Abstract

Verticillium wilt, primarily caused by the fungus Verticillium dahliae , results in substantial crop losses and ecological damage worldwide. During infection, V. dahliae forms a specialized infection structure called the hyphopodium, which penetrates host root cells. Although calcium (Ca 2+ ) influx into hyphopodia is essential for hyphopodium-mediated root penetration and subsequent colonization, the mechanism that maintains Ca 2+ homeostasis in hyphopodia during pathogenesis remains unknown. Here, we show that the transcription factor Veh1 negatively regulates hyphopodium-mediated root penetration by modulating Ca 2+ homeostasis during plant infection. Deletion of Veh1 increased hyphopodium formation, elevated Ca 2+ accumulation in hyphopodia, activated VdCrz1-mediated Ca 2+ signaling, and enhanced virulence. We further demonstrated that Veh1 directly regulates VdMid1 , which encodes a putative Ca 2+ influx component, and VdMcu1 , which encodes a mitochondrial Ca 2+ uniporter that promotes mitochondrial Ca 2+ sequestration. Through this opposing regulation, Veh1 coordinates cytosolic Ca 2+ dynamics during hyphopodium-mediated infection. Indeed, deletion of VdMid1 reduced Ca 2+ accumulation in hyphopodia and attenuated virulence, whereas deletion of VdMcu1 had the opposite effects. Together, these results uncover a Veh1–VdMid1/VdMcu1 regulatory circuit that maintains Ca 2+ homeostasis during hyphopodium-mediated plant penetration and provide mechanistic insights into fungal pathogenesis.