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[EN] Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator., This work has been financially supported by ANR (PATRIC project, grant ANR-13-KBBE-0001) (to BP), the Regional Council of Bourgogne Franche-Comte (PARI grant 2016-9201AAO050S016 36 and FEDER grant BG0005888) and INRA for the funding of Justine Claverie's PhD (grants 2015-9201AAO048502578 and 29000907), by the Gatsby Charitable Foundation (to CZ), and by the Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/G024936/1 (ERA-PG PRR CROP (to CZ).

International audience; In nature, plants are constantly exposed to potentially pathogenic microbes such as bacteria, fungi, oomycetes or viruses. However, plants have developed effective immune systems triggering various defence reactions against invading pathogens upon the perception of pathogen-associated molecular patterns (PAMPs; Dodds and Rathjen, 2010). The recognition of these conserved microbial signatures is ensured by Pattern Recognition Receptors (PRRs) which also detect plant endogenous molecules released during pathogen invasion, called damage-associated molecular patterns (DAMPs; Boller and Felix, 2009). Chitin, a fungal cell wall component, is a well-known PAMP that triggers defence responses in many mammal and plant species. The aim of the study was to determine the effects of chito-oligosaccharides on grapevine’s immunity and identify the receptor(s) involved in the perception of chito-oligosaccharides in grapevine. Material and methods Grapevine cells (Vitis vinifera cv Gamay) were cultivated as described in Gauthier et al. (2014). Arabidopsis thaliana plants from wild-type (WT) Columbia (Col-0), mutant and transgenic lines were grown in vitro for two weeks in controlled conditions for defence responses or in jiffy peat pellets in a controlled growth chamber for four weeks for protection assays. Grapevine cells or Arabidopsis plants were treated with water, chitin, chitosan (Elicityl, 0.1 g/l for cells and 1 g/l for plants) or flagelline (10 μM) taken as a positive control. ROS production and cytosolic Ca2+ variations ([Ca2+]cyt) in grapevine cells were performed according to Dubreuil-Maurizi et al. (2010) after elicitor treatments, by measuring the chemiluminescence of luminol for H2O2 production and using apoaequorin expressing cells to detect variations of [Ca2+]cyt. Protein extraction, SDS-PAGE and western blotting for MAPK phosphorylation analysis were carried out as previously described (Trdà et al., 2014). RNA extraction and quantitative real-time PCR were performed using primers for the Highlights • Two Pattern Recognition Receptors (PRRs) VvLYK1-1 and VvLYK1-2 participate in the signaling of chito-oligosaccharides in grapevine • VvLYK1-1 is involved in powdery mildew resistance amplification of defence marker genes (CHIT4C, STS1-2, PAL, RBOHD, and FRK1). Two days after elicitor treatment, Botrytis cinerea and Plasmopara viticola infections were performed on grapevine plants. For protection assays to Erysiphe necator, leaves were infected, maintained on agar medium in the incubator and then sampled at 0, 4, 8, 12 and 24 hours post inoculation. Results and discussion In grapevine cells, chitin treatment induced a rapid and transient increase in free [Ca2+]cyt that peaked at 2 min but not chitosan, even if the basal level remained higher during the whole experiment. Both chito-oligosaccharides did not trigger any H2O2 production contrary to the flagelline epitope flg-22. Chitin and chitosan treatment induced the phosphorylation of two MAPKs with relative molecular masses of 45 and 49 kDa in grapevine cells but chitosan activated the phosphorylation of these two MAPKs longer than the chitin treatment. The expression of defence marker genes activated by different elicitors was then followed by qPCR. Among them, both chito-oligosaccharides induced the expression of four grapevine defence genes encoding an acidic chitinase (CHIT4C), a stilbene synthase (STS1-2), a phenylalanine ammonia lyase (PAL) and a respiratory burst oxidase homolog D (RBOHD), 1 hour post-treatment (hpt). The efficacy of chitin- and chitosan-induced immunity was investigated in Vitis vinifera leaf discs infected by the necrotrophic fungus B. cinerea or with the biotrophic oomycete P. viticola, the causal agents of gray mold and downy mildew, respectively. If chitin pretreatment induced a low but significant resistance against these pathogens, chitosan reduced very significantly the B. cinerea lesion diameter and the P. viticola sporulation. Taken toghether, these results demonstrate that grapevine perceives chitin and chitosan suggesting that at least one PRR for chito-oligosaccharides perception exists. To identify the receptor of chito-oligosaccharides in grapevine, the grapevine family of LysM receptor like kinases was characterised and three proteins, respectively named VvLYK1-1, VvLYK1-2 and VvLYK1-3, showed a close relation to Arabidopsis CERK1/LYK1 (Chitin-Elicitor Receptor Kinase 1) and the rice ortholog CERK1. By functional complementation of the Arabidopsis cerk1/lyk1 mutant, impaired in chitin perception and signalling, we demonstrated that VvLYK1-1 and VvLYK1-2 are involved in the signalling of chito-oligosaccharides in Vitis vinifera. Moreover, VvLYK1-1 plays a key role in basal resistance against the grapevine powdery mildew causal agent E. necator.

