., 2018).

However, these yield loss estimates might vary depending on the host plant, infecting strains, soil management, year and weather conditions.

Nonetheless, the RSSC is a threat to crop production because it can affect many crops from various families, and because of the extreme persistence of the pathogen in the soil, as it can survive several years in the soil or infect weeds, creating a reservoir of pathogen that can be transmitted to subsequently sown crops (Elphinstone, 1996). Moreover, its genetic diversity favoured its presence worldwide, with a major presence in tropical areas although the presence of some strains of R. solanacearum have been reported in Europe (Figure 10). With a relatively low occurrence in Europe, R. solanacearum is considered as a quarantine pathogen by the European and mediterranean Plant Protection Organisation, in order to prevent the endemic establishment of this pathogen on the continent which could cause significant yield and economic losses (Bragard et al., 2019).

• B.2. The control methods against the RSSC are diverse but not entirely satisfactory Due to the major economic and yield impact of the RSSC, researchers and companies are continually trying to find a way to control and breed cultivars resistant to the bacterial wilt disease. Many control strategies have been experimented in the laboratory, or to a lesser extent in the field, with varying degrees of success and associated limitations, making the management of bacterial wilt a complex challenge (Huet, 2014;Mamphogoro et al., 2020;Yuliar et al., 2015). Chemical control, although being one of the most effective control methods against bacterial wilt, is not considered as a sustainable control method as ignorance and incorrect application of pesticides can be harmful to the environment, farmers and consumers. Other known control methods to reduce the presence of the RSSC in soils are physical measures including soil solarisation, biofumigation, crop rotation or prophylactic measures that rely on the use of healthy seeds, water and soil free of the RSSC (Huet, 2014;Mamphogoro et al., 2020).

Alternative control methods are based on the use of biological agents, natural antimicrobial compounds, or the use/selection of resistant cultivars to minimise the incidence of bacterial wilt. The use of biological agents such as non-pathogenic strains of the RSSC, antagonistic bacteria, or bacteriophages has been and is still being studied in an attempt to reduce the virulence of RSSC strains (Álvarez and Biosca, 2017;Hanemian et al., 2013;Mamphogoro et al., 2020). For instance, preinoculation of A. thaliana with a T3SS-deficient RSSC strain results in increased resistance that is likely due to competition between the strains (Feng et al., 2012a).

Other studies have focused on the use of bacteriophages that can infect various RSSC species. For example, it has been shown that the Jumbo phage RsoM2USA can infect strains of three different RSSC species and decrease the virulence of one RSSC strain in tomato plants when co-inoculated with that strain (Ahmad et al., 2021). However, although these control methods represent powerful strategies to limit the development of bacterial wilt, most of these studies were carried out A under laboratory conditions and would require further investigation like conducting similar experiments on crops under field conditions and assessing the long-term effects of these treatments on the environment. Another recent study assessed the antimicrobial properties of three hydroxycoumarins against RSSC bacteria, which are natural secondary metabolites that accumulate in plants when challenged with pathogens (Yang et al., 2021). The authors showed that these hydroxycoumarins induce transcriptomic changes in a strain of R. pseudosolanacearum, resulting in cell membrane destabilisation and bacterial cell death (Yang et al., 2021). While the antimicrobial effects of hydroxycoumarins could represent a sustainable natural resource for the control of bacterial wilt, many factors remain to be investigated before these metabolites can be used in the field. Currently, the most efficient disease control method against RSSC remains the use of resistant or tolerant cultivars even though this method is geographically and climatically restricted (Huet, 2014). Indeed, the selection of resistant cultivars to RSSC strains is limited because it has to combine sustainable resistance with specific agronomic traits such as yield and most of the time, resistance is negatively correlated with yield. Thus, the control of bacterial wilt remains a great challenge and for the time being, the best measures remain the local use of resistant cultivars and prophylactic measures to prevent the establishment of the RSSC in the soil, but methods using biocontrol agents seem promising and could represent potent alternative disease control measures to fight bacterial wilt disease.

• B.3. Infection process and virulence determinants of the RSSC RSSC bacteria have a relatively simple life cycle with an infection phase in planta and a saprophytic phase in the soil (Figure 11A). Present in the soil, the bacteria are attracted to root exudates by chemotaxis, attach to roots, enter the roots via lateral root emergence points and wounds and move to the xylem vessels where they thrive (Caldwell et al., 2017;Lowe-Power et al., 2018;Vasse, 1995). Once they reach the xylem, which occurs about 24 hours after inoculation in tomato, the bacteria form a biofilm and secrete exopolysaccharides (EPS) that can obstruct the xylem sap flow (Caldwell et al., 2017;Kang et al., 2002). In tomato, typical infection symptoms include wilting of aerial parts, brown discolouration of the xylem, and oozing from cut stems (Genin, 2010). Plants continue to wilt until they die, resulting in the return of bacteria to the soil, where they can survive for 2-3 years as saprophytes, and/or spread via irrigation water. In potato, spread can also occur through the transport of infected tubers (Bragard et al., 2019). In Arabidopsis, it takes ~3 days for wilting symptoms on leaves to be visible (Figure 11B).

Several factors that play a role during infection are called virulence determinants.

Virulence determinants are factors promoting pathogen infection. Among the virulence determinants of RSSC bacteria are motility, chemotaxis, EPS, the Type 2

Secretion System (T2SS) and its associated cell-wall degrading enzymes, as well as the T3SS and associated effectors (Genin and Denny, 2012). It seems quite logical that motility and chemotaxis are virulence determinants of RSSC bacteria, since the bacteria need to locate potential hosts in the soil, move towards them, and reach the xylem vessels. To do this, they can use swimming motility with their polar flagellum to locate roots in the soil, and twitching motility, which requires Type IV pili, to attach to and move along surfaces notably in xylem vessels (Lowe-Power et al., 2018). Once in contact with the host roots, the bacteria penetrate through the cell layers to reach the xylem vessels, with the help of their T2SS and Type II cell-wall degradation enzymes. Notably, mutants deficient in cellulolytic enzymes and T2SS induced delayed symptoms in tomato plants, suggesting the importance of penetrating plant tissues to reach xylem vessels for successful infection (Liu et al., 2005). In addition, secreted EPS are also a major virulence factor for RSSC bacteria. Indeed, compared to the WT strain, EPS-defective mutants are less virulent on eggplant and tobacco and spread less efficiently in tomato stems, suggesting that EPS may be involved in plant colonisation by the pathogen (Kao et al., 1992;Saile et al., 1997).

C. Focus on key virulence determinants of the RSSC: theType III effectors

• C.1. The RSSC possesses a large Type III effector (T3E) repertoire

Another main virulence determinant of the RSSC is the T3SS without which colonisation and symptom development of the host plant infected with the pathogen is strongly impacted (Vasse et al., 2000). The T3SS allows the secretion of T3Es in host cells, which can alter specific cellular processes to promote pathogen infection. RSSC strains have the particularity to possess a wide range of T3Es, with an average of 64 T3E encoding genes compared to 20-30 in P. syringae or X. campestris (Landry et al., 2020;Sabbagh et al., 2019). The RSSC T3Es repertoire was catalogued under a unified nomemclature in 2013, assigning a name starting with Rip for Ralstonia injected proteins, and an online repertoire (EffectorK) compiling T3Es from different RSSC strains is available with tools to compare the presence, absence and copy number of T3E encoding genes between different strains (Peeters et al., 2013). Landry and colleagues have provided a good synthesis of what has been described in the litterature on the different T3Es in the RSSC until recent years. Among the different T3Es categorised, about 50 of them have been characterised with a relative level of detail, from virulence or avirulence capacity in host species to functional and molecular characterisation (Landry et al., 2020). Redundancy has been noted between different T3Es of the RSSC, which could favour adaptation of pathogens to changing environments while maintaining an efficient infection capacity (Chen et al., 2014;Ghosh and O'Connor, 2017;Solé et al., 2012).

It has long been assumed that T3Es are important mainly for the virulence of the pathogen in the early stages of infection (Alfano and Collmer, 2004). However, recent studies tend to show that T3Es might be important throughout the whole infection process, even in late stages of the disease (Monteiro et al., 2012;de Pedro-Jové et al., 2021). Indeed, one of these recent studies demonstrated the dynamic expression of RSSC virulence determinants during infection in potato, including T3Es, and compared their expression pattern at different infection stages tumefaciens (Jeon et al., 2020). Interestingly, a single effector can be an avirulence factor in various host species or cultivars. This is the case for RipP1 (formerly PopP1), which triggers HR responses in three different Nicotiana species but is also known to be an avirulence factor in Petunia, since the cultivar St40 loses its resistance to GMI1000 when lacking RipP1 (Lavie et al., 2002;Poueymiro et al., 2009). Other examples are RipAT and RipAV, whose transient expression via A.

tumefaciens in planta revealed that these two effectors elicit HR in many lettuce cultivars, and in some pepper and tomato cultivars (Wroblewski et al., 2009). It can be noted that these two effectors elicit HR in common lettuce and pepper cultivars indicating that several effectors can be avirulence factors in the same cultivar.

When several RSSC effectors are avirulence factors in the same host species or cultivars, it can create difficulties for functional characterisation of the effectors, as there is a redundancy in triggering ETI. Typical example of RSSC effectors that are avirulent factors in the same species are RipB, RipP1 and RipAA (formerly AvrA)

which are avirulent factors in N. benthamiana (Nakano and Mukaihara, 2019;Poueymiro et al., 2009). RSSC strains mutated in ripP1 or ripAA triggered a weaker HR than that induced by the WT strain, and a ΔripP1 ripAA double mutant triggered almost no HR (Poueymiro et al., 2009). Later, another study revealed that another effector, RipB, acts as a major avirulence factor in N. benthamiana and that RipP1

and RipAA are minor avirulence factors (Nakano and Mukaihara, 2019). Indeed, inoculation with a ΔripB mutant strain led to wilt symptoms in contrast to the tobacco-avirulent WT strain for which N. benthamiana is resistant. However, wilt symptoms induced by a ΔripB mutant were much reduced compared to a tobaccovirulent strain, suggesting that other factors are involved in avirulence, but to a lesser extent. This prompted the authors of the study to investigate the virulence of a ΔripB ripP1 ripAA triple mutant in N. benthamiana, which recovered full virulence compared to the tobacco-virulent strain (Nakano and Mukaihara, 2019).

Thus, in N. benthamiana, RipB, RipP1 and RipAA are avirulence factors that contribute to ETI to a different extent. This implies that working on these effectors is more difficult due to the redundancy in their ETI activation function and therefore requires working on multiple mutants to avoid such redundancy. In addition, RipAW and RipAR can act as PTI suppressors since their Agrobacterium-mediated transient expression in N. benthamiana suppressed flg22-induced PTI responses, like ROS burst and activation of defence genes. Also, RipAW PTI-suppression effect seems to depend on its ubiquitin ligase activity, as transient expression of a RipAW catalytic mutant did not induce suppression of these PTI responses (Nakano et al., 2017).

With the examples of RipAR, RipAW, and RipN, we saw that, some RSSC T3Es can suppress PTI responses via different enzymatic activities, and presumably by acting at different levels within the plant cell. For other effectors, we know more about their effects on specific signalling pathways and on particular cellular mechanisms.

C.2.3. RSSC effectors affecting hormone signalling pathways

During infection, RSSC can face plant defences, some of which are controlled by hormone signalling pathways, including SA-related pathway. Therefore, to promote infection, manipulation of hormone signalling pathways by T3Es represents a potent virulence startegy.

For instance, RipAL is a chloroplastic T3E that can manipulate JA biosynthesis likely to inhibit SA-triggered defences (Nakano and Mukaihara, 2018). This effector has been demonstrated to suppress PTI responses in N. benthamiana leaves such as ROS burst and expression of PTI-related genes. Moreover, RipAL possesses a putative lipase domain that presents homology with the lipase domain of a plant chloroplastic phospholypase known to be involved in JA biosynthesis. Surprisingly, suppression of PTI responses depends on its enzymatic domain as a RipAL catalytic mutant no longer inhibits PTI responses. The authors also demonstrated that transient expression of RipAL in N. benthamiana induced JA accumulation and decreases SA levels, which depends on RipAL catalytic activity. In addition, this effector behaves as a virulence factor in pepper. Indeed, inoculation of pepper leaves with a RRSC strain mutated in ripAL led to lower bacterial growth and leaves did not accumulate JA compared to leaves inoculated with the WT strain (Nakano and Mukaihara, 2018). This study then revealed that the RSSC uses RipAL lipase activity to stimulate JA production that in turn antagonistically represses SA signalling, resulting in the dampening of plant immune responses.

Other effectors, RipG1 and RipG3, also alter SA signalling pathway to dampen plant defence responses (Medina-Puche et al., 2020). These RSSC T3Es are chloroplastic effectors belonging to the RipG family (former GALA family, comprising 7 members) and whose transient expression in N. benthamiana leaves seems to dampen the cytoplasmic calcium burst following flg22 treatment.

Moreover, transgenic A. thaliana expressing RipG1 and RipG3 had reduced expression levels of SA-responsive genes after flg22 treatment and were more susceptible to Pst DC3000, suggesting that these effectors alter SA-related defences. However, they did not suppress flg22-triggered immunity since ROS burst in these transgenic A. thaliana was not altered (Medina-Puche et al., 2020).

While these T3Es seem to manipulate SA-related defences, further research is needed to better understand the precise mechanisms of the SA signalling pathway manipulation and the benefit to RSSC bacteria, although this might be challenging as the RipG family is functionnally redundant (Cunnac et al., 2004).

C.2.4. Cellular processes altered through the nuclear activities of RSSC effectors

Some RSSC T3Es exert their virulence functions in the plant nucleus. RipAB is a nuclear targeted effector acting as a major virulence factor for the RSSC in potato (Zheng et al., 2019). In addition, transgenic expression of RipAB in potato plants led to a transcriptional reprogramming that resulted in the down-regulation of many genes including Ca 2+ signalling genes like calmodulin-encoding genes. Also, these transgenic lines showed attenuated PAMP-triggered ROS burst associated with an enhanced suceptibility. The authors hypothesised that RipAB could act either as a TF-like protein that could repress specific Ca 2+ signalling-related genes or by interacting with Ca 2+ signalling components, leading to indirect down-regulation of Ca 2+ signalling related genes (Zheng et al., 2019). However, many uncertainties remain regarding the precise action of RipAB in the nucleus and require further investigation.

The RipTAL effectors are known to alter cellular processes by binding DNA and inducing transcription of target genes. Brg11, also called RSc1815 or RipTALI-1, is a RipTAL from the RSSC strain GMI1000 that has been best documented in litterature so far (de Lange et al., 2013;Macho et al., 2010;Schandry et al., 2016;Wu et al., 2019). This effector participates in the virulence of the RSSC in eggplant et al., 2018). RSSC-mediated delivery of Brg11 in tomato increases agmatine and putrescine levels in planta (Wu et al., 2019). This raises the following question: why a component presumably linked to plant defences could benefit RSSC bacteria?

The authors noticed that inoculation of plants with RSSC bacteria lacking brg11 did not affect RSSC bacterial growth and that tomato plants defective in the Brg11targeted ADCs favour Pst DC3000 growth in planta but not RSSC bacterial growth.

Considering that Brg11 inhibits Pst DC3000 growth but not RSSC bacterial growth, the authors hypothesised that RSSC bacteria might use Brg11 to manipulate transcription and translation of polyamine biosynthesis regulators to compete with other pathogens that might not tolerate polyamine-related defence (Wu et al., 2019).

D. PopP2, a major virulence factor of the RSSC

Another effector acts at the nuclear level and has been shown to be a major virulence factor of the RSSC. This effector, well studied in the team, is PopP2 (RipP2) which deserves its own section as it is part of my thesis project. share around 42% similarity overall, suggesting that they are evolutionarily distant (Lavie et al., 2004). While the representation of popP genes in the RSSC reveals that they are present mainly in R. pseudosolanacearum strains (phylotypes I and III) rather than in R. solanacearum strains (phylotype II), the representation of PopP2 among the different strains in the RSSC is more dispersed among the different species (Lavie et al., 2004). Indeed, PopP2 is either present or absent in strains belonging to the same species. For instance, PopP2 is present in R.

pseudosolanacearum strain GMI1000 (phylotype I), originally isolated from tomato in Guyana but is absent from strain Rd15 which was originally isolated from radish in Taiwan and belongs to the same species and phylotype as GMI1000 (Lavie et al., 2004). In R. solanacearum phylotype IIB, PopP2 can be found in the Po82 strain isolated from potato in Mexico, but is not present in the MOLK2 strain, isolated from banana in Indonesia (Lavie et al., 2004;Peeters et al., 2013). However, host range does not condition the possibility of finding PopP2 in a RSSC strain. For instance, various strains originally isolated from tomato do or do not possess PopP2, like R. pseudosolanacearum CMR15 (phylotype III) from Cameroun and R.

solanacearum K60 (phylotype IIA) from the United States, which do and do not possess PopP2, respectively (Lavie et al., 2004;Peeters et al., 2013). Even in a restricted geographical area, PopP2 is not necessarily found in all strains present there. For example, in the Republic of Korea, among several strains isolated in the early 2000s that infect pepper and tomato, some strains do not possess this effector (Segonzac et al., 2017).

While PopP2 was first shown to be an avirulence factor in some host species/cultivars (see section D.2.1.), it also contributes to the virulence of RSSC bacteria in others. In particular, PopP2 has been shown to contribute to the virulence of GMI1000 in A. thaliana as a popP2-mutated strain induces fewer symptoms than the WT strain (Le Roux et al., 2015). Also, PopP2 contributes to bacterial fitness, i.e. the ability of the pathogen to grow in a competitive environment, in different crops such as tomato, bean and eggplant (Macho et al., 2010). So, PopP2 represents a major virulence factor for RSSC bacteria in many strains, and in various host species and its virulence functions are mainly based on its enzymatic activity. (Ma and Ma, 2016). As an important virulence factor for the RSSC, the enzymatic activity and how PopP2 targets host components have been well studied. PopP2 is a nuclear targeted effector whose enzymatic function is necessary for its virulence and avirulence activities (Le Roux et al., 2015;Tasset et al., 2010). As an acetyltransferase, the acetylation process used by PopP2 and the other YopJ acetyltransferase effectors has been envisioned as a "ping-pong model" (Figure 13B, Ma and Ma, 2016). In this model, the enzyme acetylates its substrate in the presence of the acetyl-coenzyme A (acetyl-CoA) cofactor in two steps. First, the enzyme uses acetyl-CoA to auto-acetylate and then transfer the acetyl group to the substrate. To activate their acetyltransferase activity, some YopJ effectors need the host cofactor inositol hexaphosphate (InsP6), an abundant metabolite in eukaryotic cells, which was hypothesised to induce changes in the effector conformation (Ma and Ma, 2016). Recent studies based on the analysis of crystal structures of PopP2 catalytic unit in complex or not with InsP6, acetyl-CoA or one of its substrates, revealed that InsP6 could enhance the acetyltransferase activity of PopP2 (Xia et al., 2021;Zhang et al., 2017b). In fact, PopP2 acetylation process could follow the ping-pong model.

NLS

Indeed, it has been shown that PopP2 auto-acetylates on a specific lysine residue and this auto-acetylation is required for its activities (Tasset et al., 2010).

Moreover, the reaction intermediate between PopP2 and acetyl-CoA was crystalised, supporting the two-step mechanism described in the ping-ping model (Zhang et al., 2017b). Also, crystal structures of the PopP2 catalytic unit in complex with InsP6 and/or its substrate revealed that InsP6 induces conformational changes in PopP2 including (i) opening of the binding pocket for Ac-CoA and (ii)

structural rearrangement in a fold-switch motif located between PopP2 regulatory domain and its substrate recognition helix (Xia et al., 2021;Zhang et al., 2017b).

This secondary-structure switch triggered by InsP6 enables PopP2 to become active and stabilises the interaction with its substrate (Xia et al., 2021) (Figure 13C). It is still unclear why such an activation process was evolved by bacterial pathogens. The authors hypothesised that the YopJ family effectors in their active state may be toxic to the bacteria producing them. To avoid potentially deleterious effects due to their enzymatic activity, these secreted effectors are completely latent until they are delivered into host cells where the InsP6 co-factor makes the enzyme active and regulates binding to its substrates. In A. thaliana, some ecotypes are resistant to the RSSC GMI1000 strain and it has been shown that this resistance is conferred by the NLR-encoding gene RRS1-R, behaving as a recessive resistance gene, and present in Nd-1 and Ws-2 accessions (Deslandes et al., 2002). In the Col-0 susceptible accession, the RRS1 protein is coded by the dominant RRS1-S allele. Later, it was shown that RRS1-R-mediated resistance was triggered by the effector PopP2, which interacts with and relocates RRS1 proteins in the nucleus (Deslandes et al., 2003). Actually, RRS1-R works in cooperation with RPS4, a second TIR NLR. The RPS4/RRS1 NLR pair confers resistance to 3 different pathogens: the bacterial pathogens RSSC producing PopP2, P. syringae expressing AvrRps4, and the fungal pathogen Colletotrichum higginsianum (Narusaka et al., 2009). The crystal structures of the TIR domains of RPS4 and RRS1 revealed that these two NLRs form a heterodimer via interaction of their TIR domains which is necessary for recognising two unrelated effectors, PopP2 and AvrRps4 (Williams et al., 2014). The main particularity of RRS1 resides in its C-terminus that contains a WRKY DNA-binding domain, which is found in WRKY TFs and known to bind specific cis-regulatory elements called the W-boxes (Deslandes et al., 2002;Rushton et al., 2010). Both RRS1-S and RRS1-R have a WRKY domain at the C-term but RRS1-R C-term has 90 additional residues compared to RRS1-S C-term (Deslandes et al., 2002).

Moreover, PopP2 recognition mechanism by RRS1-R/RPS4 has been elucidated

(Figure 14). Indeed, after relocating RRS1-R to the nucleus, PopP2 was shown to acetylate the WRKY domain of RRS1-R on a specific lysine residue, the second lysine in the conserved WRKYGQK heptad motif of the WRKY domain, leading to a loss of affinity between the WRKY domain and DNA. PopP2 acetylation of RRS1-R WRKY domain is the trigger for activation of RPS4-dependent immunity (Le Roux et al., 2015;Sarris et al., 2015). Activation of RRS1-R/RPS4-mediated resistance depends on the acetyltransferase activity of PopP2 since a catalytic mutant of PopP2 fails to acetylate RRS1-R and does not trigger resistance in planta (Le Roux et al., 2015;Tasset et al., 2010). Surprisingly, PopP2 also acetylates RRS1-S on the same key Lysine residue and this leads to its dislodging from DNA, suggesting that the inability of RRS1-S to activate a robust immune response in presence of PopP2

is not linked to a change in its ability to bind DNA (Le Roux et al., 2015;Sarris et al., 2015). Thus, at this stage, while the mechanism of PopP2 recognition by RRS1-R became clearer, the precise activation process of the immune-complex RRS1-R/RPS4 and downstream signalling remained elusive.

Subsequent studies have attempted to better understand the activation of this immune complex, including the involvment of particular PopP2 subdomains or the role of RRS1 PTMs in the activation of the immune complex. For instance, PopP2

possesses an Ethylene-responsive element binding factor associated Amphiphilic Repression (EAR) motif (LxLxLxL) within its catalytic unit, which is involved in PopP2 protein stability in planta and that is required in its avirulence functions since mutation of this motif results in a loss of recognition by RPS4/RRS1-R (Segonzac et al., 2017). This motif is normally involved in the recruitment of transcriptional co-repressors but it remains to be determined whether PopP2 can recruit such proteins.

Regarding RRS1-R, a recent study reported how phosphorylation of RRS1 might regulate its function (Guo et al., 2020). Indeed, RRS1-R but not RRS1-S C-terminus is phosphorylated in planta. This phosphorylation might participate in RRS1-R auto-inhibition and in PopP2 recognition. Activation of the RRS1-R/RPS4 complex seems to rely on increased proximity between RRS1 N-term and C-term triggered by interaction with matching effectors, inducing a decrease in affinity between the TIR domains of RRS1 and RPS4, which are known to be responsible for the inhibition of the complex. Phosphorylation of RRS1-R C-term at a specific Threonine, Thr1214, seems important for RRS1-R repression, and acetylation by PopP2 at nearby sites including Lys1221 seems to compete with Thr1214 phosphorylation and explain the derepression of RRS1-R/RPS4 complex (Guo et al., 2020). Thus, the fact that PopP2 is recognised by RRS1-R but not by RRS1-S might be due to phosphorylation of RRS1-R C-term but not of RRS1-S. Indeed, phosphorylation of RRS1-R C-term confers a greater proximity between RRS1-R Nterm and C-term, which is then enhanced by interaction with PopP2 leading to derepression of the RRS1-R/RPS4 complex. In RRS1-S, as the C-term part is not phosphorylated, the proximity between RRS1-R N-term and C-term is less pronounced, and the interaction with PopP2 does not increase this proximity sufficiently to activate the complex. The difference in phosphorylation status between RRS1-R and RRS1-S might lie in the 90 residues lacking in RRS1-S that could act as kinase docking domain (Guo et al., 2020).

D.2.2. PopP2 is also an avirulence factor in crops

Interestingly, PopP2 has been shown to be an avirulence factor also in crop species such as tomato, bean and eggplant (Macho et al., 2010;Pensec et al., 2015) Interestingly, other WRKY TFs, including WRKY8, WRKY18, WRKY28, WRKY22 and WRKY53 were acetylated by PopP2 (Le Roux et al., 2015;Sarris et al., 2015). In the absence of RRS1-R/RPS4 recognition, lysine-acetylation of multiple WRKY transcription factors by PopP2 dislodges them from their DNA-binding sites and disables their trans-activating functions needed for defence gene expression (Figure 14) (Le Roux et al., 2015). This essentially dampens host basal resistance, allowing rapid spread of the pathogen inside tissues and leading to the complete wilting of infected plants.

Therefore, RRS1-R with its integrated WRKY domain serves as a direct sensor of PopP2 virulence activity upon infection. This WRKY domain in RRS1-R can be viewed as an effector target 'decoy' which betrays the resistance-suppressing actions of PopP2 on its operational targets, the defensive WRKY transcription factors. Molecular integration of a WRKY decoy domain within the RRS1-R/RPS4 receptor complex creates an effective 'radar' for a powerful pathogen virulence activity which cannot be easily dispensed with by the pathogen. The observed fusion of further potential effector target decoy domains with NLR receptors in different plant species suggests a fundamental mechanism in plants for increasing receptor recognition 'space' by integrating in immune receptors molecular decoys that mimick the true virulence targets of pathogen effectors (Grund et al., 2019;Kroj et al., 2016). This also provides promising avenues for engineering receptors that could effectively intercept disease-promoting activities of agronomically important crop pathogens.

4-Apart from WRKY defensive TFs, PopP2 also targets bromodomain-containing Arabidopsis epigenetic readers.

To elucidate other virulence activities of PopP2, a YopJ family acetyltransferase that is exclusivey targeted to the plant nucleus, a Y2H screening was undertaken several years ago to identify Arabidopsis proteins other than WRKY proteins that could be manipulated by PopP2. This screening was performed using a A. thaliana cDNA library composed of all genes expressed in resistant and susceptible seedlings challenged for 24 hours with the RSSC strain GMI1000 (Deslandes, unpublished data). After several rounds of screening, among the various candidate PopP2-interacting partners identified was a protein containing a bromodomain

(BRD) and an extra-terminal domain: GTE11 from the General Transcription factor group E (GTE) family.

A. Arabidopsis GTE proteins contain a bromodomain, a domain that allows recognition of acetylated lysine residues GTE11, and its closest member regarding BRD protein sequence GTE9, are members of the GTE family, also sometimes referred to as the Bromodomain and Extra-Terminal domain (BET) family, as members of this family harbour two particular protein domains: a N-term bromodomain and a C-term extra-terminal domain (Pandey, 2002). The bromodomain (BRD) is a specific module of ~110 aa that allows the recognition of acetylated lysines mainly on histone tails but also on

Asp (N)

other proteins; and the extra-terminal domain is a domain allowing protein-protein interactions (Filippakopoulos and Knapp, 2012;Wang et al., 2021). Mechanistically, BRD (whose crystal structure from many animal BRD-containing proteins was solved (see Figure 15)) can recognise acetylated lysine residues via a central hydrophobic pocket in which the acetylated lysine can anchor to a conserved asparagine residue (Wang et al., 2021). The specificity of the bromodomain for a type of acetylation is protein dependent (Zaware and Zhou, 2019).

BRD-containing proteins have been extensively studied in animals since they were

shown to be involved in the development of various human diseases, including cancers (Wang et al., 2021). Thanks to this knowledge, we know that animal BRDcontaining proteins can be found in different types of proteins such as HATs, histone methyltransferases, chromatin remodelling complex subunits, transcriptional regulators or ubiquitin ligases (Zaware and Zhou, 2019). Also, most BRD-containing proteins contain other domains that can function with or independently of the BRD to mediate protein functions. As they can bind acetylated histones, BRD-containing proteins are considered to be epigenetic readers and participate in PTM modification of histones, chromatin remodelling or also in transcriptional regulation as they can recruit components of the transcriptional machinery to specific gene regions (Zaware and Zhou, 2019).

Surprisingly, animal BRD-containing proteins possess between one and four BRDs while plant BRD-containing proteins possess only one (Wang et al., 2021;Zhao et al., 2018). A recent study profiled A. thaliana histone readers and found the presence of 28 BRD-containing proteins encoded by the A. thaliana genome and they all contained a single BRD (Zhao et al., 2018). The authors showed that BRDs from A. thaliana and animals shared conserved critical residues that are important for recognising acetylated lysines on histones and that two BRD-containing proteins from Arabidopsis were able to bind acetylated lysines on histone H4, suggesting that BRD function is quite conserved between animals and plants.

Furthermore, they showed that the BRD of two Arabidopsis BRD-containing proteins had a higher affinity for diacetylated peptides on histone H4 than for monoacetylated ones, suggesting that their BRD preferentially bind a combination of acetylated residues on histone tails, similar to animal BET proteins (Wang et al., 2021;Zhao et al., 2018). About ten Arabidopsis BRD-containing proteins profiled by Zhao and colleagues have been characterised and some of them are known to be subunits of chromatin remodelling complexes, such as BRM, BRD1, BRD2 and BRD13 from the BRAHMA chromatin remodelling complex or MBD9 and NPX1 from the SWR1 chromatin remodelling complex (Farrona et al., 2007;Nie et al., 2019;Yu et al., 2021). Others are HATs or components of the transcriptional machinery, like the HAT GCN5 and the RNA-polymerase II pre-initiation complex component TAF1 (Benhamed et al., 2008;Waterworth et al., 2015). Interestingly, for some of these proteins, it has been shown that their BRD can bind to acetylated histone H3 or H4 reinforcing the hypothesis that BRD is a conserved domain between animals and plants in its abilities to recognise acetylated histone tails (Nie et al., 2019;Zhang et al., 2016a;Zhao et al., 2018). All of these BRD-containing proteins appear to be involved in a variety of biological processes including flowering, ABA signalling pathway, DNA damage repair, environmental stress response, incorporation of the histone variant H2A.Z... (Farrona et al., 2011;Grasser et al., 2021;Luo et al., 2020;Peirats-Llobet et al., 2016;Waterworth et al., 2015;Zhang et al., 2017a).

Thus, BRDs, domains that allow recognition of acetylated lysine residues on histone tails in particular, are quite conserved between animal and plants, and can be found in a variety of proteins ranging from members of the chromatin remodelling complex, to components of the transcriptional machinery, to HATs. As they possess only one BRD, plant BRD-containing proteins have been assumed to act in complex to perform their functions, for example by addressing a given protein to specific promoters, or helping to recruit other components of a complex.

Among the BRD-containing proteins from A. thaliana are also members of the GTE family, to which the PopP2-targets GTE9 and GTE11 belong. Current knowledge about the biological roles of different members of this family, including GTE9 and GTE11, will be discussed in the section below.

B. GTE9 and GTE11 belong to the GTE family, a family involved in various biological and cellular processes in A. thaliana, but whose molecular functions remain unclear

The GTE family, which includes GTE9 and GTE11, is sometimes known as BET family because the members of this family possess a BRD and an extra-terminal domain like the animal BET proteins (Pandey, 2002). In Arabidopsis, the GTE family counts twelve members (Figure 16), which appear to be involved in a variety of biological processes (Figure 17), although little is known about their precise biological functions.

