E Vergne
email: emilie.vergne@inrae.fr

Phenotypic and transcriptomic analyses reveal major differences between apple and pear scab nonhost resistance Authors

Keywords: apple, pear, nonhost resistance, transcriptomics

Background. Nonhost resistance is the outcome of most plant/pathogen interactions, but it has rarely been described in Rosaceous fruit species. Apple (Malus x domestica Borkh.) is a nonhost for Venturia pyrina, the scab species attacking European pear (Pyrus communis L.). Reciprocally, P. communis is a nonhost for Venturia inaequalis, the scab species attacking apple. The major objective of our study was to compare the scab nonhost resistance in apple and in European pear, at the phenotypic and transcriptomic levels.

Results. Macro-and microscopic observations after reciprocal scab inoculations indicated that, after a similar germination step, nonhost apple/V. pyrina interaction remained nearly symptomless, whereas hypersensitive reactions were observed during nonhost pear/V. inaequalis interaction.

Comparative transcriptomic analyses of apple and pear nonhost interactions with V. pyrina and V. inaequalis, respectively, revealed considerable differences. Very few differentially expressed genes were detected during apple/V. pyrina interaction, which is consistent with a symptomless type I nonhost resistance. On the contrary, numerous genes were differentially expressed during pear/V. inaequalis interaction, as expected in a type II nonhost resistance involving visible hypersensitive reaction. Pre-invasive defense, such as stomatal closure, was detected, as well as several postinvasive defense mechanisms (apoplastic reactive oxygen species accumulation, phytoalexin production and alterations of the epidermis composition). In addition, a comparative analysis between pear scab host and nonhost interactions indicated that, although specificities were observed, two major defense lines were shared in these resistances: cell wall and cuticle modifications and phenylpropanoid pathway induction.

Conclusion.

This first deciphering of the molecular mechanisms underlying a nonhost scab resistance in pear offers new possibilities for the genetic engineering of sustainable scab resistance in this species.

Background

Apple (Malus domestica Borkh.) and European pear (Pyrus communis L.) are two closely related species belonging to the Rosaceae family. Reclassification of the Rosaceae placed both Pyrus and Malus genera in the subfamily Spiraeoideae, tribe Pyreae and subtribe Pyrinae, this subtribe corresponding to the long-recognized subfamily Maloideae [START_REF] Potter | Phylogeny and classification of Rosaceae[END_REF]. Efforts to resolve relationships within this subtribe have frequently failed, and Campbell et al [START_REF] Campbell | Phylogeny of subtribe Pyrinae (formerly the Maloideae, Rosaceae): Limited resolution of a complex evolutionary history[END_REF] concluded that the genera of this subtribe Pyreae have not diverged greatly genetically. The recent sequencing of the pear genome [START_REF] Wu | The genome of pear (Pyrus bretschneideri Redh.)[END_REF] allowed a precise comparison with the apple genome [START_REF] Velasco | The genome of the domesticated apple (Malus × domestica Borkh.)[END_REF] and led to the estimation of a divergence time between the two genera of ≈ 5.4 -21.5 million years ago. Furthermore, apple and pear genomes share similar chromosome number (n=17), structure and organization. Scab disease, caused by Venturia spp., affects several rosaceous fruit tree species. These hemibiotrophic pathogens can infect only a limited host-range during their parasitic stage, but they can overwinter as saprophytes in the leaf litter of a larger range of plant species [START_REF] Stehmann | Molecular identification of a sexual interloper: the pear pathogen Venturia pirina, has sex on apple[END_REF]. Scab disease is caused by V. inaequalis on apple, by V. pyrina (formerly named V. pirina [6]) on European pear, and by V. nashicola on Japanese (P. pyrifolia Nakai) and Chinese (P. ussuriensis Maxim) pears. Cross inoculations of Venturia spp. on different rosaceous fruit trees indicates that these pathogens are highly host specific, probably indicating a close co-evolution of these pathogens with their hosts [7].

A plant species unable to be successfully infected by all isolates of a pathogen species is considered as a nonhost for this pathogen. Nonhost interactions of Venturia spp. on apple and pear have rarely been described. Microscopic observations have been made on P. communis / V. nashicola [8] as well as M. domestica / V. pirina and P. communis / V. inaequalis [START_REF] Stehmann | Molecular identification of a sexual interloper: the pear pathogen Venturia pirina, has sex on apple[END_REF]9]. In all cases, conidia germinated and produced appressoria and runner hypheae, but failed to establish a network of stroma. No macroscopic symptoms were visible.

Because of its durability, nonhost resistance has attracted numerous studies over the last decade, which have uncovered its multiple and complex defense components. The underlying mechanisms of nonhost resistance comprise pre-invasion resistance with preformed or induced cell-wall defenses, metabolic defense with phytoanticipin or phytoalexin accumulation, pattern-triggered immunity (PTI) as well as elicitor-triggered immunity (ETI) and various signaling pathways [10]. To our knowledge, the molecular bases of scab nonhost resistance of apple and pear have never been investigated. The objectives of our study were 1) to precisely describe nonhost resistance symptoms in M. domestica / V.pyrina and P. communis / V. inaequalis interactions 2) to analyze the underlying molecular mechanisms of both nonhost interactions through a transcriptomic study 3) to compare the mechanism of host [12] and nonhost scab resistance in apple and European pear.

Results and discussion

Variable symptoms of nonhost resistance Nonhost interactions were observed in a test performed on 'Gala' and 'Conference', all inoculated by a V. pyrina strain (VP102) and a V. inaequalis strain (VI EUB05). At the macroscopic level, a total absence of sporulation was observed on all nonhost interactions (Table 1). The apple 'Gala' remained completely symptomless after V. pyrina inoculations (Fig. 1C). This is similar to the observation of Chevalier et al [9] after inoculation of 'Gala' with another V. pyrina strain. On the contrary, pear plants inoculated with V. inaequalis presented frequent pin points symptoms (Fig. 1A) and occasional chlorotic lesions (Fig. 1B). Chlorotic lesions had already been observed by Chevalier et al [9] after inoculation of the pear 'Pierre Corneille' with the V. inaequalis strain EUB04, but pin points had never been reported in this nonhost interaction. According to our observations, apple nonhost resistance could be classified as type I and pear as type II according to Mysore and Ryu [11] definition based on the absence/presence of visible HR reaction. At the microscopic level, three days after inoculation, there was no clear difference between host and nonhost interactions: the conidia of V. inaequalis and V. pyrina germinated equally on both hosts forming one or two appressoria (Fig. 1 D andF). However, 14 days after inoculation, there was a clear reaction of the plant cells in contact with the appressoria (accumulation of red autofluorescent compounds and enlargement of these cells), which could indicate very small scale hypersensitive reactions (HR) reactions (Fig. 1 E andG) in both plant species. No formation of subcuticular stroma and no conidiogenesis were observed in the nonhost interactions, contrary to the host-resistance reactions [12]. These observations are similar to the collapsed cells described by Chevalier et al [9] in apple and pear nonhost reactions, and to the rare HR-like reactions observed by Stehmann et al [START_REF] Stehmann | Molecular identification of a sexual interloper: the pear pathogen Venturia pirina, has sex on apple[END_REF] on apple inoculated by V. pyrina.

Our results indicate that the leaf surface morphology of apple and pear is equally compatible with V. pyrina and V. inaequalis conidia germination, without specific inhibition at this stage. Recognition probably occurs only at the appressorium site, leading to the cellular reactions observed. These reactions were limited to a few cells without visible symptoms in apple / V. pyrina interaction, but extended and produced macroscopic symptoms in pear / V. inaequalis interaction.

Different patterns of global gene expression in nonhost resistance in pear versus apple

Differentially expressed genes (DEGs) were analyzed by comparing transcript abundance in leaves between T0 and 24 hours post inoculation (hpi) and between T0 and 72hpi, in the nonhost interactions 'Gala' / V. pyrina VP102 and 'Conference' / V. inaequalis EUB05. In total, 60 DEGs in apple and 1857 DEGs in pear were identified, which amounts to 0.19 % of all apple genes on the apple AryANE v2.0 microarray, and 4.23 % of all pear genes on the Pyrus v1.0 microarray (Table 2). The very small number of DEGs detected in the apple nonhost interaction at 24 or 72hpi is in agreement with the total absence of macroscopic symptoms observed during this interaction.

However, at the microscopic level, small HR-like reactions were detected in the apple / V. pyrina interaction. Because theses reactions involve only a few cells in the leaves, the changes in gene expression are probably below the threshold of DEG detection applied in this experiment.

On the contrary, the number of DEGs detected during the pear / V. inaequalis interaction is in the same order of magnitude as the number of DEGs detected during pear host resistance to V. pyrina (see [12]). This is in agreement with the frequent observation of macroscopic symptoms of resistance (chlorotic lesions or pin points) in this interaction. Among the 1857 pear DEGs, 80.2 % were only detected at 24hpi and 15.4 % only at 72hpi, whereas 4.2 % were upregulated or down regulated similarly at both time points of the kinetics. Among all the pear DEGs observed at 24 and 72hpi, the proportion of up-regulated DEGs was higher (68.8 %) than the proportion of downregulated DEGs (31.2 %). Using MapMan to map the DEGs TAIR names, we observed that the main functional categories represented in this set of DEGs were similar to those observed during pear host resistance to V. pyrina (see [12]): protein, RNA, signaling, transport and cell cycle (Fig. 2).

To basically validate the transcriptomic data, 12 DEGS with varied ratios (between -1.9 and 2.9) have been tested in QPCR (Table S1), on the two biological repeats used for transcriptomic analyses.

Considering the weak number of DEGs found for apple in this study, we only tested two of them in QPCR. As seen in Table 2 for pear, at 24hpi, a majority of DEGs are up-regulated and at 72hpi, a majority of DEG are down-regulated. QPCR was then performed essentially on DEGs with positive ratios at 24phi and negative ratios at 72hpi (Table S1). The QPCR results confirmed the induced or repressed status of all tested DEGs.

Weak involvement of hormone signaling pathways classically associated to resistance Pear DEGs were found that indicate that the jasmonic acid (JA) pathway was repressed. The JA biosynthesis and metabolic conversions were reviewed by Wasternack et al [13]. In our data, at 24hpi, the first step of JA biosynthesis, that is the conversion of linoleic acid in 12-oxo-phytodienoic acid (OPDA), is compromised given the repression of six about seven lipoxygenases (LOX) (three LOX1, two LOX2 and two LOX5), the last one being induced (Fig. 3). OPDA produced in the chloroplast is then transported to the peroxisome for subsequent conversion to JA via the action of OPR3 (12oxo-phytodienoic acid reductase) and β-oxidation enzymes (reviewed in [START_REF] Li | Role of beta-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato[END_REF] and in [13]). In pear, three β-oxidation enzymes were found activated more or less rapidly: ACX4 (24hpi), MFP2 (72hpi) and the thioesterase homolog to At2g29590 (72hpi), which suggests that constitutive OPDA stocks were turned into JA. But the early and long-lasting induction of JMT and ST2A genes is in favor of a rapid conversion of JA in inactive compounds, JMT induction being reinforced by BBD1 repression (24hpi). BBD1 is actually known as a negative regulator of JMT [START_REF] Seo | Identification of a novel jasmonate-responsive element in the AtJMT promoter and its binding protein for AtJMT repression[END_REF].

The defense response depending on JA was also clearly repressed in pear (Fig. 3). The transcription activator MYC2 of JA-induced genes is known to be repressed by its interaction with JAZ proteins (reviewed in [13]), and two JAZ1 and one JAZ3 coding genes were found activated at 24hpi in pear.

UBP12 is known as a stabilizer of MYC2 [START_REF] Jeong | The deubiquitinating enzymes UBP12 and UBP13 positively regulate MYC2 levels in jasmonate responses[END_REF]. In our data, UBP12 was found repressed at 72hpi, which reinforces the inactivation of MYC2. WRKY33 is known as an activator of the JA defense pathway [START_REF] Birkenbihl | Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection[END_REF] and WRK70 [START_REF] Kaurilind | A genetic framework for H2O2 induced cell death in Arabidopsis thaliana[END_REF] or AS1 (or MYB91; [START_REF] Nurmberg | The developmental selector AS1 is an evolutionarily conserved regulator of the plant immune response[END_REF]) as inhibitors, and among JA-responsive proteins, the pathogenesis-related PR3, PR4 and PR12 act downstream MYC2 activation [START_REF] Ali | Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance[END_REF]. In our data, accordingly with the repression of the activator WRKY33 and the activation of the inhibitors WRK70 and AS1, some JA-responsive genes were also found repressed, such as the chitinase coding genes PR4 (also called HEL) and ATEP3. Furthermore, no DEGs were found for PR3 and PR12 functions. To conclude, in the nonhost interaction between pear and V. inaequalis, some JA seems to be produced, but rapidly converted in inactive compounds and the subsequent defense response is clearly repressed.

Pear DEGs were found that seems to indicate that the salicylic acid (SA) pathway was slightly engaged and rapidly repressed (Fig. 3). WRKY70 was induced at 72hpi in our data. This transcription factor is known as a negative regulator of SA biosynthesis but a positive regulator of SA-mediated defense genes in Arabidopsis ( [START_REF] Li | The WRKY70 transcription factor: a node of convergence for jasmonatemediated and salicylate-mediated signals in plant defense[END_REF]; [START_REF] Li | WRKY70 modulates the selection of signaling pathways in plant defense[END_REF]; [START_REF] Wang | A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants[END_REF]), among them PR2, PR5 but not PR1 [START_REF] Li | WRKY70 and its homolog WRKY54 negatively modulate the cell wallassociated defenses to necrotrophic pathogens in Arabidopsis[END_REF]. WRKY33 which is known as a negative regulator of SA-responsive genes [START_REF] Genot | Constitutively active Arabidopsis MAP kinase 3 triggers defense responses involving salicylic acid and SUMM2 resistance protein[END_REF], was also repressed at 72hpi in our data. PR2 and 5 are well-known anti-fungal proteins ( [START_REF] Hu | Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein[END_REF]; [START_REF] Mestre | Identification of a Vitis vinifera endo-β-1,3-glucanase with antimicrobial activity against Plasmopara viticola[END_REF]; [START_REF] Zhang | Expression of a wheat β-1,3glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel[END_REF]). At 24hpi a PR2, two PR2like, a PR5 and a PR5-like coding genes were found induced in our work, another PR2-like and two others PR5-like being repressed. The differential expression was maintained at 72hpi for only two of the activated ones. Furthermore no DEG was found for the PR1 function but three PR1-like genes were found repressed: ATPRB1 and genes homolog to At5g57625 and At4g33720. ATPRB1 was already reported as repressed by SA treatment [START_REF] Santamaria | The promoter of a basic PR1-like gene, AtPRB1, from Arabidopsis establishes an organ-specific expression pattern and responsiveness to ethylene and methyl jasmonate[END_REF]. In our data, the WRKY70 transcription factor was induced later than the induced PR genes so we could imagine that induced PR genes were activated by another precocious regulation, such as an oxidative burst (see below), rather than by WRKY70. Furthermore, WRKY70 induction seems not sufficient to enable a long lasting induction of these defense genes.

SA accumulation was also rather mixed. CBP60a [START_REF] Sreekanta | The CALMODULIN-BINDING PROTEIN60 family includes both negative and positive regulators of plant immunity[END_REF], ACA11 [START_REF] Boursiac | Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway[END_REF], EICBP.B (or CATMA1; [START_REF] Huang | The Mediator kinase module serves as a positive regulator of salicylic acid accumulation and systemic acquired resistance[END_REF]), all three coding calcium-sensor proteins, are known as negative regulators of SA accumulation and biosynthesis, as well as the light signaling factor FAR1 [START_REF] Wang | A pair of light signaling factors FHY3 and FAR1 regulates plant immunity by modulating chlorophyll biosynthesis[END_REF] or the SA glucosyltransferase UGT74F1 which convert SA in inactive SA 2-O-beta-D-glucoside or the glucose ester of SA [START_REF] Song | Overexpression of AtSGT1, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae[END_REF]. On the contrary EDS1, PAD4 (reviewed in [START_REF] Vlot | Salicylic acid, a multifaceted hormone to combat disease[END_REF]) and MKS1 [START_REF] Andreasson | The MAP kinase substrate MKS1 is a regulator of plant defense responses[END_REF] are known as positive regulators of SA accumulation. In our data, the repression of CBP60a, ACA11 and EICBP.B genes sustained a SA biosynthesis and accumulation. In addition, EDS1 activation allowed to consider a positive feedback loop likely to potentiate SA action via EDS1 cytosolic homodimers, even though PAD4 was repressed. But, as well as WRKY70 induction, the repression of the MAPK MKS1 and the activation of the light signaling factor FAR1 (2 times) or the SA glucosyltransferase UGT74F1 were in favor of less free SA. Concerning SAR, MES1 is known as required in healthy systemic tissues of infected plants to release the active SA from methyl-SA, which serves as a long-distance signal for systemic acquired resistance (SAR) [START_REF] Vlot | Salicylic acid, a multifaceted hormone to combat disease[END_REF] and ACBP6 may be involved in the generation of SAR inducing signal(s) [START_REF] Xia | Acyl CoA binding proteins are required for cuticle formation and plant responses to microbes[END_REF]. In our data, SAR seemed compromised given the repression of ACBP6 at 24hpi and MES1 at 72hpi.To conclude, in the nonhost interaction between pear and V. inaequalis, the SA pathway could be engaged but transiently and presumably reduced to the few infection sites and not spread by SAR in healthy systemic tissues.

