The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of "VQ-motif'-containing proteins to regulate immune responses

Abstract

Mitogen-activated protein kinase (MAPK) cascades play key roles in plant immune signalling, and elucidating their regulatory functions requires the identification of the pathway-specific substrates. We used yeast two-hybrid interaction screens, in vitro kinase assays and mass spectrometry-based phosphosite mapping to study a family of MAPK substrates. Site-directed mutagenesis and promoter-reporter fusion studies were performed to evaluate the impact of substrate phosphorylation on downstream signalling. A subset of the Arabidopsis thaliana VQ-motif-containing proteins (VQPs) were phosphorylated by the MAPKs MPK3 and MPK6, and renamed MPK3/6-targeted VQPs (MVQs). When plant protoplasts (expressing these MVQs) were treated with the flagellin-derived peptide flg22, several MVQs were destabilized in vivo. The MVQs interact with specific WRKY transcription factors. Detailed analysis of a representative member of the MVQ subset, MVQ1, indicated a negative role in WRKY-mediated defence gene expression - with mutation of the VQ-motif abrogating WRKY binding and causing mis-regulation of defence gene expression. We postulate the existence of a variety of WRKY-VQP-containing transcriptional regulatory protein complexes that depend on spatio-temporal VQP and WRKY expression patterns. Defence gene transcription can be modulated by changing the composition of these complexes - in part - through MAPK-mediated VQP degradation.