• B.1. GTE family proteins are involved in a variety of biological processes GTE1, also known as IMB1, is the first GTE member characterised and is involved in seed germination in A. thaliana (Duque and Chua 2003). Seed germination is controlled by pathways involving notably ABA and light, which is perceived by plant phytochromes (Yadukrishnan and Datta, 2021). In this context, by studying Arabidopsis gte1 mutant, GTE1 was shown to be a negative regulator of ABAmediated responses and a positive regulator of phytochrome A-related responses during germination (Duque and Chua, 2003). However, the mechanistic details of the role of GTE1 in these pathways remain unknown.

Subsequently, a study characterised Arabidopsis plants defective in GTE4 and showed its importance in maintaining the mitotic cell cycle (Airoldi et al., 2010).

Indeed, gte4 mutant plants are small, with scraggy leaves and fewer cells in organs than WT plants. Further investigations revealed that these mutants had a delayed activation of the cell cycle during germination, cell proliferation stopped sooner than in WT plants, which impacted on the whole plant development (Airoldi et al., 2010). The authors could then conclude that GTE4 has a role in cell cycle regulation, but the precise molecular functions of this protein require further investigation. GTE6 was shown to be involved in leaf development (Chua et al., 2005). Indeed, gte6 mutant displayed abnormally shaped leaves in mature leaves, and GTE6 overexpressing lines had elongated young leaves. It seems that the link between GTE6

and leaf shape control relies on the positive regulation by GTE6 of ASYMMETRIC LEAVES1 (AS1), a protein that controls leaf shape, and this may be due to the binding of GTE6 to AS1 promoter which correlates with an increase in histone acetylation at this promoter. Indeed, AS1 transcripts were more abundant in the GTE6 over-expressing line and less abundant in the RNAi knockdown lines and ChIP analyses revealed that GTE6 can bind AS1 promoter transcription starting site and these regions were hyperacetylated on histones H3 and H4 in the GTE6 overexpressing line compared to WT (Chua et al., 2005). Thus, GTE6 seems to regulate leaf shape via positive regulation of AS1 expression by creating a favourable environment for histone acetylation, a marker of active transcription.

Lastly, GTE10, also known as NPX1, was found to participate in the regulation of ABA-related responses (Kim et al., 2009). GTE10 is up-regulated under stress and ABA treatment, and plants mutated or over-expressing GTE10 showed altered responses to ABA compared to WT plants. Indeed, the GTE10 over-expressing line was less sensitive to ABA as seed germination and root growth of 6-day-old seedlings were not impaired on a medium containing ABA, and ABA treatment did not induce stomatal closure contrary to WT plants. On the other hand, gte10 mutant is more sensitive to ABA suggesting that GTE10 is a negative regulator of ABAmediated responses. Moreover, GTE10 over-expressing lines are less tolerant to drought stress and conversely, gte10 mutant is more resistant, which correlates with the fact that GTE10 over-expression alters stomatal closure. In addition, GTE10 seems to be a negative regulator of ABA-related responses notably by regulating ABA-related genes expression, including TFs (Kim et al., 2009).

In conclusion, members of the GTE family appear to be involved in various biological processes ranging from seed germination, cell cycle regulation, to leaf development and ABA signalling (Figure 17). However, for most of them, their role and their molecular functions remain elusive as studies have mainly focused on phenotypical characterisation of mutants. For others, some more details are available regarding their molecular function or regulation, which will be described below.

• B.2. Nuclear activities of particular GTE proteins

As BRD-containing proteins, GTE members are expected to be nuclear proteins, and this has been demonstrated for GTE1, GTE6 and GTE10 (Chua et al., 2005;Duque and Chua, 2003;Kim et al., 2009). Moreover, two GTE members, GTE10, and GTE3, have been shown to interact with acetylated histone H3, suggesting that their BRD is functional to recognise acetylated histone tails (Figure 17) (Garcia-Dominguez et al., 2008;Nie et al., 2019).

From a mechanistic point of view, the molecular functions of GTE6 and GTE10 have been described in more detail (Figure 17) (Chua et al., 2005;Kim et al., 2009;Nie et al., 2019). As described in the precedent section, GTE6-mediated regulation of leaf development has been shown to be linked to AS1 up-regulation promoted by histone acetylation on AS1 promoter (Chua et al., 2005). Regarding GTE10mediated regulation of ABA-related responses, it seems that GTE10 is involved in the up-or down-regulation of various ABA-related genes since the GTE10 overexpressing line showed differential expression of several ABA-responsive genes, including TFs and genes involved in ABA biosynthesis (Kim et al., 2009).

Furthermore, as with some animal BRD-containing proteins, GTE10 has been shown to interact with a NAC TF called TIP, and to be a transcriptional repressor of this TF when co-expressed in yeast. Thus, it seems that GTE10 negatively regulates the ABA signalling pathway by acting notably as a transcriptional repressor of ABA-inducible TFs (Kim et al., 2009).

Recently, another role of GTE10 has been described, related to chromatin remodelling and incorporation of the histone variant H2A.Z. GTE10 has been shown

to associate with the chromatin remodelling complex SWR1, which mediates the incorporation of the histone variant H2A.Z into nucleosomes (Lei and Berger, 2020;Nie et al., 2019). Indeed, when screening for interacting partners of the SWR1 subunit PIE1 by pull-down assay, various proteins were identified including GTE10 (Nie et al., 2019). Interestingly, another BRD-containing protein, MBD9, interacts with PIE1 and seems to function redundantly with GTE10 as gte10 and mbd9 single mutants do not show a distinct phenotype from WT but a gte10/mbd9 double mutant displays longer roots than WT. This phenotype seems to depend on the BRD of GTE10 since complementation with GTE10 restores the WT phenotype but complementation with a version of GTE10 that encodes a GTE10 version mutated in its BRD does not. Also, the gte10/mbd9 double mutant exhibited increased DNA methylation, similar to mutants defective in subunits of the SWR1 chromatin remodelling complex or in components involved in DNA demethylation, suggesting that these two BRD-containing proteins antagonise transcriptional silencing and prevent hypermethylation of specific genomic regions (Nie et al., 2019). Since GTE10 interacts with a subunit of the SWR1 remodelling complex, and in the gte10/mbd9 double mutant DNA methylation is increased and H2A.Z deposition at DNA hypermethylated loci is decreased, it is hypothesised that GTE10 and MBD9, by recognising acetylated histones can recruit the SWR1 chromatin remodelling complex at specific methylated genomic loci, leading to the incorporation of H2A.Z in the nucleosomes which can lead to DNA demethylation (Nie et al., 2019).

Finally, although the biological function of GTE3 and GTE5 remains unknown, a study related a way to regulate the binding of these GTEs to acetylated histones (Garcia-Dominguez et al., 2008). Indeed, GTE3 and GTE5 have been demonstrated

to interact with an Arabidopsis SUMO ligase SIZ1 by Y2H. Of the twelve members from this family in Arabidopsis, only GTE3 and GTE5 interacted with SIZ1 in yeast and were sumoylated by SIZ1 in vitro. Interestingly, after showing that GTE3 binds to acetylated histone H3, sumoylation of GTE3 led to the alteration of GTE3 binding to acetylated histone H3 in vitro, suggesting that sumoylation could be a way to regulate GTE3 binding to acetylated histone H3, and likely for GTE5 which also binds acetylated histone H3 (Figure 17) (Garcia-Dominguez et al., 2008). Few studies mention the role of GTE9 and GTE11 members, formerly called BET9 and BET10, in Arabidopsis but it seems that these GTEs are involved in BT2mediated sugar and ABA responses (Figure 17) (Misra et al., 2018). Both GTE9 and GTE11 interact in vitro with BT2, a BTB/POZ and TAZ domain protein that is thought to be part of a ubiquitin ligase complex and mediates responses to various stresses (Figure 18) (Du and Poovaiah, 2004;Mandadi et al., 2009).

Characterisation of the gte9 and gte11 mutants revealed similar phenotypic responses to bt2 mutant, i.e. greater sensitivity to ABA-and sugar-mediated inhibition of germination, and over-expression of BT2 in gte9 or gte11 mutants does not lead to resistance to ABA-and sugar-mediated germination inhibition in contrast to BT2 over-expression in the WT background, suggesting that both GTE9 and GTE11 are required for BT2 functions (Misra et al., 2018). Considering that GTE9 interacts with BT2 in vivo, that loss of function of GTE9 or GTE11 mimics the loss of BT2 in response to exogenous application of sugar or ABA by increasing ABA-and sugar-mediated inhibition of germination, it appears that GTE9 and GTE11 are negative regulators of sugar and ABA signalling pathways. In addition, GTE9 and GTE11 do not seem to affect the transcription of BT2 or ABA-responsive genes, suggesting that they may not play a role in regulating the transcription of these genes and/or that they act downstream in the signalling pathways (Misra et al., 2018). However, the molecular mechanisms underlying the role of GTE9 and GTE11 as negative regulators of ABA and sugar signalling pathways remain to be investigated.

Although GTE9 and GTE11 do not appear to be transcriptional regulators of the BT2-mediated ABA and sugar signalling pathways, this does not exclude them from acting as transcriptional regulators (Figure 17). Indeed, it has been reported in a yeast transcription activation assay that GTE11 might have a transcriptional activation activity (Du and Poovaiah, 2004). Moreover, GTE9 and GTE11 seem involved in BT2-mediated transcription activation of genes that depend on 35S enhancers (Figure 18) (Irigoyen et al., 2021). Indeed, this study investigated the impact of BT2, GTE9 and GTE11 on the expression of YUCCA1, a gene involved in auxin biosynthesis, in the A. thaliana yucca1d line, which constitutively expresses YUCCA1 through a T-DNA insertion with four copies of the 35S enhancer near the YUCCA1 gene. By characterising crosses between yucca1d lines and mutants defective in BT2, GTE9 or GTE11, it was revealed that BT2, GTE9 and GTE11 were necessary for 35S-mediated activation of YUCCA1, meaning that GTE9 and GTE11 might be transcriptional regulators acting in complex with other transcription regulators such as BT2 (Irigoyen et al., 2021). However, whether there is a link between GTE9 and GTE11 role in ABA and sugar signalling pathways and their potential transcriptional regulatory activities remains unknown. It would also be interesting to investigate whether GTE9 and GTE11 have other transcriptional regulation activities and to find out which biological processes they are linked to.

Thus, even though the functional characterisation of the different members of the GTE family in Arabidospis remains limited, we have some insight into the different biological processes in which they participate, including ABA hormone signalling, regulation of germination, cell cycle and leaf shape. Interestingly, some of them, but not many, provide information on their molecular function and how they could contribute to the regulation of different biological processes. For instance, some

GTEs interact with various nuclear processes including TFs, transcriptional regulators, or subunits of chromatin remodelling complexes, and others seem to be able to bind promoter and transcription starting regions. In any case, many of them seem to participate indirectly to the regulation of gene transcription, probably by promoting a transcriptionally active environment at specific loci, or by recruiting other nuclear components that could influence the transcriptional status of these specific regions. Also, some of the GTE members have been shown to bind acetylated histones via their BRD, suggesting that they are epigenetic readers like BET proteins in animals. In this context, GTE9 and GTE11, two GTE members behaving as negative regulators of ABA and sugar signalling pathways, were found to be targeted by PopP2. These PopP2 targets have been the subject of preliminary studies in the team, notably by Alice Delga, a former PhD student, who started to characterise these PopP2 targets, work that I continued during my PhD.

C. Preliminary results: PopP2 targets GTE9 and GTE11, two histone readers that preferentially bind tetra-acetylated H4

Alice Delga worked on the characterisation of GTE11, a potential target of PopP2 identified from a Y2H cDNA library screening using PopP2 as bait. Alice also studied the potential targeting of GTE9 as it is the closest member of GTE11 in terms of their BRD protein sequences (Figure 16) (Delga, 2015).

During her PhD, Alice D. showed that :

-Both GTE9 and GTE11 co-localise with PopP2 or its catalytic mutant PopP2-C321A in A. thaliana or N. benthamiana nuclei.

-By using a FRET-FLIM assay performed in N. benthamiana, PopP2 was found to physically interact with GTE11 and GTE9.

-Also, PopP2 and PopP2-C321A were found to promote the protein accumulation of GTE9 and GTE11 in planta, as previously observed with some WRKY TFs (Le Roux et al., 2015).

-immunoprecipitation of Lys-acetylated proteins revealed that GTE9 and GTE11 co-expressed in planta with active PopP2 are modified by acetylation. These two proteins therefore behave as additional substrates of PopP2.

-a SPOT peptide array assay performed with the BRD of GTE9 expressed as a recombinant protein revealed its preferential binding to tetra-acetylated histone H4 and that this binding was dependent on the integrity of the conserved Asparagine residue present in the BRD of GTE9 in position 214 (N214).

-The ability of GTE9 and GTE11 to interact with histone H4 was further validated in planta using FRET-FLIM assay. Strikingly, GTE9-N214A and GTE11-N206A mutants, both mutated in the conserved N residue of their BRD, were unable to physically interact with histone H4, confirming the critical role of this residue for BRD function, as previously described in bromodomain-containing animal proteins.

Thus, at this stage, we knew that PopP2 could target the BRD-containing proteins GTE9 and GTE11, which seem to behave as histone readers since they interact with histone H4 (Figure 19). Alice started to investigate the role of GTE9 and GTE11

in the plant response to the GMI1000 strain and to see whether GTE9 and GTE11

were the only GTEs targeted by PopP2. It was in this context that I started my PhD which aimed at further characterise the targeting of GTE9 and GTE11 by PopP2. ?

PhD project My PhD project aimed at further characterising the targeting of the A. thaliana BRD-containing proteins GTE9 and GTE11 by the RSSC T3E PopP2. This project was structured around two axes. The main axis was to follow up on Alice Delga's work, to determine how PopP2 can manipulate GTE9 and GTE11, whether these proteins play a role in the plant response to the RSSC, but also to better characterise their function. The second axis aimed at studying the chromatin sites visited by PopP2 in Arabidopsis, in the presence or not of the RPS4/RRS1-R immune receptor complex (Figure 19).

In the first axis, I tried to answer different questions to further characterise the targeting of GTE9 and GTE11 by PopP2:

-Does PopP2 manipulate GTE9 and GTE11, likely by altering their association with histone H4?

-Do GTE9 and GTE11 function as virulence targets playing a role in the plant response to the RSSC? -Can PopP2 target other members of the GTE protein family?

-Can GTE9 and GTE11 be targeted by other effectors or pathogens?

-What is the role of GTE9 and GTE11 at the chromatin level?

The second axis will aim at investigating which chromatin sites are visited by

PopP2 by developing molecular and genetic tools which will presented and discussed.

Chapter 1: PopP2 manipulates the BRD-containing proteins GTE9 and

GTE11 to promote infection

Prior to Alice Delga's and my PhD, GTE11 had been identified as a potential target of PopP2 in a Y2H screening using PopP2 as a bait against a cDNA library from A.

thaliana seedlings. Alice Delga started to investigate the targeting of GTE11 by PopP2, and of GTE9, its closest member regarding the BRD protein sequence, and the potential role of GTE9 and GTE11 as histone readers (Delga, 2015).

Although Alice D. generated a large amount of data, there was still a lot of uncertainty about the manipulation of GTE9 and GTE11 by PopP2 and their possible involvement in the plant response to the RSSC GMI1000 strain. I therefore continued the work already undertaken and the data obtained during my thesis were integrated into a manuscript that will be (re)submitted very soon (Comorge et al., in prep.). This manuscript is included in this Chapter 1.

To summarise the results shown in the article, many of which are the result of my thesis work, we show that PopP2 co-localises and interacts with GTE9 and GTE11 in plant cell nuclei in N. benthamiana. In addition, GTE9 BRD was found to bind preferentially to tri-and tetra-acetylated Histone H4 in vitro. This interaction with Histone H4 was further confirmed in vivo, as GTE9 and GTE11 interact with Histone H4. This interaction depends on the integrity of the BRD of GTE9 and GTE11 since substitution of the conserved asparagine residue, known to be responsible for BRD recognition of acetylated lysine residues in animal BRD-containing proteins, abrogated this interaction with Histone H4. A mass spectrometry-based proteomic analysis of GTE9 and GTE11 demonstrated both proteins behave as PopP2 substrates since they are both modified by PopP2 acetyltransferase activity on several lysine residues, some of which are located on either side of their BRD. In addition, by using a semi-quantitative FRET-FLIM assay, we demonstrated that PopP2 alters the binding of GTE9 to Histone H4 in vivo and this depends on its acetyltransferase activity. We also demonstrated that GTE9 and GTE11 overexpressing lines are more susceptible to the RSSC strain GMI1000, suggesting that

A bacterial pathogen manipulates Arabidopsis BET bromodomaincontaining epigenetic readers using a YopJ family acetyltransferase

Authors

Virginie Comorge 1, † , Alice Delga (Cui et al., 2015). This ETI is often associated with a programmed cell death (hypersensitive response) that restricts the pathogen growth around the site of infection.

A major consequence of both PTI and ETI signalling is a rapid and massive transcriptional reprogramming with overlapping sets of defence-related genes differing both in kinetics and intensity (Cui et al., 2015;Katagiri and Tsuda, 2010;Thomma et al., 2011). In Arabidopsis, these transcriptional changes involve epigenetic modifications in chromatin composition and remodelling (Ding and Wang, 2015;Latrasse et al., 2017;Ramirez-Prado et al., 2018b, 2018a;Zhu et al., 2016b). From a bacterial pathogen's perspective, subversion of host epigenome represents a potent virulence strategy to take control of gene expression.

Several animal bacterial pathogens (e.g., Listeria monocytogenes, Chlamydia trachomatis or Shigella flexneri) target the host epigenome by interfering with the fine-tuned regulations of chromatin modifications (Dong and Hamon, 2020;Silmon De Monerri and Kim, 2014). They can favour infection by inducing histone modifications to alter the accessibility of gene promoters (Bierne and Cossart, 2012;Rolando et al., 2015).

Although host epigenome modification by animal pathogens is well documented, such mechanisms remain poorly characterised in plants. To date, only few examples of pathogen interference on epigenome reprogramming have been reported, among which suppression of small RNA silencing by the oomycete Phytophthora sojae (Xiong et al., 2014) and inhibition of defence responses through modulation of histone methylation by Geminiviruses (Castillo-González et al., 2015). The P. sojae PsAvh23 effector prevents the formation of a Histone Acetyltransferase complex required for immunity activation (Kong et al., 2017). These rare examples highlight effective counter-defence mechanisms used by plant pathogens to suppress activation of host defence genes by interfering with epigenetic-related processes. However, while plant bacterial pathogens can modulate host cell reprogramming by interfering with various transcriptional and posttranscriptional regulation processes, whether they also subvert the host epigenome remains elusive.

Here, we investigated the nuclear activities of the type III effector PopP2 expressed by the root-infecting Ralstonia solanacearum. PopP2 belongs to the YopJ family of acetyltransferases (Deslandes et al., 2003;Tasset et al., 2010). PopP2 dampens basal immune responses by acetylating a key lysine residue in the WRKY DNA-binding domain of WRKY defensive transcription factor, thereby inhibiting their transactivating functions needed for defence gene expression (Le Roux et al., 2015;Sarris et al., 2015). In Arabidopsis, PopP2 is recognised by the NLR pair RPS4/RRS1-R (RESISTANCE TO PSEUDOMONAS SYRINGAE4/RESISTANCE TO RALSTONIA SOLANACEARUM1) that molecularly interact to form a pre-activation complex that is activated by PopP2 acetylation of RRS1-R WRKY domain behaving as an integrated decoy mimicking PopP2 true virulence targets (Le Roux et al., 2015;Sarris et al., 2015;Williams et al., 2014). For better understanding of PopP2 nuclear activities promoting its virulence functions, we searched for additional host components targeted by this bacterial effector. By yeast two-hybrid, we isolated GTE11 (Global Transcription Factor with Extra-terminal domain 11) as a PopP2-interacting partner candidate.

GTE11 belongs to the Bromodomain and Extra-Terminal domain (BET) family also referred to as the Global Transcription Factor with Extra-terminal domain (GTE) family (Pandey et al., 2002). Bromodomains are conserved structural modules that bind acetylated lysine residues.

They can serve as epigenetic readers by recognising specific acetylated lysine motifs on histone tails. Here, we showed that GTE11 and its closest homolog GTE9 interact with PopP2 in the plant nucleus. PopP2 acetylates several lysine residues located on either side of GTE9 and GTE11 bromodomain that binds histone H4 in vivo. Remarkably, enzymatically active PopP2 affects GTE9 binding to histone H4, indicating that PopP2 uses acetylation to regulate the association of GTE9 to chromatin. Moreover, transgenic overexpression of GTE9 and GTE11

in Arabidopsis enhances bacterial wilt disease development in a PopP2-dependent manner. This study identifies a virulence strategy employed by a plant pathogenic bacterium that promotes infection through manipulation of BET bromodomain-containing proteins using a typeIIIsecreted acetyltransferase. 1.

ET Brd Brd ET Brd PopP2-CFP PopP2-CFP+YFP PopP2-CFP+GTE9-YFP PopP2-CFP+GTE11-YFP Brd C321A-CFP+ GTE9-YFP PopP2-CFP+ GTE9-YFP GTE9-YFP C321A-CFP PopP2-CFP nuclei (nb) C RESULTS

PopP2 interacts with GTE11 and GTE9 in yeast

To progress in the elucidation of the functions of PopP2 in planta, we searched for Arabidopsis components interacting with PopP2. To this end, we performed a yeast-two-hybrid (Y2H) screening using PopP2 as bait for a prey cDNA library derived from Arabidopsis Col-0 seedlings. We identified one positive prey clone (designated as Pip8 for PopP2-interacting partner 8) corresponding to a partial cDNA encoding for the Arabidopsis protein GTE11 protein (At3g01770, also designated by BET10) lacking its 26 N-terminal residues. The full-length GTE11 cDNA that encodes for a protein of 620 amino acids was amplified and used as a prey clone to reconfirm interaction with PopP2 in Y2H (Fig 1A-B). GTE11 (for General Transcription factor with an Extra-terminal domain 11) belongs to a family of 12 members in Arabidopsis (GTE1 to GTE12). Also referred as Bromodomain and Extra Terminal domain (BET) proteins, GTEs are characterised by the presence of two types of domains, the bromodomain (Brd) and the extra-terminal domain (ET) (Fig 1B). Present in a broad range of nuclear proteins comprising histone acetyltransferases, methyltransferases, helicases, transcriptional coactivators or nuclear-scaffolding proteins, BRDs are protein modules that exclusively recognise acetylation motifs (Muller et al., 2011). As previously published (Airoldi et al., 2010), a phylogenetic analysis of the GTE members indicates that GTEs can be divided into three groups with GTE11 belonging to a group with four yet uncharacterised GTE genes (GTE9, GTE8, GTE10, and GTE12). This group includes the GTE9 protein whose sequence has 63% identity and 72% similarity with GTE11. Since GTE9 represents the closest paralog of GTE11, we therefore included GTE9 in our study to determine whether it might also be targeted by PopP2. Use of full-length GTE9 cDNA (encoding for a protein of 688 amino acids) as prey clone in yeast-two-hybrid revealed it can also interact with PopP2 (Fig 1A-B).

GTE11 and GTE9 co-localise and physically interact with PopP2 in the plant cell nucleus

As bona fide interactors of PopP2, GTE9 and GTE11 were hypothesised to localise with PopP2 in the plant nucleus where they should interact. We first investigated the subcellular localisation of GTE9 and GTE11. As expected, GTE11 and GTE9 C-terminally fused with a yellow fluorescent protein (GTE9-YFP and GTE11-YFP, respectively) and transiently expressed in N. benthamiana were found to co-localise with PopP2-CFP as well as with catalytically inactive PopP2-C321A-CFP in the plant nucleus . Note that in a certain proportion of nuclei (with a rate of about 20 to 50% depending on the experiments), PopP2-CFP or C321A-CFP were found to be relocalized in subnuclear foci containing .

The nuclear localisation of GTE11 and GTE9 and our Y2H data strongly suggest they can physically interact in planta with PopP2. To test this hypothesis, we used a Förster resonance energy transfer (FRET) analysed by fluorescence lifetime imaging microscopy (FLIM) approach aimed at monitoring protein interactions in living cells. PopP2-CFP was used as a FRET donor and transiently expressed in N. benthamiana cells either alone or with GTE11-YFP or GTE9-YFP, both fusion proteins serving as FRET acceptors. In case of interaction, the fluorescence lifetime of PopP2-CFP was expected to decrease significantly due to close proximity with its interacting partners fused with YFP, producing transfer of energy (FRET) between the donor (CFP) and acceptor (YFP) fluorophores. In the presence of GTE11-YFP or GTE9-YFP, a significant decrease of the PopP2-CFP lifetime was observed, indicating a physical interaction between the tested proteins (Fig 1D, Supp Table 1). Notably, these interactions could only be detected when the co-expressed proteins accumulated in subnuclear foci. To verify that the decrease in PopP2-CFP lifetime was not due to a change in PopP2 nuclear environment triggered by GTE9 or GTE11 or influenced by their nuclear overaccumulation, PopP2-CFP was co-expressed either with GTE11-3HA or YFP alone, the former being theoretically able to interact with PopP2 but unable to serve as acceptor and vice versa for the latter. No decrease in PopP2-CFP lifetime was observed neither upon co-expression with GTE11-3HA nor YFP in N. benthamiana (Supp Table 1), thus confirming the physical associations detected between GTE9 and GTE11 with PopP2 in the nucleus. The catalytically inactive PopP2-C321A mutant (PopP2-C321A-CFP) was found also to interact with these two GTE proteins, indicating that the main Cys321 catalytic residue of PopP2 is dispensable for the interaction (Supp Table 1). Note that similar FLIM-FRET measurements were obtained in A. thaliana seedlings, confirming the protein interactions detected in N. benthamiana used as a heterologous expression system (Supp Table 1).

PopP2 acetylates GTE9 and GTE11

Considering that GTE9 and GTE11 physically interact with PopP2 in the nucleus, these two therefore represent putative substrates of PopP2 acetyltransferase activity. We first hypothesise that, similar to what was previously observed with WRKY transcription factors (Le Roux et al., (A) GTE11-3Flag and GTE9-3Flag were transiently expressed with 3HAtagged active PopP2 or C321A catalytic mutant in N. benthamiana leaves. Samples were harvested at 48hpi. Protein extracts were immunoblotted with anti-Flag (-Flag) and anti-HA antibodies (-HA) (Input). Immunoprecipitation of Lys-acetylated proteins was done in presence of an anti-AcK antibody on protein A-agarose beads (IP -Ac-K). The immuno-precipitated GTE9 and GTE11 proteins were detected after immunoblotting with -Flag antibodies (Ac-GTE9 and Ac-GTE11, respectively). Ponceau S staining of total proteins indicates equal loading of the samples in the input. This experiment was repeated three times with similar results. (B) Mapping of the PopP2-acetylated lysine residues in GTE9 and GTE11 protein sequences. Conserved residues between GTE9 and GTE11 are shaded in grey. The bromodomain module of GTE9 and GTE11 is boxed in orange (BD GTE9 : position 130-240; BD GTE11 : position 122-232). The truncated forms of GTE9 and GTE11 containing the bromodomain are surrounded by a dotted line (BRD9: position 112-246 in GTE9; BRD11: position 105-238 in GTE11). The conserved Asp residue lying in the acetyl-lysine binding pocket of GTE9 and GTE11 bromodomain in boxed in green. The acetylated lysine residues reproducibly identified by MS-based proteomics in GTE9 and GTE11 in presence of active PopP2 are in red and boxed in yellow. The MS-based proteomic data used to design panel B are presented in Supplementary Tables 2 and3. B specificity (Dhalluin et al., 1999;Filippakopoulos et al., 2012) The in silico modelling of Brd GTE9 and Brd GTE11 using Phyre 2.0 revealed a strong homology with the solved crystal structure of second bromodomain of Brd2 (Brd2(2) hereafter) that belongs to the cluster II of human Brd families (Filippakopoulos et al., 2012). Overall, the structural elements of canonical Brds are well conserved in Brd GTE9 and Brd GTE11 modules (position 130 to 240 and position 122 to 232, respectively) (Supp Fig 3A). Notably, the canonical asparagine residue present in most Brds and that is required for the recognition of acetyl-lysine (Kac) residues (Owen et al., 2000) is also present in Brd GTE9 and Brd GTE11 modules (residues N214 in GTE9 and N206 in GTE11, respectively, see Fig 2B). Together, these structural data based on homology modelling strongly suggest that Brd GTE9 and Brd GTE11 are capable of interacting with acetyl-Lys residues, and more specifically with acetylated histone tails.

To identify potential substrate(s) of Brd GTE9 and Brd GTE11 , we used a SPOT peptide array approach that relies on the measurement of the affinity of recombinant Brds to all the possible combinations of acetylated histone tails. Because the four core histones (H2A, H2B, H3 and H4) are highly conserved across eukaryotes in terms of sequence and structure, we used histonepeptide arrays covering all possible Kac sites of the human histones and which have already been successfully used to identify interactions sites for a large sets of human Brds (Filippakopoulos et al., 2012). In this assay, truncated forms of GTE9 and GTE11

encompassing their Brd module (position 112-246 for GTE9 and position 105-238 for GTE11, hereinafter called BRD9 and BRD11, respectively, Figs 1B-2B) were subcloned into a bacterial expression system in frame with a N-terminal His6 tag. We focused our investigation on His6-BRD9 since His6-BRD11 soluble form was more difficult to produce. As a negative control, we also produced the recombinant His6-BRD9-N214A mutant in which the highly conserved N214 residue of GTE9 predicted to be necessary for interaction with Kac was substituted with an Alanine residue (Supp Fig 3B). Ni-NTA affinity purified His6-BRD9 and His6-BRD9-N214A were sequentially incubated on a spot peptide array containing the various combinations of acetylated histone peptides. We identified preferential binding of His6-BRD9 with peptides corresponding to tri-and tetra-acetylated H4 (AcK5, AcK8 and AcK12 for tri-acetylated H4 and AcK5, AcK8, AcK12 and AcK16 for tetra-acetylated H4, respectively) (Fig 3A and Supp Table 4). On the contrary, the His6-BRD9-N214A mutant showed a much reduced affinity for tri-and tetra-acetylated H4 peptides. These data demonstrate the functionality of the Brd module of GTE9 that preferentially binds to acetylated tails of histone H4 and, in particular, that the conserved residue N214 is required for these interactions, as previously described for yeast and human BRDs (Filippakopoulos et al., 2012) . 5.

E(%)=3,3±0,9 E(%)=3,1±1,1 E(%)=2,1±0,6 E(%)=22±2,3 E(%)=16±1,8 E(%)=6,7±1,2
GTE9 and GTE11 physically interact with histone H4 in planta BRD11, given its high similarity with BRD9 (85.9%), most likely has similar binding capabilities to acetylated H4 peptides. To further validate these properties in vitro and to probe their biological relevance, we next examined whether the interaction of histone H4 with fulllength GTE9 and GTE11 proteins could be recapitulated in planta.

As was successfully demonstrated for human Brds in living cells (Kanno et al., 2004), we performed a FRET-FLIM assay using H4 N-terminally fused with CFP (CFP-H4) and transiently expressed in N. benthamiana cells either alone or with GTE9-YFP and GTE11-YFP.

As a FRET donor, CFP-H4 expressed alone displayed a mean CFP lifetime of 2.945 ns (Fig 1). This is consistent with our SPOT peptide array data that showed that His6-BRD9 did not bind to acetylated H3 peptides (Fig 3A). Together, these results confirm the ability of GTE9 and GTE11 to interact with histone H4 in vivo and highlighted the critical role of the conserved Asp residue in their Brd module for this interaction.

3B, Supp
PopP2 targets the Brd module of GTE9 and GTE11

We exploited the inability of GTE9-N214A and GTE11-N206A to bind H4 to determine whether the acetylation of GTE9 and GTE11 by PopP2 requires a functional Brd module. Thus, the GTE9-N214A and GTE11-N206A mutants were C-terminally fused with a 3HA tag and expressed either alone or with PopP2-3HA or C321A-3HA in N. benthamiana.

