MPK3 mediated phosphorylation of WRKY48 down regulates CIPK6 expression during PstDC3000 challenge in Arabidopsis

Abstract

WRKY transcription factor (TF) family are one of the key regulators of plant immune responses, functioning as both activators and suppressors depending on the pathogen encountered. Among these, WRKY48, a class IIC TF, functions as a negative regulator of pattern-triggered immunity (PTI) in Arabidopsis thaliana. Our study explicates the regulatory mechanisms underlying the function of WRKY48, revealing its role in repressing the expression of CIPK6, a negative regulator of immune response in Arabidopsis, during Pseudomonas syringae DC3000 (PstDC3000) infection. This suppression is mediated through MPK3-dependent phosphorylation of WRKY48. In vitro phosphorylation assays demonstrated that MPK3 primarily phosphorylates WRKY48 at serine residues S35 and S40. The phosphorylated WRKY48 predominantly localizes to the nucleus, enhancing its DNA-binding affinity and trans-repressive activity at specific W-box motifs in the CIPK6 promoter. Whereas invivo dual luciferase shows that unphosphorylated WRKY48 (S26S35S40 / A26A35A40 ) unable to repress luciferase activity of CIPK6 promoter. The bioinformatics analysis identified two WRKY-binding sites (P1 and P2) on the CIPK6 promoter and electrophoretic mobility shift assays confirms the preferential binding of WRKY48 to the P1 site. Unlike other WRKY TFs (WRKY28 and WRKY8) in the same subgroup, WRKY48 exhibited unique specificity, highlighting the distinct regulatory roles within the WRKY family. Functional studies demonstrated that phosphorylation of WRKY48 enhances its ability to suppress CIPK6 both transcriptionally and translationally, leading to increased reactive oxygen species (ROS) levels and decreased susceptibility to PstDC3000. The reduced expressions of PTI marker genes and ROS-associated genes in wrky48 mutants were associated with enhanced resistance in wrky48 and cipk6 mutants and emphasizes the critical role of WRKY48 in fine-tuning immune responses. Our findings identify a novel MPK3-WRKY48-CIPK6 signaling module that connects calcium-dependent pathways to MAPK signalling to modify plant immunity. HighlightsO_LIWRKY48, a class IIC transcription factor, functions as a negative regulator of pattern-triggered immunity (PTI) in Arabidopsis thaliana during Pseudomonas syringae DC3000 (PstDC3000) infection. C_LIO_LIWRKY48 suppresses the expression of CIPK6, a negative regulator of immune signaling, in response to bacterial infection. C_LIO_LIMAP kinase MPK3 phosphorylates WRKY48 at serine residues S35 and S40, enhancing its nuclear localization and transcriptional repressive activity C_LIO_LIPhosphorylated WRKY48 shows increased binding affinity to W-box motifs in the CIPK6 promoter, especially at the P1 binding site, as confirmed by electrophoretic mobility shift assays (EMSA). C_LIO_LIUnlike WRKY28 and WRKY8, WRKY48 exhibits unique binding specificity, suggesting functional diversification within WRKY subgroup IIC. C_LIO_LISuppression of CIPK6 by phosphorylated WRKY48 leads to: Elevated ROS accumulation, Reduced PTI marker gene expression, Increased susceptibility to PstDC3000 C_LI