Calcium influx and reactive oxygen species (ROS) production act as secondary messengers and lead to stomatal closure Early responses of plants upon pathogen perception include calcium influx and ROS production, which both act as secondary messengers ( [START_REF] Boller | A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern recognition receptors[END_REF], reviewed in [10]). Three pear DEGs were found that indicate early increased cytosolic calcium level. The CSC (Calcium permeable Stress-gated cation Channel) ERD4 (found two times) and the two glutamate receptors GLR3.4 and GLR2.7, are known as calcium permeable channels ( [START_REF] Vincill | Ca(2+) conduction by an amino acid-gated ion channel related to glutamate receptors[END_REF]; [START_REF] Hou | DUF221 proteins are a family of osmosensitive calcium-permeable cation channels conserved across eukaryotes[END_REF]). They were induced at 24hpi in our data. An increased cytosolic calcium level can lead to a pre-invasive defense response by stomatal closure and promote the post-invasive defense response ROS accumulation [START_REF] Fonseca | Genes involved in nonhost disease resistance as a key to engineer durable resistance in crops[END_REF].

Calcium influx has been reported to promote stomatal closure through the regulation of potassium flux and the activation of anion channels in guard cells (reviewed in [10]). The stomata closure is known to be induced via the inhibition of inward potassium currents which is achieved via activation of calcium dependent protein kinases (CDPK) such as CPK13 and CPK8/CDPK19 ( [START_REF] Ronzier | CPK13, a noncanonical Ca2+-dependent protein kinase, specifically inhibits KAT2 and KAT1 shaker K+ channels and reduces stomatal opening[END_REF]; [START_REF] Zou | Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling and H2O2 homeostasis in stomatal guard cells under drought stress[END_REF]); but also via activation of CBL1 of the CBL1-CIPK5 complex, which activates the GORK potassium outward channel [START_REF] Förster | Wounding-induced stomatal closure requires jasmonate-mediated activation of GORK K(+) channels by a Ca(2+) sensor-kinase CBL1-CIPK5 complex[END_REF]. CPK13, CPK8/CDPK19 and CBL1 were all activated at 24 hpi in our data.

A NADPH oxidase RBOHB (respiratory burst oxidase homologs, RBOH) is early and long-lasting induced in the pear/V. inaequalis nonhost interaction suggesting a rapid and maintained apoplastic ROS production. Indeed, the apoplastic ROS are mainly produced by plasma membrane localized NADPH oxidases, cell wall peroxidases and amine oxidases [START_REF] Kadota | Regulation of the NADPH oxidase RBOHD during plant immunity[END_REF]. In addition, posttranslational regulation of RBOH is required for its activation and ROS production. Calcium, phosphatidic acid, and direct interactors such as Rac1 GTPase and RACK1 (Receptor for Activated C-Kinase 1) have been reported to be positive regulators of RBOHs (reviewed in [START_REF] Adachi | Kinase-mediated orchestration of NADPH oxidase in plant immunity[END_REF]). For example, the Rac-like/ROP GTPase ARAC3 is known to interact with a RBOH to promote ROS production [START_REF] Zhai | AtROP6 is involved in reactive oxygen species signaling in response to iron-deficiency stress in Arabidopsis thaliana[END_REF]. In our data, RBOHB activity was also supported by the presence of positive regulators such as Rac-like/ROP GTPase. The three Rac-like/ROP GTPase ARAC1, ARAC3 and the homolog of At4g03100 were induced at 24hpi. CDPKs such as CPK1 are also known to activate RBOHs in response to increased cytosolic calcium level [START_REF] Gao | Bifurcation of Arabidopsis NLR immune signaling via Ca²⁺-dependent protein kinases[END_REF]. But repression of CPK1 in our data seems to indicate that this way of activation did not function.

In response to abscisic acid (ABA) or microbe-associated molecular pattern (MAMP) immunity, stomatal closure is known to be regulated by apoplastic ROS production (reviewed in [START_REF] Qi | Apoplastic ROS signaling in plant immunity[END_REF]) and cysteine-rich receptor-like kinases (CRK) are also known to be elements between ROS production and downstream signaling leading to stomatal closure, sometimes activated (CRK10), sometimes inhibited (CRK2 and CRK29; [START_REF] Bourdais | CRK consortium largescale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress[END_REF]). Three DEGs coding for CRK were found in our data and the repression of CRK2 and CRK29 (found two times) was consistent with the stomata closure previously found, but the repression of CRK10 (found two times) was not. Beyond closure, inhibition of stomatal development could be seen as an extreme defense. YODA (found two times) and MPK6 (found two times) MAPKs belong to a pathway involved in the negative regulation of stomata development [START_REF] Sun | Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling[END_REF].

These two genes were early induced in our data.

To conclude, in pear/V. inaequalis nonhost interaction, a calcium influx leads to the development of the stomatal closure pre-invasive defense, but also promotes a post-invasive defense: apoplastic ROS accumulation. Apoplastic ROS, acting themselves as messengers, come to strengthen the stomatal closure (Fig. 4).

Transcription factors and sphingolipids maintain HR under control

ROS are known to mediate cellular signaling associated with defense-related gene expression, hypersensitive response (HR) i. e. the programmed cell death (PCD) at the site of infection during a pathogen attack, and phytoalexin production [START_REF] O'brien | Reactive oxygen species and their role in plant defence and cell wall metabolism[END_REF]. Arabidopsis thaliana RCD1 regulator has been proposed to positively regulate cell death in response to apoplastic ROS by protein-protein interactions with transcription factors (reviewed in [START_REF] Brosché | Transcriptomics and functional genomics of ROS-induced cell death regulation by RADICAL-INDUCED CELL DEATH1[END_REF]) and WRKY70 and SGT1b were identified as cell death positive regulators functioning downstream of RCD1 [START_REF] Brosché | Transcriptomics and functional genomics of ROS-induced cell death regulation by RADICAL-INDUCED CELL DEATH1[END_REF]. RCD1 and WRKY70 genes were found induced in our data, at 24hpi and 72hpi respectively.

In Arabidopsis, the F-box protein CPR1, in association with the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex, targets for degradation NLR (nucleotide-binding domain and leucine-rich repeats containing proteins) resistance protein such as SNC1, RPM1 or RPS2, to prevent overaccumulation and autoimmunity (reviewed in [START_REF] Cheng | Stability of plant immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box (SCF)-mediated protein degradation[END_REF]). A Skp1-like (ASK19; 72hpi) gene and CPR1 (24hpi) gene were found induced in our data. A gene coding for RPM1 function was also found repressed at 24hpi. These results are in favor of the hypothesis that NLR receptors do not take part in the HR development observed in the pear/V. inaequalis nonhost interaction (Fig. 1A). In addition, the induction of an AtSerpin1 gene homolog at 24hpi (found two times) in our data is consistent with that hypothesis. Indeed, AtMC1 is a pro-death caspase-like protein required for full HR mediated by intracellular NB-LRR immune receptor proteins such as RPP4 and RPM1 [START_REF] Coll | Arabidopsis type I metacaspases control cell death[END_REF] and AtSerpin1 is a protease inhibitor which block AtMC1 self-processing and inhibit AtMC1-mediated cell death [START_REF] Lema-Asqui | AtSERPIN1 is an inhibitor of the metacaspase AtMC1-mediated cell death and autocatalytic processing in planta[END_REF].

The differential expression of two others components of the proteasome pathway is in favor of an HR development: the induction of the RIN3 ubiquitin E3 ligase (24hpi) and the repression of the BRG3 ubiquitin E3 ligase (24hpi). Indeed, RIN3 is known as positive regulator of RPM1 dependent HR [START_REF] Kawasaki | A duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1-and RPS2-mediated hypersensitive response[END_REF].

And BRG3 is known as a negative regulator of HR in plant/necrotrophic pathogen interactions [START_REF] Luo | The Arabidopsis Botrytis Susceptible1 interactor defines a subclass of RING E3 ligases that regulate pathogen and stress responses[END_REF].