Immunoprecipitations were carried out using an anti-Ac-K antibody and immunoblots were probed with an anti-HA. In the presence of PopP2, but not C321A, a signal corresponding to GTE9-N214A-3HA and GTE11-N206A-3HA proteins was detected, indicating these two mutants, despite their inability to interact with H4, were acetylated by PopP2 (Fig 4A and Supp Fig 4A). Interestingly, in a reproducible manner, the signal corresponding to the acetylated forms of GTE9-N214A and GTE11-N206 was stronger than that obtained with wild-type GTE9 and GTE11 (Fig 4A,Supp Fig 4A). Furthermore, this signal intensity did not correlate to the accumulation level of the co-expressed proteins. One explanation is that PopP2 acetylation of GTE9 and GTE11 may be favoured when their bromodomain is unable to bind acetylated Lys residues, making it more easily accessible to PopP2. In such a scenario, we hypothesised that the Brd module of GTE9 and GTE11 is a target of PopP2, similar to the WRKY domain present in WRKY transcription factors that has been characterised as a direct substrate of PopP2 enzymatic activity (Le Roux et al., 2015;Sarris et al., 2015). As hypothesised, a physical interaction between PopP2 and BRD9 was detected in Y2H by PopP2 as bait and BRD9 as prey

(AD-BRD9) (Fig 4B)
. This indicated that the Brd module of GTE9 was sufficient for interaction with PopP2. Unexpectedly, no interaction was detected between BRD11 and PopP2 in yeast.

We next checked whether BRD9 was modified by PopP2 acetylation in planta. MS-based proteomic analysis of immuno-purified BRD9-YFP co-expressed with catalytically active PopP2 led to the identification of three acetylated Lys residues that were previously detected in full-length GTE9 (Supp Tables 2 and3). In a similar assay performed on BRD11-N206A-YFP, these three Lys residues that are conserved between GTE9 and GTE11 were also found to be acetylated only in presence of active PopP2 (Supp Tables 23, Fig 2B). Together, our data demonstrated that PopP2 targets specific Lys residues at conserved positions on either side of the bromodomain of GTE9 and GTE11 (GTE9 K126/K241/K246 and GTE11 K118/K233/K238 ), and provided compelling evidence this functional domain represents a molecular target of PopP2.

PopP2 alters the ability of GTE9 to interact with Histone H4 in planta

Given that PopP2 acetylates several Lys residues located on either side of the bromodomain of GTE9, we hypothesised that PopP2 could interfere with its ability to interact with H4. To test this hypothesis, we used a FRET-FLIM imaging approach to quantitatively measure GTE9histone H4 interactions in the nucleus of living plant cells in presence or absence of PopP2. The mono-exponential lifetime of the eGFP used as a donor allows the measurement of a FRET efficiency containing quantitative information about the fraction of the donor bound to the acceptor that can be directly extracted from FRET-FLIM data (Albertazzi et al., 2009 drastically to 6.6%, reflecting a significant decrease in the proportion of GTE9 interacting with histone H4 (p-value<0.05) (Fig 4C, Supp Table 5). Notably, this reduction is dependent on PopP2 enzymatic activity since only a slight reduction of FRET efficiency was observed with catalytically inactive PopP2-C321A, likely due to the binding of PopP2-C321A to GTE9 that could interfere, to a lesser extent than active PopP2, with the GTE9/H4 association. As control, we used GTE9-N214A which behaves as a PopP2 substrate but is unable to interact with histone H4 (Figs 4A and 3B). We thus checked that the GFP lifetime of GTE9-N214A-eGFP coexpressed with mCherry-H4 was not influenced by the presence of active PopP2 (Fig 4C, Supp Table 5). Together, these data demonstrate that active PopP2 affects the ability of GTE9 to interact with histone H4 in vivo, and therefore indicate that PopP2 uses acetylation to regulate GTE9 bromodomain histone-binding activity.

Overexpression of GTE9 and GTE11 contributes to PopP2-mediated virulence in Arabidopsis

Given that both GTE9 and GTE11 behave as epigenetic readers whose acetylation by PopP2 alters their association with Histone H4, they likely represent virulence targets of this bacterial effector. To determine whether GTE9 and GTE11 are required for PopP2-mediated virulence in Arabidopsis, we studied the phenotypical response of gte9-1 and gte11-1 knockout lines (previously described in Misra et al., 2018) to the R. solanacearum GMI1000 strain (Fig 5A).

Note that these two mutant lines are in Col-0 susceptible background carrying the RRS1-S allele that is unable to recognise PopP2 and activate the RPS4/RRS1-R-dependent immunity. The altered gene expression of GTE9 and GTE11 in the corresponding null mutant lines was confirmed by . Kinetic of wilting disease symptom development in gte9-1 and gte11-1 was indistinguishable from that of Col-0 control plants, indicating that loss of either GTE9 or GTE11 gene expression does not affect the plant response to the GMI1000 strain (Fig 5A). However, we cannot rule out that the lack of altered phenotypic response of gte9-1 and gte11-1 mutants to the GMI1000 strain may be due to functional redundancy between members of the GTE protein family.

Next, we studied the effect of the overexpression of GTE9 and GTE11 in Col-0. For this experiment, GTE9 and GTE11 cDNAs were fused either to a C-terminal 3Flag or 3HA epitope tag sequence for transgenic overexpression in Col-0. For each construct, one transgenic T2 line was selected for phenotypical characterisation. Overexpression of 3HA and 3Flag epitopetagged GTE9 and GTE11 was confirmed by RT-qPCR and immunoblot analysis (Supp Fig 5B- D). After root-inoculation with the GMI1000 strain, the selected GTE9 and GTE11

+ RPS4/RRS1-R Ws-2 Pf0-1 Col-0 gte9-1 gte11-1 Ws-2 Col-0 gte9-1 gte11-1 - * * * * PopP2
overexpressing lines showed increased susceptibility compared to Col-0, indicating these two PopP2 targets can modulate the plant response to R. solanacearum (Fig 5B and Supp Fig 5E).

To determine whether this increased susceptible response was dependent on PopP2 acetyltransferase activity, one representative 3Flag-tagged line for GTE9 and GTE11 was rootinoculated with a GMI1000 popP2 knock-out strain expressing either wild-type PopP2 or the GTE9 and GTE11 are not involved in the RPS4/RRS1-R-dependent immunity triggered by PopP2

We also tested whether GTE9 and GTE11 could play a role in the PopP2-triggered immunity which involves the RPS4/RRS1-R NLR pair in Arabidopsis. Since the gte9-1 and gte11-1 null mutants are in Col-0 accession which carries the RRS1-S natural variant that does not trigger resistance to PopP2 (Deslandes et al., 2002), these two mutants were transformed with a genomic clone encompassing the RPS4 and RRS1-R 5' and 3' regulatory sequences from the resistant Ws-2 accession ( RPS4/RRS1-R Ws-2 ). Wild-type PopP2 and its catalytic mutant PopP2-C321A were delivered in plant leaves through the TT3S of the non-pathogenic Pseudomonas fluorescens Pf0-1 strain. In Ws-2, PopP2 but not its catalytic mutant triggers a cell death response which represents a proxy for activation of RPS4/RRS1-R-dependent immunity in Arabidopsis (Sohn et al., 2014). In Col-0, gte9-1 and gte11-1 plants transgenically expressing the RPS4/RRS1-R Ws-2 NLR pair, PopP2 delivered from Pf0-1 triggered a cell death response similar to that observed in Ws-2, whereas the untransformed genotypes did not show any response (Fig 6A). This indicates that GTE9 and GTE11 are not genetically required for RPS4/RRS1-R-dependent immunity. Note that expression levels of transgenic RRS1-R Ws-2 was checked by RT-qPCR in the different genotypes considered (Supp Fig S6). We further tested if GTE9 or GTE11 may play a role in Ralstonia-delivered PopP2 recognition. To do so, the gte9- and prey (AD-fusion) plasmid combinations were spread on non-selective (-TL) or selective media (-HTLA). Growth on SD-HTLA medium indicate an interaction between the tested proteins. BD-p53 (bait) and AD-T (prey) were used as a positive control. This experiment was conducted at least two times with similar results. (B) In addition to GTE9 and GTE11, PopP2 acetylates GTE3, GTE5, and GTE8 in planta. All GTE protein members (GTE1 to GTE12) tagged with a 3HA epitope tag were transiently expressed with 3Flag-tagged active PopP2 or C321A catalytic mutant in N. benthamiana leaves. Samples were harvested at 48hpi. Protein extracts were immunoblotted with anti-HA (-HA) and anti-Flag (-Flag) antibodies (Input). Ponceau S staining of total proteins indicates equal loading of the samples in the input. Lysacetylated proteins were immunoprecipitated with an anti-Ac-K antibody (IP -Ac-K). Immunoblot analysis of immunoprecipitated proteins with an anti-HA antibody reveals the presence of Lys-acetylated GTE3, GTE5, GTE8, GTE9 and GTE11 in presence of active PopP2 but not C321A. Immunoblots corresponding to GTE1/GTE2/GTE3/GTE4, GTE5/GTE6/GTE7GTE8, and GTE9/GTE10/GTE11/GTE12 represent, respectively, three independent experiments carried out under identical conditions. This experiment was conducted three times with similar results.

1 and gte11-1 mutant lines expressing the RPS4/RRS1-R WS-2 NLR pair were root-inoculated with the GMI1000 strain. Similar to the complemented Col-0 line, these two transgenic lines displayed a resistance phenotype, with almost no wilting symptoms at 10 dpi (Fig 6B). Taken together, these data indicated that GTE9 and GTE11 are not required for RPS4/RRS1-Rdependent immunity, although we cannot exclude that GTE9 and GTE11 inactivation in gte9-1 and gte11-1, respectively, could be compensated by other members of the GTE gene family with overlapping functions.

PopP2 targets multiple members of the GTE family

Considering that PopP2 targets both GTE9 and GTE11 by acetylating several lysine residues in the close environment of their bromodomains and that bromodomains of GTE members are well conserved, we wondered if PopP2 targets other members of the GTE family in Arabidopsis. For this, we first performed a yeast two-hybrid interaction test. The full-length cDNA of each of the other ten GTE members was cloned into a prey-plasmid for testing interaction pairs with PopP2 as bait. In addition to GTE9 and GTE11, a protein interaction was detected only between PopP2 and GTE6, although all prey proteins were correctly expressed in the yeast cells (Fig 7A and Supp Fig 7A). GTE6 belongs to subgroup I of GTE members and shares only 13.2% of identity and 24.9% of similarity with GTE11.

Since some protein interactions can be missed in Y2H due to inherent limitations in the techniquee.g. improper folding of the hybrid proteins, we took advantage of our in planta acetylation assay to test for acetylation of the different GTE members by PopP2. All the GTE members C-terminally fused to a 3HA epitope tag were transiently expressed in N. benthamiana either alone or with 3HA-tagged PopP2 or PopP2-C321A. Immunoprecipitations were performed with an anti-Ac-K antibody and purified proteins were subjected to immunoblot analysis with an anti-HA antibody. A signal corresponding to acetylated forms of GTE3, GTE5

and GTE8 was detected only in presence of active PopP2, indicating they also behave as PopP2

substrates (Fig 7B). For GTE10, a very weak signal was detected but not reproducibly in the three independent acetylation assays performed. Taken together, our data show that at least 5 of the 12 GTE family members can be modified by PopP2 enzymatic activity. Notably, several of the Lys residues acetylated by PopP2 in GTE9 and GTE11 (GTE9 K81/K126/K241 and GTE11 K72/K118/K233 ) (Supp Tables 2 and3) are also present on either side of the bromodomain of GTE3, GTE5, and GT8 (GTE3 K79/K114/-, GTE5 K87/K127/-, and GTE8 K95/-/K280 ) (Supp Fig 7B ).

They could therefore represent key lysine residues targeted by PopP2 to interfere with the molecular functions of these GTEs. Interestingly, protein accumulation level of GTE1, GTE3, GTE5, GTE8, GTE9, GTE10, and GTE11 show a significant increase in presence of PopP2 or PopP2 catalytic mutant (Fig 7B ), as previously observed with several WRKY transcription factors targeted by PopP2 (Le Roux et al., 2015), suggesting that PopP2, independently of its enzymatic activity, might promotes their accumulation.

DISCUSSION

Plants have evolved a sophisticated and robust immune system to protect themselves from infection by a vast majority of pathogens present in their environment. To evade host immune responses and promote infection, pathogens have developed various virulence strategies, among which subversion of host epigenetic mechanisms plays a prominent role. In this study, we describe a previously unknown mechanism used by the bacterial type III effector PopP2 acetyltransferase to promote infection in Arabidopsis by targeting members of the "Bromoand Extra Terminal domains" (BET) family (also known as General Transcription factor Group E, GTE). Within the BET/GTE family, GTE9 and GTE11 behave as histone readers via their single bromodomain that represents a substrate for PopP2 enzymatic activity (Figs 1-4). Our study showed that catalytically active PopP2 alters the ability of GTE9 to interact with histone H4 in vivo (Fig 4C). Thus, PopP2 might represent a bacterial epigenetic regulator that uses acetylation to dissociate bromodomain-containing proteins from chromatin.

All members of the Arabidopsis GTE proteins are characterised by the presence of a single bromodomain and an extra-terminal (ET) domain, the latter likely serving as a platform for anchoring different proteins or complexes to chromatin (Florence and Faller, 2001;Pandey et al., 2002). Evolutionary conserved between plants and animals, bromodomains are structural modules found in many chromatin-and transcription-associated proteins that have the ability to recognise acetylated lysine residues on histone tails and other transcription-associated proteins, such as transcription factors and co-factors. This activity allows bromodomains to play a key role in many acetylation-mediated protein-protein interactions, ranging from recruiting substrate for histone acetyltransferases (HATs) to assisting in the assembly of multi-protein complexes in chromatin involved in the transcriptional activation or repression of genes (Smith and Zhou, 2016).

Recently, the systemic profiling of a wide variety of histone readers in Arabidopsis showed that the recognition of acetylated histone peptides by the bromodomain is highly conserved between plants and humans, suggesting the conserved structural basis for hyper-acetylation chromatin signal readout (Zhao et al., 2018). By virtue of their association with histone H4 in vivo (Fig 3B) which requires the conserved Asparagine residue located in the BC loop of the Bromodomain of GTE9 and GTE11 (GTE9 N214 and GTE11 N206 , Supp Fig 3A), these two proteins can be considered as epigenetic readers of histone lysine acetylation, an epigenetic mark that is central to epigenetic control of gene transcription.

Over the past decade, the subversion of host epigenetic mechanisms has emerged as an effective virulence strategy employed by various pathogens to defeat host immunity. Inhibition of defence gene expression relies on pathogen interference with various histones marks, including histone methylation (Castillo-González et al., 2015;Rolando et al., 2013) and histone acetylation. Although various animal pathogens effectors were found to cause changes in epigenetic histone acetylation marks (Grabiec and Potempa, 2018), examples from plant pathogens are rarer. The PsAvh23 effector from Phytophthora sojae was shown to repress the expression of defence-related genes and promote infection in soybean by modulating GmGCN5-mediated histone acetylation. PsAvh23, competitively binds ADA2, a member of the Histone acetyltransferase complex Spt-ADA-GCN5-Acetyltransferase (SAGA). This disrupts the SAGA complex formation leading to a decrease in H3K9 acetylation levels at defence-related gene loci (Kong et al., 2017). One other effector, PsAvh52 from Phytophthora sojae, binds to the soybean GmTAP1 transacetylase and relocates it into the nucleus where it could acetylate histones H2A and H3 to promote host susceptibility (Li et al., 2018). In Arabidopsis, the 32E03 effector from the sugar beet cyst nematode (Heterodera schachtii) inhibits histone deacetylases including HDT which is involved in the regulation of rRNA gene expression through chromatin modifications (Vijayapalani et al., 2018). From a pathogen's point of view, manipulation of bromodomain-containing proteins acting as chromatin adaptors could also represent a potent virulence strategy to interfere with host transcriptional reprogramming. To date, the only example of manipulation of a bromodomain-containing protein by a pathogen is the hijacking of the Bromodomain-containing protein 4's activity by Papillomaviruses (PVs). Brd4 from human is a dynamic cellular chromatin-binding factor and transcriptional regulator that recruits sequence-specific transcription factors and chromatin modulators to control target gene transcription (Rahman et al., 2011;Wu et al., 2013) . PVs have evolved to exploit the activity of Brd4 to create a facilitating environment for the viral life cycle. Through physical interaction with the major viral regulatory protein E2 of PVs, Brd4 The binding of BRD-containing proteins to acetylated histones can be regulated by acetylation of lysine residues flanking their BRD, causing intramolecular interactions and altering their interaction with acetylated histones. (A) In yeast, the SWI/SNF chromatin remodelling complex association to acetylated histones can be regulated by acetylation. The Snf2 subunit of this complex can be acetylated by the Gcn5 HAT on lysine residues flanking its BRD likely facilitating intramolecular interactions, inhibiting its ability to interact with H4 (Kim et al., 2010).

(B) We propose a similar mechanism used by PopP2 to alter the association GTE9-H4. Indeed, PopP2 acetylates lysines residues flanking GTE9 BRD which might cause intramolecular interactions and the alteration of its interaction with H4. contributes to replication initiation, viral gene transcription, and viral genome segregation and maintenance of PVs (Iftner et al., 2017).

In this study, we identified GTE9 and GTE11 as substrates of PopP2 (Fig 2). Within these proteins, their bromodomains represent a molecular target of this bacterial effector (Figs 4B, Supp Tables 23). Remarkably, PopP2 acetylation of GTE9 is indeed accompanied with an alteration of GTE9 binding to histone H4 in vivo (Fig 4C). Among the acetyl residues identified in GTE9 and GTE11, several of them are also present in three other members of the GTE family, GTE3, GTE5 and GTE8, which behave as additional PopP2 substrates (Fig 7B and Supp Fig 7B). We therefore predict a scenario in which PopP2 uses acetylation to cause dissociation of GTE proteins from chromatin by allowing intramolecular interactions between their own bromodomain and adjacent acetyl-lysine residues. Such mechanism of bromodomain inactivation through acetylation-mediated intramolecular interactions was already reported (Kim et al., 2010;VanDemark et al., 2007). For example, the Gcn5 histone acetyltransferase regulates the dissociation of the SWI/SNF chromatin remodelling complex from acetylated histones by acetylating the Snf2 subunit at two lysine residues. This facilitates intramolecular interaction between the two acetyl lysine residues and the Snf2 bromodomain, inhibiting its ability to interact with acetylated histones (Figure 20) (Kim et al., 2010). It remains to determine whether the bromodomain of GTE proteins represents a ubiquitous substrate of PopP2 enzymatic activity, as was previously described with the WRKY DNA-binding domain of WRKY transcription factors (le Roux et al, 2015). In addition, the protein sequences surrounding the bromodomain of GTE proteins could mediate selective targeting by PopP2.

Overall, the molecular functions of the different GTE family members remain elusive. Some of them were already shown to be involved in important physiological and developmental processes. GTE1 (as known as IMB1) plays a role in the promotion of seed germination by both negatively and positively regulating the abscisic acid (ABA) and phytochrome A (phyA) transduction pathways, respectively (Duque and Chua, 2003). GTE4 is involved in the activation and the maintenance of cell division in the meristems (Airoldi et al., 2010), while GTE6 controls leaf development (Chua et al., 2005). Previously, GTE9 and GTE11 have been described as essential for BT2-mediated sugar and ABA responses. BT2 is a BTB-domain protein that regulates responses to various stress, metabolic conditions and hormones in Arabidopsis. Through physical association with BT2, GTE9 and GTE11 were hypothesised to mediate responses to ABA and sugar signals (Misra et al., 2018). More recently, both GTE9 and GTE11 were shown to be required for BT2-mediated regulation of cauliflower mosaic virus 35S enhancer activity in Arabidopsis (Irigoyen et al., 2021). Given that GTE11 activates transcription via its C-terminus extra-terminal domain in yeast (Du and Poovaiah, 2004), and as evidenced by genetic studies, authors proposed that BT2 assembles in a complex containing GTE9, GTE11 and Cullin3-based ubiquitin ligase that may serve as a scaffold and promote interactions among transcriptional regulators including the GTE proteins.

Our study revealed a previously undescribed role for GTE9 and GTE11, both of which represent and H4 in the promoter region of one of its target gene (Chua et al., 2005). GTE6 is thought to locally increase histone acetylation by binding to acetylated histones and preventing the action of histone deacetylases. Similarly, GTE9 and GTE11 could also shield acetylated histone 4 from deacetylation or increase the level of acetylation by interacting with histone acetyltransferases. Rather than the deregulation of as yet uncharacterised signalling pathways caused by overexpression of GTE9 and GTE11, our data indicate that it is the targeting of these two bromodomain-containing proteins by catalytically active PopP2 that creates a favourable context for infection by the pathogen (Fig 5C). How manipulation of GTE9 and GTE11 by PopP2 can promote pathogen infection remains to be elucidated. PopP2-triggered dissociation of GTE9 and GTE11 from chromatin might locally affect transcriptional regulation of gene expression, for example by preventing activation of defence-related genes. However, such a mechanism would not explain how GTE over-expression could increase plant's susceptibility to infection. An alternative hypothesis could thus be that over-accumulation of GTE9 and GTE11 at specific loci may help PopP2 to target these chromatin regions more efficiently or make them more accessible to the effector.

Of the 12 GTE members considered in this study, only GTE3, GTE5, GTE8, GTE9 and GTE11 could be detected as Lys-acetylated by PopP2 (Figs 2 and 7B). GTE6, which does not seem to be acetylated by PopP2, is however able to interact with (Fig 7A). From this observation, we infer that PopP2 might target GTE family members in different ways. Some of them would be dissociated from the chromatin by acetylation whereas others could serve as chromatin adaptors allowing PopP2 to anchor at specific loci. From these targeted chromatin sites, PopP2 could affect host gene expression, probably through the recruitment of negative transcriptional regulators thanks to its transcriptional repressor motif (Segonzac et al., 2017). In the future, an investigation of the chromatin sites hosting GTE proteins should reveal key genetic loci whose transcriptional regulation is affected by PopP2 to promote pathogen infection.

The ability of PopP2 to target GTE and to modify their ability to bind histones makes this bacterial effector an enzyme acting as a regulator of bromodomain activities. Further investigation on the crystal structure of protein complexes containing PopP2 and GTE proteins or their bromodomain and/or acetylated histone peptides will help explain how PopP2 interferes with the activity of these epigenetic readers. Such studies could also pave the way for the engineering and/or optimisation of protein enzymes capable of selectively regulating the association of bromodomain-containing proteins to chromatin, as done by bromodomain inhibitors. (Bouyssié et al., 2020) http://www.matrixscience.com/

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents used in this study should be directed to and will be fulfilled by the Lead Contact, Laurent Deslandes (laurent.deslandes@inrae.fr).

EXPERIMENTAL MODELS

Arabidopsis

Arabidopsis thaliana Col-0 and Ws-2 plants, and null mutants (gte9-1 (Salk_0119044C) gte11-1 (Salk_059327C), previously described by Misra et al., 2018 were grown in short days (8h light/16h dark cycle) at 22°C (60% relative humidity, 125 µE/M2/s fluorescent illumination).

Transgenic lines were generated by using the floral dip transformation method (Clough and Bent, 1998).

Nicotiana benthamiana

Nicotiana benthamiana (Nb) plants were sown on soil and grown at 24°C under long day photoperiod (16 h light/8 h dark) with 60% relative humidity. Leaves of 4 to 5 week-old plants were used for Agrobacterium tumefaciens-mediated transient expression for Immunoblotting, colocalisation, or FRET-FLIM assays.

Bacterial strains

Ralstonia solanacearum (RSSC) strains were spread on solid φ medium containing 5 g/L glucose and 0.004% TTC and grown for 3 days at 28°C. Bacteria were grown in liquid φ medium at 28°C under shaking overnight with appropriate antibiotics (popP2+PopP2 and popP2+PopP2-C321A were grown in presence of gentamicin (5 µg/mL) and tetracyclin (5 µg/mL) (Tasset et al., 2010). Pseudomonas fluorescens (Pf0-1) expressing the PopP2 variants (wild-type and catalytically inactive mutant) were grown on King's B plate supplemented with antibiotics (chloramphenicol 30 µg/mL, tetracycline 5 µg/mL and gentamicin 15 µg/mL) at 28°C overnight. Bacterial cells were centrifugated and resuspended in infiltration buffer (10 mM MgCl2) at OD600=0.2.

METHODS

Plasmid construction

The oligonucleotides used in the experiments presented in this article are listed in Supp Table 6. The full-length GTE1 (At2g34900), GTE2 (At5g10550), GTE3 (At1g73150), GTE4 (At1g06230), GTE5 (At1g17790), GTE6 (At3g52280), GTE7 (At5g65630), GTE8 (At3g27260), GTE9 (At5g14270), GTE10 (At5g63320), GTE11 (At1g01770) and GTE12 (At5g46550) cDNA clones were PCR amplified from Col-0 cDNA using PrimeSTAR Max DNA polymerase (Takara Bio). The coding sequences of Histone 4 and Histone 3 were amplified from Col-0 genomic DNA using PrimeSTAR Max DNA polymerase (Takara Bio) and PCR products were recombined in pDONR207 to obtain pENTR207-Histone4 and pENTR207-Histone3, respectively. The sequence corresponding to mCherry-Histone4 was generated by two-step PCR. Briefly, overlapping mCherry and Histone4 fragments were amplified using mCherry-Fw/mCherry-H4R and mCherry-H4F/H4-Rev primer pairs, respectively. Full-length mCherry-Histone4 sequence was generated by using the two PCR fragments as templates with AttB1-mCherry-H4 and AttB2-mCherry-H4. All PCR products flanked with AttB1 and AttB2 recombination sites were recombined in pENTR plasmid to generate the relevant pENTR clones. To generate GTE9-N214A and GTE11-N206A mutants, the corresponding mutations were introduced in pENTR207-GTE9 and pENTR207-GTE11 plasmids by site-directed mutagenesis using the PrimeStar HS DNA polymerase (Takara Bio Inc., Otsu Japan). The truncated cDNA clones corresponding to BRD9, BRD9N-N214A, BRD11, and BRD11N-206A were PCR amplified using pENTR207-GTE9, pENTR207-GTE9-N214A, pENTR207-GTE11 and pENTR207-GTE11-N206A as templates. All DNA constructs were sequence-verified. The inserts cloned in pENTR vectors were then recombined in pDEST vectors via LR reaction (Invitrogen). The expressed proteins in planta labelled with a -3HA, -3Flag, -CFP, or -YFP tag were generated by LR reaction in the following plasmid constructs:

(i) pAM-PAT-35S-GWY-3HA, -3Flag, -YFP, or -CFP or (ii) pBIN-35S-GWY-3HA, -3Flag, -YFP or -CFP.

A. thaliana stable transformation

Agrobacterium tumefaciens strains carrying the binary vectors (pAM-PAT-35S-GTE9-3Flag and pAM-PAT-35S-GTE11-3Flag in GV3103 strain; pBIN-35S-GTE9-3HA, pBIN-35S-GTE11-3HA in GV3101 strain; RPS4g/RRS1-Rg in pB7FWG2-D35S-GWY-eGFP) were grown on solid LB medium with appropriate antibiotics for 2 days at 28°C. Bacteria were then cultured in YEB liquid medium (supplemented with antibiotics; gentamicin (15 µg/mL) and carbenicillin (25 µg/mL) for pAM-PAT-based vectors, tetracyclin (5 µg/mL) and kanamycin (25 µg/mL) for pBIN-based vectors, gentamicin (15 µg/mL) and spectinomycin (50 µg/mL) for pB7-based vectors) at 28°C for 12 hours. Cells were centrifugated and resuspended in a sucrose solution (50 g/L) with 0.02% Silwet-L77.

A. thaliana Col-0, gte9-1 (Salk_0119044C) and gte11-1 (Salk_059327C) were transformed with Agrobacterium tumefaciens using the floral dip method (Clough and Bent, 1998). Primary transformant (T1) were selected on MS medium supplemented with phosphinothricin (10 µg/mL) (Duchefa) for pAM-PAT and pB7-based binary vectors and with Kanamycin (50 ug/mL) for pBIN-based binary vectors. T2 transgenic lines were genotyped by PCR and accumulation of transgenically expressed proteins was verified by immunoblot.

Genomic DNA extraction

Genomic DNA extractions were done using ~100 mg of plant material. After grinding, samples were incubated at 65°C 15 min in CTAB extraction buffer (100 mM Tris-HCl pH7.5; 1.4 M NaCl; 20 mM EDTA pH 7.5; 2% CETAB (Hexadecyltrimethyammonium bromide)). Tubes were agitated 15 min with 500 µL of chloroform and centrifugated 5 min at 13200 rpm. Supernatant (500-700 µL) was transfered in a new tube and mixed with 0.8 volume isopropanol.

After centrifugation (13200 rpm, 1 min), pelleted DNA was washed twice with 700 µL ethanol 70%, centrifugated 13200 rpm 3 min, dried and resuspended in 50 µL water with RNAse (1 µg/mL).

RNA extraction, reverse transcription and RT-qPCR

RNA extractions were performed using the Nucleospin RNAII kit (Macherey-Nagel) and following manufacturer's instructions. Reverse transcription was made with Transcriptor Reverse Transcriptase (TRT, Roche). A pre-mix of 7.25 µL (1 µL Oligo (dT)17 at 1 µg/µL; 0.25 µL TRT ; 4 µL TRT buffer; 2 µL dNTP-Mix at 10 mM) was added to a final volume of 20 µL containing 1 µg of total RNA. Samples were incubated for 30 minutes at 55°C. Reverse transcription was stopped by incubating the samples at 85°C for 10 minutes. After 5 min on ice, samples were stored at -20°C. RT-qPCR were performed with a LightCycler 480 II machine (Roche Diagnostics) with Roche reagents. Expression of AtEF1-α (At5g60390) was used to normalise the expression values. The comparative Ct method (2 ΔCt ) was used to represent the data (Schmittgen and Livak, 2008). Mean ΔCt were calculated from three technical replicates for three biological replicates (three independently grown sets of plants). The mean relative expressions from the biological replicates +/-SD was represented with ggplot2 package from R. Statistical analyses were performed on ΔCt values to assess the significance of the difference in the mean ΔCt between the different genetic lines. We applied either a one-way ANOVA when the assumptions about the underlying distribution of the data were fulfilled or a kruskaltest when they were not.

Root-inoculation assays in Arabidopsis

For root-inoculation assays, bacteria (Rs) were grown in a liquid culture at 28°C overnight and diluted in water at DO600=0.01. Four-week-old A. thaliana plants were soaked in this bacterial solution for 10 min, transferred on soil and incubated in a growth chamber (12h day/12h night, 27°C during the day/26°C at night, 75% relative humidity). Symptoms were scored daily with the following disease scale: 0 = no symptoms, 1 = 25% leaves wilt, 2 = 50%, 3 = 75% and 4 = 100%. For each independent inoculation, between 24 and 32 plants from different lines in Arabidopsis thaliana ecotypes were root-inoculated with GMI1000 strain or mutant strains of R. solanacearum and wilting symptom developments were scored for 10 days.

For each presented graph, disease scoring data were analysed from two to four independent inoculations with R software as previously described (Aoun et al., 2020). Briefly, we used a mixed model (MIXED procedure in SAS v. 9.4; SAS Institute Inc.) as follow:

𝑑𝑖𝑠𝑒𝑎𝑠𝑒 𝑖𝑛𝑑𝑒𝑥𝑖𝑗𝑐 = 𝜇 + 𝑏𝑙𝑜𝑐𝑘𝑖 + 𝑎𝑐𝑐𝑒𝑠𝑠𝑖𝑜𝑛𝑗 + 𝑐𝑜𝑣𝐶𝑜𝑙𝑐 + 𝜀𝑖𝑗𝑐
Where μ is the overall mean of the phenotypic data, "block" considers environmental conditions between the independent experiments included in the model, "accession" correspond to the tested genetic lines, "covCol" is a covariate accounting for tray effects within blocks and "ε" is the residual term. "block" was considered as a fixed factor and "accession" as a random factor.

We tested the significance of the different accessions by likelihood ratio test of model with and without this effect. With p-value<0.05, the lines were considered to have a different disease index reflecting differential response to the pathogen.

Transient expression in N. benthamiana and in A. thaliana

For transient expression in N. benthamiana leaf epidermal cells, A. tumefaciens strains grown in liquid YEB medium containing appropriate antibiotics. Cells were harvested by centrifugation and resuspended in infiltration medium (10 mM MES pH5.6, 10 mM MgCl2, 150 μM acetosyringone) at OD600=0.25. For co-expression, each bacterial suspension carrying individual constructs was adjusted to OD600 = 0.25 in the final mix for infiltration. After incubation at room temperature for 1 h, bacteria were infiltrated into the leaves of 4-week-old N. benthamiana plants using a needleless syringe. Plants were incubated for 36-48h in growth chambers under controlled conditions. Agrobacterium-based transformation for transient gene expression in Arabidopsis Col-0 seedlings was performed as previously described (Li et al., 2009).

