Arabidopsis immune receptor FLAGELLIN SENSING2 (FLS2) rapidly forms a complex to activate pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) upon perception of the bacterial protein flagellin. The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALINGKINASE1 (BSK1) interacts with FLS2 and is critical for the activation of PTI. However, it is unknown how BSK1 transduces signals to activate downstream immune responses. We identified Mitogen-Activated Protein Kinase Kinase Kinase5 (MAPKKK5) as a potential substrate of BSK1 by whole-genome phosphorylation analysis. In addition, we demonstrated that BSK1 interacts with and phosphorylates MAPKKK5. In the bsk1-1 mutant, the Ser-289 residue of MAPKKK5 was not phosphorylated as it was in the wild type. Similar to the bsk1 mutant, the mapkkk5 mutant displayed enhanced susceptibility to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000, and to the fungal powdery mildew pathogen Golovinomyces cichoracearum. Phosphorylation of the Ser-289 residue is not involved in MAPKKK5-triggered cell death, but is critical for MAPKKK5-mediated resistance to both bacterial and fungal pathogens. Furthermore, MAPKKK5 interacts with multiple MAPK kinases, including MKK1, MKK2, MKK4, MKK5, and MKK6. Overall, these results indicate that BSK1 regulates plant immunity by phosphorylating MAPKKK5, and suggest a direct regulatory mode of signaling from the immune complex to the MAPK cascade.

Perception of pathogen-associated molecular patterns (PAMPs) by plant cell surface-localized pattern-recognition receptors (PRRs) triggers the first line of plant innate immunity. In Arabidopsis thaliana, the receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALING KINASE1 (BSK1) physically associates with PRR FLAGELLIN SENSING2 and plays an important role in defense against multiple pathogens. However, how BSK1 transduces signals to activate downstream immune responses remains elusive. Previously, through whole-genome phosphorylation analysis using mass spectrometry, we showed that phosphorylation of the mitogen-activated protein kinase (MAPK) MPK15 was affected in the bsk1 mutant compared with the wild-type plants. Here, we demonstrated that MPK15 is important for powdery mildew fungal resistance. PAMPs and fungal pathogens significantly induced the phosphorylation of MPK15 Ser-511, a key phosphorylation site critical for the functions of MPK15 in powdery mildew resistance. BSK1 physically associates with MPK15 and is required for basal and pathogen-induced MPK15 Ser-511 phosphorylation, which contributes to BSK1-mediated fungal resistance. Taken together, our data identified MPK15 as a player in plant defense against powdery mildew fungi and showed that BSK1 promotes fungal resistance in part by enhancing MPK15 Ser-511 phosphorylation. These results uncovered a mechanism of BSK1-mediated disease resistance and provided new insight into the role of MAPK phosphorylation in plant immunity.

Plants manage the high cost of immunity activation by suppressing the expression of defense genes during normal growth and rapidly switching them on upon pathogen invasion. TGAs are key transcription factors controlling the expression of defense genes. However, how TGAs function, especially in monocot plants like rice with continuously high levels of endogenous salicylic acid (SA) remains elusive. In this study, we characterized the role of OsTGA5 as a negative regulator of rice resistance against blast fungus by transcriptionally repressing the expression of various defense-related genes. Moreover, OsTGA5 repressed PTI responses and the accumulation of endogenous SA. Importantly, we showed that the nucleus-localized casein kinase II (CK2) complex interacts with and phosphorylates OsTGA5 on Ser-32, which reduces the affinity of OsTGA5 for the JIOsPR10 promoter, thereby alleviating the repression of JIOsPR10 transcription and increasing rice resistance. Furthermore, the in vivo phosphorylation of OsTGA5 Ser-32 was enhanced by blast fungus infection. The CK2 α subunit, depending on its kinase activity, positively regulated rice defense against blast fungus. Taken together, our results provide a mechanism for the role of OsTGA5 in negatively regulating the transcription of defense-related genes in rice and the repressive switch imposed by nuclear CK2-mediated phosphorylation during blast fungus invasion.