Sphingolipids are involved in the control of PCD, either as structural components of membranes but also as initiators in the cell death regulatory pathway. According to Huby et al [START_REF] Huby | Sphingolipids: towards an integrated view of metabolism during the plant stress response[END_REF], free ceramides and long chain/sphingoid base components (LCBs) are able to trigger cell death, via ROS production, whereas their phosphorylated counterparts, ceramide phosphates and long chain base phosphate components (LCB-Ps) promote cell survival. The induction of PCD by LCB is based on the activation of protein kinases, among them MPK6 [START_REF] Saucedo-Garcia | MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis[END_REF]. As already mentioned, MPK6 was found early induced in our data and we found numerous DEGs in the nonhost interaction between pear and V. inaequalis that indicate the presence of free ceramides and LCB, which possibly participate to the HR development. Free LCB presence is demonstrated by the activations of SBH1 (24hpi), SLD1 (24hpi) and another sphingolipid ∆8 long-chain base desaturase homolog to At2g46210 (24hpi; found two times), and their relative conversion in ceramides is demonstrated by the differential expressions of the ceramide synthases LOH2 (repressed at 24hpi) and LOH3/LAG13 (induced at 24 and 72hpi). LCB non-conversion in phosphorylated counterparts is shown by the AtLCBK1 repression (72hpi) and free ceramides maintenance is attested by their non-conversion in glycosyled ones given the repression of a glucosyl ceramide synthase homolog to At2g19880 (24hpi).

The differential expression of numerous known regulators of HR in our data is again consistent with the HR phenotype observed. The mechanosensor MSL10 and the calmodulin-activated Ca 2+ pump (autoinhibited Ca 2+ -ATPase [ACA]) ACA11 were found engaged: at 24hpi MSL10 was induced and ACA11 was repressed. MSL10 is known as a positive regulator of cell death [START_REF] Veley | Arabidopsis MSL10 has a regulated cell death signaling activity that is separable from its mechanosensitive ion channel activity[END_REF] and ACA11 is known as a negative regulator of SA-dependent programmed cell death [START_REF] Boursiac | Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway[END_REF]. Their modulation is linked with the noticed calcium influx discussed above ( [START_REF] Boursiac | Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway[END_REF]; [START_REF] Guerringue | Sensing and transducing forces in plants with MSL10 and DEK1 mechanosensors[END_REF]). The participation of the SA pathway in the development of the hypersensitive response could also be supported by the repression of EDR1 (at 72hpi). Indeed, the MAPKKK EDR1 is known as a negative regulator of the SA-dependent HR (reviewed in [START_REF] Zhao | EDR1 physically interacts with MKK4/MKK5 and negatively regulates a MAP kinase cascade to modulate plant innate immunity[END_REF]).

Three other regulators of HR were found modulated in our data. The transcription factor AS1 (MYB91) was found induced at 24hpi. It is known as a positive regulator of HR and implicated in JA pathway (reviewed in [START_REF] Kaurilind | A genetic framework for H2O2 induced cell death in Arabidopsis thaliana[END_REF]). The transcription factor WRKY40 was found repressed at 72hpi. It is known as a negative regulator of HR [START_REF] Lee | An Arabidopsis NAC transcription factor NAC4 promotes pathogen-induced cell death under negative regulation by microRNA164[END_REF] and implicated in PTI [START_REF] Najafi | PAMP-INDUCED SECRETED PEPTIDE 3 modulates immunity in Arabidopsis[END_REF]. Another negative regulator of HR is the lipid-binding domains containing protein VAD1 [START_REF] Khafif | An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens[END_REF]. It was found repressed at 72hpi. The behavior of two others genes in our data seems to indicate that the developed HR was contained and not carried away due to too much intracellular ROS production and damages. The function UGT73B3 and CAT2 were thus activated (24hpi). UGT73B3 and CAT2 are known as restrictors of HR expansion via their action in ROS scavenging (CAT2; [START_REF] Mittler | Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection[END_REF]) or in detoxification of ROS-reactive secondary metabolites (UGT73B3; [START_REF] Simon | The secondary metabolism glycosyltransferases UGT73B3 and UGT73B5 are components of redox status in resistance of Arabidopsis to Pseudomonas syringae pv. tomato[END_REF]).

To conclude, in pear/V. inaequalis nonhost interaction, HR was spread out, in link with the calcium influx, but especially following apoplastic ROS production and ROS production via free sphingolipids accumulation and not via NLR receptors. Furthermore, the behavior of not less than height regulators indicate that the developed HR is under control (Fig. 4).

Cell wall carbohydrates content and cuticle composition are altered

The first obstacle encountered by host as well as nonhost pathogens attempting to colonize plant tissues is the plant cell wall, which is often covered with a cuticle. Preinvasive penetration barrier, as a preformed physical barrier, or as the onset place of defensive signaling pathways, is considered an important factor, especially in nonhost resistance in which non adapted pathogens normally fail to penetrate nonhost plant cells when blocked by the cell wall ([10]; [START_REF] Fonseca | Genes involved in nonhost disease resistance as a key to engineer durable resistance in crops[END_REF]). Plant cell wall alterations, of the carbohydrates or the phenolic components, either by impairing or overexpressing cell wallrelated genes, have been demonstrated to have a significant impact on disease resistance and/or on abiotic stresses (reviewed in [START_REF] Bacete | Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses[END_REF] and [START_REF] Miedes | The role of the secondary cell wall in plant resistance to pathogens[END_REF]).

We found numerous genes related to the cell wall with a modified expression during nonhost interaction between pear and V. inaequalis, among them about thirty related to the biosynthesis or the modification of carbohydrates. These genes are presented in table 3, except those related to the lignin and other phenolic compounds, which will be discussed later. We saw in particular several genes related to cellulose (8) and even more genes related to pectin [START_REF] Li | Role of beta-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato[END_REF] but no genes related to callose.

Concerning these particular carbohydrate components, the model proposed by Bacete et al [START_REF] Bacete | Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses[END_REF] is as follows. Firstly, alterations in cellulose biosynthesis from primary or secondary cell wall trigger specific defensive responses, such as those mediated by the hormones JA, ET or abscisic acid (ABA), activate biosynthesis of antimicrobial compounds, but also might attenuate pattern triggered immunity (PTI) responses. Secondly, alterations of cell wall pectins, either in their overall content, their degree of acetylation or methylation, activate specific defensive responses, such as those regulated by JA or SA, and trigger PTI responses, probably mediated by damage-associated molecular patterns like oligogalacturonides. Thus, even though our results do not completely support a role of these genes, we think that the modified expression of cell wall related genes during nonhost interaction between pear and V. inaequalis is meaningful. growth and assembling R *I: induced, R: repressed Concerning the cuticle layer, most cuticles are composed largely of cutin, an insoluble polyester of primarily long-chain hydroxy fatty acids. This lipophilic cutin framework is associated with hydrophobic compounds collectively referred to as waxes. The cuticle is also thought to contain varying amounts of intermingled cell wall polysaccharides and sometimes also a fraction termed cutan (reviewed in [START_REF] Fich | The plant polyester cutin: biosynthesis, structure, and biological roles[END_REF]). Cutin monomers are synthesized by the modification of plastid-derived 16C and 18C fatty acids in the endoplasmic reticulum (ER), yielding variously oxygenated fatty acidglycerol esters referred to as monoacylglycerols, which polymerize upon arrival at the growing cuticle (Fig. 5, reviewed in [START_REF] Fich | The plant polyester cutin: biosynthesis, structure, and biological roles[END_REF]).

C16 and C18 fatty acids are also important precursors of cuticular wax synthesis (Fig. 5). Upon transport to the ER, the C16 and C18 fatty acids are extended to form very-long-chain fatty acids (VCLFAs; C>20), and this extension is carried out by the fatty acid elongase (FAE) complex located on the ER membrane. The very-long-chain FAs are then converted into the varied cuticular waxes (primary alcohols, aldehydes, alkanes, secondary alcohols, ketones) by many ways (reviewed in [72]). Interestingly, we found three genes upregulated 24hpi belonging to the FAS (fatty acid synthase) chloroplastic complex implicated in the production of the C16 precursor (Fig. 5): ACCD, FabG and MOD1 (found two times). ACCD encodes the carboxytransferase beta subunit of the Acetyl-CoA carboxylase complex which catalyzes the first committed step in fatty acid synthesis: the carboxylation of acetyl-CoA to produce malonyl-CoA. FabG and MOD1 are respectively a β-ketoacyl ACP-reductase and an enoyl-ACP-reductase which catalyze respectively the conversion of acetoacetyl-ACP into β-hydroxyacyl-ACP and the second reductive step from enoyl-ACP to butyryl-ACP (reviewed in [72]).

