Plants deploy numerous cell surface-localized pattern-recognition receptors (PRRs) to perceive host- and microbe-derived molecular patterns that are specifically released during infection and activate defense responses. The activation of the mitogen-activated protein kinases MPK3, MPK4, and MPK6 (MPK3/4/6) is a hallmark of immune system activation by all known PRRs and is crucial for establishing disease resistance. The MAP kinase kinase kinase (MAPKKK) MEKK1 controls MPK4 activation, but the MAPKKKs responsible for MPK3/6 activation downstream of diverse PRRs and how the perception of diverse molecular patterns leads to the activation of MAPKKKs remain elusive. Here, we show that two highly related MAPKKKs, MAPKKK3 and MAPKKK5, mediate MPK3/6 activation by at least four PRRs and confer resistance to bacterial and fungal pathogens in The receptor-like cytoplasmic kinases VII (RLCK VII), which act downstream of PRRs, directly phosphorylate MAPKKK5 Ser-599, which is required for pattern-triggered MPK3/6 activation, defense gene expression, and disease resistance. Surprisingly, MPK6 further phosphorylates MAPKKK5 Ser-682 and Ser-692 to enhance MPK3/6 activation and disease resistance, pointing to a positive feedback mechanism. Finally, MEKK1 Ser-603 is phosphorylated by both RLCK VII and MPK4, which is required for pattern-triggered MPK4 activation. These findings illustrate central mechanisms by which multiple PRRs activate MAPK cascades and disease resistance.

Pattern recognition receptors (PRRs) are plasma membrane-localised proteins that sense molecular patterns to initiate pattern-triggered immunity (PTI). Receptor-like cytoplasmic kinases (RLCKs) function downstream of PRRs to propagate signal transduction via the phosphorylation of substrate proteins. The identification and characterisation of RLCK-regulated substrate proteins are critical for our understanding of plant immunity.We showed that SHOU4 and SHOU4L are rapidly phosphorylated upon various patterns elicitation and are indispensable for plant resistance to bacterial and fungal pathogens. Protein-protein interaction and phosphoproteomic analysis revealed that BOTRYTIS-INDUCED KINASE 1, a prominent protein kinase of RLCK subfamily VII (RLCK-VII), interacted with SHOU4/4L and phosphorylated multiple serine residues on SHOU4L N-terminus upon pattern flg22 treatment. Neither phospho-dead nor phospho-mimic SHOU4L variants complemented pathogen resistance and plant development defect of the loss-of-function mutant, suggesting that reversible phosphorylation of SHOU4L is critical to plant immunity and plant development.Co-immunoprecipitation data revealed that flg22 induced SHOU4L dissociation from cellulose synthase 1 (CESA1) and that a phospho-mimic SHOU4L variant inhibited the interaction between SHOU4L and CESA1, indicating the link between SHOU4L-mediated cellulose synthesis and plant immunity.This study thus identified SHOU4/4L as new components of PTI and preliminarily revealed the mechanism governing SHOU4L regulation by RLCKs.