Niemann-Pick Type C Proteins Are Required for Sterol Transport and Appressorium-Mediated Plant Penetration of Colletotrichum orbiculare

Many biotrophic and hemibiotrophic fungal pathogens use appressoria to directly penetrate the host plant surface. In the cucumber anthracnose fungus Colletotrichum orbiculare, differentiation of appressoria requires a proper G(1)/S cell cycle progression, regulated by the GTPase-activating protein complex CoBub2-CoBfa1 and its downstream GTPase CoTem1. To explore the mechanisms by which the CoTem1 cascade regulates plant infection, we screened for CoTem1 interaction factors and identified a Niemann-Pick type C2 homolog (CoNpc2). Niemann-Pick type C proteins NPC1 and NPC2 are sterol-binding proteins required for sterol export from lysosomes (vacuoles) in humans and yeasts. We showed that CoNpc2 colocalized with CoNpc1 in late endosomes and vacuoles and that disruption of its gene resulted in aberrant sterol accumulation in vacuoles and loss of sterol membrane localization, indicating that NPC proteins are engaged in sterol transport in C. orbiculare. For appressorium infection, sterol transport and proper distribution mediated by CoNpc1 and CoNpc2 are critical for membrane integrity and membrane curvature with actin assembly, leading to penetration peg emergence and appressorial cone formation. Our results revealed a novel mechanism by which NPC proteins regulate appressorium-mediated plant infection.