In the ER, the four functions we found related to waxes biosynthesis in our data were repressed at 24hpi: KCS4 (found two times), CER1 and CER3, or 72hpi: ECR/CER10. KCS4 and ECR/CER10 belong to the FAE complex ( [START_REF] Joubès | The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling[END_REF]; [START_REF] Lee | Advances in the understanding of cuticular waxes in Arabidopsis thaliana and crop species[END_REF]). The last two genes are implicated in aldehydes (CER1) and alkanes (CER1 and 3) generation (reviewed in [72]). On the contrary, the eight genes we found connected to cutin biosynthesis were induced at 24hpi except a gene homolog to At5g14450, which was induced at 72hpi. One of them is a glycerol-3-phosphate acyltransferase (GPAT) coding gene: GPAT8, which catalyzes the transfer of a fatty acid from coenzyme A (CoA) to glycerol-3-phosphate (Fig. 4; reviewed in [START_REF] Fich | The plant polyester cutin: biosynthesis, structure, and biological roles[END_REF]). GPAT8 function in cutin formation has been functionally confirmed in association with GPAT4 [START_REF] Li | Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers[END_REF]. The seven others genes code GDSL-lipases enzyme (At1g28600, At1g28660, At1g54790, At3g16370, At3g48460, AtCUS4: At4g28780, At5g14450), some of which have been shown to function as cutin synthase (Fig. 4; [START_REF] Yeats | Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants[END_REF]; reviewed in [START_REF] Fich | The plant polyester cutin: biosynthesis, structure, and biological roles[END_REF]) and polymerize monoacylglycerols.

We also found induced respectively at 24 and 72hpi two genes involved in waxes and cutin biosynthesis positive regulation: MYB16 and SHN1. The SHN genes (SHN1-SHN3), a set of three largely redundant APETALA 2 family transcription factors from A. thaliana, are regulators of floral cutin and epidermal cell morphology. SHN1 is regulated by the MYB family transcription factor MYB106, which, along with its paralog MYB16, controls many aspects of cuticle and epidermis formation in A. thaliana (reviewed in [START_REF] Cui | Dissecting abscisic acid signaling pathways involved in cuticle formation[END_REF] and [START_REF] Fich | The plant polyester cutin: biosynthesis, structure, and biological roles[END_REF]).

Cutin and cuticular waxes play an important role in plant-insect and plant-microbe interactions.

Numerous Arabidopsis mutants in cutin and waxes biosynthetic or transport genes, such as Acyl-CoA binding proteins (ACBP), show varying degrees of cuticle impairment, alterations in cutin and/or wax composition, and defects in SAR (reviewed in [72]). We found ACBP6 repressed at 24hpi. That repression is not inconsistent with the previously described amplification of cutin biosynthesis and polymerization, given that acbp6 KO mutation is not associated with a defect in that pathway [START_REF] Xia | Acyl CoA binding proteins are required for cuticle formation and plant responses to microbes[END_REF].

That repression is also consistent with the SAR repression observed above as the acbp6 KO mutant show compromised SAR [START_REF] Xia | Acyl CoA binding proteins are required for cuticle formation and plant responses to microbes[END_REF].

To conclude, our analysis of nonhost pear/V. inaequalis interaction identified an alteration of the cuticle composition with more cutin and less waxes synthesis. The increase in cutin polymerization could lead to a thickening of the cuticular layer to prevent fungus penetration via its appressoria.

Secondary metabolism leads to G unit lignin polymerization and simple coumarin or hydrocinnamic acid amine phytoalexins synthesis

As distinguished from primary metabolism, plant secondary metabolism refers to pathways and small molecule products of metabolism that are non-essential for the survival of the organism. But they are key components for plants to interact with the environment in the adaptation to both biotic and abiotic stress conditions. Plant secondary metabolites are usually classified according to their chemical structure. Several groups of large molecules, including phenolic acids and flavonoids, terpenoids and steroids, and alkaloids have been implicated in the activation and reinforcement of defense mechanisms in plants (reviewed in [START_REF] Yang | Response of plant secondary metabolites to environmental factors[END_REF]).

Terpenoids and steroids, or isoprenoids, are components of both the primary and secondary metabolisms in cells, and mono-, tri-, sesqui-and polyterpenes are considered as secondary metabolites (reviewed in [START_REF] Tetali | Terpenes and isoprenoids: a wealth of compounds for global use[END_REF]). Our results on pear identified seven DEGs and five DEGs belonging to the chloroplastic methylerythritol posphate (MEP) and to the cytosolic mevalonic acid (MVA) pathway of isoprenoids production respectively (Table 4), which results, among others compounds, in tri-and sesquiterpenes secondary metabolites. The majority of these genes contribute to produce primary metabolites according to Tetali [START_REF] Tetali | Terpenes and isoprenoids: a wealth of compounds for global use[END_REF]. Except SMT2, that we found induced at 24hpi, there is no report concerning a putative implication of others genes in plant biotic resistance. SMT2 is involved in sterols production and smt2 mutation was reported to compromise bacterial resistance in Nicotiana benthamiana [START_REF] Wang | Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast[END_REF]. The hypothesis is that sterols regulate plant innate immunity against bacterial host and nonhost infections by regulating nutrient efflux into the apoplast. V. inaequalis is an hemi biotrophic pathogens which colonizes only the apoplast compartment since the beginning of the interaction. SMT2 strong relative induction in our data could indicate that a similar mechanism of nutrient efflux regulation via sterols could take place to limit the fungus growth in pear nonhost resistance against V. inaequalis. In our data, the other DEGs that were linked to the secondary metabolism belong to the phenylpropanoid pathway production (Fig. 6). Among them we found four genes belonging to the flavonoid production, all repressed, at 24hpi (DFR and DRM6) or 72hpi (TT7 and UGT71D1). DFR (dihydroflavonol reductase) is involved in flavan-3,4-ol production and TT7 (flavonoid 3' hydroxylase) in dihydroquercetin production from dihydro-kaempferol, and UGT71D1 (glucosyl transferase) in quercetin-glycoside production from quercetin (TAIR database; https://www.arabidopsis.org/index.jsp). DMR6 (flavone synthase) is involved in flavone production from naringenin [START_REF] Ferreyra | The identification of maize and Arabidopsis type I FLAVONE SYNTHASEs links flavones with hormones and biotic interactions[END_REF]. Thus flavonoid production does not seem to be favored, which is not consistent with the induction of MYB12 at 24hpi, but consistent with MYB4 induction at 72hpi. MYB12 is actually known as a positive regulator of flavonol biosynthesis in pear and apple fruits ( [START_REF] Wang | MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana)[END_REF]; [START_REF] Zhai | The MYB transcription factor PbMYB12b positively regulates flavonol biosynthesis in pear fruit[END_REF]) whereas MYB4 is known as a negative regulator of this biosynthetic pathway [START_REF] Wang | Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis[END_REF].

Concerning the production of monolignols, precursors of lignin synthesis, some genes were found induced, others repressed. We found CYP98A3 and CAD9 (found two times) induced at 24hpi and HCT, CCR1 and a gene homolog to At2g23910 (found two times, one time repressed at 72hpi) repressed at 24hpi Fig. 6). Lignification is obtained by cross-linking reactions of the lignin monomers or by polymer-polymer coupling via radicals produced by oxidases such as peroxidases [START_REF] Fernández-Pérez | Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana[END_REF] and laccases [START_REF] Zhao | Laccase is necessary and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis[END_REF]. However, while peroxidases are able to oxidize monolignols to produce H, G and S units of lignin, laccases only generate G units [START_REF] Fernández-Pérez | Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana[END_REF]. In our data, we found two laccases induced at 24hpi: LAC11 (found two times, one time induced at 24 and 72hpi) and 17 (found two times), and three peroxidases repressed at 24hpi: PRX17, PER47 and PRX52 (also repressed at 72hpi), which can be linked to lignin biosynthetic process (Fig. 6). According to Zhao et al. [START_REF] Zhao | Laccase is necessary and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis[END_REF], LAC11 and 17, along with LAC4, play a critical role in lignification, and their results suggests that peroxidase and laccase do not serve redundant functions in lignification, at least in the vascular tissues of the stem and root of Arabidopsis. Participation in lignin formation has also been proved for PRX17 [START_REF] Cosio | The class III peroxidase PRX17 is a direct target of the MADS-box transcription factor AGAMOUS-LIKE15 (AGL15) and participates in lignified tissue formation[END_REF], PER47 [START_REF] Tokunaga | Analysis of expression profiles of three peroxidase genes associated with lignification in Arabidopsis thaliana[END_REF] and PRX52 [START_REF] Fernández-Pérez | The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units[END_REF]. But there are currently no reports about a possible involvement of all these genes in lignification linked to biotic or abiotic stresses. Concerning non-host resistance, two reports describe lignin accumulation/deposition involvement: one in apple fruit [START_REF] Vilanova | The infection capacity of P. expansum and P. digitatum on apples and histochemical analysis of host response[END_REF] and the other one in cowpea [START_REF] Fink | Early defence responses of cowpea (Vigna sinensis L.) induced by non-pathogenic rust fungi[END_REF]. In the latest, authors showed that preferentially generated lignin units in this nonhost interaction are G units, just as it seems to be the case in our pear / V. inaequalis study. To summarize, it is tempting to think that modifications of expression observed for genes linked to lignin polymerization are relevant for the pear nonhost resistance against V. inaequalis, but further functional analysis should be conducted to conclude.