Protein Extraction and Immunoblot Analysis

Plant material (4 discs of 8 mm diameter harvested 48 hours post-infiltration for N.

benthamiana samples; 20 seedlings (10 day-old) for Arabidopsis samples) were grinded and resuspended in 300 µL of Laemmli Buffer (2X). Protein extracts were then denatured for 3 min at 95°C, centrifugated at 13000 rpm for 1 min and then analysed by immunoblotting (SDS- for detection of anti-GFP and anti-mCherry; goat anti-mouse IgG2a-HRP (Bio-Rad; dilution 1:5000 for detection of anti-Ac-K antibody). Immunodetections were performed using Clarity

Western ECL substrate reagent (Bio-Rad).

Fluorescence microscopy

Colocalisation assays were performed as described previously (Tasset et al., 2010). Briefly, A.

tumefaciens strains carrying CFP-and YFP-tagged constructs were co-infiltrated in the leaves of 4-week-old N. benthamiana plants. Leaf samples were taken for imaging between 36-48 hpi.

CFP and YFP fluorescence were analysed with laser confocal microscopy (TCS SP2-AOBS and CLSM SP8, Leica, Germany) using a water-immersed objective (63x/1.20 Numerical Aperture, PL APO or 25x/0.95 Numerical Aperture, Fluotar VISIR). A laser excited CFP fluorescence at λ = 458 nm and recorded in one channel of the confocal microscope (emission spectra: 470-510 nm). YFP was excited at λ = 514 nm and recorded (emission spectra: 525-585 nm). Images were acquired sequentially with the software Leica LCS version 2.61 and LAS X.

Yeast two-hybrid

Constructs for yeast two-hybrid analysis were prepared in the MatchMaker GAL4 two-hybrid system (Clontech) using the vectors pGBKT7-and pGAD-derived vectors to express bait and prey proteins, respectively. The pDEST pGAD-GWY vector was generated by ligating a Gateway cassette (Frame C) in the pGADT7 vector blunt-ended (klenow fill-in) after Plasmid DNAs were extracted with the Wizard DNA plasmid purification kit (Promega). Coexpression of BD-p53 (bait, pGBKT7-p53 vector, Clontech) and AD-T (prey, pGADT7-T vector, Clontech) was used as positive control on selective medium. Bait and prey proteins were tagged with a c-Myc and an HA epitope tag, respectively. Bait and prey constructs were introduced in AH109 Saccharomyces cerevisae strain (Clontech) according to the manufacturer's instructions. Transformed cells were spread on SD-TL medium and incubated 3 days at 30°C. Colonies were then dropped by dilution gradient on different media: nonselective (SD-TL) and selective (SD-TLHA) and incubated between 3-5 days at 30°C. Protein expression in yeast was evaluated following incubation of cultures under shaking at 30°C overnight in SD-TL media. Cultures were processed for protein extraction according to a lithium acetate (Liac)/NaOH pre-treatement protocol as previously described (Zhang et al., 2011). Samples were denatured 3 min at 95°C in Laemmli buffer (2X) before separation by SDS-PAGE and immunoblotting with anti-c-Myc-HRP and anti-HA-HRP conjugated antibodies.

FRET-FLIM assays

The FRET-FLIM system we used is made of a mode-locked Ti:sapphire IR femto laser (Tsunami; Spectra Physics, model n°3941) which is the IR laser excitation beam. A Diodepumped laser (Spectra Physics, model Millenia-Pro 10sJ) modulates the energy of the laser.

The laser emits pulses of light thanks to a lock-to-clock electronics module (Spectra Physics, model n°3955) and a pulse selector combined with its electronics module (Spectra Physics, model n°3980 and 3986) adjust the pulse frequency at 2 MHz. The laser system is linked to an inverted microscope acquiring images via the streak camera system. It consists of a streak camera (Hamamatsu, model n°C4334-21) and a CCD camera (Hamamatsu, model C8800-53C). 1) or with a bi-exponential fit function for cells expressing both donor and acceptor, revealing FRET (Equation 2): Equation 1:

𝐼(𝑡) = 𝛼𝐷 • 𝑒 -( 𝑡 -𝑡 ) ⁄ 𝑟𝐷 + 𝐵 Equation 2 : 𝐼(𝑡) = 𝛼𝐷 • 𝑒 -( 𝑡 -𝑡 ) ⁄ 𝑟𝐷 + 𝛼𝐷𝐴 • 𝑒 -( 𝑡 -𝑡 ) ⁄ 𝑟𝐷𝐴 + 𝐵 𝑡 0 is

MS-based proteomic analyses

For immunoprecipitation of 3Flag-and YFP-tagged proteins, between 3g and 5g of N.

benthamiana leaves transiently expressing the relevant constructs were harvested 48 hours after infiltration. Leaf samples were grinded in liquid nitrogen and homogenised in 30-50 mL of extraction buffer (50 mM Tris-HCl pH7.5; 150 mM NaCl; 10 mM EDTA; 10 mM sodium butyrate; 1X Protease Inhibitor Cocktail; 1% triton). After centrifugation (14000 rpm, 10 min, 4°C), the supernatant was filtered with miracloth to get rid of the waste material. 50 µL of the supernatant (crude extract) was denatured 3 min at 95°C. The remaining supernatant was incubated 2 hours at 4°C with 100 µL of anti-Flag M2 affinity gel (Sigma) or GFP-trap agarose (Chromotek). After centrifugation (13200 rpm, 5 min, 4°C) we discarded the supernatant.

Beads were washed 3 times in new 1.5 mL-tubes with 1 mL of extraction buffer (with 0.2% triton). Fixed proteins on the beads were then denatured 3 min at 95°C. Immunoprecipitated proteins were separated by SDS-PAGE before Coomassie blue staining.

The bands containing the protein of interest were cut out and submitted to in-gel digestion using trypsin (modified, sequencing purity, Promega), as previously described (Casabona et al., 2013). The resulting peptides were analysed by online nanoliquid chromatography coupled to MS/MS (respectively Ultimate U3000, Dionex, and LTQ-Orbitrap Velos pro, Thermo Fisher Scientific, for the 3FLAG constructs, and UltiMate 3000 RSLCnano and Q-Exactive Plus, Thermo Fisher Scientific, for the YFP constructs). For this purpose, peptides were sampled on Carbamidomethyl (C, fixed), Acetyl (Protein N-term, variable), Acetyl (K, variable), Phospho (S/T/Y, variable), and Oxidation (M, variable). The Proline software (Bouyssié et al., 2020) was used for the compilation, grouping, and filtering of the results (conservation of rank 1 peptides, peptide length ≥ 6 amino acids, peptide score ≥ 25, and minimum of one specific peptide per identified protein group). Proline was then used to perform a compilation, grouping and spectral counting-based comparison of the protein groups identified in the different samples. MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019) with the dataset identifier PXD029367. The acetylation sites were taken into account if their localisation probability was above 75%.

SPOT peptide array

Recombinant proteins were expressed in Rosetta cells (Novagen) transformed with pDUET-His6-BRD9 or pDUET-His6-BRD9-N214A. Briefly, for each construct, bacteria were precultured in 50 mL liquid L medium with appropriate antibiotics (carbenicillin (50µg/mL) and chloramphenicol (30 µg/mL)) at 37°C for 12 hours under stirring (180 rpm). The precultures were diluted in 1L liquid L medium (ration 1:100, with antibiotics) at 37°C under stirring (180 rpm). When the OD600nm reaches 0.6, incubator temperature was decreased at 18°C. One hour later, protein expression was induced with 0.2 mM IPTG and cells were incubated overnight at 18°C under stirring. Cultures were centrifuged (6500 rpm, 20 min, 4°C)

and pellets were resuspended in lysis buffer (500 mM NaCl, 20 mM HEPES pH7.5, 10 mM Imidazole, 1 mM TCEP, 1:1000 protease inhibitor cocktail) (for 10 g of bacterial pellet, 100 mL lysis buffer was added). After bacterial resuspension, cells were sonicated for 21 min (35% amplitude, 5 seconds ON and 10 seconds OFF) at 4°C. Samples were then centrifuged (16000 rpm, 1 hour, 4°C). For each sample, 5 mL Ni (Nickel) beads beforehand balanced were added to the supernatant. Samples were stirred 1 hour at 20 rpm at 4°C and then poured in affinity column. 50 µL of the flow-through was denatured 3 min at 95°C in 50 µL Laemmli buffer (4X).

Proteins bound to the Ni beads were eluted with buffer A (500 mM NaCl, 5% glycerol, 20 mM HEPES pH7.5) added with different Imidazole concentrations (1 st fraction: 30 mM, 2 nd fraction: 60 mM, 3 rd fraction: 90 mM, 4 th fraction: 120 mM, 5 th fraction: 240 mM, 6 th fraction: 300 mM) at 4°C. For all the eluted fractions, 50 µL aliquots were mixed with 50 µL Laemmli buffer (4X)

and denatured at 95°C for 3 min. Samples were loaded on SDS-PAGE gel. After migration, the gel was stained with Coomassie Blue for 1 hour and washed overnight in sterile nanopure water.

Fractions with the most concentrated and the purest proteins were selected for the next steps.

Purified proteins were then concentrated with successive centrifugations (3500 rpm, 3 min, 4°C) using an Amicon Ultra-15 centrifugal spin (10 kDa). Concentrated proteins were then separated according to their size by exclusion chromatography, and collected fractions were analysed with Q-TOF. 50 µl of the fractions containing the protein of interest were mixed with 50 µL Laemmli buffer (4X) and denatured at 95°C for 3 min. After migration on SDS-PAGE gel and Coomassie Blue staining, fractions in which our protein was detected were selected for Q-TOF analysis. The SPOT array was performed as previously described (Filippakopoulos et al., 2012). Briefly, peptides corresponding to the N-terminal tails of the histones H2A, H2B, H3 and H4 (non-acetylated or acetylated on different lysine residues) were synthetised on a cellulose membrane thanks to a MultiPep SPOT peptide arrayer (Intavis). His6-tagged BRD9 and BRD9-N214A were added at a 1 mM final concentration and blots were developed with the ECL kit (Thermo Scientific) following the manufacturer's instructions.

Pseudomonas fluorescens-mediated delivery of PopP2 in Arabidopsis

For Pf0-1-mediated delivery, full-length PopP2 and PopP2-C321A recombined in the pBBR-AvrRps4prom-GWY-3HA gateway destination vector were used as previously described (Le Roux et al., 2015). Transformed Pf0-1 cells were spread on King's B agar supplemented with 6mM MgS04 and with appropriate antibiotics (tetracycline 5 µg/mL, chloramphenicol 30 µg/mL, and gentamicin 15 µg/mL). Bacteria were resuspended in 500 µL MgCl2 (10 mM) and centrifuged (8000 rpm, 1 min). Bacteria were resuspended in 500 µL MgCl2 (10 mM) and centrifuged again (8000 rpm, 1 min) to wash out antibiotics. Bacterial pellet was resuspended in 500 µL MgCl2 (10 mM) solution and bacterial density was adjusted to OD600=0,2 (OD600nm = 1 means 1.75 x 10 8 bacteria/mL). Three-week-old plants were placed in a humid environment 14 hours before infiltration to facilitate stomata opening. Bacteria were infiltrated in the plant leaves with a needleless syringe. ~12 leaves were infiltrated for each plant tested and the PopP2tiggered cell death was observed 48 hours after infiltration.

Quantification and statistical analysis

For FRET-FLIM analyses, significance of the difference between the donor lifetimes in the absence and in the presence of acceptor was assessed by a Student's t test. Mass-spectrometry based quantitative proteomics to assess acetylated residues on peptides parameters regarding precursor and fragment mass error tolerances were set at respectively at 10 ppm and 0.6 Da for LTQ-Orbitrap Velos Pro data, and 10 and 20 ppm for Q-Exactive Plus data. For the compilation, grouping, and filtering of the results (conservation of rank 1 peptides, peptide length ≥ 6 amino acids, peptide score ≥ 25, and minimum of one specific peptide per identified protein group), the Proline software (Bouyssié et al., 2020) was used. Proline was then used for compilation, grouping and spectral counting-based comparison of the protein groups identified in the different samples. The acetylation sites were considered accurate when their localisation probability was above 75%. For the Rs inoculations, disease symptom scoring was analysed and plot with R software. Graphs show least-square means +/-Standard Error of the LS means represented as linear plots with points at each day when symptoms were scored. Significance of the difference of disease index LS means at a given day between different genotypes was assessed with a one-way ANOVA performed on a mixed model generated from the data as previously used (Aoun et al., 2020). At a given day, significance of the difference between LS means was set at p-value<0.05. For RT-qPCR, data were analysed and represented with R software using the comparative Ct method (2 ΔCt ) (Schmittgen and Livak, 2008). Mean ΔCt were calculated from three technical replicates for three biological replicates (three independently grown sets of plants). The mean relative expressions from the biological replicates +/-SD was represented in barplots. ΔCt values were used to assess the significance of the difference in ΔCt between the different genetic lines. A one-way ANOVA was applied when the assumptions about the underlying distribution of the data were fulfilled, or a kruskaltest when they were not.

Data and code availability

All originally unprocessed photos of immunoblots (a) Mean lifetime, , in nanoseconds (ns). For each nucleus, average fluorescence decay profiles were plotted and fitted with exponential function using a non linear square estimation procedure and the mean lifetime was calculated according to t =  a i t i ² /  a i ti with I(t) =  a i e - t/ti , (b) Δt=tD -tDA (in ns), (c) Standard error of the mean, (d) Total number of measured nuclei, (e) % FRET efficiency: E=1 -(ƮDA/ƮD), and (f) p-value of the difference between the donor lifetimes in the absence and in the presence of acceptor (Student's t test). The statistical test used was two-sided. The lifetime measurements were carried out from at least two independent expression assays performed in N. benthamiana (leaf samples were taken between 36 and 48 hours after infiltration with A. tumefaciens).

Site

GTE9-3xFLAG GTE9-YFP GTE9-BRD-YFP

SC PopP2 C321A SC PopP2 WT SC PopP2 C321A SC PopP2 WT SC PopP2 C321A SC PopP2 WT K40 0 1 0 1 K49 0 2 0 7 K81 0 1 0 3 K103 0 1 0 4 K116 0 3 1 21 2 3 K126 0 2 0 11 0 1 K240 0 1 0 3 K241 0 1 0 3 0 2 K246 0 1 0 5 0 1 K260 0 1 0 6 K273 0 2 1 4 K434 0 1 0 2 K443 0 2 0 7 K506 0 1 0 7 K523 0 1 0 1 K563 0 1 0 2 Site GTE11-3xFLAG GTE11-YFP GTE11-BRDmut-YFP SC PopP2 C321A SC PopP2 WT SC PopP2 C321A SC PopP2 WT SC PopP2 C321A SC PopP2 WT K72 0 1 0 2 K118 0 5 1 9 0 1 K179 0 1 K233 0 1 0 4 0 1 K238 0 3 0 5 0 1 K250 0 3 0 4 K260 0 1 0 3 K277 0 1 0 1 K404 0 2 0 2 K454 0 1 0 1 K474 0 1 0 1
Supplementary Table 2. Acetylation sites reproducibly identified by MS-based proteomics in GT9 and GTE11 co-expressed either with PopP2 or the catalytically inactive PopP2-C321A mutant. GTE9 and GTE11, or the bromodomains of GT9 (BRD9) and GTE11 (BRD11-N206A), were transiently co-expressed with either wild type PopP2 or PopP2-C321A in N. benthamiana and immunopurified before MS-based proteomic analyses. The table lists the acetylation sites (localisation probability ≥ 75%) identified in both 3xFLAGand YFP-tagged constructs for full-length proteins and YFP-tagged bromodomain-containing constructs (SC = spectral counts). The Lysacetylated residues whose position is conserved between GTE9 and GTE11 proteins are shaded in grey. The total list of ident ified peptides and sites is given in Supplemental Table 3. (a) Estimated values of fluorescence lifetime (in nanoseconds, ns) when there is no FRET (Ʈ1) and when there is FRET (Ʈ2). These parameters were calculated from the fluorescence intensity data after the observation of N nuclei for each combination. (b) Standard deviation, (c) Standard error of the mean, (d) Δ=Ʈ1 -Ʈ2, (e) Ʈm, the mean lifetime lifetime in nanoseconds (ns), (f) N is the number of measured nuclei, (g) p-value of the difference between the donor lifetimes and in the presence of acceptor (Student's t test) , (h) % FRET efficiency: E=(1 -(Ʈ2/Ʈ1))*100. For each nucleus, average fluorescence decay profiles were plotted and fitted with exponential function using a non-linear square estimation procedure and the mean lifetime was calculated according to Ʈm=Σαie-t/Ʈi.

-GTE12 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGTTGCGATACCCAATATT GTE12 cDNA (At5g46550) AttB2-GTE12 GGGACCACTTTGTACAAGAAAGCTGGGTCAAGGATCTCTCCTTCCTCAAG GTE12-500 TTGAGGTTAGATGGGAATCACTCAT sequencing primer GTE9-N214A-F CAATGACGTATGCTCCACCTGGTAATGATGTTTAT GTE9 N214A mutant GTE9-N214A-R TACCAGGTGGAGCATACGTCATTGCATTGCTGAAG GTE11-N206A-F TGCTATGACTTATGCCCCGTCAGATAATAATGTGTATC GTE11 N206A GTE11-N206A-R TTATCTGACGGGGCATAAGTCATAGCATTGCGGAAA AttB1-BRD9 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACGGGTCCAGGAAAGACG BRD9 AttB2-BRD9 GGGGACCACTTTGTACAAGAAAGCTGGGTCATTTAGTTCCGGATAATTTCTT AttB1-BRD11 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACTGGCCCTGGAAAAAGG BRD11 AttB2-BRD11 GGGGACCACTTTGTACAAGAAAGCTGGGTCATTTGGTTCCGGATGACTTCT AttB1-Histone4 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTCAGGAAGAGGAAAAG Histone 4 (At2g28740) AttB2-Histone4 GGGACCACTTTGTACAAGAAAGCTGGGTCAACCACCAAATCCATATAGAG AttB1-Histone3 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCTCGTACCAAGCAAA Histone 3.3 (At4g40030) AttB1-Histone3 GGGACCACTTTGTACAAGAAAGCTGGGTCTTAAGCGCGTTCACCTCTGAT mCherry-Fw ATGGTGAGCAAGGGCGAGGAGGATAA mCherry-Histone H4 mCherry-H4R TCCTCTTCCTGACATGGACCCTCCTCGAGATCTGAGTCCGGA mCherry-H4F ACTCAGATCTCGAGGAGGGTCCATGTCAGGAAGAGGAAAAGGA H4-Rev AACCACCAAATCCATATAGAGTTCTTCCTTGT AttB1-mCherry-H4 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGTGAGCAAGGGCGA AttB2-mCherry-H4 GGGACCACTTTGTACAAGAAAGCTGGGTCAACCACCAAATCCATATAGA AtEF1-F CTGGAGGTTTTGAGGCTGGTAT RT-qPCR primer AtEF1-R CCAAGGGTGAAAGCAAGAAGA RT-qPCR primer GTE11-q-2-F CGATTTGCGGATATAATCTTGA RT-qPCR primer GTE11-q-2-R TTCCTTCTCACGTTGCAGTG RT-qPCR primer GTE9-q-2&3-R CTTGACATTTTAGGATCACC RT-qPCR primer GTE9-q-2-F CATTGGTGAAAATGAACACC RT-qPCR primer RRS1-R-ex5-q-F GGGCAAAGAAATCCTCCAT RT-qPCR primer RRS1-R-ex5-q-R CTTTCGCGATGTTTCTT RT-qPCR primer
In this section, I briefly summarises the main progresses made on the identification of GTE9 and GTE11 as targets of the RSSC T3E PopP2. I also present some additional data obtained during my thesis and which, although not included in the manuscript, allow to progress on the functional characterisation of GTE9 and GTE11 and their involvement in bacterial wilt disease.

Main conclusions from the manuscript (Comorge et al., in prep.) From the article, the following conclusions can be drawn:

-PopP2 co-localises and interacts with both GTE9 and GTE11 in plant cells.

-PopP2 acetylates GTE9 and GTE11 in planta.

-GTE9 and GTE11 bind preferentially to tri-and tetra-acetylated lysine residues on histone H4 and interact with histone H4 in vivo, suggesting that they could behave as epigenetic readers.

-PopP2 acetylates multiple lysine residues in GTE9 and GTE11, some of which are located on either side of their BRD.

-PopP2 alters the association GTE9-H4 through its acetyltransferase activity.

-GTE9 and GTE11 over-expression contributes to the virulence of the GMI1000 strain of the RSSC, and it depends on PopP2-acetyltransferase activity.

-PopP2 acetylates various members of the GTE family.

Complements to the manuscript As a complement to the inoculations of gte9-1 and gte11-1 mutants with GMI1000, we also inoculated these lines with RSSC strains either lacking PopP2 (ΔpopP2)

or expressing WT PopP2 (ΔpopP2+PopP2) or PopP2 catalytic mutant (Δ previously observed in response to the reference strain GMI1000, inoculation of gte9-1 and gte11-1 mutants with aΔpopP2+PopP2 strain resulted in similar symptom development compared to the WT line (Figure 21). This confirms that the ΔpopP2+PopP2 strain behaves like GMI1000. Furthermore, the gte9-1 and gte11-1 mutants also display similar phenotypes to the WT Col-0 line in responseΔpopP2 orΔpopP2+PopP2-C321A (Figure 21). Overall, this suggests that these two knockout mutants behave like the WT line in response to R. solanacearum strains producing or not the PopP2 effector. This could support the hypothesis of functional redundancy, likely between different GTE members. However, another possible explanation could be related to the position of the T-DNA present in gte9-1 and gte11-1 mutants. Indeed, the T-DNA insertion in these mutants is located after the second exon of GTE9 and GTE11, downstream of the BRD coding region (Figure 22A). This means that gte9-1 and gte11-1 could still produce shorter transcripts encoding for truncated forms of GTE9 and GTE11 that would contain the BRD protein module. Such truncated proteins could therefore be partially functional (if they are still able to bind acetylated histone tails) and thus potentially manipulated by PopP2. To test this hypothesis, we investigated whether such truncated transcripts could be detected by RT-qPCR in gte9 -1 and gte11-1 mutants. For this, we used primer pairs that could amplify a region located upstream the BRD of GTE9 and GTE11 (pairs in darker color in Figure 22A).

Interestingly, in the gte9-1 mutant, such truncated GTE9 transcripts could be detected, even though relatively less expressed than in the WT line (Figure 22B).

These data indicate gte9-1 should not be considered a null mutant since it does produce shorter GTE9 transcripts that could lead to the production of a truncated GTE9 protein lacking its last 328 residues but still containing a BRD, and designated by GTE9 1-370 . Interestingly, transient expression of 3Flag-tagged GTE9-1-370 (or a mutant containing the N214A substitution, and designated by GTE9-N214A-1-370 -3Flag) with active PopP2 in N. benthamiana led to the detection of an acetylated form of this truncated GTE9 protein, indicating that GTE9-1-370 behaves as a PopP2 substrate, like full-length GTE9 (Figure 22C). Therefore, it is highly likely that the gte9-1 mutant produces a truncated GTE9 protein with a functional BRD and that is acetylated by PopP2. Regarding the gte11-1 mutant, it is more difficult to conclude since the selected primers amplified less GTE11 short transcripts in both gte11-1 and WT plants. Indeed, the primer pair used for amplifying the upstream region of GTE11 had a relatively low efficiency (89% compared to ~110% for the GTE11-q-2-F and GTE11-q-2-R pair), making difficult to assess properly the amplification even in the WT and to conclude for the presence of transcripts upstream the region of GTE11 encoding the BRD (Figure 22A). Nonetheless, it seems that in the gte11-1 mutant, GTE11 transcripts accumulate much less than in the WT (Figure 22B).

• Does PopP2 regulate the expression of GTE family genes?

Apart from GTE9 and GTE11, our data demonstrate that PopP2 can target other members of the GTE family by acetylation (Figure 7B, Comorge et al., in prep.) which could thus also represent PopP2 virulence targets. We next wondered whether the expression level of the GTE gene family was modulated in presence of PopP2. To investigate this question, 3-week-old A. thaliana Col-0 leaves were infiltrated with Pseudomonas fluorescens (Pf0-1) delivering or not active PopP2 or its catalytic mutant the expression level of the different GTE genes was measured by RT-qPCR in plant leaf samples at 0, 3 and 6 hpi (hours after infiltration). Figure 23 shows the mean GTE expression of the biological replicates from the two independent experiments, at the different time points and per infiltrated strain. The top panel represents GTE genes for which their proteins were acetylated by PopP2, and the bottom panel those that were not acetylated by PopP2 (Figure 23). Note Primers used for RT-qPCR analyses are listed in Table 3.

Relative gene expression

Relative gene expression also that only 10 GTE genes are represented on Figure 23 because for GTE2 and GTE11, analyses could not be performed on data combined from the two independent experiments. Analyses for these two genes were performed for each independent experiment separately and represented in Figure 24. Overall, at 3 or 6 hpi, there is no significant changes in the GTE expression levels when PopP2 is delivered or not by Pf0-1 cells in plant leaves, for all the GTE genes, encoding for the GTEs acetylated by PopP2 or not (Figures 23 and24). It can be only noted that for some GTE like GTE9 and GTE12, their expression is modulated over the time.

Similar conclusions can be drawn for GTE2 and GTE11 (Figure 24). Indeed, in both repetitions, GTE2 and GTE11 expression levels do not seem to be affected by PopP2 or its catalytic mutant at all, but GTE11 expression varies over time after infiltration with Pf0-1 cells. To conclude, delivering PopP2 or its catalytic mutant in plant leaves does not seem to affect the expression of the GTE genes, suggesting that these genes are not transcriptionally regulated in response to PopP2.

Conclusions

In this first chapter, we demonstrated that PopP2 acetylates GTE9 and GTE11 on several lysine residues, including residues flanking their BRD. This acetylation correlates with an alteration of their ability to interact with histone H4. Moreover, over-expression of GTE9 and GTE11 in the susceptible A. thaliana ecotype Col-0 resulted in an increased susceptibility in response to the RSSC strain GMI1000 and this increased susceptibility of the GTE9 and GTE11 over-expressing lines depends on the acetyltransferase activity of PopP2. Together, these data indicate that GTE9 and GTE11 represent virulence targets of PopP2. Considering that GTE9 and GTE11 are histone readers that appear to be virulence targets PopP2, it would be interesting to determine whether the increased susceptibility of the GTE9 and GTE11 over-expressing lines to GMI1000 depends on the ability of overexpressed GTE9 and GTE11 to bind acetylated histones via their BRD. Knowing this, we could link the alteration of GTE-H4 interaction by PopP2-mediated acetylation and the role of GTE9 and GTE11 as virulence targets.

Also, the fact that gte9-1 and gte11-1 mutants behave as Col-0 in response to GMI1000 or popP2-deficient strains and/or complemented with PopP2 or its catalytic mutant suggested functional redundancy between GTE9 and GTE11, as they regulate similar pathways, the ABA and sugar signalling pathways (Misra et al., 2018). This functional redundancy is even more likely as PopP2 seems to target several members of the GTE family. To overcome this functional redundancy, it would have been very informative to study the phenotypical response of a gte9-1/gte11-1 double mutant to RSSC strains producing or not PopP2. Unfortunately, despite several attempts, it was not possible to select such a gte9-1/gte11-1 double mutant from independent genetic crosses between the gte9-1 and gte11-1 single mutant lines. Nonetheless, it would not have been guaranteed that a gte9-1/gte11-1 double mutant could have overcome the potential functional redundancy since PopP2 can target additional GTE members (GTE3, GTE5 and GTE8).

Furthermore, GTE9 and GTE11 are regulators of the ABA signalling pathway, but two other GTE members, GTE1 and GTE10, appear to be also involved in the regulation of this pathway. Therefore, the double gte9-1/gte11-1 mutation may not be sufficient to overcome potential functional redundancy (Duque and Chua, 2003;Kim et al., 2009).

In addition, we formulated another hypothesis to explain why the phenotypical response gte9-1 and gte11-1 mutants to R. solanacearum was not affected, as compared to WT Col-0 plants. Since these two mutants carry a T-DNA insertion downstream of the BRD coding region, it is possible that these mutants still express truncated forms of GTE9 and GTE11 able to bind acetylated Histones thanks to their BRD (Figure 22A). We tried to assess the presence of corresponding truncated GTE9 and GTE11 transcripts in these mutant lines. We found that that there could indeed be such truncated transcripts in the gte9-1 mutant, although it is less likely in gte11-1 (Figure 22B). Consequently, and considering that a GTE9 1-370 truncated version can be acetylated by PopP2 (Figure 22C), it is possible that PopP2 targets these truncated forms. So, to determine whether the phenotype observed in the mutants is due to functional redundancy or to the presence of truncated forms of GTE9 and GTE11 that would be able to complement the mutation resulting from the T-DNA insertion, other mutants with a T-DNA insertion upstream of the BRD coding region should be tested in interaction with R. solanacearum strains.

Alternatively, we could also try to mutate both the 5' part of GTE9 and GTE11

coding regions by CRISPR/Cas9-based genome editing which is now commonly used in model plants such as A. thaliana.

In addition to GTE9 and GTE11, we have also shown that PopP2 acetylates other GTEs such as GTE3, GTE5, and GTE8 but it may affect these epigenetic readers and leaves from susceptible ecotypes in response to GMI1000 have already been published, and none of the GTE genes were found to be differentially expressed (Hu et al., 2008;Zhao et al., 2019). Another study investigated the changes in transcriptomes of the resistant ecotype Ws-2 and Ws-2 rrs1-1 mutated line after delivering PopP2 or its catalytic mutant by Pf0-1 (Sohn et al., 2014). However, their analyses were focused on transcriptional changes linked to RRS1-R and PopP2

acetyltransferase activity, but not on the presence or absence of PopP2 itself. Thus, to determine whether PopP2 affects or not the transcription of GTEs, it would still be interesting to analyse the expression of the different GTE genes in A. thaliana

Col-0 plants after infection by RRSC strains producing or not PopP2.

Finally, an important question to be answered is to what extent PopP2 uses its acetyltransferase activity to control/modulate the biological activitiy(ies) of GTEs.

Our data clearly demonstrate that active PopP2 alters the ability of GTE9 to interact with Histone H4 in planta. It is highly likely that PopP2 compromises GTE9 BRD binding abilities by acetylating critical Lysine residues around this protein module, promoting intramolecular interactions that in turn inhibits the BRD and promotes the dissociation of GTE9 from chromatin (see Discussion section in Comorge et al., in prep.). Is it true for the other GTEs members we identified as PopP2 substrates?

This remains to be determined. In this context, it would be very interesting to identify by a mass spectrometry-based analysis which lysine residues are acetylated by PopP2 in GTE3, GTE5 and GTE8. This could lead to the identification of key conserved Lysine residues in GTEs that would be specifically targeted by PopP2 to manipulate their function.

This is reminiscent of PopP2's targeting of a particular Lysine residue within the DNA-binding domain of WRKY TFs and which results in their inability to bind to their target DNA (ie defence-related genes), and consequently in the dampening of basal immune responses (Le Roux et al., 2015;Sarris et al., 2015) .

Chapter 2: Can other effectors target GTE9 and GTE11?

GTE9 and GTE11 appear to behave as histone readers and because epigenetic mechanisms regulate a wide variety of cellular processes, including immune responses, these chromatin-associated components represent promising virulence targets that can be manipulated by pathogens.

We were able to show in Chapter 1 that GTE9 and GTE11 represent PopP2 virulence targets since (i) they are acetylated by PopP2 and that (ii) overexpression of GTE9 and GTE11 leads to enhanced disease response to R.

solanacearum, which depends on the acetyltransferase activity of PopP2 (Comorge et al., in prep.).

If PopP2 were the only effector responsible for the increased susceptibility observed in the over-expressing lines in response to GMI1000, inoculation with a popP2-deficient strain (ΔpopP2 strain) should result in comparable wilting symptom development between the over-expressing lines and the WT. Strikingly, upon root-inoculation of GTE9 and GTE11 over-expressing lines with a R. solanacearum strain lacking popP2, these transgenic lines also displayed an enhanced disease response compared to WT plants (Figure 25). Therefore, the absence of PopP2 does not seem to abrogate the increased susceptibility of the GTE9 and GTE11 over-expressing lines to the RSSC. To explain this, one scenario

A BD-PopP2 BD-p53 BD-PopP1 10 AD-GTE1 AD-GTE2 AD-GTE3 AD-GTE4 AD-GTE5 AD-GTE6 AD-GTE7 AD-GTE8 AD-GTE9 AD-GTE10 AD-GTE11 AD-GTE12
AD-T 10 -1 10 -2 10 -3 10 10 -1 10 -2 10 -3 10 10 -1 10 -2 10 -3 10 10 -1 10 -2 10 -3 10 10 -1 10 -2 10 -3 10 10 -1 10 -2 10 -3 predicts that these epigenetic readers could be also targeted by other T3Es produced by the GMI1000 strain.

