HvMPK4 phosphorylates HvWRKY1 to enhance its suppression of barley immunity to powdery mildew fungus.

Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species. However, the functions of MAPK signaling pathways in crop disease resistance are largely unknown. Here we report the function of HvMKK1-HvMPK4-HvWRKY1 module in barley immune system. HvMPK4 is identified to play a negative role in barley immune response against Bgh, as virus-induced gene silencing of HvMPK4 results in enhanced disease resistance whilst stably overexpressing HvMPK4 leads to super-susceptibility to Bgh infection. Furthermore, the barley MAPK kinase HvMKK1 is found to specifically interact with HvMPK4, and the activated HvMKK1^DD variant specifically phosphorylates HvMPK4 in vitro. Moreover, the transcription factor HvWRKY1 is identified to be a downstream target of HvMPK4 and phosphorylated by HvMPK4 in vitro in the presence of HvMKK1^DD. Phosphorylation assay coupled with mutagenesis analyses identifies S122, T284, and S347 in HvWRKY1 as the major residues phosphorylated by HvMPK4. HvWRKY1 is phosphorylated in barley at the early stages of Bgh infection, which enhances its suppression on barley immunity likely due to enhanced DNA-binding and transcriptional repression activity. Our data suggest that the HvMKK1-HvMPK4 kinase pair acts upstream of HvWRKY1 to negatively regulate barley immunity against powdery mildew.