L Camborde

A Kiselev

M J C Pel

A Leru

A Jauneau

C Pouzet

B Dumas

Elodie Gaulin
email: gaulin@lrsv.ups-tlse.fr

A DEAD BOX RNA helicase from Medicago truncatula is hijacked by an RNA-binding effector from the root pathogen Aphanomyces euteiches to facilitate host infection

Keywords: effectors, RNA-helicase, plant development, ribosome biogenesis pathway, oomycete, Medicago, nucleolar stress

Microbial effectors from plant pathogens are molecules that target host components to facilitate colonization. While eukaryotic pathogens are virtually able to produce hundreds of effectors, the molecular mechanisms allowing effectors to promote infection are still largely unexplored. Here we show that the effector AeSSP1256 from the soilborne oomycete pathogen Aphanomyces euteiches is able to interact with plant RNA. Heterologous expression of AeSSP1256 delays Medicago truncatula host roots development and facilitate pathogen colonization. Transcriptomic analyses of AeSSP1256-expressing roots show a downregulation of genes implicated in ribosome biogenesis pathway. A yeast-two hybrid approach reveals that AeSSP1256 associates with a nucleolar L7 ribosomal protein and a M. truncatula RNA helicase (MtRH10) orthologous to the Arabidopsis RNA helicase RH10. Association of AeSSP1256 with MtRH10 impaired the capacity of MtRH10 to bind nucleic acids. Promoter:GUS composite plants revealed that MtRH10 is expressed preferentially in the meristematic root cells. Missense MtRH10 plants displayed shorter roots with developmental delay and are more susceptible to A. euteiches infection. These results show that the effector AeSSP1256 facilitates pathogen infection by causing stress on plant ribosome biogenesis and by hijacking a host RNA helicase involved in root development and resistance to root pathogens.

Introduction

Plant pathogens divert host cellular physiology to promote their own proliferation by producing effector proteins that interact with molecular targets [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF].

Numerous studies indicate large variation in the effector repertoire of plant pathogens suggesting that a large number of molecular mechanisms are targeted.

Oomycetes constitute a large phylum that includes important eukaryotic pathogens, and many of which are destructive plant or animal pathogens [START_REF] Kamoun | The Top 10 oomycete pathogens in molecular plant pathology[END_REF]van West and Beakes, 2014). They share common morphological characteristics with true fungi as filamentous growth, osmotrophic feeding or the presence of a cell wall, but they evolved independently [START_REF] Judelson | Metabolic diversity and novelties in the Oomycetes[END_REF]. Oomycetes are included in the Stramenopile lineage and have diatoms and brown algae as closest cousins. These filamentous microorganisms have the capacity to adapt to different environment as illustrated by their capacity to develop resistance to anti-oomycete chemicals or quickly overcome plant resistance (Rodenburg et al., 2020).

Comprehensive identification of oomycete proteins that act as effectors is challenging. Up to now, computational predictions of effector proteins have provide a fast approach to identify putative candidate effectors in oomycetes [START_REF] Haas | Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans[END_REF]Tabima and Grünwald, 2019).

Based on their predictive subcellular localization within the host cells they are classified as extracellular (apoplasmic) or intracellular (cytoplasmic) effectors. As example, RxLR and Crinklers (CRNs) constitute the two largest family of oomycetes intracellular effectors that contain hundreds of members per family (McGowan and Fitzpatrick, 2017). While oomycete effector proteins have probably different mechanisms of action, what they have in common might be the ability to facilitate pathogen development. Nonetheless, computational predictions do not give any clues regarding the putative role of theses effectors since numerous effectors are devoid of any functional domains. Therefore, biochemical and molecular studies are used to discover and confirm the functional activity of these proteins.

To promote infection oomycete intracellular effectors interfere with many host routes which include for example signaling such as MAPKinase cascades [START_REF] King | Phytophthora infestans RXLR effector PexRD2 interacts with host MAPKKK ε to suppress plant immune signaling[END_REF], phytohormone-mediated immunity (Boevink et al. 2016;[START_REF] Liu | Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis[END_REF], trafficking vesicles secretion [START_REF] Du | Phytophthora infestans RXLR effector AVR1 interacts with exocyst component Sec5 to manipulate plant immunity[END_REF] or autophagosome formation (Dagdas et al., 2016). Growing evidences point to plant nucleus as an important compartment within these interactions thanks to the large portfolio of putative nucleus-targeted effectors predicted in oomycete genomes.

The study of subcellular localization of fifty-two Phytophthora infestans RxLR effectors is the most common pattern, with 25% effectors that display a strong nuclear association (Wang et al. 2019). The CRN family was firstly reported as a class of nuclear effector from P. infestans (Schornack et al., 2010), around 50% of predicted NLS-containing CRN effectors from P. capsici showed nuclear localization (Stam et al., 2013) and numerous CRNs effectors from P. sojae such as PsCRN108, PsCRN63 or PsCRN115 harbor a nuclear localization (Song et al., 2015;Zhang et al., 2015). In agreement with this, different mechanisms of action at the nuclear level have been reported for oomycete effectors such as the alteration of genes transcription (Wirthmueller et al., 2018;Song et al., 2015;[START_REF] He | Phytophthora infestans effector SFI3 targets potato UBK to suppress early immune transcriptional responses[END_REF], the mislocalisation of transcription factor (Mclellan et al., 2013), the suppression of RNA silencing by inhibition of siRNA accumulation (Qiao et al., 2015;Xiong et al., 2014) or the induction of plant DNAdamage (Camborde et al. 2019;Ramirez-Garcés et al. 2016). However specific function has been assigned to very few effectors.

We previously use comparative genomics and predictive approaches on the Aphanomyces genus to identify putative effectors and characterized a large family of small secreted proteins (SSPs) [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF]. SSPs harbor a predicted N secretion signal, are less than 300 residues in size and devoid of any functional annotation. More than 290 SSPs are predicted in the legume pathogen A. euteiches (AeSSP) while 138 members with no obvious similarity to AeSSP members are reported in the crustacean parasite A. astaci [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF]. This specific SSP repertoire suggests its role in adaption of Aphanomyces species to divergent hosts. We have previously identified one AeSSP (AeSSP1256) based on a screen aiming to identify SSP able to promote infection of Nicotiana benthamiana plants by the leaf pathogen Phytophthora capsici. AeSSP1256 harbors a nuclear localization signal indicating its putative translocation to host nucleus. However, the function of this protein remained to be identified.

Here we report on the functional analysis of AeSSP1256 and the characterization of its plant molecular target. We show that AeSSP1256 binds RNA in planta, induces developmental defects when expressed in M. truncatula roots and promotes A. euteiches infection. This phenotype is correlated with a downregulation of a set of ribosomal protein genes. A yeast two-hybrid approach identified a host RNA helicase (MtRH10) and a L7 ribosomal protein as interactors of AeSSP1256. By FRET-FLIM analyses we reveal that AeSSP1256 co-opts MtRH10 to abolish its nucleic acid binding capacity. We provide a mechanistic explanation of this observation by demonstrating the implication of MtRH10 in roots development by generating missense and overexpressing Medicago lines. Finally we observed that silenced-MtRH10 roots are highly susceptible to A. euteiches infection like AeSSP1256-expressing roots, showing that MtRH10 as AeSSP1256 activities modify the outcome of the infection. We now present results supporting effector-mediated manipulation of a nuclear RNA helicase as a virulence mechanism during plant-eukaryotic pathogens interactions.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Results

AeSSP1256 contains RGG/RG domains and binds RNA in planta

AeSSP1256 is a member of a large family of A. euteiches effectors devoid of any predicted functional domain, except the presence of a signal peptide at the N-terminus [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF]. As showed in Figure 1A, AeSSP1256 protein is enriched in glycine residues (30% of the amino acid sequence). Analysis using the Eukaryotic Linear Motif database (Gouw et al., 2018) revealed 3 GGRGG motifs (positions 81-85;95-99 and 99-103). These motifs are variant arginine methylation site from arginine-glycine(-glycine) (RGG/RG) domains, presents in many ribonucleoproteins and involved in RNA binding [START_REF] Thandapani | Defining the RGG/RG Motif[END_REF]Bourgeois et al., 2020). We then noticed the presence of two di-RGG domains (RGG(X 0- 5 )RGG) (position 75-85 and 97-103) and one di-RG domains (RG(X 0-5 )RG) (position 123-126) corresponding to RGG or RG motifs that are spaced less than 5 residues (Chong et al., 2018). According to RGG/RG definition, those repeats occur in low-complexity region of the protein (position 60-180) (Chong et al., 2018) and are associated with di-glycine motifs and GR or GGR sequences (Figure 1A), which are also common in RGG/RG-containing proteins (Chong et al., 2018). Considering that RGG/RG domains are conserved from yeast to humans (Rajyaguru and Parker, 2012) and represent the second most common RNA binding domain in the human genome (Ozdilek et al., 2017), we therefore investigated the RNA binding ability of AeSSP1256. To test this, we performed a FRET-FLIM assay on N. benthamiana agroinfiltrated leaves with AeSSP1256:GFP fusion protein in presence or absence of Sytox Orange to check its capacity to bind nucleic acids (Camborde et al. 2017). Briefly AeSSP1256:GFP construct is transiently express in N. benthamiana leaves where it accumulates in the nucleus [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF]. Samples are collected 24h after treatment and nucleic acids labeled with the Sytox Orange dye. In presence of Sytox, if the GFP fusion protein is in close proximity (<10nm) with nucleic acids, the GFP lifetime of the GFP tagged protein will significantly decrease, due to energy transfer between the donor (GFP) and the acceptor (Sytox). To distinguish RNA interactions from DNA interactions, an RNase treatment can be performed. In the case of a specific RNA-protein interaction, no FRET acceptor will be available due to RNA degradation and the lifetime of the GFP tagged protein will then return at basal values. It appeared that GFP lifetime of AeSSP1256:GFP decreased significantly in presence of Sytox Orange as reported in table 1 and in Figure 1B, decreasing nucleic acids. After an RNase treatment, no significant difference on GFP lifetime was observed in absence (2.01 ns +/-0.02) or in presence (1.96 ns +/-0.02) of Sytox Orange, meaning that the FRET was not due to DNA interaction but was specific to RNA (table 1 andFigure 1B). These results indicate that AeSSP1256 is able to bind nuclear RNA in plant cells.