PopP2 belongs to the PopP family of RSSC effectors, which is composed of PopP1, PopP2 and PopP3. In the reference strain GMI1000, only popP1 and popP2 are functional genes, since popP3 is inactivated by the presence of an insertion sequence within the gene (Lavie et al., 2004). Given its similarities with PopP2 (24% identity, 39.5% similarity), we hypothesised that PopP1 which could represent a promising effector candidate also able to physically interact with GTE proteins, like PopP2, and responsible for the enhanced disease response of the GTE9 and GTE11 over-expressing lines to a RSSC strain lacking popP2. To test this hypothesis, PopP1 was used as a bait in a Y2H interaction assay with the twelve different A. thaliana GTE members used as prey proteins (Figure 26A). As negative control, we used the BD-p53 protein as bait. Strikingly, PopP1 was found to interact with various GTE members in yeast cells including GTE1, GTE2, GTE5, GTE6, GTE7, GTE8, GTE9 and GTE11. These data suggest that within the GTE family, PopP1 and PopP2 could interact with specific subsets of GTE proteins. The nucleocytoplasmic localisation of PopP1 (unpublished data) is consistent with a physical interaction between PopP1 and GTEs that would occur in the plant nucleus, as described with

PopP2. The targeting of different GTEs by PopP1, including GTE9 and GTE11, would explain why the GTE9 and GTE11 over-expressing lines inoculated with GMI1000 deleted from popP2 but still producing PopP1 were still more susceptible that the WT line in later stages of disease. In N. benthamiana, PopP1 recognition is mediated by the NLR NbZAR1 (Deslandes, unpublished data) but the virulence targets of PopP1 remain largely unknown, both in N. benthamiana and in A.

thaliana. The identification of GTEs proteins as putative interacting partners and substrates of PopP1 should help elucidate the virulence functions of this effector.

These findings prompted us to investigate whether GTE9 and GTE11 could represent a cellular hub targeted by effectors from different plant bacterial pathogens that would have independently evolved virulence strategies converging onto the manipulation of GTEs proteins to promote infection. To address this question, GTE9 and GTE11 over-expression lines were challenged with the virulent bacterial pathogen Pst DC3000 which contains a repertoire of T3Es with welldescribed virulence activities (Schreiber et al., 2021a). In detail, 3-week-old Arabidopsis Col-0 and lines over-expressing GTE9 or GTE11, previously used for inoculations with R. solanacearum strains in Chapter 1, were leaf-inoculated by infiltration of a Pst DC3000 suspension in order to assess their bacterial multiplication in planta at 4 dpi (Figure 27). For each genotype this experiment has been repeated independently twice (Figure 27A and 27B). Taking the results of the two independent repetitions, the conclusions are unclear. Indeed, according to the results presented in Figure 27A, the 3HA-tagged GTE9 and GTE11 over-expressing lines (GTE9-3HA and GTE11-3HA) displayed similar bacterial growth to that of Col-0, but in the second independent experiment, the GTE9-3HA line seemed to be more susceptible than Col-0 (Figure 27B). Similarly, in the second independent experiment, the GTE9-3Flag over-expressing line showed similar bacterial multiplication to Col-0; whereas in the first experiment, this line contained less bacteria than Col-0 (Figure 27B and 27A). Note also that the significant differences in bacterial loads between genotypes at 4 dpi are not 1 log difference, but closer to 0.5 log or less. To conclude, since DC3000 bacterial growth in the GTE9 and GTE11 over-expressing lines did not show a reproducible trend, it is not clear whether the GTE9 or GTE11 over-expressing lines are more susceptible to this virulent pathogen. Additional rounds of this bacterial multiplication assay will be performed soon in order to determine if the overexpression of GTE9 and GTE11 also promote infection by DC3000.

We also saw that PopP1 and PopP2 interact in yeast cells with several GTE proteins, and they both interact with GTE6, GTE9 and GTE11 (Figure 26A).

Interestingly, despite both belonging to the YopJ effector family, PopP1 and PopP2 belong to different phylogenetic groups in this family (group II and group V, respectively) (Figure 28) (Ma and Ma, 2016). This raises the question of whether other effectors from the YopJ family could target GTE proteins and this could be tested by Y2H.

As GTE9 and GTE11 are epigenetic components that probably act on a wide range of biological mechanisms, we wondered whether GTE9 and GTE11 could be targeted by other pathogen effectors. As this was not the priority of my PhD, we simply tried to see if GTE9 and GTE11 could be virulence targets of a different bacterial pathogen such as the well-described Pst DC3000. Unfortunately, in planta bacterial growth assays performed on the GTE9 and GTE11 over-expressing lines led to unclear results since the two independent experiments did not display reproducible trends (Figure 27A and 27B). In order to draw conclusions, it is necessary to repeat this experiment again. In addition, it could be interesting to study the phenotypical response of the GTE9 and GTE11 over-expressing lines challenged with other pathogens available in the lab, including bacteria and fungi.

This should help to better understand how GTE9 and GTE11 could play a role in modulating host immune responses.

Although we know that GTE11 and GTE9 are negative regulators of ABA and sugar signalling pathways and appear to be involved in the regulation of 35S enhancer activity (Irigoyen et al., 2021;Misra et al., 2018), we lack information on the precise role of GTE11 and GTE9 as epigenetic readers at the chromatin level. Identifying the chromatin regions hosting GTE9 and GTE11 could help us to elucidate (i) what are the function(s) of these epigenetic readers, and (ii) how their manipulation by

PopP2 can impact the course of the infection by RSSC in A. thaliana.

In Figure 29 and hereafter in the text, these lines are referred to as GTE9g-3HA, GTE11g-3HA, GTE9g-N214A-3HA, GTE11g-N206A-3HA lines.

For a given construct, complemented lines (hemizygous T1 lines and subsequent homozygous T3 lines) were selected. As the selection of the GTE11g-3HA homozygous lines was trickier than for GTE9g-3HA, we prioritised this chapter on the GTE9g-3HA lines. Among the homozygous lines available, two independent lines were selected for GTE9g-3HA and GTE9g-N214A-3HA (in gte9-1 genetic background) based on GTE9 expression level and immunodetection of the 3HAtagged proteins (Figure 29A and 29B, respectively). Originally, our plan was to select two lines with comparable gene expression to the WT. Unfortunately, among the available homozygous lines, only one line for GTE9g-3HA and GTE9g-N214A-3HA fulfilled this condition. The second independent line for GTE9g-3HA and GTE9g-N214A-3HA was chosen for having a relatively close GTE9 expression level to the one in the WT, but they are slightly over-expressed. These conditions are not optimal but since we plan to cross the ChIP-seq data obtained from two independent lines and we will focus on GTE9 targets identified in the two independent lines.

I generated the transgenic plants (T3 progeny) and the material (seed stock for GTE9g-3HA) was sent to our collaborators in Paris, who routinely perform ChIPseq analyses on A. thaliana seedlings. This ChIP-seq analysis should lead to the identification of peaks corresponding to particular genomic DNA regions hosting transgenically expressed 3HA-tagged GTE9. This analysis will be performed on unchallenged seedlings (uninfected context). Information provided by these results

will allow a better understanding on the role of GTE9 in planta, about their importance in specific cellular processes, but also if they seem associated with genes involved in plant response to pathogens. After obtaining the possible chromatin regions associated with GTE9, the next step will be to confirm that the targeted loci are really associated with GTE9. To do that, the idea will be to select various candidate genes and check that they are bound by GTE9 by ChIP-qPCR in the GTE9g-3HA lines. Similar analyses will be performed on the GTE9g-N214A-3HA lines in which mutated GTE9-N214A should be unable to bind those loci.

These ChIP-seq data will be crossed with PopP2-dependent RNA-seq data (showing differentially expressed genes induced by PopP2) in order to identify candidates genes hosting GTE9 and transcriptionally deregulated by PopP2 (Sohn et al., 2014). Considering that PopP2 alters the GTE9-H4 interaction likely by acetylating GTE9 around its bromodomain, further experiments could be conducted to check whether PopP2 dissociates GTE9 from particular loci and triggers its relocalisation to other genomic regions. To do so, we could consider performing ChIP-seq analyses on leaf cells expressing GTE9g-3HA and in which PopP2 or its catalytic mutant PopP2-C321A would be delivered by Pf0-1 cells. Finally, it would be very interesting to perform similar analyses on GTE11g-3HA lines and compare the different loci associated with GTE9 and with GTE11, and how PopP2 could influence their association with particular chromatin regions.

Chapter 4: Where is PopP2 addressed at the chromatin level?

Considering that PopP2 targets chromatin-associated proteins, such as WRKY TFs, GTE9 and GTE11, we wondered whether we could have a global overview of the chromatin sites visited by PopP2, and to see whether these sites differ in contexts where PopP2 is recognised or not by the immunoreceptor complex RPS4/RRS1-R (Le Roux et al., 2015).

To unravel the chromatin sites visited by PopP2, we initially planned to perform a ChIP-seq analysis by focusing only on plant cells in which PopP2 was delivered through a T3SS. To achieve this goal, we decided to use a GFP strand system which has been previously successfully used for the detection of plant nuclei hosting PopP2 delivered in a T3SS-dependent manner (Henry et al., 2017). Combined with Fluorescence-Activated Cell Sorting, plant nuclei hosting PopP2 could be separated from nuclei that do not host PopP2. Then, we would be able to perform ChIP-seq analyses on nuclei specifically hosting PopP2 to unravel the chromatin sites visited by PopP2 and determine if there are differences between genetic backgrounds that recognise or not PopP2.

I spent several months to try to set up the GFP strand system to perform ChIP-seq analyses on PopP2, but we were not able to use it in the end, as described by exploiting the ability of Pf0-1 cells to deliver PopP2 through its T3SS in leaf cells.

For this strategy, I generated a Pf0-1 strain producing the PopP2 effector Cterminally fused with a triple hemagglutinin epitope followed by the 11 th beta strand of GFP (PopP2-3HA-GFP 11 ) (Figure 30B). These two tags are useful for protein immunoprecipitation and detection, respectively.

Setting up the GFP-strand system for the detection of plant cells hosting

PopP2 and be compatible for ChIP-seq analyses

We generated Pf0-1 strains that can deliver PopP2-3HA-GFP 11 or its catalytic mutant PopP2-C321A-3HA-GFP 11 . These protein fusions seem to be delivered by the T3SS of Pf0-1 since when infiltrating N. benthamiana leaves with these Pf0-1 strains, we could detect the proteins by immunoblot 6 hours after infiltration (Figure 31A). In addition, we checked that PopP2-3HA-GFP 11 retained its avirulence function. Indeed, in wild type Ws-2 expressing the immunoreceptor pair of proteins RPS4/RRS1-R, Pf0-1-mediated delivery of GFP 11 -tagged PopP2, but not PopP2 catalytic mutant, triggered a cell death response, as previously observed with WT PopP2 tagged with only a 3HA epitope. By contrast, in rps4-21/rrs1-1 double mutant, none of the PopP2 fusion delivered by Pf0-1 were able to trigger such a cell death response (Figure 31B). together, these data indicate that PopP2-3HA-GFP 11 retains its avirulence function in triggering activation of the RPS4/RRS1-R immune receptor complex.

The next step was to check that PopP2-3HA-GFP 11 delivered by Pf0-1 in transgenic Arabidopsis expressing GFP 1-10 could be detected. Despite multiple attempts, and even though PopP2-3HA-GFP 11 seems to be delivered in plant cells, it was not possible to detect any GFP fluorescence signal, 6 hours after infiltration of Pf0-1 cells. Since this GFP strand system approach was not successful and timeconsuming, we decided to switch to a more classical ChIP-seq approach.

Alternatives to the GFP-strand system by using a more classical ChIP-seq approach This approach is based on the use of tagged versions of PopP2 that would be still delivered by Pf0-1 cells and the immunoprecipitations will be performed on the whole infiltrated leaf material (containing a mixture of cells hosting PopP2 and cells that do not host PopP2 at all). In order to generate robust ChIP-seq data, two different tagged versions of PopP2 have been generated (PopP2-3HA and PoPP2-3Flag), so that we could cross the outputs from two ChIP-seq performed independently using different affinity resins (HA vs Flag). This would increase the reliability of the results provided by the ChIP-seq analyses. That is why we planned to use Pf0-1 strains expressing WTPopP2 or its catalytic mutant, both of them Cterminally fused either with 3HA or 3Flag epitope tag. The strains expressing PopP2-3HA or PopP2-C321A-3HA were already available in the lab, and I generated the strains expressing PopP2-3Flag or PopP2-C321A-3Flag. Both

PopP2-3HA and PopP2-3Flag retained their avirulence functions as evidenced by the cell death response they triggered in Ws-2 plants (Figure 31B). We could also follow the kinetics of PopP2-3HA and PopP2-3Flag accumulation in A. thaliana and N. benthamiana leaves (Figure 31C). Both fusion proteins were detected by immunoblot already 2 hours after infiltration. For the ChIP-seq experiments, we decided to consider the "4 hours after infiltration" (hpi) timepoint since previously performed RNA-seq analyses revealed a large subset of differentially expressed genes in presence of PopP2 (Sohn et al., 2014). Unfortunately, these ChiP-seq analyses have been delayed and we hope to have the results available in the near future.

Once/if the system to deliver PopP2 in A. thaliana plantlets is set up, we will be able to investigate the chromatin sites visited by PopP2 in Ws-2 and in rps4-21/rrs1-1. ChIP-seq analyses will reveal peaks associated with PopP2 in a context when it is recognised in planta or not, and will show chromatin regions and genes visited by PopP2 after mapping these peaks on A. thaliana genome. Crossing these data with RNA-seq data obtained from the same material would allow us to identify the chromatin regions associated with PopP2 and for which genes are differentially expressed because of PopP2 in a resistant or susceptible background. In addition, by comparing data obtained with the catalytic mutant of PopP2, we should be able to determine whether its acetyltransferase activity enables PopP2 to reach specific chromatin regions.

Altogether, these results could allow us to draw a picture of the impact of PopP2 Recent studies related the importance of the plant epigenome in the regulation of plant defences (Ding and Wang, 2015;Hannan Parker et al., 2022;Zhu et al., 2016b). As a way to counter the activation of host defence, parasites have evolved ingenious ways to manipulate the host epigenome to promote infection via effectors. It is quite well documented for animal pathogens, for which some effectors have been described to manipulate histone readers, histone marks and chromating remodelling (Dong and Hamon, 2020;Ramirez-Prado et al., 2018b).

While less examples are documented in plant pathogens, some effectors from plant parasites were shown to manipulate the plant epigenome. They include mainly diverse effectors from viruses, fungi and nematodes (Castillo-González et al., 2015;Kong et al., 2017;Li et al., 2018;Vijayapalani et al., 2018). Examples of manipulation of the plant epigenome by bacterial pathogens are scarce, even though some effectors from P. syringae have been shown to inhibit plant RNA silencing to promote infection (Navarro et al., 2008).

In this context, my PhD project focused on characterising the targeting of A.

thaliana histone readers by a T3E from a plant bacterial pathogen. More precisely, pursuing the work from Alice Delga, my project aimed at further characterising the targeting of the BRD containing proteins GTE9 and GTE11 by the RSSC T3E PopP2

acetyltransferase by trying to answer several questions (Figure 32). We wondered how PopP2 could manipulate GTE9 and GTE11 and demonstrated that PopP2

acetylates both of them on several lysine residues, including residues flanking their BRD, among which common residues shared by the two proteins (Figure 2B, Comorge et al., in prep.). We also showed that PopP2 alters the interaction between GTE9 and H4, involving its acetyltransferase activity (Figure 4C,Comorge et al.,in prep.). As GTE9 and GTE11 are targeted by PopP2, we next wondered whether they could represent PopP2 virulence targets playing a role in the plant response to the RSSC. Whereas gte9 and gte11 mutant line did not show any alteration of their phenotypical response to the GMI1000 strain, we showed that A. thaliana GTE9 and GTE11 over-expressing lines were more susceptible than WT plants to the RSSC producing active PopP2 (Figure 5B and 5C,Comorge et al.,in prep.). This suggests that RRSC uses PopP2 to intefere with GTE9 and GTE11 functions and promote infection. As GTE9 and GTE11 belong to a family of 12 proteins in Arabidopsis, we investigated the potential targets of other GTE members by PopP2 and found that PopP2 can acetylate other GTE members such as GTE3, GTE5 and GTE8 (Figure 7B, Comorge et al., in prep.). Therefore, GTE proteins represents additional host components whose targeting by a YopJ family acetyltransferase helps the pathogen infect its host plant. Could GTEs be targeted by other effectors/pathogens? Notably, we were able to demonstrate that PopP1, an other YopJ family member from the RSSC, can interact with several GTE members in yeast (GTE1, GTE2, GTE5, GTE6, GTE7, GTE8, GTE9 and GTE11) (Figure 26). I also developped molecular and genetic tools aimed at unraveling the chromatin binding sites of GTE9 and GTE11 (Figure 29, ongoing).

Altogether, this work reveals that PopP2 targets by acetylation the BRD-containing proteins GTE9 and GTE11, promoting virulence of the RSSC. To our knowledge, this represents the first example of a T3E from a plant pathogenic bacteria manipulating histone readers. This work adds more evidence about pathogens targeting host epigenetic components in plants to promote virulence. Interestingly, it seems that PopP2 can target various GTE proteins and so might PopP1. Similarly to some virulence targets that represent a susceptibility hub for P. syringae (Schreiber and Lewis, 2021;Schreiber et al., 2021b), the targeting of the GTE proteins may also represent a susceptibility hub for the RSSC.

While this project paves the way towards the characterisation of the manipulation of histone readers by a plant bacterial pathogen, more questions arise and would be worth investigating. We demonstrated that PopP2 alters the interaction between GTE9 and histone H4 and that it depends on PopP2 acetyltransferase activity and we know that PopP2 acetylates lysine residues surrounding GTE9 and GTE11 BRD.

Similarly to the regulation of the yeast SWI/SNF subunit Snf2 by intramolecular interaction of its BRD with flanking Lysine residues acetylated by a HAT (Kim et al., 2010), we proposed that PopP2 could trigger similar intramolecular interaction between GTE9 BRD and surrounding acetylated lysine residues (Figure 20). It would be interesting to unravel which acetylated lysine residues are responsible for the alteration of the GTE9-H4 interaction. For that, we could generate GTE9 variants mutated on some of the lysine residues normally targeted by PopP2 that could become insentive to PopP2 manipulation. The ability of these "refractory"

GTE9 variants to bind in planta Histone H4 in presence of PopP2 could be easily monitored by using FRET-FLIM assays.

In addition, we showed that GTE9 and GTE11 over-expression promotes the RSSC virulence, but the link between the alteration of the interaction GTE-H4 by acetylation by PopP2 and its impact on the overall course of infection with RSSC is still not established. One way to answer this question would be to study the phenotypical response to RSSC of A. thaliana transgenic lines over-expressing GTE9-N214A and GTE11-N206A mutants, both of which being impaired in their histone reader activity. Alternatively, if we identify "refractory" GTE9 variants that could not be manipulated anymore by PopP2, we could generate corresponding A.

thaliana over-expressing lines and test their phenotypical response to the RSSC.

We also discovered that PopP2 can acetylate other GTE proteins. This suggests that PopP2 could target various members of the GTE family, as previously observed for many WRKY factors that belong to the same family of TFs. It's then tempting to imagine that PopP2 could target GTE3, GTE5 and GTE8 in a similar way than GTE9 and GTE11, by acetylating residues flanking their BRDs. It would be informative to investigate by MS/MS-based analyses which lysine residues are acetylated by PopP2 in the other targeted GTEs, and see whether key conserved lysine(s) can be identified. In complement, since GTE3 was found to interact with acetylated H3, we could assess whether PopP2 is also able to compromise the ability of GTE3 to interact with H3 in planta (Garcia-Dominguez et al., 2008).

But more than being a protein family targeted by a bacterial effector, GTE proteins could represent a susceptibility hub. It has already been described in the litterature that some host proteins could represent a hub for different effectors from the same pathogen, or from different pathogens (Mukhtar et al., 2011;Schreiber and Lewis, 2021;Schreiber et al., 2021b;Weßling et al., 2015). Here, we showed that PopP1 also interacts in yeast with several GTE proteins, suggesting that GTE proteins could represent a susceptibility hub for different effectors from the RSSC.

Considering that PopP1 is also a YopJ family acetyltransferase, assessing if PopP1 can acetylate certain GTE proteins (in planta acetylation assays) would allow us to determine whether PopP1 and PopP2 could cooperate together for efficient manipulation of histone readers. Moreover, as PopP1 and PopP2 belong to the YopJ family of effectors, but in different subgroups, the fact that both effectors can target or interact with GTE proteins raises the question of whether other YopJ family effectors from plant pathogenic bacteria, such as P. syringae or X.

campestris, also manipulate GTE proteins too (Figure 28). This raises the question of whether YopJ family acetyltransferases produced by animal pathogenic bacteria could also exert their virulence functions by manipulating epigenetic readers. incubated for 30 minutes at 55°C. Reverse transcription was stopped by incubating the samples at 85°C for 10 minutes. After 5 min on ice, samples were stored at -20°C. RT-qPCR were performed with a LightCycler 480 II machine (Roche Diagnostics) with Roche reagents. Expression of AtEF1-α (At5g60390) was used to normalise the expression values. The comparative Ct method (2 ΔCt ) was used to represent the data (Schmittgen and Livak, 2008). Mean ΔCt were calculated from three technical replicates for three biological replicates (three independently grown sets of plants). The mean relative expressions from the biological replicates +/-SD was represented with ggplot2 package from R.

Statistical analyses were performed on ΔCt values to assess the significance of the difference in the mean ΔCt between the different genetic lines or conditions.

We applied either a one-way ANOVA when the assumptions about the underlying distribution of the data were fulfilled or a kruskal-test when they were not.

Root-inoculation assays in Arabidopsis

For root-inoculation assays, bacteria (Rs) were grown in a liquid culture at 28°C overnight and diluted in water at DO 600 =0.01. Four-week-old A. thaliana plants were soaked in this bacterial solution for 10 min, transferred on soil and incubated in a growth chamber (12h day/12h night, 27°C during the day/26°C at night, 75% relative humidity). Symptoms were scored daily with the following disease scale: 0 = no symptoms, 1 = 25% leaves wilt, 2 = 50%, 3 = 75% and 4 = 100%. For each independent inoculation, between 24 and 32 plants from different lines in Arabidopsis thaliana ecotypes were root-inoculated with GMI1000 strain or mutant strains of R. solanacearum and wilting symptom developments were scored for 10 days.

For each presented graph, disease scoring data were analysed from three independent inoculations with R software as previously described (Aoun et

Protein Extraction and Immunoblot Analysis

Plant material (4 discs of 8 mm diameter harvested 48 hours post-infiltration for N.

benthamiana samples; 20 seedlings (10 day-old) or 8 leaf discs of 4mm diameter for Arabidopsis samples) were grinded and resuspended in 300 µL of Laemmli Buffer (2X). Protein extracts were then denatured for 3 min at 95°C, centrifugated at 13000 rpm for 1 min and then analysed by immunoblotting (SDS-PAGE).

Transferred proteins were visualised by Ponceau S red staining. Membranes were blocked in a 2% milk TBS-T (Tris Buffer Saline-Tween 20; 50 mM Tris-HCl pH7.5, 150 mM NaCl, and 0.2% Tween-20) solution before incubation. The following primary antibodies were used in this study: anti-HA-HRP (3F10; Roche; dilution 1:5000), anti-Flag-HRP (Sigma; dilution 1:5000). Immunodetections were performed using Clarity Western ECL substrate reagent (Bio-Rad).

Figures

  Figures and Tables list ....................................................................................................................... Introduction ....................................................................................................................................... 1-Molecular bases of plant-pathogen interactions ...................................................................... A. Generalities ....................................................................................................................... B. The zig-zag model: a model to summarise plant-pathogen interactions molecular bases17

•

  B.3. Effector-Triggered Immunity (ETI) .......................................................................... B.3.1. Intracellular receptors are able to recognise effector proteins .............................. B.3.1.a. Direct recognition of effectors .......................................................................... B.3.1.b. Indirect recognition of effectors: guardee and decoy models .......................... B.3.1.c. A particular type of decoy: the Integrated Decoys (ID) ..................................... B.3.2. NLRs work often in cooperation ..............................................................................

  .. • B.4. How is pathogen perception translated into immune responses? ........................ B.4.1. Ca 2+ influx and other ion fluxes ............................................................................... B.4.2. ROS burst ................................................................................................................. B.4.3. MAPK signalling cascades ........................................................................................ B.4.4. Phytohormones .......................................................................................................

  reprogramming .............................................................................. B.4.6. Hypersensitive Response and Systemic Acquired Resistance ................................. • B.5. How phytopathogens facilitate infection: focus on Effector-Triggered Susceptibility ......................................................................................................................... B.5.1. Effectors, pathogen secreted proteins aiming at disturbing cell homeostasis to favour infection ................................................................................................................. B.5.2. Effectors can manipulate various cellular processes............................................... B.5.2.a. Effectors can dampen PTI responses by targeting

  C. Adjustments and limitations to the zig-zag model ............................................................ 2-The epigenome, an emerging player of plant-pathogen interactions ...................................... A. Chromatin is a dynamic environment regulated by epigenetic factors ............................ • A.1. Chromatin structure influences gene expression .................................................. • A.2. Epigenetics, or how to study the regulation of chromatin condensation status ... B. Epigenetic mechanisms regulate plant defence against pathogens ................................. • B.1. DNA methylation mainly represses plant defence ................................................. B.1.1. What is DNA methylation? ...................................................................................... B.1.2. DNA methylation mainly represses plant immunity ............................................... • B.2. Histone marks influence chromatin condensation state leading to differential regulation of defence-related genes in response to pathogens ........................................... B.2.1. Histone marks: Post-Translational Modifications on histone tails influence chromatin condensation status .........................................................................................

  -pathogen interactions50 B.2.2.b. Histone acetylation and plant response to pathogens ..................................... • B.3. Chromatin remodelling complexes modulate chromatin density by playing with nucleosomes .......................................................................................................................... B.3.1. What are chromatin remodelling complexes? ........................................................ B.3.2. Chromatin remodelling complexes regulate plant responses to pathogens .......... • B.4. Non-coding RNAs .................................................................................................... B.4.1. Long and short non-coding RNAs are regulatory non-coding RNAs involved in many • A.1. Pathogen description ............................................................................................. • A.2. Classifying the different strains of the species complex ........................................ • A.3. A complicated taxonomy, since it is a species complex ......................................... B. Pathogenicity of the RSSC and its virulence determinants ............................................... • B.1. The RSSC is an important threat for crop production due to its wide host range and persistence in the field ................................................................................................... • B.2. The control methods against the RSSC are diverse but not entirely satisfactory .. • B.3. Infection process and virulence determinants of the RSSC ................................... C. Focus on key virulence determinants of the RSSC: theType III effectors .......................... • C.1. The RSSC possesses a large Type III effector (T3E) repertoire................................ • C.2. RSSC T3Es, diverse weapons to manipulate the host plant for the benefit of the pathogen ...............................................................................................................................

Introduction

Figure 1 .

 1 Figure 1. The Zig-zag model.Figure 2. PAMPs and DAMPs recognition by PRRs.Figure 3. Effector recognition mediated by NLRs.Figure 4. PTI and ETI downstream signalling events.Figure 5. Pathogen effectors target various cellular processes.Figure 6. Eukaryotic chromatin structure.

Figure 2 .

 2 Figure 1. The Zig-zag model.Figure 2. PAMPs and DAMPs recognition by PRRs.Figure 3. Effector recognition mediated by NLRs.Figure 4. PTI and ETI downstream signalling events.Figure 5. Pathogen effectors target various cellular processes.Figure 6. Eukaryotic chromatin structure.

Figure 3 .

 3 Figure 1. The Zig-zag model.Figure 2. PAMPs and DAMPs recognition by PRRs.Figure 3. Effector recognition mediated by NLRs.Figure 4. PTI and ETI downstream signalling events.Figure 5. Pathogen effectors target various cellular processes.Figure 6. Eukaryotic chromatin structure.

Figure 4 .

 4 Figure 1. The Zig-zag model.Figure 2. PAMPs and DAMPs recognition by PRRs.Figure 3. Effector recognition mediated by NLRs.Figure 4. PTI and ETI downstream signalling events.Figure 5. Pathogen effectors target various cellular processes.Figure 6. Eukaryotic chromatin structure.

Figure 5 .

 5 Figure 1. The Zig-zag model.Figure 2. PAMPs and DAMPs recognition by PRRs.Figure 3. Effector recognition mediated by NLRs.Figure 4. PTI and ETI downstream signalling events.Figure 5. Pathogen effectors target various cellular processes.Figure 6. Eukaryotic chromatin structure.

Figure 6 .

 6 Figure 1. The Zig-zag model.Figure 2. PAMPs and DAMPs recognition by PRRs.Figure 3. Effector recognition mediated by NLRs.Figure 4. PTI and ETI downstream signalling events.Figure 5. Pathogen effectors target various cellular processes.Figure 6. Eukaryotic chromatin structure.

Figure 7 .

 7 Figure 7. Histone acetylation correlates with active transcription.Figure 8. Pathogens manipulate the host epigenome by targeting/interfering with diverse mechanisms. Figure 9. The RSSC consists of three species. Figure 10. Worldwide distribution of the RSSC.

Figure 8 .

 8 Figure 7. Histone acetylation correlates with active transcription.Figure 8. Pathogens manipulate the host epigenome by targeting/interfering with diverse mechanisms. Figure 9. The RSSC consists of three species. Figure 10. Worldwide distribution of the RSSC.

Figure 11 .

 11 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 12 .

 12 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 13 .

 13 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 14 .

 14 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 15 .

 15 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 17 .

 17 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 18 .

 18 Figure 11. Life cycle and infection process of the RSSC. Figure 12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes.Figure 13. PopP2 is a YopJ family acetyltransferase.Figure 14. PopP2 acetylates WRKY domain-containing. Figure 15. The bromodomain, a domain that recognises acetyl-lysine residues. Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins.Figure 17. Arabidopsis GTEs are involved in various biological processes.Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2.

Figure 19 .

 19 Figure 19. How does PopP2 target the histone readers GTE9 and GTE11?

Figure 1 .

 1 Figure 1. PopP2 interacts with Arabidopsis GTE9 and GTE11 in yeast and in the nucleus of N. benthamiana cells.

Figure 2 .

 2 Figure 2. PopP2 acetylates GTE9 and GTE11. Figure 3. The bromodomain of GTE9 confers preferential binding to acetylated histone H4. Figure 4. PopP2 targets the bromodomain of GTE9 and interferes with its ability to bind histone H4. Figure 5. Targeting of GTE9 and GTE11 by PopP2 promotes wilt disease development in Arabidopsis. Figure 6. GTE9 and GTE11 are not required for RPS4/RRS1-R-mediated immunity.Figure 7. PopP2 targets several members of the GTE family.

Figure 3 .Figure 4 .

 34 Figure 2. PopP2 acetylates GTE9 and GTE11. Figure 3. The bromodomain of GTE9 confers preferential binding to acetylated histone H4. Figure 4. PopP2 targets the bromodomain of GTE9 and interferes with its ability to bind histone H4. Figure 5. Targeting of GTE9 and GTE11 by PopP2 promotes wilt disease development in Arabidopsis. Figure 6. GTE9 and GTE11 are not required for RPS4/RRS1-R-mediated immunity.Figure 7. PopP2 targets several members of the GTE family.

Figure 5 .

 5 Figure 2. PopP2 acetylates GTE9 and GTE11. Figure 3. The bromodomain of GTE9 confers preferential binding to acetylated histone H4. Figure 4. PopP2 targets the bromodomain of GTE9 and interferes with its ability to bind histone H4. Figure 5. Targeting of GTE9 and GTE11 by PopP2 promotes wilt disease development in Arabidopsis. Figure 6. GTE9 and GTE11 are not required for RPS4/RRS1-R-mediated immunity.Figure 7. PopP2 targets several members of the GTE family.

Figure 7 .