Highlights • Fragments derived from plant cell wall xyloglucans induce Arabidopsis thaliana defence responses and protection against Botrytis cinerea • Xyloglucan-triggered immunity against B. cinerea requires the phytoalexin, ethylene and jasmonic acid-dependent pathways Results and discussion Xh treatment induced a dose-dependent MAPK phosphorylation in Arabidopsis cell suspensions. From 5 to 60 min, Xh treatment induced a rapid phosphorylation of two MAPKs with relative molecular masses of 43 and 47 kDa. Treatment with Xh did not induce any free [Ca2+]cyt variations whereas OG treatment induced a rapid and transient increase in free [Ca2+]cyt that peaked after 30 sec. Xh did not trigger any H2O2 production, as observed in control cells but OG treatment induced an oxidative burst with maximal H2O2 production detected at 10 min. To investigate late defence responses, we analysed callose deposition at the site of infection by B. cinerea after elicitor treatments. Xh and OG-treatment resulted in a significant increase of callose production 3 days post infection with the pathogen. The expression of different defence genes was analysed by qPCR. Xh triggered the accumulation of PR-1, PAD3, LOX3 and ICS1 transcripts. To further investigate the efficacy of xyloglucans to induce resistance, we performed protection assays against the necrotrophic fungi B. cinerea and the biotrophic oomycete H. arabidopsidis. Xh treatment applied 48 h before pathogen infection significantly reduced both the B. cinerea lesion diameter and the H. arabidopsidis sporulation on Arabidopsis leaves. Together, these results suggest that Xh are new elicitors of Arabidopsis immunity. Interestingly, some defence responses triggered by Xh are different from those induced by OG. As Arabidopsis responds to Xh treatment, we aimed to identify some signalling components. By using a genetic approach with T-DNA mutants in different defence responses, our data indicated that the Xh-triggered immunity against B. cinerea requires the phytoalexin (cyp71A13, pad3, pad2), ethylene (etr1, ein2) and jasmonic acid-dependent pathways (dde2, lox3, coi1).These results show that Xh are recognised by Arabidopsis. In order to identify a receptor involved in Xh perception or signalling, knock-out mutants of previously known A. thaliana receptors or candidate receptors up-regulated in microarray analysis have been.

Damaged-Associated Molecular Patterns (DAMPs) are endogenous molecules released from the plant cell wall after wounding by pathogens. DAMPs are recognized by Pattern- Recognition Receptors (PRRs) that play a key role in plant immunity by mediating defense responses. The plant cell wall-derived oligogalacturonides (OG) are well characterized DAMPs that elicit plant immune responses such as MAPK activation, [Ca2+]cyt variations, H2O2 production, defense-related gene expression and enhanced resistance against Botrytis cinerea. Our study focused on a new polysaccharide component of the plant cell wall called xyloglucans (Xh) and compared the immune events triggered by OG and Xh in Arabidopsis thaliana. Our results indicated that Xh can be considered as new elicitors as they induced MAPK activation, the expression of defense-related genes, callose deposition and triggered immunity against Botrytis cinerea. By using a genetic approach, our data indicated that the Xh-triggered immunity against B. cinerea requires the phytoalexin and jasmonic aciddependent pathways.