The biosynthesis of two others types of phenylpropanoid compounds appears to be favored during pear nonhost resistance against V. inaequalis: simple coumarin on one hand and hydroxycinnamic acid amides on the other hand. We found four BGLU-like genes induced at 24hpi: BGLU42 (also induced at 72hpi), 47 and BGLC3, or 72hpi: BGLU16 (Fig. 6). These β-glucosidases could be implied in simple coumarin path production from the cinnamic acid (KEGG database). Some natural simple coumarins are known as antifungal compounds in vitro and have been developed as fungicides [START_REF] Song | Evaluation of antifungal activities and structure-activity relationships of coumarin derivatives[END_REF].

Ancient work on Hevea also reports the correlation between the resistance against pathogenic fungi and the production of some coumarins, with antifungal activity in vitro [START_REF] Giesemann | Identification of scopoletin as a phytoalexin of the rubber tree Hevea brasiliensis[END_REF]. We also found induced at 24hpi the genes AACT1/ACT1, ATPAO5 and genes homologs to At4g17830 and At4g38220 (Fig. 6).

AACT1/ACT1 catalyze the first specific step in branch pathway synthesizing hydroxycinnamic acid amides from the p-Coumaroyl CoA or the feruloyl CoA and amines agmatine or putrescine [START_REF] Muroi | Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana[END_REF].

Hydroxycinnamic acid amides are produced in response to pathogenic infections [START_REF] Muroi | Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana[END_REF] and surface exported. Hydroxycinnamic acid amides are reported to participate in Arabidopsis nonhost resistance against Phytophthora infestans via their inhibitory activity on spore germination [START_REF] Dobritzsch | MATE transporterdependent export of hydroxycinnamic acid amides[END_REF]. The three others genes belong to the arginine biosynthesis path (homologs to At4g1783 and At4g38220) and the arginine and proline metabolisms which produce the amines agmatine and putrescine (ATPAO5) (KEGG database). Agmatine is directly produced from arginine thanks to an ADC activity (arginine decarboxylase) and putrescine can be produced from spermidine thanks to a PAO activity (polyamine oxidase). ATPAO5 catalyzes the conversion of spermine in spermidine. The induction of these three last genes is therefore consistent with the hypothesis of amines production in order to enable hydroxycinnamic acid amides synthesis. The induction of C4H at 24hpi could also favor hydroxycinnamic acid amides synthesis via p-Coumaroyl CoA biosynthesis promotion. C4H

(cinnamate 4-hydroxylase) catalyzes the production of p-Coumaric acid from Cinnamic acid and p-Coumaric acid gives p-Coumaroyl CoA thanks to 4CL (4-coumarate-CoA ligase) (KEGG database).

Among the suite of defense components synthetized in nonhost as in host context, a chemical barrier can be established via accumulation of a diverse array of secondary metabolites rapidly produced upon pathogen infection, named phytoalexins, with toxic or inhibitory effects (reviewed in [10]).

Phytoalexins can be flavonoids, such as the pisatin of pea (in [START_REF] Celoy | +)-Pisatin biosynthesis: from (-) enantiomeric intermediates via an achiral 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene[END_REF]) but also varied phenylpropanoid compounds. In the nonhost interaction pear / V. inaequalis, the production of flavonoid type phytoalexins does not seem to be favored, except simple coumarin and hydroxicinnamic acid amines.

Very limited transcriptomic modulation during apple / V. pyrina nonhost interaction Only 60 DEGs were detected in the apple / V. pyrina nonhost interaction at 24 or 72hpi, in agreement with the total absence of macroscopic symptoms and few cells engaged in an HR-like reaction observed at the microscopic level. Among these 60 DEGs, 36 have no known function. Among the 24 remaining DEGs, nine DEGS could be relevant in apple / V. pyrina nonhost interaction in view of our findings in pear / V. inaequalis nonhost interaction. ORG2 (BHLH038), a putative integrator of various stress reactions [START_REF] Vorwieger | Iron assimilation and transcription factor controlled synthesis of riboflavin in plants[END_REF] was induced at 24hpi. Three genes were related to an oxidative stress: GASA10 was repressed at 24hpi and NRAMP3 and AOR were induced at 24hpi. GASA proteins have been suggested to regulate redox homeostasis via restricting the levels of OH . in the cell wall [START_REF] Trapalis | The Arabidopsis GASA10 gene encodes a cell wall protein strongly expressed in developing anthers and seeds[END_REF]. The repression of this gene is thus in favor of more OH . in the cell wall. The oxidoreductase coding gene AOR is known in the chloroplast to contribute to the detoxification of reactive carbonyls produced under oxidative stress [START_REF] Yamauchi | Chloroplastic NADPH-dependent alkenal/one oxidoreductase contributes to the detoxification of reactive carbonyls produced under oxidative stress[END_REF]. NRAMP genes function as positive regulators of ROS accumulation, especially during Arabidopsis Erwinia chrisanthemi resistance [100]. The induction (at 24 and 72hpi) of another gene suggests modifications at the cell wall level: EXP8, an expansin coding gene involved in cell wall loosening (Tair database). We also found two genes related to hormone pathways, one induced at 24hpi: WIN1 and the other one repressed at 72hpi: UBP12. WIN1 is known as a negative regulator of SA pathway [101] and UBP12 as a positive regulator of JA pathway via the stabilization of MYC2 [START_REF] Jeong | The deubiquitinating enzymes UBP12 and UBP13 positively regulate MYC2 levels in jasmonate responses[END_REF]. In link with the JA pathway, we also found TPS21 induced at 24hpi. TPS21 is involved in sesquiterpenes production and is promoted by JA signal via MYC2 [102]. TPS21 is especially involved in the jasmonate-dependent defensive emission of herbivore-induced volatiles in cranberries [103].

Finaly the last DEG we found relevant in apple / V. pyrina nonhost interaction could promote HR via ceramides accumulation. ACD11 is repressed at 24hpi in our data. In acd11 mutants, the relatively abundant cell death inducer phytoceramide rises acutely [104].

Because nonhost resistance of apple against V. pyrina is of a type I, with a very limited number of cells engaged in an HR-like reaction, it has not been possible for us to exhaustively describe how this interaction is expressed at the transcriptomic level. Further insight with more adapted technics such as laser-assisted cell picking, prior to micro arrays or RNA sequencing analysis (review in [105]) could provide more information in the future.

Comparison of pear resistances against the host pathogen V. pyrina and the nonhost pathogen V. inaequalis reported a major role of ethylene signalization during the compatible interaction between P. pyrifolia and Alternaria alternata, a necrotrophic pathogen. Finally, Xu et al.

[109] applied RNA-seq to characterize the genes of Penicillium expansum activated after infection of pear fruits. None of these studies can be directly compared to our work on host and nonhost scab pear resistance.

Concerning the recognition and early signaling steps of the interactions, many receptors and coreceptors have been found induced in the host pear resistance, especially damage-associated molecular patterns receptors such as RLK7, revealing that PTI and ETI must be engaged. We did not found evidence of the mobilization of such receptors in the pear nonhost resistance. PTI and ETI receptors are nonetheless reported as implicated in nonhost resistance (reviewed in [110] and [10]).

As we only analyzed post infection transcriptional modulations in the nonhost pear/V. inaequalis interaction (at 24 and 72hpi), one hypothesis to explain the lack of PTI and ETI receptors in our data could be that these receptors were already present as preformed defenses and not particularly induced by the infection onset. In pear nonhost interaction, the earliest signaling pathways we were able to highlight are calcium influx and apoplastic ROS production. Calcium signaling seems to be also implicated in pear host resistance, but less obviously than in nonhost resistance.

About the hormonal signaling pathways, the JA defense signaling pathway was found repressed in pear nonhost resistance but quite activated in pear host resistance. The JA/ethylene (ET) defense signaling pathway is known as an effective defense against necrotrophic fungi in Arabidopsis [111].

Thus, it is not surprising to find the JA pathway repressed in the development of the pear nonhost resistance against the hemi-biotrophic pathogen V. inaequalis. But it is very interesting to find this pathway rather induced in the development of the pear host resistance against the other hemibiotrophic pathogen V. pyrina. The SA signaling pathway is commonly seen as the classical one triggered to resist biotrophic fungi in Arabidopsis [111], but only a little engagement in pear nonhost resistance has been observed, SA signaling being repressed in pear host resistance. If this absence of SA implication is quite unexpected in pear host resistance against a hemi-biotrophic fungus, it is consistent with the report that the exact role of these key defense phytohormone is unclear in nonhost resistance and remains to be established [START_REF] Fonseca | Genes involved in nonhost disease resistance as a key to engineer durable resistance in crops[END_REF]. As shown by Tsuda et al [112], an explanation for the hormone pathways behavior in pear host resistance could be that: as both the SA and JA/ET pathways positively contribute to immunity, a loss of signaling flow through the SA pathway can be compensated by a rerouting signal through the JA/ET pathways. In addition, independently of SA signaling, but in positive connection with JA signaling, SAR seems to be engaged in distal tissues during pear host resistance. To conclude, in pear host as well as nonhost resistances, classical resistance hormones SA and JA/ET, and the correlative PR gene defenses, seems differently involved than in Arabidopsis.