 7 Figure 2. PopP2 acetylates GTE9 and GTE11. Figure 3. The bromodomain of GTE9 confers preferential binding to acetylated histone H4. Figure 4. PopP2 targets the bromodomain of GTE9 and interferes with its ability to bind histone H4. Figure 5. Targeting of GTE9 and GTE11 by PopP2 promotes wilt disease development in Arabidopsis. Figure 6. GTE9 and GTE11 are not required for RPS4/RRS1-R-mediated immunity.Figure 7. PopP2 targets several members of the GTE family.

Figure 20 .

 20 Figure 20. Intramolecular interactions between BRD and acetylated lysines regulating the binding of BRD-containing proteins to acetylated histones.

Figure 21 .

 21 Figure 21. gte9-1 and gte11-1 mutants show similar disease development as Col-0 in presence or absence of PopP2.

Figure 22 .Figure 25 .

 2225 Figure 22. Expression analysis of GTE9 and GTE11 truncated transcripts in gte9-1 and gte11-1 T-DNA insertion mutant lines. Figure 23. Expression level of GTE genes family is not affected by the presence of active PopP2 or PopP2-C321A catalytic mutant.Figure24. Expression level of GTE2 and GTE11 is not modulated by the presence of active and catalytically inactive PopP2.

Figure 26 .

 26 Figure 26. PopP1 interacts with various GTE members in yeast cells.

Figure 27 .

 27 Figure 27. The in planta growth of Pst DC3000 is not impacted by overexpression of GTE9 and GTE11.

Figure 28 .Figure 29 .Figure 30 .

 282930 Figure 28. Phylogeny of the YopJ family effectors.

Figure 31 .

 31 Figure 30. Monitoring of the spatio-temporal delivery of PopP2 in Arabidopsis cells by using the GFP strand system (principle).Figure 31. WT PopP2 C-terminally tagged with various epitopes retains its avirulence activity in Ws-2 accession.

Figure 1 .

 1 Figure1. The Zig-zag model (adapted fromJones and Dangl, 2006). The zig-zag model describes the arms-race that takes place between plants and pathogens upon interaction. Plants can detect conserved molecules from pathogens, PAMPs, through cell surface receptors, PRRs. The PAMP recognition by PRRs triggers PTI responses. During evolution, pathogens have evolved effectors capable of disturbing cellular processes to promote infection, known as ETS. Some effectors can be detected by plants through NLR (R) proteins leading to ETI responses, often associated with a programmed cell death (Hypersentive Response (HR) response). To counteract activation of host immune responses and evade recognition, pathogens evolve new effectors or delete/modify others. In turn, plants can also evolve new recognition mechanisms/capabilities to improve pathogen recognition and immunity activation.

  to basal immune responses is based on the recognition of conserved molecular patterns of the pathogen called Pathogen-Associated Molecular Patterns (PAMPs), by plant receptors located on the cell surface, called Pattern-Recognition Receptors (PRRs). PAMP recognition by a PRR leads to PAMP-Triggered Immunity or PRR-triggered Immunity (PTI). To overcome PTI, pathogens have evolved virulence factors called effector proteins that interfere with PTI. Pathogen effectors can interfere with PTI by altering various cellular mechanisms to promote infection.

  Intracellular receptors are able to recognise effector proteins Plant R proteins are intracellular receptors that trigger ETI after perception of effector proteins. These R proteins are called NOD-like receptors (NLRs) because they share similarities with Nucleotide Oligomerisation Domain receptors from mammals. In general, NLRs have three different parts: at the N-term part of the NLRs there is either a coiled-coil (CC) domain or a Toll/Interleukin1 receptor (TIR)

Figure 3 .

 3 Figure 3. Effector recognition mediated by NLRs (adapted from Nguyen et al., 2021). During plant-microbe interaction, microbes deliver effectors into plant tissue to alter diverse cellular processes. (A) Plants have evolved specialised intracellular immune receptors, the NLRs, which can specifically recognise some effectors by direct of indirect interaction. In indirect recognition mechanisms, NLRs generally guard an effector-targeted protein, whose modification triggers NLR activation. This host protein can be a guardee or a decoy. (B) & (C) NLRs tend to act cooperatively. Upon effector recognition, NLRs activate and tend to oligomerise into larger structures known as resistosomes, thereby activating ETI responses. ETI activation also depends on helper NLRs such as NRG1 and ADR1.

  NLR cluster is defined by a genomic area of less than 200 kb and containing less than eight non-NLR-encoding genes within the cluster. Across different vascular plant species, while the number of NLRs varies, the proportion of NLR clusters is relatively constant at around 60% (van Wersch andLi, 2019). It is tempting to imagine that NLR clustering is beneficial in activating plant immunity at a regulatory level or in eliminating some fitness costs, but this requires further investigation. The clustering of NLR encoding genes could allow them to be cotranscriptionally regulated, thus facilitating the assembly of NLRs into oligomeric complexes that mediate activation of immune responses; B.3.2.a. Self-association of NLRs

Figure 5 .

 5 Figure 5. Pathogen effectors target various cellular processes (adapted from Schreiber et al., 2021). To counteract PTI and promote infection, pathogens deliver effectors into plant cells to modify cellular activities to their advantage. Effectors can manipulate cellular processes via their enzymatic activities or simply by interacting with host components, regulating them positively or negatively. In this thesis manuscript, I have focused on some examples of host processes targeted by effectors such as PTI components, MAPK cascades, cytoskeleton, transcription, proteasome and ETI-related components.

  the zig-zag model, this pathogen interference is referred to ETS. I will introduce effector proteins and give several examples of manipulation of host processes by pathogens to promote infection. I will deliberately avoid examples concerning the bacterium Ralstonia solanacearum and the manipulation of the host epigenome by pathogens, as they will be detailed in later parts of this Introduction section.Pathogen effectors can either be secreted into the apoplast or translocated into the host cytoplasm. Bacterial pathogens use complex multiprotein secretion systems such as type 3 and type 4 secretion systems (T3SS, T4SS) which are complex needle-like structures allowing translocation of effectors and effectors

2-

  The epigenome, an emerging player of plant-pathogen interactions Since my PhD project focused on the functional characterisation of Arabidopsis histone readers targeted by a YopJ family acetyltransferase effector, I thought it would be useful to present in this introductory chapter a non-exhaustive overview of current knowledge about the regulation of plant defence responses by epigenetic mechanisms and the virulence strategies used by animal and plant pathogens to subvert the host epigenome.

Figure 7 .

 7 Figure 7. Histone acetylation correlates with active transcription (adapted from Kumar et al., 2021). A. Acetylation of lysine residues on histone tails is catalysed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs). Lysine acetylation neutralises the positive charge of the amine group, resulting in chromatin relaxation. B. The response of plants to development and environmental stimuli is epigenetically regulated in part by histone acetylation. Under stimuli, HATs and HDACs are induced and lead to fine regulation of their target genes by acetylation or deacetylation, promoting or decreasing their transcription, resulting in biochemical responses that will be transduced into cellular responses and trigger developmental processes or stress tolerance responses. TF: Transcription Factor.

  Altogether, these different studies demonstrate how histone mark regulators can modulate in various ways plant defence responses. Expression of some of them is increased during infection. They can also act by partially regulating hormonal pathways, NLR-encoding gene or defence gene expression. Some of them potentiate the activation of plant defence by enhancing the activation of defence genes, rendering immune responses fully effective. Others promote the expression of positive regulators of plant defences, or conversely, the repression of negative regulators. Whether histone mark regulators positively or negatively affect defences against a pathogen also depends on the nature of the pathosystem.

  B.4. Non-coding RNAs B.4.1. Long and short non-coding RNAs are regulatory non-coding RNAs involved in many biological processes Non-coding RNAs (ncRNAs) are parts of the genome that are transcribed into RNA but that do not encode proteins. They include housekeeping ncRNAs, like ribosomal RNAs and transfer RNAs, and regulatory RNAs. Regulatory ncRNAs consist of small

•

  C.1. Numerous animal pathogens have been shown to subvert host epigenome C.1.1. Pathogen targeting of host DNA, CRCs or epigenetic readers C.1.1.a. Manipulation of DNA methylation by Mycobacterium tuberculosis

Figure 8 .

 8 Figure 8. Pathogens manipulate the host epigenome by targeting/interfering with diverse mechanisms.Various effectors from plant and animal pathogens and parasites were shown to interefere with the host epigenome, supposedly to favour infection. Some can directly or indirectly modify DNA methylation and histone marks such as acetylation, methylation or phosphorylation. Others target histone marks by manipulating cytoplasmic acetyltransferase of histone mark regulators such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), or epigenetic readers. Orange: effectors from animal pathogens; green: effectors from plant parasites or pathogens.

  localises into the nucleus where it binds to specific DNA sequences and methylates cytosines outside CpG contexts, in which cytosines are followed by guanines. It can also bind to histone H3 and H4 harbouring specific

  C.1.1.c. Interfering with epigenetic readers to hijack host transcription Epigenetic readers contribute to the regulation of gene transcription by protecting the sites they recognise from adding or removing histone marks, or by helping to recruit epigenetic regulators, thus facilitating the switch of epigenetic marks. They indirectly participate in gene transcription regulation, making them a virulence target of choice for pathogens.

  subverting the host epigenome (and especially histone PTMs) are produced by bacterial animal pathogens, such examples of pathogen interference on plant epigenomes by bacterial plant pathogens and parasites are missing, with all documented studies involving only viruses, oomycetes and nematodes. During my PhD, I investigated the targeting of A. thaliana epigenetic readers by PopP2, a type III effector from the plant bacterial pathogen Ralstonia solanacearum species complex (RSSC). The following section presents the R. solanacearum species complex and focuses on the available knowledge concerning PopP2 and its biological functions.

•

  A.1. Pathogen descriptionRalstonia solanacearum is a species complex of gram-negative β-proteobacteria of the Burkholderiales order and the Ralstoniaceae family. RSSC bacteria cause bacterial wilt disease, which can be called by different names depending on the host such as potato brown rot or the Moko disease affecting banana species. The bacterial cells of R. solanacearum are 0.5-1.5µm long, rod-shaped and harbour one polar flagellum. On growth medium, spread R. solanacearum bacteria have a fluid aspect, forming flat, irregular, pearly cream-white colonies(Osdaghi, 2020). Most strains generally grow in a moist environment with an optimum temperature of 35-37°C, but others can grow at a lower temperature. • A.2. Classifying the different strains of the species complex Due to the intraspecific diversity within the RSSC, several attempts have been made to classify the different RSSC strains, either by host range, biochemical types or geographical distribution. The concept of races was introduced in 1962 and allowed the classification of the different strains according to their pathogenicity to different hosts and the phenotypic characteristics of the strains (Buddenhagen

Figure 10 .

 10 Figure 10. Worldwide distribution of the RSSC (extracted from EPPO, 2021).The RSSC is a devastating plant bacterial pathogen with a worldwide distribution. Many strains thrive in equatorial regions but other strains are adapted to more temperate climates. While in many areas the RSSC is endemic, its presence in Europe is less pronounced and for this reason it is considered a quarantine pathogen in Europe.

BFigure 11 .

 11 Figure 11. Life cycle and infection process of the RSSC (adapted from Genin 2010). (A) The RSSC is a soil-borne vascular pathogen that can infect host plants through wounds in the roots or secondary roots emerging points, and then reach the xylem vessels, where it develops. Clogging of the xylem vessels induces wilting symptoms that will eventually cause the death of the host plants. After plant death, the RSSC bacteria return to the soil where it can survive for a long period (2-3 years) or colonise weeds, thus maintaining a bacterial reservoir in the soil. (B) Wilting symptoms on Arabidopsis plant. A healthy Arabidopsis plant on the left, compared to a totally wilted Arabidopsis plant on the right.

Figure 12 .

 12 Figure12. Effectors from the RSSC interfere with plant immune responses and affect host cellular processes (adapted fromLandry et al., 2020). RSSC bacteria translocate T3Es into the plant cell to subvert the plant defences and accommodate the bacterial needs. T3Es act on different host pathways. RipAY and RipN alter the glutathione level and NADH/NAD+ ratio, respectively. RipAL, RipG1, and RipG3 target the hormone synthesis and signalling level. RipTAL binds to the plant DNA, activating the expression of shorter and more efficiently translated transcripts of arginine decarboxylase (ADC) genes, key enzymes in the biosynthesis of polyamines. This boost in the polyamine level could prevent the proliferation of Ralstonia niche competitors. The nuclear T3E RipAB inhibits the expression of Ca2+-related defence genes. In addition to these functionally characterised RSSC T3Es, other effectors involved in dampening of basal defence through as yet unknown mechanisms have been identified including RipAR, RipAW, RipAB, and RipE1. RSSC T3Es can also be perceived in planta by NLRs, leading to the activation of specific defence mechanisms, often associated with an HR. RipE1, RipAA, RipP1, RipAT, RipAV, RipAB, and RipB induce HR on several hosts. Some T3Es can modulate the activity of others and prevent their recognition by the plant surveillance system. For instance, RipAY inhibit RipE1-mediated HR.

  and presents sequence similarity to TALEs found in Xanthomonas spp., which made researchers curious as to test whether this RipTAL could act in the same way as the TALEs, i.e. activate the transcription of susceptibility genes by binding to specific EBEs on host DNA(de Lange et al., 2013; Macho et al., 2010). Indeed, Brg11 has been demonstrated to bind to the EBE of two tomato genes encoding Arginine decarboxylases (ADC) and increase their transcript accumulation in tomato, eggplant, N. benthamiana and N. tabacum, suggesting that Brg11 functions in different host plants. ADCs are enzymes involved in polyamine biosynthesis by catalysing the conversion of arginine to agmatine which can be subsequently converted into putrescine and longer polyamines like spermine and spermidine.Putrescine and other polyamines are known to participate in ROS burst activation after catabolition and have been shown to potentiate ROS burst during PTI (O'Neill

  is a T3E belonging to the PopP (RipP) family in RSSC which comprises 3 members, PopP1 (RipP1), PopP2 and PopP3 (RipP3). The three effectors share about 17-20% protein identity when compared in pairs, and they

Figure 13 .

 13 Figure 13. PopP2 is a YopJ family acetyltransferase (adapted from Xia et al., 2021 and Ma and Ma, 2016). (A) PopP2 is a RSSC T3E with a NLS (grey box) and an acetyltransferase domain consisting of a catalytic core, conserved among all YopJ family members, and a regulatory domain. (B) Acetylation of PopP2 susbrates likely follows the ping-pong model, in which PopP2 auto-acetylates using acetyl-CoA as an acetyl donor, and then transfers the acetyl group onto its substrates. (C) In host cells, YopJ effectors activation likely occurs via interaction of the catalytic domain with the eukaryotic Inositol 6-phosphate (InsP6) co-factor, resulting in conformational changes that switch the effectors from inactive to active form.

Figure 14 .

 14 Figure 14. PopP2 acetylates WRKY domain-containing proteins (adapted fromLe Roux et al., 2015 and Guo et al., 2020). PopP2 targets WRKY domain-containing proteins, including various defensive WRKY TFs and the TIR-containing NLR RRS1-R. In susceptible plants, where RRS1-R is not present, PopP2 suppresses PTI by acetylating WRKY TFs, dislodging them from DNA, thereby disturbing their trans-regulating functions required for activation of basal immune responses. In resistant plants, where RRS1-R is present, PopP2 uses the same Lys-acetylation strategy to acetylate the WRKY domain of RRS1-R, which is in complex with RPS4. This dislodges the NLR complex from DNA and leads to its activation and the initiation of ETI. Phosphorylation of specific serine and threonine residues in RRS1-R is required for recognition of PopP2 by the NLR complex RRS1-R/RPS4.

Figure 15 .

 15 Figure15. The bromodomain, a domain that recognises acetyl-lysine residues (adapted fromFilippakopoulos and Knapp, 2012). Structure of the first BRD from human BRD2 binding to acetylated H4K12. Bromodomains allows the recognition of acetylated lysine residues via a central hydrophobic pocket and mediated by anchoring to a conserved asparagine (N) residue represented with a red star. They share a similar folding with a left-handed bundle of four α-helices (αZ, αA, αB, and αC), linked by loops (ZA and BC loops).

Figure 17 .

 17 Figure 17. Arabidopsis GTEs are involved in various biological processes.Members of the GTE family are involved in various biological processes such as ABA and sugar signalling pathways, control of leaf shape, cell cycle regulation or germination. The molecular functions of the different GTEs remain mostly unknown. However, GTE6 was shown to bind to the promoter of a target gene and promotes acetylation in that region, GTE10 interacts with TFs but can also recruit CRC subunits to methylated DNA regions. GTE6 and GTE10 regulate the transcription of various target genes likely indirectly. GTE9 and GTE11 are involved in the activation of a 35S enhancer-dependent gene. Regarding the GTE BRD, the BRDs of GTE3 and GTE10 have been shown to interact with histone H3. The GTE3 BRD binding to acetylated histones, and likely GTE5 BRD, might be regulated by sumoylation. CRC: chromatin-remodelling complex

Figure 18 .

 18 Figure 18. Hypothetical model: GTE9 and GTE11 participate in the regulation of multimerised 35S enhancers with BT2 (adapted from Irigoyen et al., 2021). BT2 interacts with GTE9/GTE11 and with CULLIN3 to form a ubiquitin ligase complex. With the other components of the complex, BT2 may interact with and mark transcriptional regulators (target) for destruction by polyubiquitination or activation by monoubiquitination, resulting in reduced methylation of multimerised 35S enhancers, increasing their transcriptional activity. Other components of the ubiquitin ligase complex include UBIQUITIN (U), a UBIQUITIN conjugating enzyme (E2), a RING-box protein (RBX) that interact with CULLIN3 and CALMODULIN (CAM) protein. 35S enhancers are shown by red arrowheads, DNA methylation marks with blue flags.

Figure 19 .

 19 Figure 19. How does PopP2 target the histone readers GTE9 and GTE11?Previous work carried by former PhD student Alice Delga allowed to know that PopP2 targets the BRD-containing proteins GTE9 and GTE11 by acetylation, that these GTEs interact with H4 and that their BRDs have preferential binding to tetra-acetylated H4, suggesting that they behave as histone readers. My PhD project continued to characterise the manipulation of GTE9 and GTE11 by PopP2 but also tried to study which chromatin sites are visited by PopP2 in plants that possess or not the immune receptor pair of proteins RPS4/RRS1-R.

  GTE9 and GTE11 are PopP2 virulence targets whose manipulation promote pathogen infection. Finally, we also showed that PopP2 can modify other members of the GTE family by acetylation(Comorge et al., in prep.). For greater clarity, I specify hereafter the parts of this article to which I contributed.My contribution to the results presented in the article ranges from the result showing the alteration of GTE9 binding to H4 by PopP2, to the targeting of different GTE members by PopP2. In details, I developed in collaboration with the TRI imaging platform on campus molecular tools aimed at monitoring in living plant cells how PopP2 could alter GTE9 binding to H4 via its acetyltransferase activity. I also generated all the data related to the involvement of GTE9 and GTE11 in the plant response to the RSSC, as virulence targets and likely not involved in the RPS4/RRS1-R-mediated immunity. Finally, I also participated in the fine analysis of the mass spectrometry-related data, which allowed the precise identification of the Lys residues in GTE9 and GTE11 that are acetylated by PopP2.ManuscriptComorge et al., in prep.

Figure 1 .

 1 Figure 1. PopP2 interacts with Arabidopsis GTE9 and GTE11 in yeast and in the nucleus of N. benthamiana cells. (A) PopP2 interacts with GTE9 and GTE11 in yeast. PopP2 fused to the Gal4 DNA-binding domain (BD) was used as bait (BD-PopP2). GTE9 and GTE11 fused to the Gal4 activating domain (AD) were used as prey proteins (AD-GTE9 and AD-GTE11). The murine p53 protein was used as negative control (BD-p53) for testing interactions with GTE9 and GTE11 and as a positive control for interaction with the SV40 large T-antigen (AD-T). Yeast transformants were grown on non-selective SD/-Trp/-Leu (SD-TL) medium or selective medium SD/-Trp/-Leu/-His/-Ade (SD-TLHA). Plates were photographed at 3 days after plating. This experiment was repeated three times with similar results. (B) Schematic representation of the GTE9 and GTE11 proteins. The bromodomain module (Brd) and the extra-terminal domain (ET) are boxed in orange and in grey, respectively. Truncated forms of GTE9 and GTE11 containing their bromodomain are designated by BRD9 and BRD11, respectively. The numbers indicated correspond to the position of the residues within the GTE9 and GTE11 proteins. (C) GTE9 and GTE11 colocalise with PopP2 in the plant nucleus. PopP2-CFP was transiently expressed either alone or with GTE9-YFP or GTE11-YFP in N. benthamiana cells. Plant cells were photographed at 72hpi. Scale bars, 10 µm. (D) PopP2 physically interacts in planta with GTE9 and GTE11. CFP lifetime distribution of PopP2 fused with CFP (PopP2-CFP) and expressed with GTE11-YFP (bottom), GTE9-YFP (middle) and YFP (top). Histograms shows the distribution of nuclei (number) according to PopP2-CFP lifetime classes in the absence (green bars) or presence (orange and red bars) of the co-expressed YFP fusion proteins. The scanned nuclei correspond to those used for FRET-FLIM measurements presented in Supplementary Table1.

Figure 2 .

 2 Figure 2. PopP2 acetylates GTE9 and GTE11. (A) GTE11-3Flag and GTE9-3Flag were transiently expressed with 3HAtagged active PopP2 or C321A catalytic mutant in N. benthamiana leaves. Samples were harvested at 48hpi. Protein extracts were immunoblotted with anti-Flag (-Flag) and anti-HA antibodies (-HA) (Input). Immunoprecipitation of Lys-acetylated proteins was done in presence of an anti-AcK antibody on protein A-agarose beads (IP -Ac-K). The immuno-precipitated GTE9 and GTE11 proteins were detected after immunoblotting with -Flag antibodies (Ac-GTE9 and Ac-GTE11, respectively). Ponceau S staining of total proteins indicates equal loading of the samples in the input. This experiment was repeated three times with similar results. (B) Mapping of the PopP2-acetylated lysine residues in GTE9 and GTE11 protein sequences. Conserved residues between GTE9 and GTE11 are shaded in grey. The bromodomain module of GTE9 and GTE11 is boxed in orange (BD GTE9 : position 130-240; BD GTE11 : position 122-232). The truncated forms of GTE9 and GTE11 containing the bromodomain are surrounded by a dotted line (BRD9: position 112-246 in GTE9; BRD11: position 105-238 in GTE11). The conserved Asp residue lying in the acetyl-lysine binding pocket of GTE9 and GTE11 bromodomain in boxed in green. The acetylated lysine residues reproducibly identified by MS-based proteomics in GTE9 and GTE11 in presence of active PopP2 are in red and boxed in yellow. The MS-based proteomic data used to design panel B are presented in Supplementary Tables2 and 3.

Figure 3 .

 3 Figure3. The bromodomain of GTE9 confers preferential binding to acetylated histone H4. (A) BRD9 binds preferentially to tetra-acetylated histone peptides from H4. A recombinant His6-tagged protein corresponding to a truncated form of GTE9 containing its bromodomain (BRD9) was used for screening against an array of single/multiple acetylated peptides that cover all possible acetylation sites in H2A, H2B, H3 and H4. Non-acetylated peptides are indicated in grey. Values are representative of two independent experiments. The BRD9-N214A mutant (in which the conserved Asparagine residue of the acetyl-lysine binding pocket was substituted with an Alanine) was used as a negative control (6His-BRD9-N214A). See also Supplementary Table4. (B) GTE9 binds to histone H4 in vivo, but not to histone H3. On the left, CFP lifetime distribution of histone H4 fused with CFP (CFP-H4) and transiently expressed either alone or with GTE9-YFP or GTE9-N214A-YFP. Histograms shows the distribution of nuclei (number) according to CFP-H4 lifetime classes in the absence (green bars) or presence of co-expressed GTE9-YFP (orange bars) or GTE9-N214A-YFP (light green bars). On the right, CFP lifetime distribution of histone H3 fused with CFP (CFP-H3) and transiently expressed either alone or with GTE9-YFP. Histograms shows the distribution of nuclei (number) according to CFP-H3 lifetime classes in the absence (light blue bars) or presence of co-expressed GTE9-YFP (dark blue bars).The scanned nuclei in (B) correspond to those used for FRET-FLIM measurements presented in Supplementary Table1.

Figure 4 .

 4 Figure 4. PopP2 targets the bromodomain of GTE9 and interferes with its ability to bind histone H4. (A) GTE9-3HA and GTE9-N214A-3HA were transiently expressed with 3HA-tagged active PopP2 or C321A catalytic mutant in N. benthamiana leaves. Samples were harvested at 48hpi. Protein extracts were immunoblotted with anti-HA (-HA) (Input).Immunoprecipitation of Lys-acetylated proteins was done in presence of an anti-AcK antibody on protein A-agarose beads (IP -Ac-K). The immunoprecipitated GTE9 and GTE9-N214A proteins were detected after immunoblotting with an -HA antibody. Ponceau S staining of total proteins indicates equal loading of the samples in the input. This experiment was repeated three times with similar results. (B) (A) PopP2 interacts with the bromodomain of GTE9 (BRD9). PopP2 fused to the Gal4 DNA-binding domain (BD) was used as bait (BD-PopP2). BRD9, BRD9-N214A, BRD11 and BRD11-N206A fused to the Gal4 activating domain (AD) were used as prey proteins. The murine p53 protein was used as negative control (BD-p53) for testing interactions with the different prey proteins and as a positive control for interaction with the SV40 large T-antigen (AD-T). Yeast transformants were grown on non-selective SD/-Trp/-Leu (SD-TL) medium or selective medium SD/-Trp/-Leu/-His/-Ade (SD-TLHA). Plates were photographed at 4 days after plating. This experiment was performed three times with similar results. (C) PopP2 alters the physical interaction between GTE9 and Histone 4. On the left, GFP lifetime distribution of GTE9 fused with eGFP (GTE9-eGFP) and transiently co-expressed with mCherry-Histone4 in absence or in presence of PopP2-3HA or C321A-3HA. Histograms shows the frequency of nuclei according to GTE9-eGFP lifetime classes with Tau1 corresponding to the lifetime of the free donor and Tau2 corresponding to the lifetime of the donor bound to the acceptor. The scanned nuclei in (C) correspond to those used for FRET-FLIM measurements presented in Supplementary Table5.

Figure 5 .

 5 Figure 5. Targeting of GTE9 and GTE11 by PopP2 promotes wilt disease development in Arabidopsis. (A) gte9-1 and gte11-1 null mutant are not affected in their phenotypical response the GMI1000 strain of R. solanacearum. The graph shows least-square means +/-Standard Error of the LS means from 4 independent inoculations of ~24 plants (n total = 93 plants for Col-0 and 96 plants for gte9-1 and gte11-1). (B) Transgenic overexpression of GTE11-3Flag or GTE9-3Flag in Col-0 plants enhances the development of bacterial wilt disease symptoms. The graph shows leastsquare means +/-Standard Error of the LS means from 2 and 3 independent inoculations of ~24-32 plants for GTE11-3F #265 and GTE9-3F #283, respectively (n total = 88 plants for Col-0, 64 plants for GTE11-3F #265 and 82 plants for GTE9-3F #283). (C) The enhanced susceptible response of GTE11-3Flag and GTE9-3Flag overexpressing lines to GMI1000 strain is dependent on catalytically active PopP2. The graph shows least-square means +/-Standard Error of the LS means from 3 independent inoculations of ~24 plants respectively (inoculation with ΔpopP2+PopP2: n total = 69 plants for Col-0 and 71 plants for GTE11-3F #265 and GTE9-3F #283; inoculation with ΔpopP2+PopP2-C321A: n total = 66 plants for Col-0 and 72 plants for GTE11-3F #265 and GTE9-3F #283. In A, B, and C, a, b and c denote significant difference between two lines at a certain DAI (one-way ANOVA performed on the mixed model described in the methods, p-value < 0.05).

Figure 6 .

 6 Figure 6. GTE9 and GTE11 are not required for RPS4/RRS1-R-mediated immunity. (A) Pseudomonas fluorescens (Pf0-1)-delivered wild-type PopP2 triggers a cell death response in Col-0, gte9-1 and gte11-1 plants transgenically expressing the RPS4 and RRS1-R NLR genes from the Ws-2 ecotype. Leaves of four week-old Arabidopsis plants were infiltrated with Pf0-1 strain delivering either PopP2 or PopP2-C321A or no effector. The photographs were taken at 48 hours post infection (hpi). The red * shows leaves with cell death response triggered by PopP2 recognition. This experiment was conducted three times with similar results. (B)The RPS4/RRS1-R-dependent immunity in response to the GMI1000 strain of R. solanacearum is not affected by gte9-1 or gte11-1 null mutations. Four week-old Col-0, gte9-1 and gte11-1 plants complemented with the RPS4/RRS1-R genomic clone from Ws-2 display a resistant phenotype upon root-inoculation with the GMI1000 strain of R. solanacearum, compared to that of wild-type Col-0, and untransformed gte9-1 and gte11-1 plants showing pronounced wilting disease symptoms. A disease score was attributed to every plant for 10 days after inoculation (DAI). 0 = no symptoms; 1 = 25% wilted leaves; 2 = 50% wilted leaves, 3 = 75% wilted leaves and 4 = 100% wilted leaves. The graph shows least-square means +/-Standard Error of the LS means from 3 independent inoculation assays. a and b denote significant difference between two lines at a certain DAI (one-way ANOVA performed on the mixed model described in the methods, p-value < 0.05). Left panel, gte9-1 and gte11-1 mutant lines in susceptible Col-0 background with ~24 inoculated plants in each independent experiment (n = 64 plants for Col-0, 69 plants for gte9-1 and gte11-1). Right panel, gte9-1 and gte11-1 mutant lines complemented with the genomic clone RPS4/RRS1-R from the Ws-2 resistant accession, with ~24 inoculated plants in each independent experiment (n = 68 plants for Col-0, gte9-1 and gte11-1).

  PopP2-C321A catalytic mutant (popP2+PopP2 and popP2+PopP2-C321A, respectively) (Fig5C). The GTE9-3Flag and GTE11-3Flag overexpressing lines remained more susceptible to popP2+PopP2 but responded to popP2+PopP2-C321A similarly to wild-type Col-0 plants. This result demonstrates that the enhanced susceptibility of GTE9 and GTE11 overexpressing lines relies on PopP2 acetyltransferase activity, and is therefore biologically relevant to the virulence function of PopP2. Overall, these results indicate that GTE9 and GTE11 are virulence targets of PopP2 and whose manipulation promotes wilt disease development in Arabidopsis.

Figure 7 .

 7 Figure 7. PopP2 targets several members of the GTE family. (A) Yeast two-hybrid assay to test the interaction between PopP2 and the different GTE proteins from Arabidopsis thaliana. Yeast cells transformed with different bait (BD-fusion)and prey (AD-fusion) plasmid combinations were spread on non-selective (-TL) or selective media (-HTLA). Growth on SD-HTLA medium indicate an interaction between the tested proteins. BD-p53 (bait) and AD-T (prey) were used as a positive control. This experiment was conducted at least two times with similar results. (B) In addition to GTE9 and GTE11, PopP2 acetylates GTE3, GTE5, and GTE8 in planta. All GTE protein members (GTE1 to GTE12) tagged with a 3HA epitope tag were transiently expressed with 3Flag-tagged active PopP2 or C321A catalytic mutant in N. benthamiana leaves. Samples were harvested at 48hpi. Protein extracts were immunoblotted with anti-HA (-HA) and anti-Flag (-Flag) antibodies (Input). Ponceau S staining of total proteins indicates equal loading of the samples in the input. Lysacetylated proteins were immunoprecipitated with an anti-Ac-K antibody (IP -Ac-K). Immunoblot analysis of immunoprecipitated proteins with an anti-HA antibody reveals the presence of Lys-acetylated GTE3, GTE5, GTE8, GTE9 and GTE11 in presence of active PopP2 but not C321A. Immunoblots corresponding to GTE1/GTE2/GTE3/GTE4, GTE5/GTE6/GTE7GTE8, and GTE9/GTE10/GTE11/GTE12 represent, respectively, three independent experiments carried out under identical conditions. This experiment was conducted three times with similar results.

Figure 20 .

