Veh1 modulates Ca²⁺ dynamics through VdMid1 and VdMcu1 to control hyphopodium-mediated infection in Verticillium...

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 (Ca²⁺) influx into hyphopodia is essential for hyphopodium-mediated root penetration and subsequent colonization, the mechanism that maintains Ca²⁺ homeostasis in hyphopodia during pathogenesis remains unknown. Here, we show that the transcription factor Veh1 negatively regulates hyphopodium-mediated root penetration by modulating Ca²⁺ homeostasis during plant infection. Deletion of Veh1 increased hyphopodium formation, elevated Ca²⁺ accumulation in hyphopodia, activated VdCrz1-mediated Ca²⁺ signaling, and enhanced virulence. We further demonstrated that Veh1 directly regulates VdMid1, which encodes a putative Ca²⁺ influx component, and VdMcu1, which encodes a mitochondrial Ca²⁺ uniporter that promotes mitochondrial Ca²⁺ sequestration. Through this opposing regulation, Veh1 coordinates cytosolic Ca²⁺ dynamics during hyphopodium-mediated infection. Indeed, deletion of VdMid1 reduced Ca²⁺ 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 Ca²⁺ homeostasis during hyphopodium-mediated plant penetration and provide mechanistic insights into fungal pathogenesis.