AeSSP1256 impairs M. truncatula root development and susceptibility to A. euteiches

To check whether expression of AeSSP1256 may have an effect on the host plant, we transformed M. truncatula (Mt) roots, with a native version of GFP tagged AeSSP1256. As previously observed [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF], confocal analyses confirmed the nuclear localization of the protein in root cells, with accumulation around the nucleolus as a perinucleolar ring (Figure 2A) despite the presence of a signal peptide [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF].

Anti-GFP western blot analysis on total proteins extracted from transformed roots confirmed the presence of GFP-tagged AeSSP1256 (46.7 kDa expected sizes) (Figure 2B). We noticed the presence of a second band around 28 kDa, which is probably free GFP due to the cleavage of the tagged protein. AeSSP1256:GFP transformed plants showed delayed development (Figure 2C), with total number of roots and primary root length per plant being significantly lower than values obtained with GFP control plants (Figure 2D). As previously observed in N. benthamiana, when a KDEL-endoplasmic reticulum (ER) retention signal is added to the native AeSSP1256 construct [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF], AeSSP1256:KDEL:GFP proteins mainly (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

accumulates in the ER (Supplemental Figure 1A-C) and roots showed no significant differences in development as compared to GFP control roots (Supplemental Figure 1D andE). In contrast a construct devoid of a native signal peptide (SP) shows that the proteins accumulated in root cell nuclei (Supplemental Figure 1B), leading to abnormal root development, with symptoms similar to those observed in presence of the AeSSP1256:GFP construct, including shorter primary root and lower number of roots (Supplemental Figure 1D andE). Altogether these data show that within the host, AeSSP1256 triggers roots developmental defects thanks to its nuclear localization.

To investigate whether AeSSP1256 modifies the outcome of the infection, AeSSP1256transformed roots were inoculated with A. euteiches zoospores. RT-qPCR analyses at 7, 14 and 21 days post inoculation were performed to follow pathogen development. At each time of the kinetic, A. euteiches is more abundant in M. truncatula roots expressing the effector than in GFP control roots (respectively 1.5, 3 and 5 times more) (Figure 1E). This indicates that roots are more susceptible to A. euteiches in presence of AeSSP1256. Transversal sections of A17-transformed roots followed by Wheat-Germ-Agglutinin (WGA) staining to detect the presence of A. euteiches, showed that the pathogen is still restricted to the root cortex either in the presence or absence of AeSSP1256 (Supplemental Figure 2). This phenotype is similar to the one observed in the natural A17 M. truncatula tolerant line infected by A. euteiches [START_REF] Djébali | Partial Resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes[END_REF]. This data suggests that defence mechanisms like protection of the central cylinder [START_REF] Djébali | Partial Resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes[END_REF] are still active in AeSSP1256expressing roots.

AeSSP1256 affects the expression of genes related to ribosome biogenesis

To understand how AeSSP1256 affects M. truncatula roots development and facilitates A.

euteiches infection, we performed expression analyses by RNASeq using AeSSP1256expressing roots and GFP controls roots. 4391 genes were differentially express (DE) between the two conditions (p adjusted-value <10 -5 ) (Supplemental Table 1a). Enrichment analysis of 'Biological process' GO-terms showed the presence of 'ribosome biogenesis' and 'organonitrogen compound biosynthetic, cellular amide metabolic' processes terms among the most enriched in AeSSP1256 roots as compared to GFP-expressing roots (Supplemental

Table 1b

). We noticed that over 90% of DE-genes from 'ribosome biogenesis' and 'translation' categories are downregulated in AeSSP1256-expressing roots, suggesting that expression of the effector within the roots affects ribosome biogenesis pathway (Supplemental Table 1a). To evaluate whether expression of AeSSP1256 mimics infection of M. truncatula by A. euteiches infection through downregulation of genes related to ribosome biogenesis, we analyzed RNASeq data previously generated on the susceptible F83005.5 M. truncatula line nine days after root infection [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF]. As shown on the Venn diagram depicting the M. truncatula downregulated genes in the different conditions (Figure 3A, Supplemental Table1c), among the 270 common downregulated genes between AeSSP1256-expressing roots and susceptible F83-infected lines, 58 genes (>20%) are categorized in the 'ribosome biogenesis' and 'translation' GO term (Figure 3B). We next selected seventeen M. truncatula genes to confirm the effect via qRT-PCR. First, we selected ten A. thaliana genes related to plant developmental control (i,e mutants with shorter roots phenotype) (Supplemental Table 1d) by Blast searches (>80% identity) in A17 line r5.0 genome portal (Pecrix et al., 2018). In addition, seven nucleolar genes coding for ribosomal and ribonucleotides proteins and related to the 'ribosome biogenesis' in M. truncatula were selected for expression analysis based on KEGG pathway map (https://www.genome.jp/keggbin/show_pathway?ko03008) (Supplemental 2b).

The second candidate is a predicted DEAD-box ATP-dependent RNA helicase measurements on these nuclei confirm the interaction between AeSSP1256 and the Medicago RNA helicase (Figure 4B), with a mean CFP lifetime of 2.86 ns +/-0.02 in absence of the effector protein, to 2.53 ns +/-0.03 in presence of AeSSP1256:YFP (Table 2). mean life-time in nanoseconds (ns). (b) s.e.m.: standard error of the mean. (c) N: total number of measured nuclei. (d) E: FRET efficiency in % : E=1-(DA/D). (e) p-value (Student's t test) of the difference between the donor lifetimes in the presence or absence of acceptor.

To confirm this result, co-immunoprecipitation assays were carried out. A GFP:MtRH10 construct was co-transformed with AeSSP1256:HA construct in N. benthamiana leaves. As expected, the localization of GFP:MtRH10 protein in absence of AeSSP1256 was nucleocytoplasmic while it located around the nucleolus in the presence of the effector (Supplemental Figure 4B). Immunoblotting experiments using total proteins extracted from infiltrated leaves (24hpi) showed that AeSSP1256:HA proteins were co-immunoprecipitated with GFP:MtRH10, but not with the GFP alone (Figure 4C). These data indicate that AeSSP1256 associates with MtRH10 in the nucleus. To go further we checked the stability of the two proteins when expressed alone or in combination in N. benthamiana cells during 72 hours. While GFP:MtRH10 was still detected at 72h after agroinfiltration, it started to be degraded 48hpi (Figure 4D). Expression of the effector alone is stable along the time. In contrast, when the two proteins are co-expressed, GFP:MtRH10 is almost entirely processed at 48h, and no more detectable at 72h (Figure 4D), suggesting that the effector enhance instability of its host target. Taken together, these results strongly suggest an interaction between AeSSP1256 and two type of components, a ribosomal protein and a nuclear RNA helicase from M. truncatula.

AeSSP1256 alters the RNA binding activity of MtRH10

DEAD-box RNA helicases are RNA binding proteins involved in various RNA-related processes including pre-rRNA maturation, translation, splicing, and ribosome assembly [START_REF] Jarmoskaite | DEAD-box proteins as RNA helicases and chaperones[END_REF]. These processes are dependent to the RNA binding ability of the proteins. Therefore we checked whether MtRH10 is able to bind nucleic acids in planta using FRET-FLIM assays as described previously. As reported in Table 3 and in Figure 5A, GFP lifetime of GFP:MtRH10 decreased in presence of the acceptor, from 2.32 ns +/-0.02 to 2.08 ns +/-0.03 due to FRET between GFP and Sytox, confirming as expected that MtRH10

protein is bounded to nucleic acids. To evaluate the role of AeSSP1256 on the function of MtRH10 we reasoned that the effector may perturb its binding capacity since it is required for the activity of numerous RH protein family [START_REF] Jankowsky | RNA helicases at work: binding and rearranging[END_REF]. We then co-expressed the GFP:MtRH10 construct with AeSSP1256:HA in N. benthamiana leaves and performed FRET-FLIM assays. Measurements made in nuclei where both proteins are detected due to the re-localization of MtRH10 indicated that GFP lifetime of GFP:MtRH10 remained unchanged with or without Sytox (2.3 ns in both conditions) showing that MtRH10 was not able to bind nucleic acids in the presence of the effector (Table 3 andFigure 5B). These data reveal that AeSSP1256 hijacks MtRH10 binding to RNA, probably by interacting with MtRH10. 6B).

MtRH10 is expressed in meristematic root cells and its deregulation in M. truncatula impacts root architecture and susceptibility to A. euteiches infection

We also noticed the presence of brighter dots in the nucleolus corresponding probably to the where MtRH10 is silenced to 2 to 3 times as compared to GFP control roots, qPCR analyses revealed approximately 5 to 10 times more of the pathogen at 7, 14 and 21 dpi (Figure 7).

Taken together these infection assays show that MtRH10 is involved in conferring basal resistance to A. euteiches at the root level.

Discussion

Protein effectors from filamentous plant pathogens such as fungi and oomycetes facilitate host colonization by targeting host components. However the molecular mechanisms that enhance plant susceptibility to the pathogen are still poorly understood. Here we report that the A.

euteiches AeSSP1256 RNA-binding effector facilitate host infection by downregulating expression of plant ribosome-related genes and by hijacking from its nucleic target MtRH10, a Medicago nuclear RNA-helicase (RH). Thus the current study unravels a new strategy in which pathogenic oomycete triggers plant nucleolar stress to promote infection.

