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Plants are always found together with bacteria and other microbes. Although plants can be attacked by phytopathogenic bacteria, they are more often engaged in neutral or mutualistic bacterial interactions. In the soil, plants associate with rhizobia or other plant growth promoting rhizosphere bacteria; above ground, bacteria colonise plants as epi- and endophytes. For mounting appropriate responses, such as permitting colonisation by beneficial symbionts while at the same time fending off pathogenic invaders, plants need to distinguish between the "good" and the "bad". Plants make use of proteins containing the lysin motif (LysM) for perception of N-acetylglucosamine containing carbohydrate structures, such as chitooligosaccharides functioning as symbiotic nodulation factors or bacterial peptidoglycan. Moreover, plant hydrolytic enzymes of the chitinase family, which are able to cleave bacterial peptidoglycan or chitooligosaccharides, are essential for cellular signalling induced by rhizobial nodulation factors during symbiosis as well as bacterial peptidoglycan during pathogenesis. Hence, LysM receptors seem to work in concert with hydrolytic enzymes that fine-tune ligand availability to either allow symbiotic interactions or trigger plant immunity.

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis. The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions., This work was supported by Agence Nationale de la Recherche ANR-2010-JCJC-1608 and ANR-14-CE19-0014 (to D.P.); Investissement d’Avenir Infrastructures Nationales en Biologie et Santé program (ProFI project, ANR-10-INBS-08); and by King Abdullah University of Science and Technology BAS/1/1062-01-01 (to H.H.). The Institute of Plant Sciences Paris-Saclay benefits from the support of the LabEx Saclay Plant Sciences (ANR-10-LABX-0040-SPS). M.A.-T. was supported by a Humboldt-Capes fellowship.

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members and for some a specific function in resistance towards a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance towards the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea. As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP-treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP-treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis PLD family consists of 12 members and for some a specific function in resistance towards a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLDγ1, but not its close homologs PLDγ2 and PLDγ3, is specifically involved in plant immunity. Genetic inactivation of PLDγ1 resulted in increased resistance towards the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea. As pldγ1 mutant plants responded with elevated levels of reactive oxygen species to MAMP-treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pldγ1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are unlikely the cause for the enhanced immunity in the pldγ1 line. Instead, the plasma-membrane-attached PLDγ1 protein colocalized and associated with the receptor-like kinases BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLDγ1 and BIR2 was further promoted upon MAMP-treatment. Hence, we propose that PLDγ1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.One-sentence summaryA phospholipase D is a novel negative regulator of plant immunity and forms complexes with regulatory receptor-like kinases.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N‐acetyl‐glucosamine) oligomers, we here identify six‐subunit‐long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll‐like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size‐dependent system of immuno‐modulation that appears conserved in plants and humans. Since blocking of the chitin‐TLR2 interaction effectively prevents chitin‐mediated inflammation in vitro and in vivo, our study highlights the chitin‐TLR2 interaction as a potential target for developing novel therapies in chitin‐related pathologies and fungal disease.

Chitin is a highly abundant polysaccharide and linked to fungal infection and asthma. Unfortunately, its polymeric structure has hampered the identification of immune receptors directly binding chitin and signaling immune activation and inflammation, because purity, molecular structure and molarity are not well definable for a polymer typically extracted from biomass. Therefore, by using defined chitin (N-acetyl-glucosamine) oligomers, we identified six subunit long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR) 2 as the primary fungal chitin receptor on human and murine immune cells. Chitin oligomers directly bound TLR2 with nanomolar affinity and showed both overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Conversely, chitin oligomers shorter than 6 subunits were inactive or showed antagonistic effects on chitin/TLR2-mediated signaling, hinting to a size-dependent sensing/activation system unexpectedly conserved in plants and humans. Since blocking the chitin-TLR2 interaction effectively prevented chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.