Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens.

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

Convergent evolution of plant pattern recognition receptors sensing cysteine-rich patterns from three microbial kingdoms.

The Arabidopsis thaliana Receptor-Like Protein RLP30 contributes to immunity against the fungal pathogen Sclerotinia sclerotiorum. Here we identify the RLP30-ligand as a small cysteine-rich protein (SCP) that occurs in many fungi and oomycetes and is also recognized by the Nicotiana benthamiana RLP RE02. However, RLP30 and RE02 share little sequence similarity and respond to different parts of the native/folded protein. Moreover, some Brassicaceae other than Arabidopsis also respond to a linear SCP peptide instead of the folded protein, suggesting that SCP is an eminent immune target that led to the convergent evolution of distinct immune receptors in plants. Surprisingly, RLP30 shows a second ligand specificity for a SCP-nonhomologous protein secreted by bacterial Pseudomonads. RLP30 expression in N. tabacum results in quantitatively lower susceptibility to bacterial, fungal and oomycete pathogens, thus demonstrating that detection of immunogenic patterns by Arabidopsis RLP30 is involved in defense against pathogens from three microbial kingdoms.

The grapevine LysM receptor-like kinase VvLYK5-1 recognizes chitin oligomers through its association with VvLYK1-1.

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1.

Activation of plant pattern-triggered immunity (PTI) relies on the recognition of microbe-derived structures, termed patterns, through plant-encoded surface-resident pattern recognition receptors (PRRs). We show that proteobacterial translation initiation factor 1 (IF1) triggers PTI in Arabidopsis thaliana and related Brassicaceae species. Unlike for most other immunogenic patterns, IF1 elicitor activity cannot be assigned to a small peptide epitope, suggesting that tertiary fold features are required for IF1 receptor activation. We have deployed natural variation in IF1 sensitivity to identify Arabidopsis leucine-rich repeat (LRR) receptor-like protein 32 (RLP32) as IF1 receptor using a restriction site-associated DNA sequencing approach. RLP32 confers IF1 sensitivity to rlp32 mutants, IF1-insensitive Arabidopsis accessions and IF1-insensitive Nicotiana benthamiana, binds IF1 specifically and forms complexes with LRR receptor kinases SOBIR1 and BAK1 to mediate signaling. Similar to other PRRs, RLP32 confers resistance to Pseudomonas syringae, highlighting an unexpectedly complex array of bacterial pattern sensors within a single plant species.

The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.

The Arabidopsis thaliana LysM-containing Receptor-Like Kinase 2 is required for elicitor-induced resistance to pathogens.

In Arabidopsis thaliana, perception of chitin from fungal cell walls is mediated by three LysM-containing Receptor-Like Kinases (LYKs): CERK1, which is absolutely required for chitin perception, and LYK4 and LYK5, which act redundantly. The role in plant innate immunity of a fourth LYK protein, LYK2, is currently not known. Here we show that CERK1, LYK2 and LYK5 are dispensable for basal susceptibility to B. cinerea but are necessary for chitin-induced resistance to this pathogen. LYK2 is dispensable for chitin perception and early signalling events, though it contributes to callose deposition induced by this elicitor. Notably, LYK2 is also necessary for enhanced resistance to B. cinerea and Pseudomonas syringae induced by flagellin and for elicitor-induced priming of defence gene expression during fungal infection. Consistently, overexpression of LYK2 enhances resistance to B. cinerea and P. syringae and results in increased expression of defence-related genes during fungal infection. LYK2 appears to be required to establish a primed state in plants exposed to biotic elicitors, ensuring a robust resistance to subsequent pathogen infections.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered 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.

ABA-Dependent Salt Stress Tolerance Attenuates Botrytis Immunity in Arabidopsis.