AeSSP1256 is an effector from the oomycete root pathogen A. euteiches previously shown to enhance oomycete infection [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF]. Despite the absence of any functional domain, in silico RGG/RG RNA-binding motif prediction (see for review [START_REF] Thandapani | Defining the RGG/RG Motif[END_REF]) prompt us to show by FRET/FLIM analysis that the secreted AeSSP1256 effector is an RNA-binding protein (RBP). RNAs play essential role in cell physiology and it is not surprising that filamentous plant pathogens may rely on RNA-dependent process to control host infection (for review see [START_REF] Göhre | RNA biology in fungal phytopathogens[END_REF]Pedersen et al., 2012)). Moreover RBPs are key players in the regulation of the post-transcriptional processing and transport of RNA molecules (Yang et al., 2018). However to our knowledge only three examples of RBPs acting as virulence factor of plant pathogens are known. This includes the glycine-rich protein MoGrp1 from the rice pathogen Magnaporthe oryzae [START_REF] Gao | A glycine-rich protein MoGrp1 functions as a novel splicing factor to regulate fungal virulence and growth in Magnaporthe oryzae[END_REF], the UmRrm75 of Ustilago maydis (Rodríguez-Kessler et al., 2012) and the secreted ribonuclease effector CSEP0064/BEC1054 of the fungal pathogen Blumeria graminis which probably interferes with degradation of host ribosomal RNA (Pennington et al., 2019). This situation is probably due to the absence of conventional RNA-binding domain which render this type of RBP undetectable by prediction algorithms. The future studies that will aim to unravel the atlas of RNA-binding effector in phytopathogens should not only rely on computational analysis but will have to use functional approaches such as crystallization of the protein to validate function as performed with CSEP0064/BEC1054 effector (Pennington et al., 2019) screening method like the RNA interactome capture (RIC) assay develops in mammals (Castello et al., 2012) or FRET/FLIM assays to detect protein / nucleic acid interactions (Camborde et al., 2017).

We observed that when expressed inside roots of the partially resistant Jemalong A17 M.

truncatula line, AeSSP1256 triggers developmental defects such as shorter primary roots and delay in root development. Defects in roots development and retarded growth are typical characteristics of auxin-related and ribosomal proteins mutants reported in Arabidopsis (Ohbayashi et al., 2017;Wieckowski and Schiefelbein, 2012). In addition, those composite Medicago promote infection of A. euteiches. This modification in the output of the infection is highly relevant since we previously observed that M. truncatula quantitative resistance to A.

euteiches is correlated to the development of secondary roots (Rey et al., 2016). This activity is dependent on the nucleolar rim localization of AeSSP1256, closed to the nucleolus.

The nucleolus is a membrane-free subnuclear compartment essential for the highly complex process of ribosome biogenesis organized in three domains including the fibrillar center that contain rDNA, which are not yet engaged in transcription (reviewed in (Shaw and Brown, 2012). Ribosome biogenesis is linked to cell growth and required coordinated production of processed ribosomal RNA (rRNA), ribosomal biogenesis factors and ribosomal proteins (RP).

In the nucleolus, ribosome biogenesis starts with the transcription of pre-rRNAs from rRNA genes, followed by their processing and assembly with RPs into two ribosome subunits (ie small and large subunit). In animals, perturbation of any steps of ribosome biogenesis in the nucleolus can cause a nucleolar stress or ribosomal stress which stimulates specific signaling pathway leading for example to arrest of cell growth (Pfister, 2019). The nucleolar rim localization of AeSSP1256 within the host cells suggested that this effector could interfere with ribosome biogenesis pathway to facilitate infection. This speculation was further strengthened by RNAseq experiments which showed that, within A17-roots, AeSSP1256 downregulated numerous genes implicated in ribosome biogenesis pathway, notably ribosomal protein genes. This effect was also detected in susceptible F83 M. truncatula lines infected by A. euteiches indicating that AeSSP1256, mimics some A.euteiches effects during roots invasion.

An Y2H approach led to the identification of putative AeSSP1256 plant targets and all but one correspond to predicted nuclear M. truncatula proteins. By a combination of multiple experiments as FRET-FLIM to detect protein/protein interactions, a L7 ribosomal protein (MtrunA17_Chr4g0002321) and a DExD/H box RNA helicase ATP-dependent (MtrunA17_Chr5g0429221) were confirmed as AeSSP1256-interacting proteins. The DExD/H (where x can be any amino acid) box protein family include the largest family of RNA-helicase (RH). Rather than being processive RH, several DExD/H box proteins may act as 'RNA chaperone' promoting the formation of optimal RNA structures by unwinding locally the RNA (for review see [START_REF] Fuller-Pace | DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation[END_REF]). These proteins are of major interest due to their participation to all the aspects of RNA processes such as RNA export and translation, splicing but the most common function of these proteins is in ribosome biogenesis including assembly [START_REF] Jarmoskaite | DEAD-box proteins as RNA helicases and chaperones[END_REF]. Specific function of RH is probably due to the presence of a variable C-terminal 'DEAD' domain in contrast to the well conserved Nterminal 'helicase core' domain (for review see [START_REF] Fuller-Pace | DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation[END_REF]). This structural organization was detected in the MtRH10. This M. truncatula protein corresponds to the ortholog of the nucleolar human DDX47 (Sekiguchi et al., 2006), the nuclear yeast RRP3

(O' Day, 1996) and the nucleolar Arabidopsis AtRH10 RNA-helicases, all involved in ribosome biogenesis [START_REF] Liu | Function of plant DExD/H-Box RNA helicases associated with ribosomal RNA biogenesis[END_REF][START_REF] Matsumura | A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis[END_REF], and the nucleolar rice OsRH10 (TOGR1) involved in rRNA homeostasis (Wang et al. 2016).

Like its human ortholog DDX47 (Sekiguchi et al., 2006), MtRH10 possesses a bipartite nuclear transport domain which can function as a nuclear localization signal (NLS) and two nuclear export signal (NES), and thereby it probably shuttles between the cytoplasm and the nucleus as reported for many others RNA helicases involved in rRNA biogenesis and splicing function (Sekiguchi et al. 2006;Wang et al. 2009). Fluorescence analysis showed a relocalization of the nucleocytoplasmic MtRH10 in the nucleoli periphery, when it is transiently co-express with AeSSP1256 in N. benthamiana cells. The change in MtRH10 distribution suggests that the interaction between the two proteins caused a mislocation of MtRH10 that can probably affect its activity. We thereby check the nucleic acid binding capacity of MtRH10 by FRET-FLIM approach. The decrease in the lifetime of GFP revealed the ability of MtRH10 to bind nucleic acids. Knowing that both proteins display the same properties, we further provided evidence that the presence of AeSSP1256 effector inhibits the nucleic binding capacity of MtRH10. This mechanism was also reported for the RNA-binding HopU1 effector from the plant bacterial pathogen Pseudomonas syringae which associates to the glycin-rich RNA binding 7 protein (GRP7) of Arabidopsis to abolish GRP7 binding to immune gene transcripts (ie FLS2 receptor, (Nicaise et al., 2013)). Here we cannot exclude that AeSSP1256 also blocks the putative helicase activity of MtRH10, but we favored an inhibitory mechanism of AeSSP1256 on MtRH10 activity as complex and at least in part due to both protein-protein interaction and nucleic acid interaction with the two proteins.

Interestingly, we also noticed that co-expression of both proteins led to decrease in MtRH10 probably due to degradation of the protein. While this observation warrants further analyses, this effect is reminiscent of other effector activities which destabilize their targets (for review see [START_REF] Langin | Microbial effector proteins -A journey through the proteolytic landscape[END_REF]).

Plant genomes encode a large variety of DExD/H RH family in comparison to other organisms and numerous studies have shown that several are associated through their activity with plant development, hormone signaling or responses to abiotic stresses (for review see [START_REF] Liu | Function of plant DExD/H-Box RNA helicases associated with ribosomal RNA biogenesis[END_REF]). Very few studies reported that DExD/H RH could also be involved in biotic stresses, like responses to pathogens. One example is the DExD/H RH OsBIRH1 from rice that enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae through activation of defense-related genes [START_REF] Li | OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress[END_REF]. A recent study on oomycete reports the binding of the Phytophthora sojae RxLR PSR1 effector to a putative nuclear DExD/H RH. Although the affinity for nucleic acids was not evaluated for the RH, association of both partners promote pathogen infection by suppressing small RNA biogenesis of the plant (Qiao et al., 2015). Here we showed that MtRH10 knockdown tolerant A17 lines supported higher-level accumulation of A. euteiches in contrast to overexpressed MtRH10 lines, indicating the importance of MtRH10 for M. truncatula roots defense against soil-borne pathogens.

This works reveals that MtRH10 expression is restricted at the root apical meristematic zone (RAM) where cells divide (ie, primary and lateral roots). Missense MtRH10 roots harbor defects in the primary root growth and reduced number of roots. Longitudinal sections in elongation zone (EDZ) of these composite roots show a significant reduction in the size and shape modification of cortical cells indicating that MtRH10 is required for normal cell division. Defect in primary roots elongation is also detected in silenced AtRH10 and OsRH10 mutant [START_REF] Matsumura | A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis[END_REF]Wang et al. 2016). Thus MtRH10 plays a role on Medicago root development as its orthologs OsRH10 and AtRH10. At the cellular level we also observed in AeSSP1256-expressing roots, reduction in cell size in elongation zone, with defects in cell shape and in adhesion between cells of the cortex, maybe due to a modification of the middle lamella (Zamil and Geitmann, 2017). Thus AeSSP1256 triggers similar or enhanced effect on host roots development as the one detected in defective MtRH10 composite plants, supporting the concept that the activity of the effector on MtRH10 consequently leads to developmental roots defects. Several reports have indicated that

Arabidopsis knockout of genes involved in rRNA biogenesis or in ribosome assembly cause abnormal plant development including restriction and retardation in roots growth (Ohtani et al., 2013;[START_REF] Huang | The DEAD-Box RNA Helicase AtRH7/PRH75 participates in pre-rRNA processing, plant development and cold tolerance in Arabidopsis[END_REF][START_REF] Huang | A DEAD-box protein, AtRH36, is essential for female gametophyte development and is involved in rRNA biogenesis in Arabidopsis[END_REF]. These common features suggest the existence of a common mechanism that regulate growth in response to insults of the ribosome biogenesis pathway, known as nucleolar stress response (for review see (Ohbayashi and Sugiyama, 2018)). How plant cells sense perturbed ribosome biogenesis and nucleolar problems is still an open question (Sáez-Vásquez and Delseny, 2019), but the ANAC082 transcription factor from Arabidopsis can be a ribosomal stress response mediator (Ohbayashi et al., 2017). In addition the recent report on the activity of the nucleolar OsRH10 (TOGR1, MtRH10 ortholog) implicated in plant primary metabolism through is activity on rRNA biogenesis, suggests that metabolites may play a role in this process. Finally our current study indicates that nuclear RNA-binding effector like AeSSP1256, by interacting with MtRH10, can act as a stimulus of the ribosomal stress response.

