Kss1 of Verticillium dahliae regulates virulence, microsclerotia formation, and nitrogen metabolism

Verticillium dahliae causes destructive vascular wilt diseases on more than 200 plant species, including economically important crops and ornamental trees worldwide. The melanized microsclerotia (MS) enable V. dahliae to survive for years in soil, thus the fungus is especially difficult to control once it has become established. Previously, we found that the mitogen activated protein kinase VdSte11 (MAPKKK) plays key roles in MS formation, penetration, and virulence in V. dahliae. In this study, two MAPK homologs of the yeast Ste7p and Kss1p were identified and characterized in V. dahliae. Deletion of VdSte7 or VdKss1 reuslted in severe defects in melaninized MS formation and virulence. Furthermore, phosphorylation assays demonstrated that VdSte11 and VdSte7 can phosphorylate VdKss1 in V. dahliae. Proteomic analysis revealed a significant change in sterol biosynthesis with a fold change of ≥ 1.2 after the deletion of VdKss1. In addition, phosphoproteomic analysis showed that VdKss1 was involved in the regulation of nitrogen metabolism. Finally, we identified VdRlm1 as a potentially downstream target of VdKss1, which is involved in regulating ammonium nitrogen utilization. This study sheds light on the network of regulatory proteins in V. dahliae that affect MS formation and nitrogen metabolism.