In plants leucine-rich repeat receptor kinases (LRR-RKs) located at the plasma membrane play a pivotal role in the perception of extracellular signals. For two of these LRR-RKs, the brassinosteroid receptor BRI1 and the flagellin receptor FLS2, interaction with the LRR receptor-like kinase BAK1 (BRI1-associated receptor kinase 1) was shown to be required for signal transduction. Here we report that FLS2⅐BAK1 heteromerization occurs almost instantaneously after perception of the ligand, the flagellin-derived peptide flg22. Flg22 can induce formation of a stable FLS2⅐BAK1 complex in microsomal membrane preparations in vitro, and the kinase inhibitor K-252a does not prevent complex formation. A kinase dead version of BAK1 associates with FLS2 in a flg22-dependent manner but does not restore responsiveness to flg22 in cells of bak1 plants, demonstrating that kinase activity of BAK1 is essential for FLS2 signaling. Furthermore, using in vivo phospholabeling, we are able to detect de novo phosphorylation of both FLS2 and BAK1 within 15 s of stimulation with flg22. Similarly, brassinolide induces BAK1 phosphorylation within seconds. Other triggers of plant defense, such as bacterial EF-Tu and the endogenous AtPep1 likewise induce rapid formation of heterocomplexes consisting of de novo phosphorylated BAK1 and proteins representing the ligand-specific binding receptors EF-Tu receptor and Pep1 receptor 1, respectively. Thus, we propose that several LRR-RKs form tight complexes with BAK1 almost instantaneously after ligand binding and that the subsequent phosphorylation events are key initial steps in signal transduction.One of the central themes in cell biology is the sensing of extracellular chemical signals through cell surface receptors: How does the event of receptor-ligand interaction on the outside of the cell activate a signal transduction chain in the inside of the cell? In higher plants, the most prominent class of membrane receptors is formed by proteins with intracellular serine/ threonine-type protein kinases. These receptors account for ϳ2.5-4% of all proteins encoded by the genome of a plant (1). Despite their importance, there is still little experimental evidence on the molecular activation mechanisms of plant transmembrane receptor kinases. Current models are based on the precedent of animal receptor tyrosine kinases where ligand binding causes receptor tyrosine kinases to form homo-or hetero-oligomers, followed by transphosphorylation (2). In the case of the epidermal growth factor receptor, these phosphorylation events occur within 60 s of receptor activation (3).The best studied plant transmembrane receptor kinase is BRI1, the receptor for the brassinosteroid growth hormones (4). BRI1 is one of the 224 members of LRR-RKs 2 in Arabidopsis (5). Upon ligand binding BRI1 interacts with a second LRR receptor-like kinase named BAK1 (6, 7). Two further well characterized plant LRR-RKs are the flagellin receptor FLS2 (flagellin sensing 2) (8) and the EF-Tu receptor (EFR) (9). FLS2 perceives a generally conser...

Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGNs) are major components of bacterial cell walls that possess immunity-stimulating activities in metazoans and plants. Here we show that PGN sensing and immunity to bacterial infection in Arabidopsis thaliana requires three lysin-motif (LysM) domain proteins. LYM1 and LYM3 are plasma membrane proteins that physically interact with PGNs and mediate Arabidopsis sensitivity to structurally different PGNs from Gram-negative and Gram-positive bacteria. lym1 and lym3 mutants lack PGN-induced changes in transcriptome activity patterns, but respond to fungus-derived chitin, a pattern structurally related to PGNs, in a wild-type manner. Notably, lym1, lym3, and lym3 lym1 mutant genotypes exhibit supersusceptibility to infection with virulent Pseudomonas syringae pathovar tomato DC3000. Defects in basal immunity in lym3 lym1 double mutants resemble those observed in lym1 and lym3 single mutants, suggesting that both proteins are part of the same recognition system. We further show that deletion of CERK1, a LysM receptor kinase that had previously been implicated in chitin perception and immunity to fungal infection in Arabidopsis, phenocopies defects observed in lym1 and lym3 mutants, such as peptidoglycan insensitivity and enhanced susceptibility to bacterial infection. Altogether, our findings suggest that plants share with metazoans the ability to recognize bacterial PGNs. However, as Arabidopsis LysM domain proteins LYM1, LYM3, and CERK1 form a PGN recognition system that is unrelated to metazoan PGN receptors, we propose that lineage-specific PGN perception systems have arisen through convergent evolution.

Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

Peptide signals have emerged as an important class of regulators in cell-to-cell communication in plants. Several families of small, secreted proteins with a conserved C-terminal Pro-rich motif have been identified as functional peptide signals in Arabidopsis thaliana. These proteins are presumed to be trimmed proteolytically and undergo posttranslational modifications, such as hydroxylation of Pro residues and glycosylation, to form mature, bioactive signals. Identification and matching of such ligands with their respective receptors remains a major challenge since the genes encoding them often show redundancy and low expression restricted to a few cells or particular developmental stages. To overcome these difficulties, we propose the use of ectopic expression of receptor genes in suitable plant cells like Nicotiana benthamiana for testing ligand candidates in receptor output assays and in binding studies. As an example, we used the IDA peptide HAE/HSL2 receptor signaling system known to regulate floral organ abscission. We demonstrate that the oxidative burst response can be employed as readout for receptor activation by synthetic peptides and that a new, highly sensitive, nonradioactive labeling approach can be used to reveal a direct correlation between peptide activity and receptor affinity. We suggest that these approaches will be of broad value for the field of ligand-receptor studies in plants.

SummaryWe characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1.In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS).We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression.We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling.