CERK1 is a lysine motif-containing plant pattern recognition receptor for chitin and peptidoglycan. Chitin recognition by OsCERK1 triggers rapid engagement of a rice MAP kinase cascade, which leads to defense response activation. How the MAP kinase cascades are engaged downstream of OsCERK1 remains obscure. Searching for host proteins that interact with Xoo1488, an effector of the rice pathogen Xanthomonas oryzae, we identified the rice receptor-like cytoplasmic kinase, OsRLCK185. Silencing OsRLCK185 suppressed peptidoglycan- and chitin-induced immune responses, including MAP kinase activation and defense-gene expression. In response to chitin, OsRLCK185 associates with, and is directly phosphorylated by, OsCERK1 at the plasma membrane. Xoo1488 inhibits peptidoglycan- and chitin-induced immunity and pathogen resistance. Additionally, OsCERK1-mediated phosphorylation of OsRLCK185 is suppressed by Xoo1488, resulting in the inhibition of chitin-induced MAP kinase activation. These data support a role for OsRLCK185 as an essential immediate downstream signaling partner of OsCERK1 in mediating chitin- and peptidoglycan-induced plant immunity.

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4DD-induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.

WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

Plants, as sessile organisms, survive environmental changes by prioritizing their responses to the most life-threatening stress by allocating limited resources. Previous studies showed that pathogen resistance was suppressed under abiotic stresses. Here, we show the mechanism underlying this phenomenon. Phosphorylation of WRKY45, the central transcription factor in salicylic-acid (SA)-signalling-dependent pathogen defence in rice, via the OsMKK10-2–OsMPK6 cascade, was required to fully activate WRKY45. The activation of WRKY45 by benzothiadiazole (BTH) was reduced under low temperature and high salinity, probably through abscisic acid (ABA) signalling. An ABA treatment dephosphorylated/inactivated OsMPK6 via protein tyrosine phosphatases, OsPTP1/2, leading to the impaired activation of WRKY45 and a reduction in Magnaporthe oryzae resistance, even after BTH treatment. BTH induced a strong M. oryzae resistance in OsPTP1/2 knockdown rice, even under cold and high salinity, indicating that OsPTP1/2 is the node of SA-ABA signalling crosstalk and its down-regulation makes rice disease resistant, even under abiotic stresses. These results points to one of the directions to further improve crops by managing the tradeoffs between different stress responses of plants.

The Rac/Rop GTPase OsRac1 plays an essential role in rice immunity. However, the regulatory genes acting downstream of OsRac1 are largely unknown. We focused on the RAI1 gene, which is up-regulated in suspension cells expressing a constitutively active form of OsRac1. RAI1 encodes a putative basic helix-loop-helix transcription factor. A microarray analysis of cells transformed with an inducible RAI1 construct showed increased expression of PAL1 and OsWRKY19 genes after induction, suggesting that these genes are regulated by RAI1. This was confirmed using RAI1 T-DNA activation-tagged and RNA interference lines. The PAL1 and OsWRKY19 genes were also up-regulated by sphingolipid and chitin elicitors, and the RAI1 activation-tagged plants had increased resistance to a rice blast fungus. These results indicated that RAI1 is involved in defense responses in rice. RAI1 interacted with OsMAPK3 and OsMAPK6 proteins in vivo and in vitro. Also, RAI1 was phosphorylated by OsMAPK3/6 and OsMKK4-dd in vitro. Overexpression of OsMAPK6 and/or OsMAPK3 together with OsMKK4-dd increased PAL1 and OsWRKY19 expression in rice protoplasts. Therefore, the regulation of PAL1 and OsWRKY19 expression by RAI1 could be controlled via an OsMKK4-OsMAPK3/6 cascade. Co-immunoprecipitation assays indicated that OsMAPK3 and OsRac1 occur in the same complex as OsMAPK6. Taken together, our results indicate that RAI1 could be regulated by OsRac1 through an OsMAPK3/6 cascade. In this study, we have identified RAI1 as the first transcription factor acting downstream of OsRac1. This work will help us to understand the immune system regulated by OsRac1 in rice and its orthologs in other plant species.