This work established a connection between the ribosome biogenesis pathway, a nuclear

DExD/H RH, root development and resistance against oomycetes. Our data document that the RNA binding AeSSP1256 oomycete effector downregulated expression of ribosome-related genes of the host plant. The effector hijacked MtRH10, a nuclear DExD/H RH involved in root development, to promote host infection. This work not only provides insights into plantroot oomycete interactions but also reveals the requirement of fine-tuning of plant ribosome biogenesis pathways for infection success.

Material and Methods

Plant material, microbial strains, and growth conditions M. truncatula A17 seeds were in vitro-cultured and transformed as previously described (Boisson-Dernier et al., 2001;[START_REF] Djébali | Partial Resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes[END_REF]. A. euteiches (ATCC 201684) zoospore inoculum were prepared as in (Badreddine et al., 2008). For root infections, each plant was inoculated with a total of 10µl of zoospores suspension at 10 5 cells.ml -1 . Plates were placed in growth chambers with a 16h/8h light/dark and 22/20°C temperature regime. N. benthamiana plants were grown from seeds in growth chambers at 70% of humidity with a 16h/8h light/dark and 24/20°C temperature regime. E.coli strains (DH5α, DB3.5), A. tumefaciens (GV3101::pMP90) and A. rhizogenes (ARQUA-1) strains were grown on LB medium with the appropriate antibiotics.

Construction of plasmid vectors and Agrobacterium-mediated transformation

GFP control plasmid (pK7WGF2), +SPAeSSP1256:GFP and +SPAeSSP1256:YFP (named AeSSP1256:GFP and AeSSP1256:YFP in this study for convenience) and minus or plus signal peptide AeSSP1256:GFP:KDEL constructs were described in [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF].

Primers used in this study are listed in Supplemental Table 3 Using pENTR/ D-TOPO:AeSSP1256, described in [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF], AeSSP1256 was transferred by LR recombination in pAM-PAT-35s::GTW:3HA for co-immunoprecipitation and western blot experiments to create a AeSSP1256:HA construct and in pUBC-RFP-DEST [START_REF] Grefen | A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies[END_REF] to obtain a AeSSP1256:RFP construct for FRET-FLIM analysis. For

RNAi of MtRH10 (MtrunA17_Chr5g0429221), a 328 nucleotides sequence in the 3'UTR was amplified by PCR (see Supplemental Table 3), introduced in pENTR/D-TOPO vector and LR cloned in pK7GWiWG2(II)-RedRoot binary vector (http://gateway.psb.ugent.be/) to obtain RNAi MtRH10 construct. This vector allows hairpin RNA expression and contains the red fluorescent marker DsRED under the constitutive Arabidopsis Ubiquitin10 promoter (http://gateway.psb.ugent.be/), to facilitate screening of transformed roots. For MtRH10 promoter expression analyses, a 1441nt region downstream of the start codon of MtRH10 gene was amplified by PCR (see Supplemental Table 3), fused to β-glucuronidase gene (using pICH75111 vector [START_REF] Engler | A golden Ggate modular cloning toolbox for plants[END_REF]) and inserted into pCambia2200:DsRED derivative plasmid [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF] by Golden Gate cloning to generate PromoterMtRH10:GUS vector.

Generation of M. truncatula composite plants was performed as described by (Boisson-Dernier et al., 2001) using ARQUA-1 A. rhizogenes strain. For leaf infiltration, GV3101 A.

tumefaciens transformed strains were syringe-infiltrated as described by [START_REF] Gaulin | The CBEL glycoprotein of Phytophthora parasitica var-nicotianae is involved in cell wall deposition and adhesion to cellulosic substrates[END_REF].

Cross-section sample preparation for confocal microscopy

M. truncatula A17 plants expressing GFP or AeSSP1256:GFP constructs were inoculated with A. euteiches zoospores 21 days after transformation as indicated previously. Roots were harvested 21 days post inoculation, embedded in 5% low-melting point agarose and cutted using a vibratome (VT1000S; Leica, Rueil-Malmaison, France) as described in [START_REF] Djébali | Partial Resistance of Medicago truncatula to Aphanomyces euteiches is associated with protection of the root stele and is controlled by a major QTL rich in proteasome-related genes[END_REF]. Cross-sections were stained using Wheat Germ Agglutin (WGA)-Alexa Fluor 555 conjugate (Thermo Fischer; W32464), diluted at 50 μg/ml in PBS for 30min to label A.

euteiches.

RNA-Seq experiments

Roots of composite M. truncatula A17 plants expressing GFP or AeSSP1256:GFP constructs were harvested one week later after first root emergence. Before harvest, roots were checked for GFP-fluorescence by live macroimaging (Axiozoom, Carl Zeiss Microscopy, Marly le Roi, France) and GFP-positive roots were excised from plants by scalpel and immediately frozen in liquid nitrogen. Four biological replicates per condition were performed (GFP vs AeSSP1256-expressing roots), for each biological replicate 20-40 transformed plants were used. Total RNA was extracted using E.Z.N.A.® total RNA kit (Omega bio-tek) and then purified using Monarch® RNA Cleanup Kit (NEB). cDNA library was produced using MultiScribe™ Reverse Transcriptase kit using mix of random and poly-T primers under standard conditions for RT-PCR program. Libraries preparation was processed in GeT-PlaGe genomic platform (https://get.genotoul.fr/en/; Toulouse, France) and sequenced using Illumina HiSeq3000 sequencer. The raw data was trimmed with trmigalore (version 0.6.5) (https://github.com/FelixKrueger/TrimGalore) with cutadapt and FastQC options, and mapped to M. truncatula cv. Jemalong A17 reference genome V. 5.0 (Pecrix et al., 2018) using Hisat2 (version 2.1.0) [START_REF] Kim | Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype[END_REF]. Samtools (version 1.9) algorithms fixmate and markdup [START_REF] Li | The sequence alignment/map format and SAMtools[END_REF] were used to clean alignments from duplicated sequences. Reads were counted by HTseq (version 0.9.1) (Anders et al., 2015) using reference GFF file. The count files were normalized and different expression were quantified using DESeq2 algorithm [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF], false-positive hits were filtered using HTS filter (Rau et al., 2013). GO enrichment were done using ErmineJ [START_REF] Lee | ErmineJ: tool for functional analysis of gene expression data sets[END_REF] and topGO (Alexa and Rahnenfuhrer 2020) software. RNASeq experiments on F83005.5 (F83) susceptible plants infected by A. euteiches and collected nine days after infection are described in [START_REF] Gaulin | Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation[END_REF].

RNA extraction and qRT-PCR

RNA was extracted using the E.Z.N.A ® Plant RNA kit (Omega Bio-tek). For reverse transcription, 1µg of total RNA were used and reactions were performed with the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems and cDNAs obtained were diluted 10 fold. qPCR reactions were performed as described in (Ramirez-Garcés et al., 2016) and conducted on a QuantStudio 6 (Applied Biosystems) device using the following conditions: 10min at 95°C, followed by 40 cycles of 15s at 95°C and 1min at 60°C. All reactions were conducted in triplicates.

To evaluate A. euteiches's infection level, expression of Ae α-tubulin coding gene (Ae_22AL7226, [START_REF] Gaulin | Transcriptome of Aphanomyces euteiches: new oomycete putative pathogenicity factors and metabolic pathways[END_REF]) was analyzed and histone 3-like gene and EF1 gene of M. truncatula (Rey et al., 2013) were used to normalize plant abundance during infection.

For Aphanomyces infection in plant over-expressing GFP, AeSSP1256:GFP or GFP:MtRH10, cDNAs from five biological samples were analyzed, given that a sample was a pool of 3 to 5 plants, for each time point, on three independent experiments, representing 45 to 75 transformed plants per construct. M. truncatula roots were harvested 7, 14 and 21 dpi. For missense MtRH10 experiments, downregulation of MtRH10 gene was first verified using cDNAs from five biological samples, given that a sample was a pool of 5 plants, harvested 21 days post transformation. For A. euteiches inoculation, three biological samples were analyzed, given that a sample was a pool of 3 plants, for each time point, on two independent experiments, representing around 50 transformed missense MtRH10 plants. Relative expression of Ae α-tubulin or MtRH10 helicase genes were calculated using the 2 -∆∆Ct method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[END_REF]. For qPCR validation of RNAseq experiment, cDNAs from five biological replicates (pool of three plants) of AeSSP1256-expressing roots were extracted 21

Yeast Two Hybrid assays

An ULTImate Y2H™ was carried out by Hybrigenics-services (https://www.hybrigenicsservices.com) using the native form of AeSSP1256 (20-208 aa) as bait against a library prepared from M. truncatula roots infected by A. euteiches. The library was prepared by Hybrigenics-services using a mixture of RNA isolated from uninfected M. truncatula F83005.5 (+/-12%), M. truncatula infected with A. euteiches ATCC201684 harvested one day post infection (+/-46%) and M. truncatula infected with A. euteiches harvested six days post infection (+/-42%). This library is now available to others customers on Hybrigenicsservices. For each interaction identified during the screen performed by Hybrigenics (65 millions interaction tested), a 'Predicted Biological Score (PBS)' was given which indicates the reliability of the identified interaction. The PBS ranges from A (very high confidence of the interaction) to F (experimentally proven technical artifacts). In this study we kept eight candidates with a PBS value from 'A and C' for validation.