Plants have evolved adaptive measures to cope with abiotic and biotic challenges simultaneously. Combinatorial stress responses require environmental signal integration and response prioritization to balance stress adaptation and growth. We have investigated the impact of salt, an important environmental factor in arid regions, on the Arabidopsis innate immune response. Activation of a classical salt stress response resulted in increased susceptibility to infection with hemibiotrophic Pseudomonas syringae or necrotrophic Alternaria brassicicola, and Botrytis cinerea, respectively. Surprisingly, pattern-triggered immunity (PTI)-associated responses were largely unaffected upon salt pre-treatment. However, we further observed a strong increase in phytohormone levels. Particularly, abscisic acid (ABA) levels were already elevated before pathogen infection, and application of exogenous ABA substituted for salt-watering in increasing Arabidopsis susceptibility toward B. cinerea infection. We propose a regulatory role of ABA in attenuating Botrytis immunity in this plant under salt stress conditions.

The BIR2/BIR3-Associated Phospholipase Dγ1 Negatively Regulates Plant Immunity.

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 of these, a specific function in resistance toward 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 toward 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.

A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis.

The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar. Therefore we investigated whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. We identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved.

Interplay of plant glycan hydrolases and LysM proteins in plant-Bacteria interactions.

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.

Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1-dependent differences.

Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine-rich repeat receptor kinases (LRR-RK) FLS2 and EFR, and the LRR receptor protein (LRR-RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR-RK and LRR-RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP-mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23-regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2-signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP-type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size.

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.

Sensing Danger: Key to Activating Plant Immunity.

In both plants and animals, defense against pathogens relies on a complex surveillance system for signs of danger. Danger signals may originate from the infectious agent or from the host itself. Immunogenic plant host factors can be roughly divided into two categories: molecules which are passively released upon cell damage ('classical' damage-associated molecular patterns, DAMPs), and peptides which are processed and/or secreted upon infection to modulate the immune response (phytocytokines). We highlight the ongoing challenge to understand how plants sense various danger signals and integrate this information to produce an appropriate immune response to diverse challenges.

Peptidoglycan Isolation and Binding Studies with LysM-Type Pattern Recognition Receptors.

In the last decade, more and more plant receptors for complex carbohydrate structures have been described. However, studies on receptor binding to glycan ligands are often hampered due to the technical challenge to obtain pure preparations of homogeneous carbohydrate ligands such as bacterial peptidoglycan (PGN) in amounts suitable for studying protein-glycan interactions. Also, most approaches rely on the availability of defined soluble ligands, which in the case of glycans can rarely be synthesized but have to be purified from the respective microorganism. In this chapter, we describe the purification of complex PGN from sources such as gram-positive bacteria, from which PGN isolation is facilitated due to its larger content in their cell wall. Insoluble PGN can subsequently be used in simple carbohydrate pull-down assays to test for interaction with plant proteins. In this respect, lysin motif (LysM)-domain containing proteins are of particular interest. All plant receptors described to date to be involved in the perception of N-Acetylglucosamine-containing ligands (such as PGN or chitin) have been shown to belong to this protein class. Thus, this chapter will also include the production of recombinant LysM proteins to analyze their PGN interaction.

Isolation of Novel MAMP-like Activities and Identification of Cognate Pattern Recognition Receptors in Arabidopsis thaliana Using Next-Generation Sequencing (NGS)-Based Mapping.

Activation of pattern-triggered plant immunity requires recognition of microbe-derived molecular patterns (MAMPs) by plant-encoded pattern recognition receptors (PRRs). Many plant PRRs are found in selected plant genera only. Transfer of single PRRs or of cassettes expressing several PRRs (PRR stacking) across plant genus boundaries offers the potential to boost disease resistance by improving pathogen recognition features in economically important crop plants. The success of such an approach is most dependent on the availability of a large number of plant PRRs. Here, an efficient method for the identification of novel PRRs in the model plant Arabidopsis thaliana (hereafter, Arabidopsis for simplicity) is described. This method takes advantage of natural variation in microbial pattern sensitivity among hundreds of Arabidopsis accessions currently available. Identification of pattern-sensitive as well as pattern-insensitive accessions facilitates next-generation sequencing (NGS)-assisted mapping of PRRs. This approach is potentially applicable to the identification of PRRs that recognize patterns of any chemical nature. © 2017 by John Wiley & Sons, Inc.