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 G1/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. IMPORTANCE Fungal morphogenesis requires accurate cell cycle progression. Two-component GTPase-activating protein (GAP) CoBub2-CoBfa1 interacts with downstream GTPase CoTem1 and is required for G1/S progression to establish plant infection in Colletotrichum orbiculare. To understand the pathogenicity related functions of CoTem1 downstream, we identified a Niemann-Pick type C2 homolog (CoNpc2) as a novel physical interaction factor with CoTem1. Whereas NPC proteins (NPC1 and NPC2) are essential for sterol homeostasis in humans and yeasts, their functions in plant invasion by pathogenic fungi have remained unclear. In this study, we show that CoNPC1 and CoNPC2 play a critical role in intracellular sterol transport and that appropriate sterol distribution is required for membrane integrity and membrane curvature with actin assembly that leads to appressorium-mediated plant penetration and pathogenicity of C. orbiculare. Our findings suggest the importance of sterol distribution in fungal morphogenesis during plant infection.