Analysis of amino acid sequence of MtRH10

Conserved motifs and domains of DEAD-box RNA helicase were found using ScanProsite tool on ExPASy web site (https://prosite.expasy.org/scanprosite/). MtRH10 putative NLS motif was predicted by cNLS Mapper with a cut-off score of 4.0 [START_REF] Kosugi | Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs[END_REF]

Co-immunoprecipitation assay

Co-immunoprecipitation was performed on N. benthamiana infiltrated leaves expressing GFP, GFP:MtRH10 or AeSSP1256:HA tagged proteins. Total proteins were extracted with GTEN buffer and quantified by Bradford assay. 50µg of total proteins were incubated 3H at 4°C with 30µl of GFP-Trap Agarose beads (Chromotek; gta-20) under gentle agitation for GFP-tagged protein purification. After four washing steps with GTEN buffer containing 0,05% Tween-20, beads were boiled in SDS loading buffer.

Confocal microscopy

Scanning was performed on a Leica TCS SP8 confocal microscope. For GFP and GFP variant recombinant proteins, excitation wavelengths were 488 nm (GFP) whereas 543nm were used for RFP variant proteins. Images were acquired with a 40x water immersion lens or a 20x

water immersion lens and correspond to Z projections of scanned tissues. All confocal images were analyzed and processed using the Image J software.

Cytological observations of transformed roots

Roots of composite plants expressing GFP, AeSSP1256:GFP, GFP:MtRH10 or RNAi MtRH10 were fixed, polymerized and cutted as described in (Ramirez-Garcés et al., 2016).

NDPview2 software was used to observe longitudinal root sections of GFP or missense MtRH10 plants and to measure RAM size. Image J software was used for all others measurements. Average RAM cells size were estimated by measuring all the cells from a same layer from the quiescent center to the RAM boundary. Mean values were then calculated from more than 200 cells. In the elongation zone (EDZ) of GFP, AeSSP1256:GFP or missense MtRH10 roots, cell area and cell perimeter were measured in rectangular selection of approximately 300x600µm (two selections per root). To obtain a normalized cell perimeter, each cell perimeter is proportionally recalculated for a of 500µm² area standard cell. To estimate cell shape differences, considering that cortical cells in EDZ of GFP control roots are mostly rectangular, we measured the perimeter bounding rectangle (PBR), which represent the smallest rectangle enclosing the cell. Then we calculated the ratio perimeter / PBR.

Rectangular cells have a perimeter / PBR ratio close to 1. Three roots per construct from three independent experiments were used.

FRET / FLIM measurements

For protein-protein interactions, N. benthamiana agroinfiltrated leaves were analysed as described in (Tasset et al., 2010). For protein-nucleic acid interactions, samples were treated as described in (Camborde et al., 2017;[START_REF] Escouboué | Preparation of plant material for analysis of protein-nucleic acid interactions by FRET-FLIM[END_REF]. Briefly, 24 h agroinfiltrated leaf discs were fixed with a 4% (w/v) paraformaldehyde solution. After a permeabilization step of 10 min at 37°C using 200 µg/ml of proteinase K (Thermo Fisher; 25530049), nucleic acid staining was performed by vaccum-infiltrating a 5 µM of Sytox Orange (Thermo Fisher; S11368) solution. For RNase treatment, foliar discs were incubated 15 min at room temperature with 0.5 mg/ml of RNAse A (Merck; R6513) before nucleic acid staining. Then fluorescence lifetime measurements were performed in time domain using a streak camera as described in (Camborde et al., 2017).

  mean life-time in nanoseconds (ns).(b) s.e.m.: standard error of the mean.(c) N: total number of measured nuclei.(d) E: FRET efficiency in %: E=1-(DA/D).(e) p-value (Student's t test) of the difference between the donor lifetimes in the presence or absence of acceptor.

(

  MtrunA17_Chr5g0429221), related to the human DDX47 RNA helicase and the RRP3 RH in yeast. Blast analysis revealed that the closest plant orthologs were AtRH10 in Arabidopsis thaliana and OsRH10 in Oryza sativa. Consequently the M. truncatula protein target of AeSSP1256 was named MtRH10. The conserved domains of DEAD-box RNA helicase are depicted in the alignment of MtRH10 with DDX47, RRP3, AtRH10, OsRH10 proteins (Supplemental Figure 4A) (Schütz et al., 2010; Gilman et al., 2017). MtRH10 CFP-tagged fusion protein harbors nucleocytoplasmic localization when transiently express in N. benthamiana cells (Figure 4A), in accordance with the presence of both putative nuclear export signals (NESs) (position 7-37; 87-103; 261-271) and nuclear localization signal (NLS) sequences (position 384-416). When MtRH10 is co-expressed with YFP-tagged version of AeSSP1256, the fluorescence is mainly detected as a ring around the nucleolus, indicating a partial relocalisation of MtRH10 to the AeSSP1256 sites (Figure 4A). FRET-FLIM

  Figure 6D, the perimeter/PBR ratio in GFP roots is close to 1 and significantly different than

  For each nucleus, fluorescence lifetime of the donor (GFP recombinant protein) was experimentally measured in the presence and absence of the acceptor (Sytox Orange). FRET efficiency (E) was calculated by comparing the lifetime of the donor in the presence ( DA ) or absence ( D ) of the acceptor: E=1-( DA ) / ( D ). Statistical comparisons between control (donor) and assay (donor + acceptor) lifetime values were performed by Student t-test. For each experiment, nine leaf discs collected from three agroinfiltrated leaves were used.Accession NumbersTranscriptomic data are available at the National Center for Biotechnology Information (NCBI), on Gene Expression Omnibus (GEO) under accession number[GEO:GSE109500] for RNAseq corresponding to M. truncatula roots (F83005.5 line) infected by A. euteiches (9dpi) and Sequence Read Archive (SRA) under accession number PRJNA631662 for RNASeq samples corresponding to M. truncatula roots (A17) expressing either a GFP construct or a native AeSSP1256:GFP construct. SRA data will be release upon acceptation of the manuscript.(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: AeSSP1256 is a RNA-binding protein(A) AeSSP1256 protein sequence that shows the signal peptide (underlined), GGRGG boxes (red), RGG domains (bolt, underlined and linked), RG motifs (bolts with asterisks) predicted with Eukaryotic Linear Motif Prediction[START_REF] Gouw | The eukaryotic linear motif resource -2018 update[END_REF]. (B) One day after agroinfection of N. benthamiana leaves with a AeSSP1256:GFP construct, infiltrated area are collected for FRET-FLIM analysis to detect protein/nucleic acid interactions as described byCamborde et al., 2018. Without RNAse treatment and in presence of nucleic acids dye Sytox Orange, the AeSSP1256:GFP lifetime decreases to shorter values, indicating that the proteins bounded to nucleic acids (top panel). After RNase treatment, no significant decrease in the GFP lifetime was observed in presence of Sytox Orange, indicating that AeSSP1256:GFP proteins were bounded specifically to RNA (bottom panel). Histograms show the distribution of nuclei (%) according to classes of AeSSP1256:GFP lifetime in the absence (blue bars) or presence (orange bars) of the nucleic acids dye Sytox Orange. Arrows represent GFP lifetime distribution range.

  Figure 4: AeSSP1256 interacts and re-localizes the nuclear MtRH10 RNA Helicase around the nucleolus (A) Confocal analyses on N. benthamiana agroinfiltrated leaves. The CFP:MtRH10 candidate presents a nucleocytoplasmic localization when expressed alone (Left panel), and is re-localized in the nucleus, mostly around nucleolus, in the presence of AeSSP1256:YFP proteins (Right panels). Pictures were taken at 24h post agroinfection. Scale bars: 10µm. (B) FRET-FLIM experiments indicate that CFP:MtRH10 and AeSSP1256:YFP proteins are in close association when co-expressed in N. benthamiana cells. Histograms show the distribution of nuclei (%) according to classes of CFP:MtRH10 lifetime in the absence (blue bars) or presence (green bars) of AeSSP1256:YFP. Arrows represent CFP lifetime distribution range. (C) Co-immunoprecipitation experiments confirm the direct association of the two proteins. Upper panel: anti-GFP and anti-HA blots confirm the presence of recombinant proteins in the input fractions. Lower panel: anti-GFP and anti-HA blots on output fractions after GFP immunoprecipitation. Arrows indicate the corresponding proteins. (D) anti-GFP and anti-HA blots on N. benthamiana leaf extracts expressing the GFP:MtRH10 alone or in combination with AeSSP1256:HA protein after 24, 48 or 72h post agroinfection. Arrows indicate the corresponding proteins. Note that GFP:MtRH10 is degraded faster in presence of AeSSP1256:HA.

Figure 5 :Figure 6 :

 56 Figure 5: AeSSP1256 inhibits RNA binding activity of MtRH10 (A) FRET-FLIM experiments on N. benthamiana cells expressing GFP:MtRH10 in presence or absence of nucleic acids dye Sytox Orange. In presence of Sytox Orange, the GFP:MtRH10 lifetime decreases to shorter values, indicating that the proteins bounded to nucleic acids. (B) In presence of AeSSP1256:HA, when GFP:MtRH10 is re-localized around the nucleolus and interacts with AeSSP1256, no significant decrease in the GFP lifetime was observed in presence of Sytox Orange, meaning that the relocalized GFP:MtRH10 proteins were not able to interact with nucleic acids. Histograms show the distribution of nuclei (%) according to classes of GFP:MtRH10 lifetime in the absence (blue bars) or presence (orange bars) of the nucleic acids dye Sytox Orange. Arrows represent GFP lifetime distribution range.

Figure 7 :

 7 Figure 7: Deregulation of MtRH10 helicase gene expression in Medicago truncatula impacts Aphanomyces euteiches susceptibility

Table 1 :

 1 FRET-FLIM measurements for AeSSP1256:GFP with or without Sytox Orange

	Donor	Acceptor	 a	sem (b) N (c)	E (d)	(e) p-value
	AeSSP1256:GFP	-	2.06	0.020	78	-	-
	AeSSP1256:GFP	Sytox	1.84	0.026	77	11	1.34E -09
	AeSSP1256:GFP	-(+ RNase)	2.01	0.026	50	-	-
	AeSSP1256:GFP	Sytox (+ RNase)	1.96	0.027	50	2.6	0.17

Table 1d). As shown on Figure 3C, all

	of the
	selected genes from M. truncatula are downregulated in presence of AeSSP1256, supporting
	the RNAseq data. Altogether these expression data show that the effector by itself mimics
	some effects induced by pathogen infection of the susceptible F83 line. At this stage of the
	study, results point to a perturbation of the ribosome biogenesis pathway of the host plant by
	the AeSSP1256 effector.

AeSSP1256 targets a DEAD-box RNA helicase and a L7 ribosomal protein

  To confirm the Y2H results, we first expressed AeSSP1256 and candidates in N. benthamiana cells to observe their subcellular localization and performed FRET-FLIM experiments to validate protein-protein interactions. Only two candidates showed co-localization with

	AeSSP1256, a L7 ribosomal protein (RPL7, MtrunA17_Chr4g0002321) and a predicted RNA
	helicase (RH) (MtrunA17_Chr5g0429221). CFP-tagged version of RPL7 displays a nucleolar
	localization, with partial co-localization areas in presence of AeSSP1256 (Supplemental
	Figure 3A, Table 2b). FRET-FLIM measurements confirmed the interaction of RPL7:CFP
	protein with AeSSP1256:YFP effector (Supplemental Figure 3B, Table 2b), with a mean
	CFP lifetime of 2.83 ns +/-0.03 in absence of the SSP protein, leading to 2.46 ns +/-0.03 in
	presence of AeSSP1256:YFP (Supplemental Table
	To decipher how AeSSP1256 can affect ribosome biogenesis pathway of the host plant and
	knowing that numerous RNA-binding proteins interact with protein partners, we searched for
	AeSSP1256 host protein targets. For this, a Yeast two hybrid (Y2H) library composed of
	cDNA from M. truncatula roots infected with A. euteiches was screened with the mature form
	of the effector. Eight M. truncatula coding genes were identified as potential protein targets
	(Supplemental Table 2a), all these genes but one (a lecithin retinol acyltransferase gene)
	correspond to putative nuclear proteins in accordance with the observed subcellular
	localization of AeSSP1256.

Table 2 :

 2 FRET-FLIM measurements of CFP:MtRH10 in presence or absence of

	AeSSP1256:YFP
	Donor	Acceptor	 a	sem (b) N (c)	E (d)	(e) p-value
	CFP:MtRH10	-	2.86	0.023	50	-	-

Table 3 :

 3 FRET-FLIM measurements for GFP:MtRH10 with or without Sytox Orange, in

	presence or in absence of AeSSP1256:HA
	Donor	Acceptor	 a sem (b) N (c)	E (d) (e) p-value
	GFP:MtRH10	-	2.32 0.020	60	-	-
	GFP:MtRH10	Sytox Orange	2.08 0.027	60	10.3	1.30E -10
	GFP:MtRH10 (relocalized)	-(+ AeSSP1256:HA)	2.30 0.023	60	-	-
	GFP:MtRH10 (relocalized)	Sytox Orange (+ AeSSP1256:HA)	2.30 0.020	60	0	0.789

mean life-time in nanoseconds (ns).

(b)

s.e.m.: standard error of the mean.

(c)

N: total number of measured nuclei.

(d)

E: FRET efficiency in % : E=1-(DA/D).

(e)

p-value (Student's t test) of the difference between the donor lifetimes in the presence or absence of acceptor.

  To characterize the function of MtRH10, we firstly consider the expression of the gene by .toulouse.inra.fr/k/legoo/), Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and MedicagoEFP browser on Bar Toronto (http://bar.utoronto.ca/efpmedicago/cgibin/efpWeb.cgi). No variability was detected among the conditions tested in the databases and we do not detect modification of MtRH10 expression upon A. euteiches inoculation in our RNAseq data. To go further in the expression of the MtRH10 gene, transgenic roots expressing an MtRH10 promoter-driven GUS (-glucuronidase) chimeric gene were generated. GUS activity was mainly detectable in meristematic cells, at the root tip or in lateral emerging roots (Figure6A) suggesting a role in meristematic cell division. We

	mining	public	transcriptomic	databases	including	Legoo	(https://lipm-

browserscomplete MtRH10 analyses by overexpressing a GFP-tagged version in Medicago roots. The observation by confocal analyses of the subcellular localization of MtRH10 confirms its nucleocytoplasmic localisation as previously observed in N. benthamiana cells (Figure

Acknowledgements

The authors would like to thanks the GeT-PlaGe genomic platform (https://get.genotoul.fr/en/; Toulouse, France) for RNASeq studies ; H. San-Clemente and M. Aguilar for statistical analysis help (LRSV, France) ; S. Courbier and A. Camon for their assistance in cloning steps. This work was supported by the French Laboratory of Excellence project "TULIP" (ANR-10-LABX-41; ANR-11-IDEX-0002-02) and by the European Union's Horizon 2020 Research and Innovation programme under grant agreement No 766048.

author/funder.

Supplemental Data

The following supplemental data are available:

Supplemental

FRET-FLIM validation of CFP:L7RP candidate

Supplemental Table 3: List of primers used in this study

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Contributions

LC designed, performed molecular approaches on AeSSP1256 and wrote the manuscript, AK prepared and analyzed the RNAseq-experiments performed in this study and wrote the manuscript, AJ and LC performed FRET/FLIM analyses, CP and LC developed confocal studies, ALR performed cross and longitudinal sections studies and analyzed roots architecture of the different samples, MJCP prepared and analyzed yeast two hybrid assay, performed candidates cloning. BD analyzed the data and wrote the manuscript. EG conceived, designed, and analyzed the experiments, managed the collaborative work, and wrote the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.


Student Member, IEEE Marco Cabbia

Member, IEEE Chandan Yadav

Member, IEEE Marina Deng

Sebastien Fregonese

Magali De Matos

Senior Member, IEEE Thomas Zimmer

Sébastien Fregonese

Magali De Matos

Silicon Test Structures Design for Sub-THz and THz Measurements

Keywords: TRL Calibration, Characterization, EM Simulation, HFSS, THz, Test Structures

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I. INTRODUCTION

I N order to provide cost-effective and compact terahertz (THz) and sub-THz electronic systems for various applications in the millimeter wave and sub-millimeter wave ranges, integrated circuits (ICs) based on silicon-germanium heterojunction bipolar transistor (SiGe HBTs) are gaining much attention for their compelling compatibility with silicon very large scale integration (VLSI) technologies. The advances in silicon process technology and improved figures of merit of SiGe HBTs (f T /f max of 505/720 GHz, respectively) [START_REF] Heinemann | SiGe HBT with f T /fmax of 505 GHz/720 GHz[END_REF] outperform CMOS transistors, offering higher power densities and better analog performance, and promise to make this technology a viable option for ultra-high-speed wireless communications, satellites, industrial sensors, automotive radars, person scanning for security, medical imaging, etc. [START_REF] Shur | Terahertz sensing technology[END_REF]- [START_REF] Pawar | Terahertz technology and its applications[END_REF].

To ensure a successful design of a high frequency electronic circuit, it is crucial to provide an accurate compact model description of the active (i.e. HBTs) and passive elements (capacitances, inductances, resistances, etc.) based on experimental data. At frequencies beyond 110 GHz, that is a particularly troublesome requirement to satisfy due to the parasitic electrical contributions of the measurement set-up and external environment. For this reason, current researches focus on calibration and de-embedding techniques to obtain accurate measurements to extract correct model parameters [START_REF] Fregonese | Comparison of On-Wafer TRL Calibration to ISS SOLT Calibration With Open-Short De-Embedding up to 500 GHz[END_REF]- [START_REF] Williams | Calibrations for Millimeter-Wave Silicon Transistor Characterization[END_REF].

The calibration algorithms such as TRL, mTRL, LRM, LRRM, SOLT, etc. are usually followed by a de-embedding step, which should be accurate and not introduce additional errors [START_REF] Pourchon | From measurement to intrinsic device characteristics: Test structures and parasitic determination[END_REF]- [START_REF] Deng | Small-signal characterization and modelling of 55-nm SiGe BiCMOS HBT up to 325 GHz[END_REF]. For practical reasons, calibrations on Impedance Standard Substrate (ISS) combined with openshort de-embedding are commonly used in industry, even though they lack of accuracy at higher frequencies [START_REF] Potereau | Limitations of On-Wafer Calibration and De-Embedding Methods in the Sub-THz Range[END_REF]. For Chandan Yadav is with the National Institute of Technology, Calicut, India. Earlier, he was associated with the IMS laboratory, University of Bordeaux, France. All other authors are with the IMS Laboratory (e-mail: marco.cabbia@ims-bordeaux.fr, thomas.zimmer@ims-bordeaux.fr). frequencies above 110 GHz, it has been shown that on-wafer calibration is the main method of choice compared to conventional off-wafer calibration [START_REF] Fregonese | Comparison of On-Wafer TRL Calibration to ISS SOLT Calibration With Open-Short De-Embedding up to 500 GHz[END_REF], [START_REF] Rumiantsev | Comparison of onwafer multiline TRL and LRM+ calibrations for RF CMOS applications[END_REF]. For this reason, onwafer TRL (Thru-Reflect-Line) calibration has been chosen for this work: it is well suited to measurements at sub-millimeter wavelengths generated by probes with small pitch, since it relies on transmission lines rather than lumped elements, which cannot be considered as non-distributed elements at such high frequencies [START_REF] Williams | Calibration-Kit Design for Millimeter-Wave Silicon Integrated Circuits[END_REF].