The carbohydrate content of the cell-wall is modified in response to the attacks by the pathogens.

Regarding cell-wall and cuticle, in pear host as well as nonhost resistances, important modifications were highlighted. Similar modifications affected the cellulose and mainly the pectin contents, but no callose production was observed. Regarding cuticle, waxes production was induced in host resistance whereas it was repressed in nonhost resistance, in favor of cutin production / polymerization, which was also induced in host resistance. To conclude, as a first obstacle encountered by host as well as nonhost pathogens attempting to colonize plant tissues, the plant cell wall and its cuticle seem to play a foreground role in pear host as well as nonhost resistances.

Finally, the production of secondary metabolites and phenylpropanoids compounds in particular, seems to be a major line of defense, in pear host as well as nonhost resistances, but with divergences. If lignin and flavonoid productions are preponderant in pear host resistance against V.

pyrina, lignin implication in pear nonhost resistance is less clear and flavonoids production is obviously repressed. But the biosynthesis of two other types of phenylpropanoid-derived phytoalexins appears to be favored during pear nonhost resistance: simple coumarin on one hand and hydroxycinnamic acid amides on the other hand.

The comparative analysis between a host and a nonhost resistance in pear shows that, even though specificities are observed, the two major defense lines engaged are shared: the cell wall and its cuticle on one hand, the secondary metabolism with the phenylpropanoid pathway on the other hand. Moreover, these defenses seem deployed largely independently of the SA signaling pathway, widely recognized as the main defense hormone against biotrophic pathogens.

Conclusion

As far as we know, our work is the first one published regarding a transcriptomic analysis of postinfections events of a nonhost resistance to Venturia sp. in apple and pear. Velho and Stadik [113] recently published a detailed description of the apple / Colletotrichum higginsianum nonhost resistance, highlighting the accumulation of callose at the sites of penetration of the fungus. But no data on gene expression was included. Here, our molecular work on apple / V. pyrina nonhost resistance remains preliminary and in order to allow a deeper deciphering, further analyses must be considered with the aid of tools adapted to this type I nonhost resistance with very few cells engaged in an HR-like reaction, only visible at a microscopic level. In pear, this deciphering allowed us to show that nonhost resistance against V. inaequalis is a type II one, which involves enough pathogen penetration in plant tissue to trigger visible HR and develops post-invasive defenses.

To summarize our findings on pear with a notion of cascading effect, we can propose the following scenario (Fig. 4): once V. inaequalis presence is recognized by pear, a calcium cellular influx is induced and leads to the development of a pre-invasive defense, the stomatal closure, but also promotes an early post-invasive defense, an apoplastic ROS accumulation. Apoplastic ROS, acting themselves as ubiquitous messengers, come to reinforce the stomatal closure but also mediate cellular signaling resulting in two post-invasive defenses: HR development at infection sites, along with phytoalexin (simple coumarin and hydroxicinnamic acid amines) production. The observed alterations of the epidermis composition (cellulose, pectin, lignin for the cell wall, and cutin for the cuticle), are presumed to strengthen this physical barrier and can be seen as the development of another pre-invasive defense. The calcium (action on pectin reviewed in [114]) and the ROS (action on lignin, [115]; [116]; action on cuticle, [117]) have been linked to some type of epidermis modifications and may participate in the proceeding of these defense in pear / V. inaequalis nonhost interaction.

Nonhost resistance is defined as the resistance of an entire plant species against a specific parasite or pathogen [118] and is seen as the most durable resistance of plant.

Material and methods

Biological material

Apple plants from the cultivar 'Gala' and pear plants from the cultivar 'Conference' were chosen because of their susceptibility to V. inaequalis and V. pyrina, respectively. The apple and pear genotypes were multiplied in vitro, rooted and acclimatized in greenhouse as described previously

Transcriptomics experiment

Leaf samples were immediately frozen in liquid nitrogen and kept at -80°C until analysis. Sampling concerned the youngest expanded leaf of each plant labeled the day of the inoculation. Each sample is a pool of leaves from three different plants and two biological repeats (n=2) have been made by condition (genotype x treatment x time). Leaf samples taken just before inoculation (T0) and at 24 and 72hpi, were then used to perform transcriptomics analyses.

For RNA extraction, frozen leaves were ground to a fine powder in a ball mill (MM301, Retsch, Hann, Germany). RNA was extracted with the kit NucleoSpin RNA Plant (Macherey Nagel, Düren, Germany) according to the manufacturer's instructions but with a modification: 4% of PVP40 (4 g for 100 ml)

were mixed with the initial lysis buffer RAP before use.

QPCR validation of transcriptomic data

In order to validate transcriptomic data, QPCR was performed on a selection of gene/sample associations. First-strand cDNA was synthesized using total RNA (2.0 μg) in a volume of 30 μl of 5× buffer, 0.5 μg of oligodT15 primer, 5 μl of dNTPs (2.5 mM each), and 150 units of MMLV RTase (Promega, Madison, WI, USA). The mixture was incubated at 42°C for 75 min. Quantitative RT-PCR (QPCR) was then performed. Briefly, 2.5 µl of the appropriately diluted samples were mixed with 5 µl of PerfeCTa SYBR Green SuperMix for iQ kit (Quantabio, Beverly, MA, USA) and 0.2 or 0.6 µl of each primer (10 µM) in a final volume of 10 µl. Primers were designed with Primer3Plus, their volumes were according to their optimal concentration (determined for reaction efficiency near to 100%; calculated as the slope of a standard dilution curve; [134]). Accessions, primer sequences and optimal concentrations are indicated in Table S1. The reaction was performed on a CFX Connect Real-Time System (BIO-RAD, Hercules, CA, USA) using the following program: 95°C, 5 min followed by 40 cycles comprising 95°C for 3 s, 60°C for 1 min. Melting curves were performed at the end of each run to check the absence of primer-dimers and nonspecific amplification products. Expression levels were calculated using the ΔΔCT method [135] and were corrected as recommended in

Table 1 :

 1 Scab qualitative note of pear and apple lines inoculated with V. pyrina and V. inaequalis. .Percentage of plants in the different classes of symptoms, 42 days after inoculationClass of symptomsV. pyrina strain VP102V. inaequalis strain EUB05 'absence of symptoms Class 1: hypersensitivity (pin points) Class 2: resistance (chlorotic lesions, slight necrosis, crinkled aspect) Class 3a: weak resistance (necrotic or chlorotic lesions with occasional very light sporulation) Class 3b: weak susceptibility (clearly sporulating chlorotic or necrotic lesions Class 4: susceptibility (sporulation only)

  CYP98A3 encode a C3H (coumarate 3-hydroxylase), CAD9 encode a CAD (cinnamyl alcohol dehydrogenase), HCT is an hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, CCR1 encode a CCR (cinnamoyl-CoA reductase) and At2g23910 encode a CCR-related protein. (TAIR and KEGG databases (https://www.genome.jp/kegg/)).

  Perchepied et al[12] performed a detailed transcriptomic analysis of the host resistance of pear against V. pyrina strain VP102, deployed in a transgenic pear bearing the well-known apple Rvi6 resistance gene against V. inaequalis. Comparing this work to our gives us the rare opportunity to analyze similarities and differences between a host and a nonhost resistance in the same plant. Only four transcriptomic studies involving pear/pathogen interactions have been published so far. Yan et al[106] reported the modulation of expression of 144 pear genes after fruit treatment by Meyerozyma guilliermondii, an antagonistic yeast used for biocontrol of natural pear fruit decay.Zhang et al [107] similarly reported the modulation of expression of 1076 pear genes after treatment with Wickerhamomyces anomalus, another biocontrol agent. Using RNA-seq,Wang et al. [108]

Fig. 1 :Fig. 2 :

 12 Fig. 1: Macro-and microscopic observations of nonhost interactions.

Fig. 3 :

 3 Fig. 3: DEGs involved in hormonal pathways during pear/V. inaequalis non-host interaction.

Fig. 4 :

 4 Fig. 4: Scenario of major events observed while three first days of pear/V. inaequalis non-host

Fig. 5 :

 5 Fig. 5: Main DEGs involved in cutin and wax biosynthesis during pear/V. inaequalis non-host

Fig. 2

 2 Fig. 2

  Fig.3

  Fig. 6

Table 2 . Number of DEGs identified during apple and pear nonhost response to V. pyrina and V. inaequalis

 2

		'Gala' / VP102	'Conference' / EUB05
		24hpi	72hpi	24hpi	72hpi
	Total # of DEGs*	49	11	1570	364

Table 3 : Main DEGs related to cell wall carbohydrates synthesis/modification detected during non- host interaction pear/V. inaequalis.

