Cytoplasmic interaction of LysM receptors contributes to the formation of symbiotic receptor complex.

Receptor complex formation at the cell surface is a key step to initiate downstream signaling but the contribution of this process for the regulation of the direction of downstream responses is not well understood. In the plant-microbe interactions, while CERK1, an Arabidopsis LysM-RLK, mediates chitin-induced immune responses, NFR1, a Lotus homolog of CERK1, regulates the symbiotic process with rhizobial bacteria through the recognition of Nod factors. Concerning the mechanistic insight of the regulation of such apparently opposite biological responses by the structurally related RLKs, Nakagawa et al. previously showed that the addition of YAQ sequence, conserved in NFR1 and other symbiotic LysM-RLKs, to the kinase domain of CERK1 switched downstream responses from defense to symbiosis using a set of chimeric receptors, NFR1-CERK1s. These results indicated that such a subtle difference in the cytoplasmic domain of LysM-RLKs could determine the direction of host responses from defense to symbiosis. On the other hand, it is still not understood how such structural differences in the cytoplasmic domains determine the direction of host responses. We here analyzed the interaction between chimeric NFR1s and NFR5, a partner receptor of NFR1, by co-immunoprecipitation (Co-IP) of these proteins transiently expressed in Nicotiana benthamiana. These results indicated that the cytoplasmic interaction between the LysM-RLKs is important for the symbiotic receptor complex formation and the YAQ containing region of NFR1 contributes to trigger symbiotic signaling through the successful formation of NFR1/NFR5 complex.

AtCERK1 Phosphorylation Site S493 Contributes to the Transphosphorylation of Downstream Components for Chitin-Induced Immune Signaling.

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.

Autophosphorylation site Y428 is essential for the in vivo activation of CERK1.

Autophosphorylation of PRR is a critical event for the activation of immune signaling in plant. However, the detailed function of these phosphorylation sites is still not well understood. We analyzed the function of an autophosphorylation site of Arabidopsis CERK1, Y428, in immune signaling. Biochemical characterization of CERK1 mutants transiently expressed in N. benthamiana indicated that Y428 plays a crucial role for the in vivo activation of CERK1, differently from the previous observation by the in vitro kinase assay with its cytoplasmic domain. Similar discrepancy between in vitro and in vivo kinase assay was also reported for the corresponding phosphorylation site of EFR, suggesting that these conserved tyrosine residues play important roles for the activation of both RD and non-RD RLKs.

Plant immunity and symbiosis signaling mediated by LysM receptors.

Plants possess the ability to recognize microbe-associated molecular patterns (MAMPs) and PAMPs through the PRRs, and initiate pattern-triggered immunity. MAMPs are derived from cell-envelope components, secreted materials and cytosolic proteins from bacteria, oomycetes or fungi, and some MAMPs play a similar function in the innate immunity in mammals. Chitin is a representative fungal MAMP and triggers defense signaling in a wide range of plant species. The chitin receptors CEBiP and CERK1 on the plasma membrane have LysM (lysin motif) in their ectodomains. These molecules play an important role for the defense responses in rice and Arabidopsis, strictly recognizing the size and acetylated form of chitin oligosaccharides. However, related LysM receptors also play major roles for the signaling in root nodule and arbuscular mycorrhizal symbiosis. This review summarizes current knowledge on the molecular mechanisms of the defense and symbiosis signaling mediated by LysM receptors, including the activation steps of chitin-induced defense signaling downstream of LysM receptors.

Autophosphorylation of Specific Threonine and Tyrosine Residues in Arabidopsis CERK1 is Essential for the Activation of Chitin-Induced Immune Signaling.

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.

Selective regulation of the chitin-induced defense response by the Arabidopsis receptor-like cytoplasmic kinase PBL27.

Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.