A limitation for the use of TRL is that, in real-world applications, the structures are positioned in a wafer side by side, with small spacing between each element, in order to save space. The effect of the coupling due to the adjacent structures on the measurements of the device-under-test (DUT) is therefore non-negligible [START_REF] Phung | Parasitic effects and measurement uncertainties in multi-layer thin-film structures[END_REF]- [START_REF] Phung | Influence of Microwave Probes on Calibrated On-Wafer Measurements[END_REF], and may result in some artifacts such as resonances (dips and oscillations) in the calibrated S-parameters of the DUT. Thus, it is crucial to reduce the impact of such coupling as well as the probeto-probe crosstalk and the coupling between probes and the back-end-of-line (BEOL) environment, eventually providing a reliable guide to test structure layout designers.

In the following sections, we will first present a discussion about the technology used to fabricate the test structures of the two separate runs, a description of the layout design of each run including the differences between them, and the methodology to validate the measurements through EM simulation models in detail. Next, the topic will focus on a comparison between the measurements and the EM simulation results of the capacitances of a relevant test structure, open-M1. To provide a better insight on the different behaviors of the two fabrication runs, the layout design used in a previous run will be suitably modified to reflect the design of the newest run, and will be simulated with and without neighboring structures. The electric field contours of both designs will be shown to observe the physical phenomena taking place in different layout configurations.

II. STRUCTURES AND METHODOLOGY A. On-Wafer Calibration Standards

For the on-wafer TRL calibration, the test structures and HBTs have been designed [START_REF] Deng | Design of Silicon On-Wafer Sub-THz Calibration Kit[END_REF] and fabricated with STMicroelectronics BiCMOS 55-nm (B55) technology [START_REF] Chevalier | A 55 nm triple gate oxide 9 metal layers SiGe BiCMOS technology featuring 320 GHz f T / 370 GHz fmax HBT and high-Q millimeter-wave passives[END_REF]. All the structures lay on a silicon substrate and consist of 8 metal layers (M1-M8) and corresponding vias in the BEOL (Fig. 1). On top of M8, which is used for the microstrip line design, aluminum ground-signal-ground (G-S-G) contact pads are realized. In addition to the on-wafer TRL calibration

Published in: IEEE Transactions on Electron Devices (Early Access ; Nov. 2020) kit described in [START_REF] Deng | Design of Silicon On-Wafer Sub-THz Calibration Kit[END_REF] (labelled "Run-1" from now on), a new mask with the same layer sequence but a different layer layout has also been designed and fabricated (labelled "Run-2"). The on-wafer TRL calibration standards 'thru', 'reflect' and 'lines' of Run-1 and 2 are shown in panels (a)-(f) of Fig. 2, respectively. In both runs, two lines are used in the on-wafer TRL calibration up to 500 GHz where the longer line is named 'line 110 GHz' and shorter line is named 'line 500 GHz', with a range of validity from 20 GHz to 500 GHz. The dimensions of each line and thru for both runs are listed in Table I. In the on-wafer TRL calibration for each run, 'line 110 GHz' is used up to 110 GHz while 'line 500 GHz' is used in the 140-500 GHz range. For the reflect standard, a structure called 'pad-open' acts as an open circuit at the RF pads' plane, thus providing high reflection for the incident waves. Moreover, to perform an impedance correction, one extra 'pad-load' structure consisting of two loads at port-1 and port-2, including pads and M8 access line, is considered. After performing the on-wafer TRL calibration, the reference plane is set after the RF pads at the edge of the access line, as shown in Fig. 2(b) and Fig. 2(e) on the pad-open. In addition to the on-wafer TRL calibration kit structures, two test structures are dedicated to the de-embedding of the transistor accesses. These two test structures are called open-M1 and short-M1 and, as their names suggest, they provide an open/short circuit at the contact plane at M1, with the same access design as the transistor. They are representative because of their elaborate BEOL and importance in the transistor measurements, thus the former is chosen as our DUT (Fig. 34).

SiO 2 ε R =3.3 SiO 2 ε R =4.3 Si 3 N 4 ε R =8.1

B. Differences Between Run-1 & Run-2

Giving more insight on the masks, we can list the following differences:

• Run-1 presents a 10-µm-thick silicon dioxide (SiO 2 )

"ring" extending from the silicon substrate to the top , where an open circuit is located (see detail). The "pad shield" is located at the back of the RF pads and pointed out explicitly. The shades in a darker tone of pink are small cubic "dummy" structures, that have been added to respect the density rules. of M8 and surrounding all the test structures for an electrical isolation [START_REF] Deng | Design of Silicon On-Wafer Sub-THz Calibration Kit[END_REF]: this element was needed to make Run-1 compliant to process design rules, while maintaining a proper distance between structures. In [START_REF] Williams | A prescription for sub-millimeter-wave transistor characterization[END_REF], it is indicated as a "rule of thumb" to avoid the use of slots in microstrip grounds for calibration structures, since they may generate excess coupling between structures. However, this recommendation has not been studied in detail by simulation with probe models, as it will be done in the following. Moreover, oftentimes this advice does not seem to be taken in the industrial environment; • in Run-1, the ground plane extends from M1 to M4, while in Run-2, the ground is provided by M1 only (Fig. 4); • the microstrip line realized on the top metal layer (M8) is 5.8/7.7 µm thick in Run-1/Run-2, and its distance to the ground plane is 4.9/5.6 µm;

• the RF pads are designed to have compatibility for either 50-or 100-µm pitch probes. The associated access lines after the RF signal pads are 4/15 µm, for Run-1 and Run-2, respectively. The signal pads are 14 % longer in Run-2 than Run-1, but 7 % narrower and the signalground pad gaps are respectively 16/20 µm. The to-edge distance between the closest signal pads of two adjacent structures is 120/210 µm in the x direction and 248/245 µm in the y direction. Also, the edge-to-edge distance between the closest ground pads of two adjacent structures is 172/180 µm in the x direction and 45/15 µm in the y direction. The RF pads have surface areas of 35×27 µm 2 and 40×25 µm 2 ; • the characteristic impedance is corrected to approximately 50 Ω in both designs. Two important additions on the Run-2 layout (see Fig. 3): 1) a shielding behind the signal pads [START_REF] Seelmann-Eggebert | On the Accurate Measurement and Calibration of S-Parameters for Millimeter Wavelengths and Beyond[END_REF], [START_REF] Yadav | Impact of on-Silicon De-Embedding Test Structures and RF Probes Design in the Sub-THz Range[END_REF]; 2) a M1-to-M8 ground volume that connects all the ground pads together and is seen at M8 as a boundless plane. We call this novel layout "continuous ground plane" to stress on its ubiquity among the structures on the die, its intent being to reduce the probe-to-substrate EM coupling and coupling with neighbors. In Fig. 5, we show a top view of a portion of the wafer where the test structures layout is visible. In this picture, the open-M1 structures for Run-1 (Fig. 5(a)) and Run-2 (Fig. 5(b)) are surrounded by their respective neighbors (the "oxide ring" is visible in white in Fig. 5(a)). For Run-1, the structures are aligned in columns and rows. Structures in Run-2 are placed differently: a particular design in staggered rows is used in order to reduce EM coupling between the RF probes and the adjacent structures.

C. EM Simulation Procedure

Calibration methods and de-embedding techniques are indispensable tools to remove the parasitic contributions from measurement data; nevertheless, the associated error models only approximately represent the true environment. Consequently, electromagnetic (EM) simulations are carried out in Ansoft's High-Frequency Structure Simulator (HFSS) as part of the analysis of layouts and measurement verification. After creation of the 3D models of each structure, simulation is carried out much like described in [START_REF] Yadav | Impact of on-Silicon De-Embedding Test Structures and RF Probes Design in the Sub-THz Range[END_REF]. Thanks to the optical microscope imaging, several G-S-G probe models replicating the GGB® Picoprobe RF probes with a 50/100 µm pitch for each band up to 500 GHz have been designed in HFSS [START_REF] Yadav | Importance and requirement of frequency band specific RF probes EM models in sub-THz and Thz measurements up to 500 GHz[END_REF].

D. Measurement Setup

Four sets of measurements have been carried out to cover the whole spectrum up to 500 GHz. In the 1-110 GHz frequency range, measurements are performed with an Agilent E8361A Vector Network Analyzer (VNA), with an extender (N5260-60003) in the 67-110 GHz range; 140-220 GHz, 220-325 GHz and 330-500 GHz measurements were performed with a ZVA24 VNA from Rhode & Schwartz with Rhode & Schwartz extenders (ZC220, ZC330, and ZC500, respectively). The RF probes used in measurement are from Picoprobe, with 50 µm pitch, except for Run-2 in 1-110 GHz band measurements where Picoprobe probes with 100 µm pitch are used. The on-wafer TRL calibration is performed using raw measurement data of the DUT, open-M1, provided by the VNA, where the TRL calibration with impedance correction has been applied [START_REF] Williams | Transmission line capacitance measurement[END_REF], [START_REF] Marks | Characteristic impedance determination using propagation constant measurement[END_REF], setting the reference plane at the end of the small access lines after the pad (see in Fig. 2(b)).

III. RESULTS AND DISCUSSION

A. Open-M1 up to 500 GHz -Run-1 vs. Run-2

In Fig. 6, all the capacitances of open-M1 are shown for both Run-1 and 2: they are related to port-1 (C 11 ), port-2 (C 22 ), and the coupling capacitance between the port-1 & 2 (C 12 ). The measurements are very well replicated by simulation. It is worth noticing that particularly at very high frequency, the measurements and the corresponding simulations with probes follow the intrinsic simulation, thus confirming the efficiency of the on-wafer TRL calibration. Overall, the Run-2 measurements appear to be more consistent and have very good band-to-band continuity; the lower capacitance at port-1, which is, for that matter, predicted by the intrinsic simulation, is due to the smaller metallic M1 layer surface and the higher distance between the base metal contact and ground. In the 140-220 GHz band, though, both the measurement and the simulation with probes diverge from the trend indicated by the intrinsic simulation, particularly the port-to-port capacitance (C 12 ), which becomes clearly nonphysical. This fact may be traced back to the port-1 to port-2 crosstalk between the two RF probes due to their design [START_REF] Yadav | Importance and requirement of frequency band specific RF probes EM models in sub-THz and THz measurements up to 500 GHz[END_REF]. It has been showed that a coplanar probe with a much simpler design does not present this behavior in this band [START_REF] Yadav | Impact of on-Silicon De-Embedding Test Structures and RF Probes Design in the Sub-THz Range[END_REF]. We can also clearly see that values of open-M1 turn out to be generally positive for Run-2 (except in the 140-220 GHz range), while Run-1 measured values become negative from 240 GHz on. This more physical behavior with respect to Run-1 can be attributed to a different configuration of each of the BEOL layers and the continuous ground plane, which provides a different environment around the DUT, more distance from the neighboring structures and less coupling through the pad shield.

B. EM Simulation -Run-2 vs. Altered Run-2

To further investigate the influence of the DUT environment, we consider altered versions of the reference Run-2 layout. By this, we mean to alter the Run-2 design to match the default Run-1 design by introducing spot modifications in order to eventually understand and compare the layout impact. Any direct comparison, though, would be pointless, since the layout of each layer of the BEOL has deeply changed from Run-1 to 2. We study the capacitances of the following configurations:

• "Single": In Fig. 7 ring surrounds each isolated structure, much like in Run-1, and the pad shield is removed.

• "Neighbors" (for short, "Neigh"): In Fig. 7(d)-7(f), all the structures have been simulated with their actual corresponding adjacent structures (just the closest neighbors are taken into account, as the electromagnetic impact of more distant structures on the DUT would be negligible), and capacitances of open-M1 are shown. The pad-topad distance between each DUT and its neighbors is brought to the same as Run-1 (45 µm). Two different configurations are considered for these plots too:

-"Ref": the reference, where every Run-2 structure is not surrounded by any ideal infinite plane of dielectric and metal, but by its actual neighbors, in their actual position, as they are on the wafer; -"Align" and "Align+ring": the altered version, where the neighbors are also present but are aligned in columns and rows. For simplicity, the common chosen neighbor is only one: pad-open, since this structure has proved to be more prone to EM coupling, thus representing a "worst case" situation. In "Align+ring", the oxide ring is added, like in Run-1: therefore, this represents a "pseudo Run-1" case. From Fig. 7(a)-7(c), the effect of the oxide ring is visible. Overall, the curves show small fluctuations around the reference (mostly on C 12 ), and larger deviation from the intrinsic curves at low frequency. In Fig. 7(d)-7(f), the contribution of neighbors is also considered. As we can see, the curves representing the reference case with neighbors, the orange curves in Fig. 7(d)-7(f), keep close to measurements, as expected, but the curves in the case with no neighbors (blue curves in Fig. 7(a)-7(c)), do not deviate considerably: in fact, they differ less than 1 fF all over the spectrum, except in the 1 -110 GHz band, where deviation slightly exceeds this value. This important result yields to the conclusion that by the optimized design of Run-2, the impact of the neighbors is considerably reduced. Also, this fact makes us confident on the use of "single" structures instead of models with adjacent structures around them for EM simulations, which greatly reduces simulation time and complexity. The alteration of the neighboring environment specified in Fig. 7(d)-7(f) by the light blue and pink curves also provides some interesting insights. If we first consider the case where the structures have just been aligned ("align"), capacitances C 11 and C 22 keep above the reference configuration ("ref") in all the considered frequency spectrum; the coupling is reduced thanks to the pad shielding and the staggered configuration. Note that, on the other hand, C 12 is only very moderately affected. The addition of the oxide ring ("align+ring") disturbs the trends of the port capacitances generating a small ripple, but more strongly the trend of C 12 , which is similar in shape to C 12 in the case of a single structure with oxide ring (Fig. 7(a)-7(c)). Therefore, on C 12 the effect of the alignment is negligible compared to the one produced by the oxide ring while, on the contrary, closer and aligned neighbors affect more the capacitances between the ports and the substrate, with an additional capacitance adding up to the one which is already present (offset on C 11 and C 22 ).

We observe now the electric field 3D contour obtained by HFSS. In the backside view of the DUT and its neighbors (Fig. 8), we see the E-field concentrating on the DUT and beneath the probe corresponding to port-1, where the field is excited, and the most intense E-field is efficiently confined inside the space created by the pad shield. The field contour has the same shape either with or without the presence of adjacent structures (Fig. 8(a), 8(b)). When the dielectric ring is present (Fig. 8(c)), the E-field is heavily affected. The intensity of the field increases around the DUT and below the excitation probe and we can clearly see the field densifying around every adjacent structure. In Fig. 9, the penetration inside the silicon substrate is well depicted. Where no oxide ring is present (Fig. 9(a) and Fig. 9(b)), the E-field is only slightly visible in the substrate region below the DUT. Also, no difference can be noticed in the field shape when the neighbors are considered, thanks to the continuous ground plane. On the other hand, when aligned neighbors are in place (Fig. 9(c)), the field is free to couple below the DUT with all the neighbors through the rings.

Fig. 8(d), 9(d) introduce yet another configuration. This aims to represent a more dense layout design approach of test structures: the neighbors in this case are in staggered row but no continuous ground plane is present. In fact, no metal volume is connecting the structures, which are only surrounded by the dielectic. Also, their mutual distance is even more reduced, in order to create the most compact design: the area occupancy is reduced of a quarter. The electric field in Fig. 8(d) consequently scatters and permeates all the neighboring structures more intensly than in Fig. 9(c). Fig. 9(d), on the other hand, shows that the probe-to-substrate coupling is reduced (since no metal is under the probe), but the field uncontrollably propagates through the lossy silicon substrate; however, while the coupling can be removed by calibration, the dispersion of the field in the substrate is harder to correct, making it more prone to measurement errors.

IV. CONCLUSION

In this work, we presented S-parameters measurements of a test structure creating an open circuit at M1 up to 500 GHz fabricated in two separate runs using the STMicroelectronics' B55 technology. The comprehensive EM simulation analysis is presented to analyze the measurement data behavior. The impact of the layout design improvements of Run-2, the socalled continuous ground plane, is highlighted on the open-M1 characteristics through measurement and EM simulation analysis. Shielding structure, staggered row placement and removing the dielectric ring result in a more confined energy flow and avoiding all artifacts on any capacitance, as confirmed by simulation of the altered Run-2 layout. Also, the electric field contour reinforces our motivation in the use of the boundless ground plane, whereas a widespread approach leads to uncontrolled coupling with neighbors and the substrate. It is worth to point out that the presented results are valid not only for the particular case of open-M1, but for any test structure with similar layout design and neighbors displacement.

Fig. 1 .

 1 Fig. 1. Cross-sectional side view of a transmission line of fabricated onwafer TRL calibration kit where dielectric constants levels used in simulation are shown (Run-2). The G-S-G RF pad configuration at M8 is shown in orange, with signal trace in the center. The ground plane is at M1.

Fig. 2 .

 2 Fig. 2. Top view of the on-wafer TRL calibration test structures. Run-1&2, respectively: (a),(d) thru, (b),(e) pad-open, (c),(f) line 500 GHz. In pad-open, post-calibration reference planes are shown with black dashed line.

P a d S h ie ldFig. 3 .

 3 Fig. 3. Open-M1 3D model (Run-2) for transistor de-embedding. From access lines in M8 (pink), metals (yellow) alternate with vias (black) down to M1, where an open circuit is located (see detail). The "pad shield" is located at the back of the RF pads and pointed out explicitly. The shades in a darker tone of pink are small cubic "dummy" structures, that have been added to respect the density rules.

Fig. 4 .

 4 Fig. 4. Open-M1 structure for transistor de-embedding. Cross section details from Run-1 (a) and Run-2 (b) show metal layers from M1 (yellow) to M8 (pink) in color, while vias are in black. Ground plane is up to M4 (Run-1) or M1 (Run-2). Dimetric 3D details from Run-1 (c) and Run-2 (d) are also shown. At M1 a space between port-1 and 2 contacts provides an open circuit. In Run-1, the M8 cubic connection bridges the emitter contact (not visible due to perspective) to the M1-M4 ground plane.

Fig. 5 .

 5 Fig. 5. Top layout view of the open-M1 (DUT: name in red) surrounded by its neighbors in Run-1 (a) and Run-2 (b). The same test structures of the central column are repeated at the right for another transistor (with different emitter size).

Fig. 6 .

 6 Fig. 6. All the capacitances measurement, intrinsic HFSS and complete structure simulations of the open-M1 for both Run-1 and 2. These capacitances are related to port-1 (C 11 ), port-2 (C 22 ), and the coupling capacitance between the port-1 and port-2 (C 12 ).

Fig. 7 .

 7 Fig. 7. Run-2 measurement vs. simulation. Single structure with and without SiO 2 ring (from (a) to (c)), DUT with neighbors (adjacent structures) in the actual "staggered row" configuration, with aligned neighbors, and in a "pseudo Run-1" configuration (aligned structures with a 10 µm SiO 2 ring around them).

  (a)-7(c), all the structures (DUT and calibration standard 'thru', 'pad-open' and 'line 110/500 GHz') have first been simulated as isolated structures laying on planes emulating infinite dielectric layers and ground. The DUT, open-M1, is subsequently calibrated with the corresponding isolated calibration standards. We consider two different configurations: -"Ref": the reference, where each Run-2 isolated structures are modelled; -"Ox ring": the altered version, where a 10-µm SiO 2

Fig. 8 .

 8 Fig. 8. HFSS-simulated electric field contour (back view) for DUT transistor-open at 500 GHz: single structure (a), neighbors (b), aligned neighbors with oxide ring (c), and neighbors in a more dense configuration (no continuous ground plane) (d).

Fig. 9 .

 9 Fig. 9. HFSS-simulated electric field contour (side view) for DUT transistor-open at 500 GHz: single structure (a), neighbors (b), aligned neighbors with oxide ring (c), and neighbors in a more dense configuration (no continuous ground plane) (d).

TABLE I .

 I Dimensions of thru and lines in each run.

	Test Structure Name	Run-1	Run-2
	Thru	35 µm	65 µm
	Line 110 GHz	365 µm	595 µm
	Line 500 GHz	115 µm	185 µm