Abstract: Phytopathogens develop specialized infection-related structures to penetrate plant cells during infection. Different from phytopathogens that form appressoria or haustoria, the soil-borne root-infecting fungal pathogen Verticillium dahliae forms hyphopodia during infection, which further differentiate into penetration pegs to promote infection. The molecular mechanisms underlying the regulation of hyphopodium formation in V. dahliae remain poorly characterized. Mitogen-activated protein kinases (MAPKs) are highly conserved cytoplasmic kinases that regulate diverse biological processes in
eukaryotes. Here we found that deletion of VdKss1, out of the five MAPKs encoded by V. dahliae, significantly impaired V. dahliae hyphopodium formation, in vitro penetration, and pathogenicity in cotton plants. Constitutive activation of MAPK kinase (MAPKK) VdSte7 and MAPK kinase kinase (MAPKKK) VdSte11 specifically activate VdKss1. Deletion of VdSte7 or VdSte11 resulted in a phenotype similar to that of the mutant with VdKss1 deletion. Thus, this study demonstrates that VdSte11-VdSte7-VdKss1 is a core MAPK cascade that regulates hyphopodium formation and pathogenicity in V. dahliae.