 20 Figure 20. Intramolecular interactions between BRD and acetylated lysines regulating the binding of BRD-containing proteins to acetylated histones.The binding of BRD-containing proteins to acetylated histones can be regulated by acetylation of lysine residues flanking their BRD, causing intramolecular interactions and altering their interaction with acetylated histones. (A) In yeast, the SWI/SNF chromatin remodelling complex association to acetylated histones can be regulated by acetylation. The Snf2 subunit of this complex can be acetylated by the Gcn5 HAT on lysine residues flanking its BRD likely facilitating intramolecular interactions, inhibiting its ability to interact with H4(Kim et al., 2010). (B) We propose a similar mechanism used by PopP2 to alter the association GTE9-H4. Indeed, PopP2 acetylates lysines residues flanking GTE9 BRD which might cause intramolecular interactions and the alteration of its interaction with H4.

  PopP2 virulence targets since their transgenic overexpression in Col-0 plants enhances wilting disease development in response to R. solanacearum infection(Fig 5B and Supp Fig 5E). In Arabidopsis, transgenic overexpression of GTE6 increases the acetylation states of histones H3

  PAGE). Transferred proteins were visualised by Ponceau S red staining. Membranes were blocked in a 2% milk TBS-T (Tris Buffer Saline-Tween 20; 50 mM Tris-HCl pH7.5, 150 mM NaCl, and 0.2% Tween-20) solution before incubation. The following primary antibodies were used in this study: anti-Acetylated Lysine (Ac-K-103, Cell Signaling Technology; dilution 1:2000), anti-HA-HRP (3F10; Roche; dilution 1:5000), anti-GFP (mouse monoclonal; Roche; dilution 1:3000), anti-mCherry (mouse monoclonal; Sigma; dilution 1:2000), anti-Flag-HRP (Sigma; dilution 1:5000). The appropriate horseradish peroxidase-conjugated secondary antibody was applied to the membranes: goat anti-mouse IgG-HRP (Bio-Rad; dilution 1:10000)

  linearisation with NdeI and XhoI restriction enzymes. The pDEST pGBG-GWY vector was generated by ligating a Gateway cassette (Frame C) in the pGBKT7 vector blunt-ended (klenow fill-in) after linearisation with NdeI and SalI restriction enzymes. The cDNA corresponding to GTE1 to GTE12 cloned into the relevant pENTR plasmids were recombined in the pGAD-GWY vector. The coding sequence of PopP2 in pENTR-PopP2 was recombined in pGBG-GWY (bait) vector by LR reaction (Gateway, Invitrogen). All the pDEST plasmids were introduced in E. coli cells (DH5α, Stratagene) by electroporation (1mm tank, 1.8 kV, 25 µF and 200 Ohms). Transformed cells were spread on solid LB medium with appropriate antibiotic selection (carbenicillin 50 µg/mL for pGAD vectors, kanamycin 50 µg/mL for pGBG vectors).

  data were obtained by leaf observation of N. benthamiana or A. thaliana in which transient expression was carried out (leaf samples were harvested at 48 to 72 hpi). Data were analysed with the TAU_POGRAPHY-FLIM software v. 3.1 allowing the acquisition of the fluorescence lifetime of the donor fluorophore, the fluorescence intensity over the time and the proportion of nuclei in relation with the fluorescence lifetime. The measured fluorescence decay is either fit with a mono-exponential fit function for cells expressing donor alone or cells that have a negligible FRET level (Equation

  the time origine and B a constant that takes in account the background level of photons. τ D is the lifetime of the donor alone and τ DA the lifetime of the donor in presence of the acceptor. α D and α DA represent the contribution of each exponential in the fluorescence decay. It is then possible to calculate the mean lifetime ( m ), the apparent FRET efficiency (EF) and the fraction of donor bound to the acceptor (β): m =   i  2 /   i  i ; 𝐸F = 1 -(𝑟𝐷𝐴/𝑟𝐷) ; 𝛽 = 𝛼𝐷𝐴 / 𝛼𝐷𝐴+𝛼𝐷Immunoprecipitation of GTE proteins acetylated on lysine residuesFor immunoprecipitation of lysine-acetylated 3HA-tagged GTE proteins in denaturing conditions, plant protein samples were obtained from N. benthamiana leaves (4 discs of 8 mm diameter harvested 48 hours post-infiltration) transiently expressing the relevant constructs.Samples were grinded and homogenised in 300 µL of denaturing Buffer (50 mM Tris-HCl pH7.5, 10 mM sodium butyrate, 5 mM EDTA, 2 mM DTT, 1x Protease Inhibitor Cocktail, 1% SDS) and denatured 3 min at 95°C. The extract was centrifuged at 13000 rpm for 2 min at room temperature. 25µL of the supernatant (crude extract) were denatured in 25 µL of Laemmli buffer (4X). The remaining supernatant was mixed with 10 mL of immunoprecipitation buffer (IP1 buffer: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 10 mM sodium butyrate, 5 mM EDTA, 2 mM DTT, 1% Triton). This mix was incubated for 1 hour at 4°C under stirring with 1 µL of anti-acetylated lysine antibody (Cell Signalling). Then, 25 µL of agarose protein A agarose were added to the mix and incubated for 2 hours at 4°C under stirring. Agarose beads were washed three times with 800 µL of IP1 buffer and subsequently denatured for 3 min at 95°C in 40 µL of Laemmli buffer (2X) before analysing the immunoprecipitated proteins by immunoblotting (SDS-PAGE).For immunoprecipitation of lysine-acetylated GTE9/GTE11 in non-denaturing conditions, plant protein samples were obtained from N. benthamiana leaves (4 discs of 8 mm diameter harvested 48 hours post-infiltration) transiently expressing the relevant constructs. Samples were grinded and homogenised in IP2 buffer (50 mM Tris-HCl pH7.5; 150 mM NaCl; 10 mM EDTA; 0.2% Triton; Protease Inhibitor Cocktail 1X; 10 mM sodium butyrate; 2 mM DTT).Tubes were centrifugated 1 min at 14000 rpm at 4°C and 50 µL of the supernatant (crude extract) was denatured in 50 µL of Laemmli buffer (2X) at 95°C for 3 min. A pre-clearing was done by adding 25 µL of Agarose protein A to the remaining supernatant for 10 minutes at 4°C under stirring. After centrifugation (13200 rpm, 1 min), the supernatant was transferred in a new tube and incubated with 1 µL of anti-acetylated lysine antibody (Cell Signaling) for 1 hour at 4°C under stirring. Then, 25 µL of agarose protein A were added to the mix and incubated for 2 hours at 4°C under stirring. Beads were washed twice with 800 µL of IP2 buffer and subsequently denatured for 3 min at 95°C in 40 µL of Laemmli buffer (2X) before analysing the IPs by immunoblotting (SDS-PAGE).

  a precolumn (300 μm x 5 mm PepMap C18, ThermoFisher Scientific) and separated by a 25min gradient in a 75 μm x 150 mm column (PepMap C18, 3µm, Thermo Scientific) for the 3FLAG constructs, or a 60-min gradient in a 75 μm x 250 mm column (Reprosil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch), for the YFP constructs. The MS and MS/MS data were acquired by Xcalibur (Thermo Fisher Scientific). Peptides and proteins were identified by Mascot (version 2.7.0.1, Matrix Science) through concomitant searches against the Uniprot database (Nicotiana benthamiana taxonomy, June 2021 download), specific sequences of tagged proteins (homemade), and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin, etc.). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set at respectively at 10 ppm and 0.6 Da for LTQ-Orbitrap Velos Pro data, and 10 and 20 ppm for Q-Exactive Plus data. Peptide modifications allowed during the search were:

Figure 21 .

 21 Figure 21. gte9-1 and gte11-1 mutants show similar disease development as Col-0 in presence or absence of PopP2. The graphs show least-square means +/-Standard Error of the LS means from 3 independent inoculations of ~24 plants respectively (inoculation with ΔPopP2: n total = 72 plants for Col-0 and gte11-1 and 70 for gte9-1; with ΔPopP2+PopP2: n total = 72 plants for Col-0 and gte11-1 and 70 for gte9-1; with ΔPopP2+PopP2-C321A: n total = 72 plants for Col-0, 71 for gte9-1 and 70 for gte11-1). In A, B and C, a denotes no significant difference between two lines at a certain DAI (one-way ANOVA performed on the mixed model described in the methods, p-value < 0.05).

Figure 23 .

 23 Figure 23. Expression level of GTE genes family is not affected by the presence of active PopP2 or PopP2-C321A catalytic mutant. Expression level of GTE genes in Col-0 leaves infiltrated with Pf0-1 (pfo), Pf0-1 PopP2-3HA (pfo P2) or Pf0-1 PopP2-C321A-3HA (pfo CA). The relative gene expression levels were measured by RT-qPCR on leaf discs from 3-week-old plants at 0, 3 or 6 hours post-infiltration and normalised to the expression level of AtEF1-α. Mean values +/-SD from 2 independent experiments of 3 biological replicates (n=6; p-value=0.05, a, b, and c show significative difference in mean ΔCt for a given gene at each combination of T and strain, based on two-way ANOVA or kruskal-test).Primers used for RT-qPCR analyses are listed in Table3.

  only at a post-translational level protein level since PopP2 does not seem to modulate the expression level of the GTEs upon Pf0-1-mediated delivery of PopP2 in Arabidopsis(Figures 23 and 24). However, such a conclusion should be taken with caution. Indeed, to assess whether PopP2 modulates the transcription of the different GTE genes, we used an engineered pathogen effectors delivery assay (Pf0-1-mediated delivery) allowing the delivery of PopP2 or its variants in plant cells through a T3SS and we measured the expression of the different GTE genes at 3 hpi and 6 hpi. During the natural infection process used by the RSSC, PopP2 is likely secreted and injected in root and leaf tissues which are colonised by this bacterial pathogen. In other words, the spatio-temporal delivery of PopP2 by the RSSC in root-inoculated Arabidopsis plants might be quite different from that obtained with Pf0-1 cells infiltrated in leaf cells. So, to determine if PopP2 does affect or not the transcription of GTE members, we should study the expression of the different GTE genes in A. thaliana Col-0 plants over the kinetic of infection with RSSC strains producing or not PopP2. Transcriptomic analyses of A. thaliana roots

Figure 25 .

 25 Figure 25. Disease wilting symptom developement is accelarated in GTE9 and GTE11 overexpressing lines in response to a RSSC strain lacking the popP2 gene. GTE9-3Flag and GTE11-3Flag over-expressing lines are more susceptible to a RSSC strain lacking popP2 in late stages of disease. The graphs show least-square means +/-Standard Error of the LS means from 3 independent inoculations of ~24 plants respectively (n total = 70 plants for Col-0, GTE9-3Flag and GTE11-3Flag). a, b and c denote significant difference between two lines at a certain DAI (one-way ANOVA performed on the mixed model described in the methods, p-value < 0.05).

Figure 26 .

 26 Figure 26. PopP1 interacts with various GTE members in yeast cells. (A).Yeast two-hybrid assay to test the interaction between PopP1 and the different GTE proteins from A. thaliana. Yeast cells transformed with different bait (BD-fusion) and prey (AD-fusion) plasmid combinations were spread on non-selective (-TL) and selective media (-HTLA). Growth on -HTLA selective medium indicates an interaction between the tested proteins. Photographs were taken at 5 days post-dilution. PopP2 (left), p53 (middle) and PopP1 (right) fused with the BD domain were used as bait proteins, respectively. White stars indicate interaction between the bait and prey proteins tested in yeast cells. This experiment has been carried out twice. (B) Protein alignement between PopP2 (488 residues) and PopP1 (388 residues). Identical residues are boxed in black. The three residues of PopP2 and PopP1 catalytic triad are boxed in red. The PopP2 Lys residue targeted by PopP2 autoacetylation activity (K383) is boxed in green (corresponding residue in PopP1 is at position 299)

Figure 30 .

 30 Figure30. Monitoring of the spatio-temporal delivery of PopP2 in Arabidopsis cells by using the GFP strand system (principle). The GFP strand system can be used to monitor effector delivery in plant cells or focus on cells that contain the effector when associated with Fluorescence-Associated Cell Sorting. (A) The GFP strand system consists in reconstituting fluorescence in cells from transgenic plants expressing GFP1-10 in which was delivered an effector fused to GFP11 (Adapted fromHenry et al., 2017). (B) We intended on using the GFP strand system for delivering PopP2-3HA-GFP11 in A. thaliana leaves via the T3SS of Pf0-1, followed by Fluorescence-Associated Cell Sorting to find the chromatin sites visited by PopP2 with ChIP-seq analyses performed on nuclei hosting PopP2.

  on the host transcriptome. It could allow us to better understand how PopP2 could influence transcriptome reprogramming through the targeting of specific chromatin regions, likely by manipulating the activity of Transcription Factors (ie defensive WRKY TFs) or epigenetic readers (ie GTE proteins).

  al., sterile water, and the extraction solutions were then plated on King B + 50 µg/mL Rifampicin agar plates. Colonies were counted 24h after incubation at 28°C. The whole experiment has been repeated twice. Transient expression in N. benthamiana For transient expression in N. benthamiana leaf epidermal cells, A. tumefaciens strains grown in liquid YEB medium containing appropriate antibiotics. Cells were harvested by centrifugation and resuspended in infiltration medium (10 mM MES pH5.6, 10 mM MgCl 2 , 150 μM acetosyringone) at OD 600 =0.25. For co-expression, each bacterial suspension carrying individual constructs was adjusted to OD 600 = 0.25 in the final mix for infiltration. After incubation at room temperature for 1 h, bacteria were infiltrated into the leaves of 4-week-old N. benthamiana plants using a needleless syringe. Plants were incubated for 36-48h in growth chambers under controlled conditions.

  • B.1. Presentation of the model .....................................................................................

• B.2. PAMP-Triggered Immunity (PTI)............................................................................. B.2.1. PAMPs are conserved microbial motifs................................................................... B.2.2. PRRs are extracellular receptors that recognise PAMPs ......................................... B.2.3. PRR/PAMP examples ...............................................................................................

Table 1 .

 1 Epigenetic factors are involved in plant defence against pathogens.

Table 2 .

 2 Pathogens can manipulate the host epigenome to favour infection.

B.5.2. Effectors can manipulate various cellular processes

	In this part, I will present some examples of effectors and how they can manipulate
	host cellular processes. This section intends on describing the virulence activities
	of diverse pathogen effectors but is by no means complete.
	B.5.2.a. Effectors can dampen PTI responses by targeting PRRs
	Some effectors can directly target PTI components to suppress basal immune
	responses. The P. syringae effector HopB1 is a threonine protease that interacts in
	Arabidopis with FLS2 and cleaves the RLK BAK1 when phoshorylated. The
	proposed model describes that upon perception of P. syringae, flg22 triggers the
	recruitment of BAK1 to FLS2, which is in complex with HopB1, and then, after BAK1
	phosphorylation, HopB1 cleaves phosphorylated-BAK1 leading to the alteration of
	early PTI responses like ROS production (Li et al., 2016).
	Another effector, HopAB2 (formerly AvrPtoB), from the same bacterium also
	targets PTI components in Arabidopsis and contributes to pathogen virulence
	(Göhre et al., 2008). HopAB2 is an E3 ubiquitin ligase that can ubiquitinate and
	catalyse the degradation of different PTI components like the RLKs FLS2 and
	CERK1 and this participates in promoting bacterial growth (Gimenez-Ibanez et al.,
	2009; Göhre et al., 2008).
	B.5.2.b. Effectors can interfere with MAPK signalling cascades
	Interference with MAPK cascades is an effective strategy as they constitute a node
	transducing signal perception into intracellular responses.

  ).

	Another example is the effector PITG20303 from the oomycete Phytophthora
	infestans. Du and colleagues demonstrated that PITG20303 dampens PTI
	responses and promotes pathogen colonisation by stabilising StMKK1, a potato
	MAPK kinase kinase acting as a negative regulator of PTI responses (Du et al.,
	2021).
	Instead of stabilising a negative regulator of plant immunity, the P. syringae
	HopZ1a acetyltransferase targets a MAPK kinase kinase that acts as a positive
	regulator to suppress plant immunity in Arabidopsis. HopZ1a interacts with MKK7,
	a positive regulator of PTI responses, acetylates it and this induces the suppression
	of all MKK7-dependent signalling, suppressing local and systemic plant immunity
	(Rufián et al., 2021).
	B.5.2.c. Effectors manipulate hormonal pathways
	Since hormone signalling pathways are important for the regulation of plant
	immune responses, it makes sense that some effectors target these pathways.
	One way to manipulate hormone pathways is to play on antagonistic pathways. In
	Arabidopsis, the effector HopX1 from P. syringae pv. tabaci 11528 is a cysteine
	protease that targets JAZ transcriptional repressors. HopX1 associates with and
	degrades several JAZ proteins. Degradation of JAZ proteins derepresses JA
	signalling pathways which antagonises the SA signalling pathway, a hormonal
	pathway detrimental to P. syringae, promoting susceptibility (Gimenez-Ibanez et
	al., 2014).
	Another way to manipulate hormone signalling pathways is to disrupt the turnover
	of key actors. The Xanthomonas euvesicatoria XopJ effector is an acetyltransferase
	with a protease activity. XopJ was shown to degrade a subunit of the pepper
	(Capsicum annuum) proteasome, Regulatory particule triple-A APTase subunit 6
	(RPT6)

B.5.3. Effectors target in their own way different pathways in a redundant manner

	To conclude, we have seen that plant pathogens have evolved different virulence
	strategies by using various effectors to subvert host processes related to defence,
	metabolism, and transcription. They can act by inducing modifications on host
	proteins like phosphorylation, uridylation, acetylation, or ubiquitination. These
	modifications can inactivate or degrade targeted proteins favouring pathogen
	infection. They can also target higher and larger levels of host protein regulation
	such as protein folding, protein turnover, master regulators of plant responses or
	even hormone biosynthesis. All these ways of manipulating plant processes are
	aimed at redirecting plant responses to promote pathogen infection.
	Interestingly, it is frequently observed that several effectors from the same
	pathogen target similar pathways. From the examples I have described, we can see
	that P. syringae uses (i) HopAB2 and HopB1 to target PTI components, (ii) HopAI1
	and HopZ1a to interfere with MAPK cascades and (iii) HopZ1a and AvrBsT to
	disturb the plant cytoskeleton. Diversifying effectors while staying redundant in the
	targeted pathway reflects how pathogens can prevent effector recognition while
	targeting key pathways to promote infection. We also note that a single effector
	can target several pathways, as HopZ1a which targets the cytoskeleton, PTI
	components and MAPK cascades. Finally, we can also notice hubs of action
	between different pathogens. For instance, AvrAC from Xcc and HopAR1 from P.
	syringae both target the kinase RIPK. Several pathogens can also specifically target
	particular pathways, which are essential for plant defence and/or for pathogen
	thriving in plants.

Table 1 . Epigenetic factors are involved in plant defence against pathogens.

 1

	There is increasing evidence in the literature that epigenetic regulators play a positive or negative
	role in plant defence against bacterial, fungal and oomycete pathogens. These include maintenance
	of DNA methylation, DNA demethylases, histone methyltransferases, histone demethylases,
	histone acetyltransferases, histone deacetylases, chromatin remodelling complexes and non-
	coding RNAs. lncRNA: long non-coding RNA; miRNA: microRNA.

Name Related epigenetic mechanism Host species Pathogen Mutant phenotype Positive or negative regulator of plant defences? Function in plant defence Reference

	DNA methylation maintenance DNA demethylases (ROS1, DML1, DML3) SDG8 IBM1 JMJ704 ELP2 HDA19 HDA9 CHR5 SWP73A TAR-191, TAR197, TAR-212 and TAR-224 ELENA1 miR472 mechanisms, such as DNA methylation, histone post-translational modifications, DNA methylation Arabidopsis thaliana Pseudomonas syringae pv. tomato (Pst) DC3000 More resistant Negative Regulate PR1 expression Dowen et al., 2012 DNA demethylation A. thaliana Fusarium oxysporum More susceptible Positive Regulate stress-responsive genes Le et al., 2014 Histone methylation A. thaliana Botrytis cinerea and Alternaria brassicicola More susceptible Positive H3K36me3 activation of JA/ET-related genes Berr et al., P. syringae More susceptible Positive H3K36me3 activation of SA-related genes Zhang et al., 2020 Maintain the NLR LAZ5 in active state through H3K36me3 Palma et al., 2010 Histone demethylation A. thaliana P. syringae More susceptible Positive Activation of PR1, PR2 and FRK1 defence genes by controlling H3K9me2 and H3K4me3 levels Chan and Zimmerli 2019 Histone demethylation Oryza sativa Xanthomonas oryzae pv. oryzae More susceptible Positive Repression of negative regulators of rice immunity like NRR, OsWRKY62 and Os-11N3 Hou et al., Histone acetylation A. thaliana B. cinerea and A. brassicicola More susceptible Positive Control levels of H3K9/14ac at defence-related genes like NPR1, PR2, EDS1 and PAD4 Wang et al., 2015 Histone deacetylation A. thaliana A. brassicicola More susceptible Positive Positive control of JA/ET-related plant defence Zhou et al., 2005 P. syringae More resistant Negative Negative control of SA-related plant defence by favouring repressive chromatin at loci like PR1 and PR2 Choi et al., 2012 Histone deacetylation A. thaliana P. syringae More resistant Negative Negative regulation of SNC1 expression upon infection Yang et al., 2020 Chromatin remodelling A. thaliana Avirulent Pst DC3000 strains and P. syringae pv. maculicola More susceptible Positive Zou et al., Chromatin remodelling A. thaliana avirulent Pst DC3000 strains More resistant Negative Suppresses directly or indirectly the transcription of several NLRs like RPS2, RRS1, RPS4 and ZAR1 Huang et al., 2021 lncRNA A. thaliana F. oxysporum More susceptible Positive Zhu et al., lncRNA A. thaliana Pst DC3000 More susceptible Positive Positive regulation of PR1 expression Seo et al., miRNA A. thaliana Pst DC3000 More resistant Negative Negatively regulates both PTI and ETI chromatin remodelling complexes and non-coding RNAs. All these epigenetic marks and associated epigenetic factors design the fate of chromatin structure and help regulate gene transcription in many stages of the plant life cycle, including growth, development and response to biotic and abiotic stresses. In Table 1 are listed some epigenetic factors with a role in plant-pathogen interactions. They will be detailed in the section below. Boccara et al., Changes in chromatin structure are regulated by a variety of epigenetic B. Epigenetic mechanisms regulate plant defence against pathogens

• B

.1. DNA methylation mainly represses plant defence B.1.1. What is DNA methylation?

Table 2 . Pathogens can manipulate the host epigenome to favour infection.

 2 Several animal pathogens are known to manipulate the host epigenome to their benefit, mainly bacteria while there are fewer examples in plant parasites, mainly from viruses, oomycetes and nematodes. Parasites and pathogens can use effectors or other proteins to manipulate epigenetic mechanisms by modifying histones or DNA, interfering with histone writers, erasers or readers, and chromatin-remodelling complexes.

	Name	Host type	Pathogen	Epigenetic targeted mechanism	Targeting mechanism	Reference
					Blocks MAPK signalling cascade, preventing
	OspF	Animal	Shigella flexneri	Epigenetic readers	phosphorylation of the epigenetic reader HP1γ leading to the alteration of the transcription of pro-	Harouz et al., 2014
					inflammatory genes
	OspF	Animal	Shigella flexneri	Histone phosphorylation	Blocks MAPK signalling cascade, preventing H3 phosphorylation at promoters of chemokines and cytokines, altering their transcription	Arbibe et al., 2007
	NUE	Animal	Chlamydia trachomatis	Histone methylation	Methylates histones H2B, H3 and H4 in vitro	Pennini et al., 2010
	BaSET	Animal	Bacillus anthracis	Histone methylation	Methylates histone H1 in infected cells	Mujtaba et al., 2014
	RomA	Animal	Legionella pneumophilia	Histone methylation	Methylates H3K14 in mammalian cells leading to a genome-wide decrease in acetylation levels notably at immune-related genes	Rolando et al., 2013
	LpqH	Animal	Mycobacterium tuberculosis	Chromatin remodelling	Prevents the recruitment of a SWI/SNF remodelling complex component at the promoter of an immune-related gene	Pennini et al., 2006
	NleC	Animal	Escherichia coli	Histone acetylation	Likely degrades the HAT p300 resulting in the alteration of Interleukin-8 secretion	Shames et al., 2011
	Rv2966c Animal	Mycobacterium tuberculosis	DNA methylation	Methylates DNA on cytosines outside CpG contexts	Sharma et al., 2015
	Rv3423 Animal	Mycobacterium tuberculosis	Histone acetylation	Acetylates H3K9 and K3K14 in vitro	Jose et al., 2016
					Inhibit the histone methyltransferase SUVH4/KYP
	TrAP	Plant	Geminiviruses Histone methylation	activity leading to a decrease in H3K9 methylation levels in the host and viral genome	Castillo-Gonzalez et al., 2015
					methylation
	PsAvh23	Plant	Phytophthora sojae	Histone acetylation

. Some phytopathogen effectors interfere with histone methylation

  While some animal pathogens have evolved effectors that can directly modify histone methylation status through their methyltransferase activity, there is no evidence yet that effectors from plant pathogens act in the same way. However,

C.2.1

this does not mean that plant pathogens cannot disturb histone methylation in plants. They can target regulators of histone methylation, such as histone methyltransferases.

TrAP proteins, encoded by the geminiviruses Tomato Golden Virus and Cabbage Leaf Curl Virus, inhibit the activity of the Arabidopsis histone methyltransferase SUVH4/KYP

(Castillo-González et al., 2015)

. By luciferase complementation imaging, co-immunoprecipitation and acceptor bleaching FRET, the authors demonstrated that TrAP proteins interact with this histone methyltransferase that catalyses the dimethylation of H3K9, among other histone methyltransferases. Furthermore, SUVH4/KYP H3K9 methyltransferase activity was inhibited by TrAP proteins in vitro. Overexpression of TrAP led to a decrease of H3K9me2 marks at specific loci in Arabidopsis. The authors also demonstrated that SUVH4/KYP is involved in viral genome methylation. Considering that host-mediated methylation of viral chromatin can limit viral replication, altering SUVH4/KYP activity by TrAP proteins could represent a virulence strategy to prevent inhibition of viral replication by SUVH4/KYP-mediated methylation (Castillo-González et al., 2015; Pumplin and Voinnet, 2013).

C.2.

2. Some phytopathogen effectors interfere with histone acetylation

	C.2.2.a. by inhibiting HAT activity through competitive binding to a subunit of a HAT
	complex
	Some effectors from the oomycete Phytophthora sojae can interfere with histone
	acetylation via various strategies. One of them, PsAvh23, is targeted to the nucleus
	where it can interfere with the subunit Alteration/Deficiency in Activation 2 (ADA2)

  that successful nematode infection might rely on the appropriate dosage of the effector 32E03, as different dosage may trigger antagonistic effects on the expression of ribosomal RNA gene and the outcome of the nematode-plant interaction(Vijayapalani et al., 2018).

	genes, indicating C.2.2.c. Favouring histone acetylation by relocating a cytoplasmic acetyltransferase
	into the nucleus
	Another P. sojae effector, PsAvh52, affects host histone acetylation by
	manipulating a host acetyltransferase (Li et al., 2018). By co-immunoprecipitations
	and pull-down assays PsAvh52 was shown to interact with GmTAP1, a soybean
	acetyltransferase that is normally localised in the cytoplasm. Nuclear relocalisation
	of GmTAP1 triggered by PsAvh52 correlates with enhanced susceptible response
	to Phytophthora infection. Also, GmTAP1, although not being a HAT, can acetylate
	lysine residues on histone tails such as H3K9 and H2AK5, two marks associated
	with active transcription
	This suggests that this effector can manipulate histone acetylation levels at
	ribosomal RNA genes to promote their derepression and successful parasitism of
	the nematode. However, at high levels, 32E03 triggers RdDM-mediated ribosomal
	DNA methylation, which correlates with repression of the tested ribosomal RNA

C.2.2.b. Inhibiting HDAC activities

Instead of targeting HAT, the effector 32E03 from the sugar beet cyst nematode Heterodera schachtii targets HDAC activities

(Vijayapalani et al., 2018)

. HDT1, identified among the interacting partners of 32E03, is a HDAC that deacetylates H3K9 and is involved in the regulation of ribosomal RNA gene expression via chromatin modifications. Assays to determine HDACs activity in seedlings expressing 32E03 revealed a decrease in HDACs activity, as did similar assays performed on nuclear extracts from WT plants treated with recombinant 32E03, suggesting that this effector manipulates host histone deacetylation. At low levels, 32E03 was shown to participate in the derepression of some ribosomal RNA genes, which correlates with histone modifications on ribosomal chromatin including an increase in acetylated H3K9 levels, and appears to favour H. schachtii parasitism.

2. Effectors that inhibit plant immune responses

  .2.2.a.Inhibiting ETI responses We have seen that some RSSC effectors can trigger ETI responses in a given host species, leading to a strong plant defence activation that will restrict plant colonisation and result in plant resistance. It is therefore not beneficial for RSSC bacteria to let ETI responses be activated. To prevent this, the RSSC has evolved some effectors that can prevent ETI triggered by other effectors. For instance, RipE1 triggers ETI responses in N. benthamiana and upon its ectopic expression in transgenic A. thaliana, which in turn can be inhibited by the effector RipAY(Sang et al., 2020). RipAY can suppress plant immune responses by degrading plant glutathione, a regulator of plant redox homeostasis with a role in plant response to environmental and biotic stresses(Sang et al., 2018). In addition to triggering ETI responses, RipE1 expression in N. benthamiana is also accompanied with glutathione accumulation in plant leaves prior to immunity activation. Interestingly, When RipN was transgenically expressed in A. thaliana, pathogen multiplication in planta was promoted and PTI responses were inhibited, such as callose deposition and activation of defence-related genes, including CBP60g, suggesting that RipN could inhibit PTI responses. This effector has a Nudix hydrolase domain, which supposedly catalyses the hydrolysis of some substrates such as the reduced and oxidised forms of nicotinamide adenine dinucleotide (NADH and NAD+ respectively), and ADP-ribose among others. In this study, the authors showed that RipN possesses pyrophosphorylase activity of ADP-ribose and NADH in vitro and destabilises NADH/NAD+ ratios in planta, which depends on RipN enzymatic activity. Furthermore, PTI inhibition also seems linked to RipN enzymatic activity since transgenic expression of a catalytically inactive version of RipN in A. thaliana did not alter callose deposition or defence-gene activation under flg22 activation(Sun et al., 2019). Thus, RipN inhibits PTI responses likely through its hydrolase activity.Other examples of RSSC T3Es that can suppress PTI responses are RipAR and RipAW, cytoplasmic T3Es possessing an atypical E3 ubiquitin ligase domain, domain that catalyses protein ubiquitination(Nakano et al 2017). Both of these T3Es have been shown to possess functional E3 ubiquitin ligase activity in vitro.

C.2.

Ctransient co-expression of RipAY and RipE1 in N. benthamiana inhibited ion leakage and associated cell-death, as well as the expression of SA-related genes such as PR1, normally triggered by RipE1, revealing that RipAY suppresses RipE1triggered immunity. Considering that RipE1-triggered immunity is inhibited by catalytically active RipAY only, it appears that RipAY may prevent RipE1-triggered immunity activation likely via its glutathione degradation activity

(Sang et al., 2020)

. C.2.2.b. Inhibiting plant basal defences Some of the RSSC T3Es are able to suppress plant basal defences, PTI, in various ways. For instance, RipN is a conserved T3E in the RSSC that has been shown to inhibit PTI via its enzymatic activity

(Sun et al., 2019)

.