 3

		Gene	Action	Expression*
		Primary cell wall
		CSLA2	synthesis	I
		PNT1	synthesis	I
	Cellulose	COBL2	deposition (GPI-anchored protein) R
		AtGH9A4	catabolism	I
		XTR7	loosening	I
	Hemi-cellulose (xyloglucan) At5g15490	synthesis	I
		At3g42180	synthesis	I
		At4g01220	synthesis	I
		GHMP kinase synthesis	I
		RHM1	synthesis	R
		PME
		At2g45220	methylesterification	I
	Pectin	PME
		At2g46930	methylesterification	I
		PME
		At3g05910	methylesterification	R
		PME
		At1g02810	methylesterification	R
		PME44	methylesterification	R

Table 4 : Main DEGs involved in biosynthetic pathways for terpenes and isoprenoids during pear/V. inaequalis non-host interaction.

 4

	Gene	Function

  Thus, understanding the molecular mechanisms underlying nonhost resistance can open up some interesting avenues to create sustainable host resistances in the same plant species. Considering pear, in order to stop the germination and entrance of hemibiotrophic host fungi such as V. pyrina, strengthening the cuticle

	initial barrier via more cutin production and cross-link, or promoting the biosynthesis of phytoalexins
	like hydroxycinnamic acid amines, appear as promising solutions, relatively easy to engineer
	regarding recent advances in biotechnology tools on this species ([119]; [120]; [121]).

  Venturia pyrina strain VP102 for apple and Venturia inaequalis strain EUB04 for pear. Symptoms were recorded at 14, 21, 28, 35 and 42 days after inoculation. The type of symptoms was scored using the 6 class-scale of Chevalier et al[128].

	([122]; [123]). For apple scab inoculation, the V. inaequalis monoconidial isolate used was EU-B05 from the European collection of V. inaequalis of the European project Durable Apple Resistance in Europe [124]. For pear scab inoculation, the monoconidial strain VP102 of V. pyrina was chosen for its aggressiveness on 'Conference' [125]. Scab inoculation procedure Greenhouse growth conditions and mode of inoculum preparation were as described in Parisi and Lespinasse [126] for apple and Chevalier et al [127] for pear. Briefly, the youngest leaf of actively growing shoots was tagged and the plants inoculated with a conidial suspension (2 × 10 5 conidia ml -1 ) Histological studies were made on samples stained with the fluorophore solophenylflavine [129]. In brief, leaf discs were rinsed in ethanol 50° before staining in a water solution of solophenylflavine 7GFE 500 (SIGMA-Aldrich, St Louis USA) 0.1% (v/v) for 10 min. The samples were first rinsed in deionized water, then in glycerol 25% for 10 min. Finally, the leaf samples were mounted on glass-slides in a few drops of glycerol 50%. They were examined with a wide-field epifluorescence microscope BH2-RFC Olympus (Hamburg, D) equipped with the following filter combination: of Microscopic observations excitation filter 395 nm and emission filter 504 nm.

  For transposable elements we used one consensus sequence for each family and a randomly peaked number of elements proportionally to their respective abundance in the genome. The microarray used in this study also have probes for coding genes of V. inaequalis but they have not been taken into account.For pear the design was done on the Pyrus communis Genome v1.0 Draft Assembly & Annotation available on GDR (https://www.rosaceae.org/species/pyrus/pyrus_communis/genome_v1.0) web site. We have downloaded the reference genome and gene predictions fasta files and structural annotation gff file the 21st of September 2015. Using home-made Biopython scripts we have extracted spliced CDS sequences with 60 nucleotides before start and after stop codons to get UTRlike sequences likely to be found on transcripts resulting in a fasta file containing 44491 sequences. Metadata for the 172 (162 for pear and 10 for apple) DEGs discussed in this work are available in TableS2and S3 (Online only).

	PCP017051 for pear, available at https://www.rosaceae.org/, with the datasets on "Pyrus communis
	v1.0 draft genome") were used to check the absence of genomic DNA contamination by PCR. The
	PCR reaction conditions were as follows: 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 60°C
	for 45 s, 72°C for 1 min, with a final extension at 72°C for 5 min. The PCR products were separated on
	a 2% agarose gel.
	Amplifications (aRNAs) were produced with MessageAmpII aRNA Kit (Ambion Invitrogen, Waltham,
	MA, USA), from 300 ng total RNA. Then 5 µg of each aRNA were retrotranscribed and labelled using a
	SuperScript II reverse transcriptase (Transcriptase inverse SuperScript™ II kit, Invitrogen, Carlsbad,
	Purity and concentration of the samples were assayed with a Nanodrop spectrophotometer ND-1000 (ThermoFisher Scientific, Waltham, MA, USA) and by visualization on agarose gel (1% (weight/volume) agarose, TAE 0.5x, 3% (volume/volume) Midori green). Intron-spanning primers (forward primer: CTCTTGGTGTCAGGCAAATG, reverse primer: CA, USA) and fluorescent dyes: either cyanine-3 (Cy3) or cyanine-5 (Cy5) (Interchim, Montluçon, France). Labeled samples (30 pmol each, one with Cy3, the other with Cy5) were combined two by two, depending on the experimental design. For each comparison two biological replicates were analyzed in dye-switch as described in Depuydt et al [130]. Paired labeled samples were then co-hybridized to Agilent microarray AryANE v2.0 (Agilent-070158_IRHS_AryANE-Venise, GPL26767 at GEO: https://www.ncbi.nlm.nih.gov/geo/) for apple, or Pyrus v1.0 (Agilent-078635_IRHS_Pyrus, GPL26768 at GEO) for pear, containing respectively 133584 (66792 sense and 66792 anti-sense probes) and 87812 (43906 sense and 43906 anti-sense probes) 60-mer oligonucleotide probes. The hybridizations were performed as described in Celton, Gaillard et al [131] using a MS 200 microarray scanner (NimbleGen Roche, Madison, WI, USA). For microarray analysis we designed two new chips. For apple we used a deduplicated probeset from the AryANE v1.0 ([131]; 118740 probes with 59370 in sense and 59370 in anti-sense) augmented by 14844 probes (7422 in sense and 7422 in anti-sense) designed on new gene annotations from Malus domestica GDDH13 v1.1 (https://iris.angers.inra.fr/gddh13 or https://www.rosaceae.org/species/malus/malus_x_domestica/genome_GDDH13_v1.1). These probes target new coding genes with UTRs when available, manually curated micro-RNA precursors similarity. This file was sent to the eArray Agilent probe design tool sense probes and filtered to remove duplicated probes. The final probeset contains 87812 unique probes targeting 1 (73612 probes) or more (14200 probes) potential transcript both in sense and anti-sense. Normalization and statistical analyses performed to get normalized intensity values have been done as in Celton, Gaillard et al [131]. For each comparison and each probe, we retrieved a ratio of the logarithms of the fluorescence intensities (one per compared sample: T0 versus 24hpi or T0 versus 72hpi in our case) and an associated p-value. The applied p-value threshold to determine DEGs (differentially expressed genes) was 0.05. Through blast analyze, a TAIR accession number (The Arabidopsis Information Resource; https://www.arabidopsis.org/; [132]) has been linked to a majority of apple or pear "probe/corresponding gene" and the couple "TAIR accession/ratio value" and transposable elements. These 60 nucleotides size increase the probability of finding specific probes on genes with high has then been used to make a global analyze of functional categories observed in the Mapman
	TCAAGGTTGGTGGACCTCTC) designed on the EF-1α gene (accession AJ223969 for apple and

(https://earray.chem.agilent.com/earray/) to generate one probe per gene prediction. Options used were: Probe Length: 60, Probe per Target: 1, Probe Orientation: Sense, Design Options: Best Probe Methodology, Design with 3' Bias. The probeset was then reverse-complemented to generate antisoftware (https://mapman.gabipd.org/homemapman.gabipd.org; [133]). The detailed analyze of DEGs has been done through TAIR and KEGG (https://www.genome.jp/kegg/) databases, and bibliography.

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Acknowledgements

The authors gratefully acknowledge the IRHS-ImHorPhen team of INRA Angers for technical assistance in plant maintenance and the technical platforms ANAN and IMAC.

Funding

This project was funded by the Synthé-Poir-Pom project (Angers University) and by the TIFON project (INRAE, department BAP).

Availability of data and materials

The datasets supporting the conclusion of this article are available in the Gene Expression Omnibus (GEO) repository [https://www.ncbi.nlm.nih.gov/geo/] with GSE159179 and GSE159180 accession numbers for apple and pear respectively.

Supplementary information

Additional File 1: Table S1

Consent for publication

This section is not applicable.

Competing interests

The authors declare that they have no competing interests