  1, † , Gaëlle Huet 1 , Cécile Pouzet 2 , Alain Jauneau 2 , Alexandra Kraut 3 , Marbella Fonseca 4,5,6 , Stefan Knapp 4,5 , Yohann Couté 3 , and Laurent Deslandes 1,*

	Upon interaction between plants and pathogenic bacteria, conserved pathogen-derived
	molecules called Pathogen Associated Molecular Patterns (PAMPs) are recognised by plant
	Pattern Recognition Receptors and trigger basal immune responses known as PAMP-Triggered
	Immunity (PTI) (Bigeard et al., 2015; Mott et al., 2014). PTI involves a signalling cascade
	leading to the induction of defence-responsive genes preventing host infection. However, PTI
	can be inhibited by virulence factors called effectors that are injected into host cells by adapted
	pathogens. PTI inhibition by such virulence strategies is called Effector-Triggered
	Affiliations Susceptibility (ETS). To counteract these virulence strategies, plants have evolved immune
	receptors (resistance proteins encoded by R genes) that can specifically detect directly or
	1 Laboratoire des Interactions Plantes-Microbes-Environnement (LIPME), INRAE, CNRS, indirectly effector activities and trigger a stronger activation of immune responses called
	Université de Toulouse, F-31326 Castanet-Tolosan, France Effector-Triggered Immunity (ETI)

2 FRAIB-TRI Imaging Platform Facilities, FR AIB, Université de Toulouse, CNRS, 31320 Castanet-Tolosan, France 3 Univ. Grenoble Alpes, INSERM, CEA, UMR BioSanté U1292, CNRS, CEA, FR2048 38000, Grenoble, France 4 Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany 5 Structural Genomics Consortium, Buchman Institute for Life Sciences, Goethe University, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany 6 Laboratório de Biologia Molecular e Genômica, Universidade Federal do Rio Grande do Norte, Natal, Brazil † Co-first author * Correspondence: laurent.deslandes@inrae.fr INTRODUCTION

  Table1). Co-expression of CFP-H4 with GTE9-YFP or GTE11-YFP led to a significant decrease of the CFP lifetime (2.432 ns and 2.455 ns, respectively). By contrast, no decrease of the CFP lifetime could be monitored when CFP-H4 was co-expressed either with

GTE9-N214A-YFP or GTE11-N206A-YFP, indicating that these two mutant proteins were unable to physically interact with histone H4 (Fig 3B, Supp Table

1

), confirming the interaction data obtained in vitro

(Fig 3A)

. To determine the specificity of the interaction detected between histone H4 and GTE9 in vivo, we used histone H3 fused to CFP (CFP-H3) as control. The mean CFP lifetime of CFP-H3 expressed alone was not statistically different from that measured in the presence of GTE9-YFP, indicating that GTE9 did not interact with H3

(Fig 3B, Supp Table

Table 1 . FRET-FLIM measurements showing that GTE9 and GTE11 interact with PopP2 and histone H4 in nuclei of N. benthamiana cells.

 1

	and yeast two-hybrid assays presented in

Table 5 . Quantitative FRET-FLIM measurements showing that catalytically active PopP2 alters the ability of GTE9 to interact with histone H4 in nuclei of N. benthamiana cells.

 5

	GTE9-eGFP	mCherry-H4	-	2.895	0.199	0.037	2.229	0.263	0.049	665	2.633	0.149	0.028	28	-	22 +/-2.3%
	GTE9-eGFP	mCherry-H4	PopP2-3HA	2.842	0.221	0.028	2.645	0.280	0.036	197	2.691	0.101	0.013	60	2.2 x 10 -9	6.7 +/-1.2%
	GTE9-eGFP	mCherry-H4	C321A-3HA	2.887	0.105	0.014	2.413	0.205	0.027	474	2.754	0.080	0.011	56	2.7 x 10 -2	16 +/-1.8%
	GTE9-N214A-eGFP mCherry-H4	-	2.784	0.084	0.015	2.689	0.090	0.017	95	2.738	0.052	0.009	30	-	3.3 +/-0.9%
	GTE9-N214A-eGFP mCherry-H4	PopP2-3HA	2.766	0.081	0.015	2.706	0.093	0.017	60	2.737	0.072	0.013	30	4.7 x 10 -1	2.1 +/-0.6%
	GTE9-N214A-eGFP mCherry-H4	C321A-3HA	2.777	0.103	0.019	2.682	0.106	0.019	95	2.731	0.046	0.008	30	8.1 x 10 -1	3.1 +/-1.1%
	Supplementary

Table S6 . Primers used in this study.

 S6

	Primer	Sequence (5' to 3')	Cloning/sequencing/RT-qPCR
	AttB1-GTE1	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTCTGTACATGTCAAGGAA	GTE1 cDNA (At2g34900)
	AttB2-GTE1	GGGACCACTTTGTACAAGAAAGCTGGGTCAGCTTTCTTAGCTCTTTTAATTG
	AttB1-GTE2	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGATCGGGCAACGTCATT	GTE2 cDNA (At5g10550)
	AttB2-GTE2	GGGACCACTTTGTACAAGAAAGCTGGGTCACACTCAAAATCTTTTCTTTCGA
	GTE2-500	TCAGGGAGCTGAAGAAGCGTCTCAA	sequencing primer
	GTE2-1000	CAAAGGGTCAAGATGTTTATTTGAT	sequencing primer
	GTE2-1500	CGTGAATCTACAAGAATTGCCTCCT	sequencing primer
	AttB1-GTE3	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCGTCTGGTCCTATAGC	GTE3 cDNA (At1g73150)
	AttB2-GTE3	GGGACCACTTTGTACAAGAAAGCTGGGTCATTACCGGTATCAGATTCATGT
	GTE3-500	TATTAAGGAGCCTATGGATTTAGGA	sequencing primer
	AttB1-GTE4	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCTTCGGAGCCTGTTAA	GTE4 cDNA (At1g06230)
	AttB2-GTE4	GGGACCACTTTGTACAAGAAAGCTGGGTCGGTCTGATCTGATCCAGATGATGA
	GTE4-500	TTGATGAAAACTCTATCAAGGAACC	sequencing primer
	GTE4-1000	AATGGAGGAGGGAGGATTCTGTCAG	sequencing primer
	GTE4-1500	TGCTATGACTTACAACCCAGAAGGA	sequencing primer
	AttB1-GTE5	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTCTTCTGAACATATATCAG	GTE5 cDNA (At1g17790)
	AttB2-GTE5	GGGACCACTTTGTACAAGAAAGCTGGGTCATTACCATTATCTGATCCACGT
	GTE5-500	AAGCACCTGTTAACAACAGGGACCT	sequencing primer
	AttB1-GTE6	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCTGACTCAGTGCCAG	GTE6 cDNA (At3g52280)
	AttB2-GTE6	GGGACCACTTTGTACAAGAAAGCTGGGTCTATGCGTGACCGCTTCGTTT
	AttB1-GTE7	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCTCCGGCTGTTTTCG	GTE7 cDNA (At5g65630)
	AttB2-GTE7	GGGACCACTTTGTACAAGAAAGCTGGGTCACATTGGGCTTCTTTTGCTTC
	GTE7-500	TCCGTCGGATCCGGAGTCGGAGAAA	sequencing primer
	GTE7-1000	GAAGCTTGATTCAGTGAAGCCGCCA	sequencing primer
	AttB1-GTE8	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGTTGAAAGTGCTGCGTT	GTE8 cDNA (At3g27260)
	AttB2-GTE8	GGGACCACTTTGTACAAGAAAGCTGGGTCGATTTCACCTTCTTCCACGTC
	GTE8-500F	GTACCTCAGGGAAGTTTGAGTCTTC	sequencing primer
	GTE8-1000F	AGAACCGGTGAAGCCACTTATGACA	sequencing primer
	GTE8-1500F	TTGTTGGGAGTCAATCTACTGGTGC	sequencing primer
	GTE8-2000F	TACCAAGCTCAGCCGAAGAAACCAG	sequencing primer
	AttB1-GTE9	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACAGAGAGAAACGGTG	GTE9 cDNA (At5g14270)
	AttB2-GTE9	GGGGACCACTTTGTACAAGAAAGCTGGGTCATCAATCTCTCCCTCTTCTA
	GTE9-500F	GTGTACATACATGAATGAACCT	sequencing primer
	GTE9-1000F	CGCTTCAGTTGGATCATCGATT	sequencing primer
	GTE9-1500F	AGTTGATTCGTAGGCTAAGACA	sequencing primer
	GTE9-2000F	TCATACTGAACCGAGTAACTTA	sequencing primer
	GTE9-2500F	TCTATTGCAGCTTCTGCATGGA	sequencing primer
	GTE9-3000F	CTGGGTTGTACTACTGATTGCT	sequencing primer
	GTE9-3500F	CAACAAGAGGATGTGAGTGGAT	sequencing primer
	GTE9-4000F	TAAATCAACTGTAGTTGGTGAC	sequencing primer
	GTE9-1350-R	TCGGTAAACTTGACATTTTAGGATCA	sequencing primer
	AttB1-GTE10	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGGTAAAGCGCGGAAACA	GTE10 cDNA (At5g63320)
	AttB2-GTE10	GGGACCACTTTGTACAAGAAAGCTGGGTCAATTTCTACATCTATATCTTCCT
	GTE10-500	AGTGATGAAGGAGTGTGAGACGTTA	sequencing primer
	GTE10-1000	CAAGATCTGATGGCATTAGAAGAAG	sequencing primer
	GTE10-1500	AGAGGATGATAGCAACAGTGAGAAG	sequencing primer
	GTE10-2000	AGACCTCCAGATGCTTAGAGCTACA	sequencing primer
	GTE10-2500	GATATGGGAGTGGAAGAAGTACATC	sequencing primer
	AttB1-GTE11	GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACTGTGAGGAACGGTG	GTE11 cDNA (At1g01770)
	AttB2-GTE11	GGGGACCACTTTGTACAAGAAAGCTGGGTCGTCGATTTCACCCTCTTCTA
	GTE11-500F	GCGCGTGCCGTTCCTGCGAT	sequencing primer
	GTE11-1000F	CATATTGAGTGTTCACTGCT	sequencing primer
	GTE11-1500F	AGAGCCAGTCACCACTTCCA	sequencing primer
	GTE11-2000F	ATGACAGATGAGGATAGAGT	sequencing primer
	GTE11-2500F	ACATCTCAACTGTCAGAACA	sequencing primer
	GTE11-3000F	CAGATGGTCATCAAGATGGT	sequencing primer
	GTE11-3500F	CCAAGGAAGCTGAAGAAGCT	sequencing primer
	GTE11-1350g-R	TGAGATGTGAGATATAGGGTGC	sequencing primer
	AttB1

• gte9-1 and gte11-1 mutants behave similarly to WT Col-0 plants in response to RSSC producing or not PopP2

Results and Discussion

Acknowledgments

Acknowledgments VC was supported by a doctoral fellowship co-funded by the Agence Nationale pour la Recherche (ANR-18-CE20-001) and the INRAE SPE department. AD and GH were supported by a research grant from the Agence Nationale pour la Recherche (ANR-10-JCJC-1706-01 (PercepTOME)). LD was supported by research grants from the Agence Nationale pour la Recherche (ANR15-CE20-0016-01 (RADAR) and ANR-18-CE20-0015 (EpiCLIPSE)). This study is set within the framework of the "Laboratoires d Excellences (LABEX)" TULIP (ANR 10 LABX 41 ) and of the "École Universitaire de Recherche (EUR)" TULIP GS (ANR 18 EURE 0019). The proteomic experiments were partially supported by Agence Nationale de la Recherche under projects ProFI (Proteomics French Infrastructure, ANR-10-INBS-08) and GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (ANR-17-EURE-0003). SK and MF are grateful for support by the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA), Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome. We thank Dr Cécile Raynaud for critical reading of the manuscript.

Abbreviations

GTE

Protein binding?

BRD

Extra terminal domain Bromodomain (interaction with Ac-K residues)

Figure 16. The GTE family in Arabidopsis counts twelve BRD-containing proteins (adapted from Airoldi et al., 2010). The General Transcription factor from group E family is a family of BRD-containing proteins that counts 12 members in A. thaliana. They belong to the BET family, characterised by a BRD, domain allowing the recognition of acetylated lysine residues, and an extra-terminal domain, thought to be important for protein-protein interactions.

2015; Sarris et al., 2015), PopP2 could also acetylate GTE9 and GTE11, likely on Lys residues.

To check this hypothesis, GTE9 and GTE11 fused to a triple Flag epitope (GTE9-3Flag and GTE11-3Flag, respectively) were co-expressed transiently in N. benthamiana with triple hemagglutinin (HA) epitope-tagged PopP2 (PopP2-3HA) or the catalytically PopP2-C321A (PopP2-C321A-3HA) (Fig 2A). Total protein extracts were then subjected to immunoprecipitation using an anti-acetyl-lysine antibody. Immunoblotting of immune complexes captured on protein A-coupled agarose beads with anti-Flag antibodies led to the detection of GTE9-3Flag and GTE11-3Flag co-expressed with catalytically active PopP2 but not with PopP2-C321A, indicating that PopP2 causes acetylation of Lys residues in GTE9 and GTE11 (Fig 2A).

To identify the Lys residues of GTE9 and GTE11 modified by PopP2, purified GTE9 and GTE11 either fused to a 3Flag or a YFP tag and co-expressed with PopP2 or PopP2-C321A

(Supp Fig 2 ) were used for mass spectrometry (MS)-based proteomic analysis. The Lysacetylation sites identified with a localisation probability ≥ 75% in both 3Flag-and YFP-tagged proteins co-expressed with wild-type PopP2 are listed in Supp Table 2 (with the sequences of acetylated peptides presented in Supp Table 3). This analysis revealed the acetylation of 16 and 10 Lys residues in GTE9 and GTE11, respectively. Notably, of the five acetylated Lys residues shared by GTE9 and GTE11, three of them are located on either side of their Brd (position 130-240 for BD GTE9 and position 122-232 for BD GTE11 ) (Fig 2B). Since PopP2 physically interacts with and acetylates multiple Lys residues of GTE9 and GTE11, these two GTE proteins therefore represent substrates of PopP2 enzymatic activity.

The bromodomain of GTE9 preferentially interacts with tri-and tetraacetylated histone H4 N-terminal tails in vitro

PopP2 acetylation of conserved Lys residues in GTE9 and GTE11 and in particular at Lys residues flanking their bromodomain (designated Brd hereafter) was intriguing. We reasoned that this targeted Lys acetylation could reflect a pathogen virulence strategy aimed at interfering with the functions of these two proteins. In an attempt to elucidate the molecular properties of GTE9 and GTE11, we scrutinised their Brd to determine whether this protein module was capable of binding acetyl-Lys residues and if so, with what specificity.

The bromodomain is a conserved region of ~110 amino acids that structurally forms four αhelices (αZ, αA, αB, αC) and two variable loops that connect helices αZ-αA (ZA loop) and αB-αC (BC loop), shaping the acetyl-lysine binding pocket and thus, contributing to substrate

Author Contributions

Declaration of interest

The authors declare no competing interests 2 and3). Expression level of GTE9 and GTE11 in Col-0, gte9-1 and gte11-1 null mutants. The relative gene expression levels were measured by RT-qPCR on leaf discs from 3-week-old plants and normalised to the expression level of AtEF1-α. The primer pairs used were, respectively, GTE9-q-2-F/GTE9-q-2&3-R for GTE9, and GTE11-q-2-F/GTE11-q-2-R for GTE11. Mean Expression level of GTE9, GTE11, and RRS1-R (top, middle and bottom, respectively) in Col-0, gte9-1 and gte11-1 mutant lines complemented or not with a genomic clone containing the coding and regulatory sequences for expression of RRS1-R and RPS4 genes from the Ws-2 accession. The relative gene expression levels were measured by RT-qPCR on leaf discs from 3-week-old plants and normalised to the transcript accumulation of AtEF1-α. Primers used were respectively GTE9-q-2-F and GTE9-q-2&3-R for GTE9, GTE11-q-2-F and GTE11-q-2-R for GTE11, and RRS1-R-ex5-q-F and RRS1-R-ex5-q-R for RRS1-R. Mean values +/-SD (n = 3; p-value = 0.05). (B) Expression level of GTE9 and GTE11 in Col-0, gte9-1 and gte11-1 mutants. The relative gene expression levels were measured by RT-qPCR on leaf discs from 3-week-old plants and normalised to the expression level of AtEF1-α. The primer pairs used were, respectively, GTE9-q-3-F and GTE9-q-3-R for GTE9 and GTE11-q-ex2-F and GTE11-q-ex2-R for GTE11. Mean values +/-SD (n = 3; p-value = 0.05, a and b show significative difference in mean ΔCt based on the one-way ANOVA or the kruskaltest).

(C) GTE9 1-370 -3Flag, GTE9-N214A 1-370 -3Flag and GTE9-N214A-3Flag were transiently expressed with 3HA-tagged active PopP2 or C321A catalytic mutant in N. benthamiana leaves. Protein extracts were immunoblotted with anti-HA and anti-Flag antibodies (Input). Immunoprecipitation of Lys-acetylated proteins was done in presence of an anti-AcK antibody on protein A-agarose beads (IP a-Ac-K). The immunoprecipitated GTE9 1-370 -3Flag, GTE9-N214A 1-370 -3Flag and GTE9-N214A-3Flag proteins were detected after immunoblotting with an anti-Flag antibody. Ponceau S staining of total proteins indicates equal loading of the samples in the input. This experiment was repeated two times with similar results.

Conclusions

To conclude this chapter, it seems that GTE9 and GTE11 are not only targeted by PopP2 but also by at least one other effector from the RSSC which also belong to the YopJ family of acetyltransferase and corresponding to PopP1 (Figure 26A and 26B). The interaction between PopP1 and GTE9 and GTE11 remains to be confirmed in planta (e.g. by FRET-FLIM assay or co-immunoprecipitations). Such assays performed in N. benthamiana through Agrobacterium-mediated transient expression could be quite challenging since PopP1 triggers a strong cell death response in Nicotiana spp. (Lavie et al., 2002;Poueymiro et al., 2009), involving the NLR NbZAR1 in N. benthamiana (Deslandes, unpublished data). To circumvent this problem, the use of the zar1-1 mutant line of N. benthamiana (Schultink et al., 2019), which is unable to recognise the Xanthomonas perforans XopJ4 effector (which shares ~61% identity with PopP1), should make possible the transient expression of PopP1, and of its putative GTE interacting partners, without triggering of cell death.

It would be also interesting to know whether GTE9 and GTE11 are virulence targets of other effectors from the RSSC. To check this, we could first study the phenotypical response of the GTE9 and GTE11 over-expressing lines to a RSSC strain lacking both PopP2 and PopP1 (popP1 popP2 strain). If the loss of both popP1 and popP2 suppresses the hyper-susceptible phenotype of the GTE9 and GTE11 over-expressing lines in response to R. solanacearum, this would mean that PopP1 and PopP2 are likely the only GMI1000 effectors manipulating these histone readers. Otherwise, it would mean that other GMI1000 effectors could target GTE9 and/or GTE11 and this could be tested by a Y2H screening using GTE9 and GTE11

as baits against a library containing the different GMI1000 effectors cloned as prey constructs (and vice versa). Chapter 3: What is the role of GTE9 and GTE11 at the chromatin level?

It has been described in the literature that GTE9 and GTE11 behave as negative regulators of BT2-mediated sugar and ABA signalling pathways, and are involved in the activation of genes by Cauliflower mosaic virus 35S (35S) multimerised enhancers (Irigoyen et al., 2021;Misra et al., 2018). However, we still lack information on (i) their precise involvement in these mechanisms, (ii) the link with their histone reader activity, and (iii) the reason of their targeting by an acetyltransferase like PopP2. To try to gather more information about the role of GTE9 and GTE11 as histone readers at the molecular level, we decided to explore their role at the chromatin level. Our objective was to identify the chromatin regions targeted by GTE9 and GTE11 thanks to a Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) approach (Veluchamy et al., 2016). This work was performed in the frame of an ANR project (ANR EpiCLIPSE) involving as scientific partners Drs Cécile Raynaud and Moussa Benhamed from Institut Paris Saclay 2 (IPS2) in Paris, as they are experts in techniques related to epigenetics in plants,

including ChIP-seq.

As a pre-requisite for this ChIP-seq analysis, we generated some transgenic lines expressing tagged versions of GTE9 and GTE11 under the control of their endogenous promoter regions. To this end, gte9-1 and gte11-1 mutant lines were complemented with GTE9 and GTE11 genomic clone, respectively, both fused to a triple hemagglutinin tag (GTE9prom::GTE9g-3HA and GTE11prom::GTE11g-3HA).

As negative controls, similar genomic constructs corresponding to GTE9 and GTE11 mutated in their BRD module (GTE9prom::GTE9g-N214A-3HA and GTE11prom::GTE11g-N206A-3HA) and making them theoretically unable to bind acetylated histone tails were also introduced in gte9-1 and gte11-1, respectively.

gte9-1 2(4) 3(1) a( 5) 2(3) 3( 4

Principle of the GFP-strand system and its uses for the detection of plant cells hosting PopP2

The GFP strand system relies on the use of a truncated GFP, with on one side the GFP first 10 beta strands, GFP 1-10 , and on the other side its last beta strand, GFP 11 .

The GFP 1-10 is not fluorescent and can be stably expressed in plants. After delivering an effector fused to GFP 11 , plant cells containing the effector show reconstituted green fluorescence (Figure 30A). This system can be used for monitoring the spatio-temporal delivery of pathogen effectors in plant cells, and has been successfully implemented to various effectors from Pseudomonas syringae in A. thaliana and N. benthamiana (Henry et al., 2017;Park et al., 2017). This system has also been successfully tested for delivering PopP2 fused to GFP 11 (PopP2-GFP 11 ) by a RSSC strain (Henry et al., 2017). Indeed, a strain mutated in popP2 (ΔpopP2) and complemented with PopP2-GFP 11 was able to deliver PopP2-GFP 11 in transgenic A. thaliana expressing 35S::GFP 1-10 , leading to the reconstitution of fluorescence in root and petiole cells hosting PopP2. Moreover, the 35S::GFP 1-10 Arabidopsis transgenic line showed similar symptom development to the WT following infection with a ΔpopP2 strains or complemented with PopP2 (popP2+PopP2-3HA), its catalytic mutant PopP2-C321A (ΔpopP2+PopP2-C321A-3HA), or PopP2-GFP 11 (ΔpopP2+PopP2-GFP 11 ). In addition, the ΔpopP2+PopP2-GFP 11 complemented strain was comparable to the ΔpopP2+PopP2-3HA complemented strain in terms of infection on susceptible or resistant A. thaliana ecotypes (Henry et al., 2017). Thus, the GFP strand system seems to be compatible with the A. thaliana -R. solanacearum pathosystem, and more precisely for studying the secretion and injection of PopP2 in plant cells.

However, ChIP-seq analyses performed on root cells in which PopP2 has been injected by the RSSC is quite challenging since ChIP-seq analyses require a relatively large amount of plant material, which might not be easily obtainable with roots. Hence, we decided to use an alternative GFP strand system-based strategy GTE11 on several lysine residues notably flanking their BRD. PopP2 alters GTE9-H4 association through its acetyltransferase activity. GTE9 and GTE11 over-expression contribute to virulence of the RSSC, and it depends on PopP2 acetyltransferase activity too. PopP2 can also acetylate other GTE: GTE3, GTE5 and GTE8. PopP1 could also target GTE proteins as it can interact with various GTE proteins. Where GTE9 and GTE11 are located at the chromatin level remains to be determined, as well as the chromatin sites visited by PopP2 (ongoing).

?

Material and Methods

EXPERIMENTAL MODELS

Arabidopsis

Arabidopsis thaliana Col-0 and Ws-2 plants, and null mutants (gte9-1 (Salk_0119044C) gte11-1 (Salk_059327C), previously described by Misra et al., 2018 were grown in short days (8h light/16h dark cycle) at 22°C (60% relative humidity, 125 µE/M2/s fluorescent illumination). Transgenic lines were generated by using the floral dip transformation method (Clough and Bent, 1998).

Nicotiana benthamiana

Nicotiana benthamiana plants were sown on soil and grown at 24°C under long day photoperiod (16 h light/8 h dark) with 60% relative humidity. Leaves of 4 to 5 week-old plants were used for Agrobacterium tumefaciens-mediated transient expression for Immunoblotting, colocalisation, or FRET-FLIM assays.

Bacterial strains

Ralstonia solanacearum (RSSC) strains were spread on solid φ medium containing 5 g/L glucose and 0.004% TTC and grown for 3 days at 28°C. Bacteria were grown in liquid φ medium at 28°C under shaking overnight with appropriate antibiotics (ΔpopP2+PopP2 and ΔpopP2+PopP2-C321A were grown in presence of gentamicin (7.5 µg/mL) and tetracyclin (5 µg/mL) (Tasset et al., 2010).

Pseudomonas fluorescens (Pf0-1) expressing the PopP2 variants (wild-type and catalytically inactive mutant) were grown on King's B plate supplemented with antibiotics (chloramphenicol 30 µg/mL, tetracycline 5 µg/mL and gentamicin 15 µg/mL) at 28°C overnight. Bacterial cells were centrifugated and resuspended in infiltration buffer (10 mM MgCl 2 ) at OD 600 =0.2.

Plasmid construction

The oligonucleotides used in the experiments presented in this article are listed in Table 3. The full-length GTE1 (At2g34900), GTE2 (At5g10550), GTE3 (At1g73150), GTE4 (At1g06230), GTE5 (At1g17790), GTE6 (At3g52280), GTE7 (At5g65630), GTE8

A. thaliana stable transformation

Agrobacterium tumefaciens strains carrying the binary vectors (pAM-PAT-35S-GTE9-3Flag and pAM-PAT-35S-GTE11-3Flag in GV3103 strain; pBIN-35S-GTE9-3HA, pBIN-35S-GTE11-3HA in GV3101 strain; pAM-PAT-35S-GTE9prom::GTE9g-3HA-GTE9term, pAM-PAT-35S-GTE9prom::GTE9g-N214A-3HA-GTE9term, pAM-PAT-D35S-GTE11prom::GTE11g-3HA-GTE11term, and pAM-PAT-D35S-GTE11prom::GTE11g-N206A-3HA-GTE11term in GV3103 strain) were grown on solid LB medium with appropriate antibiotics for 2 days at 28°C. Bacteria were then cultured in YEB liquid medium (supplemented with antibiotics; gentamicin (15 µg/mL) and carbenicillin (25 µg/mL) for pAM-PAT-based vectors, tetracyclin (5 µg/mL) and kanamycin (25 µg/mL) for pBIN-based vectors) at 28°C for 12 hours.

Cells were centrifugated and resuspended in a sucrose solution (50 g/L) with 0.02% Silwet-L77.

A. thaliana Col-0, gte9-1 (Salk_0119044C) and gte11-1 (Salk_059327C) were transformed with Agrobacterium tumefaciens using the floral dip method (Clough and Bent, 1998). Primary transformant (T1) were selected on MS medium supplemented with phosphinothricin (10 µg/mL) (Duchefa) or with Kanamycin (50 µg/mL) for pAM-PAT-and pBIN-based binary vectors, respectively. T2 transgenic lines were genotyped by PCR and accumulation of transgenically expressed proteins was verified by immunoblot.

RNA extraction, reverse transcription and RT-qPCR

RNA extractions were performed using the Nucleospin RNAII kit (Macherey-Nagel) and following manufacturer's instructions. Reverse transcription was made with

Transcriptor Reverse Transcriptase (TRT, Roche). A pre-mix of 7.25 µL (1 µL Oligo (dT) 17 at 1 µg/µL; 0.25 µL TRT ; 4 µL TRT buffer; 2 µL dNTP-Mix at 10 mM) was added to a final volume of 20 µL containing 1 µg of total RNA. Samples were 2020). Briefly, we used a mixed model (MIXED procedure in SAS v. 9.4; SAS Institute Inc.) as follow:

Where μ is the overall mean of the phenotypic data, "block" considers environmental conditions between the independent experiments included in the model, "accession" correspond to the tested genetic lines, "covCol" is a covariate accounting for tray effects within blocks and "ε" is the residual term. "block" was considered as a fixed factor and "accession" as a random factor. We tested the significance of the different accessions by likelihood ratio test of model with and without this effect. With p-value<0.05, the lines were considered to have a different disease index reflecting differential response to the pathogen.

Bacterial growth quantification of Pst DC3000

The Pst DC3000 strain was cultured on King B solid medium with 50 µg/mL

Rifampicin 2 days prior inoculation, and respread on a new King B plate with 50 µg/mL Rifampicin 1 day before inoculation. For disease assay, we proceded similarly to what described (Zipfel et al., 2004). Briefly, bacteria were collected by centrifugation and washed once with a 10mM MgCl 2 solution, and adjusted to a cell density of OD 600 =0.0003. Bacteria were infiltrated into leaves with a needleless syringe, and inoculated plants were kept under at ambiant humidity for 2h to let evaporate excess water from the leaf and then were covered with a holed transparent plastic foil to keep humidity for facilitating disease development. As a control at 0 day post-infiltration, 4 leaf discs of 4mm diameter were harvested as one biological repeat, and 2-3 repeats were taken for each genetic line.

For quantification of bacteria 4 days after DC3000 infiltration, four leaf discs from 4 different leaves of 4mm diameter were harvested as one biological repeat, and 6 repeats were taken for each genetic line. Leaf discs were ground and diluted in

Yeast two-hybrid

Constructs for yeast two-hybrid analysis were prepared in the MatchMaker GAL4 two-hybrid system (Clontech) using the vectors pGBKT7-and pGAD-derived vectors to express bait and prey proteins, respectively. The pDEST pGAD-GWY vector was generated by ligating a Gateway cassette (Frame C) in the pGADT7 Transformed cells were spread on solid LB medium with appropriate antibiotic selection (carbenicillin 50 µg/mL for pGAD vectors, kanamycin 50 µg/mL for pGBG vectors). Plasmid DNAs were extracted with the Wizard DNA plasmid purification kit (Promega). Co-expression of BD-p53 (bait, pGBKT7-p53 vector, Clontech) and AD-T (prey, pGADT7-T vector, Clontech) was used as positive control on selective medium. Bait and prey proteins were tagged with a c-Myc and an HA epitope tag, respectively. Bait and prey constructs were introduced in AH109 Saccharomyces cerevisae strain (Clontech) according to the manufacturer's instructions.

Transformed cells were spread on SD-TL medium and incubated 3 days at 30°C.

Colonies were then dropped by dilution gradient on different media: non-selective (SD-TL) and selective (SD-TLHA) and incubated between 3-5 days at 30°C.

Pseudomonas fluorescens-mediated delivery of PopP2 in Arabidopsis

For Pf0-1-mediated delivery, full-length PopP2 and PopP2-C321A recombined in the pBBR-AvrRps4prom-GWY-3HA gateway destination vector were used as previously described (Le Roux et al., 2015). For PopP2-3Flag and PopP2-C321A-3Flag delivery (both ending with a stop codon), pENTR207-PopP2-3Flag and pENTR207-PopP2-C321A-3Flag were used for LR recombination with the pBBR-AvrRps4prom-GWY-3HA gateway destination vector. For PopP2-3HA-GFP 11 or

PopP2-C321A-3HA-GFP 11 delivery, full-length PopP2 and PopP2-C321A were introduced in the pBBR-AvrRps4prom-GWY-3HA-GFP 11 gateway destination vector by using LR reaction. pBBR-derived contructs were introduced in Pf0-1 cells by tri-parental mating. Transformed Pf0-1 cells were selected on King's B agar supplemented with 6mM MgS04 and with appropriate antibiotics (tetracycline 5 µg/mL, chloramphenicol 30 µg/mL, and gentamicin 15 µg/mL). For infiltration in Arabidopsis leaves, bacteria were resuspended in 500 µL MgCl 2 (10 mM) and centrifuged (8000 rpm, 1 min). Bacteria were resuspended in 500 µL MgCl 2 (10 mM) and centrifuged again (8000 rpm, 1 min) to wash out antibiotics. Bacterial pellet was resuspended in 500 µL MgCl 2 (10 mM) solution and bacterial density was adjusted to OD600=0,2 (OD 600nm = 1 means 1.75 x 10 8 bacteria/mL). Threeweek-old plants were placed in a humid environment 14 hours before infiltration to facilitate stomata opening. Bacteria were infiltrated in the plant leaves with a needleless syringe. ~12 leaves were infiltrated for each plant tested and the PopP2-tiggered cell death was observed 48 hours after infiltration.

Quantification and statistical analysis

For the Rs inoculations, disease symptom scoring was analysed and plot with R software. Graphs show least-square means +/-Standard Error of the LS means represented as linear plots with points at each day when symptoms were scored.

190

Significance of the difference of disease index LS means at a given day between different genotypes was assessed with a one-way ANOVA performed on a mixed model generated from the data as previously used (Aoun et al., 2020). At a given day, significance of the difference between LS means was set at p-value<0.05.

For RT-qPCR, data were analysed and represented with R software using the comparative Ct method (2 ΔCt ) (Schmittgen and Livak, 2008). Mean ΔCt were calculated from three technical replicates for three biological replicates (three independently grown sets of plants). The mean relative expressions from the biological replicates +/-SD was represented in barplots. ΔCt values were used to assess the significance of the difference in ΔCt between the different genetic lines. A one-way ANOVA was applied when the assumptions about the underlying distribution of the data were fulfilled, or a kruskal-test when they were not.

For DC3000 bacterial growth analyses, data were analysed and represented with R software. Graphs show boxplots of the biological replicates from one experiment.

Significance of the difference of bacterial growth between genetic lines at 0 dpi and 4 dpi was assessed on R. A one-way ANOVA was applied when the assumptions about the underlying distribution of the data were fulfilled, or a kruskal-test when they were not.