The Small Ras Superfamily GTPase Rho4 of the Maize Anthracnose Fungus Colletotrichum graminicola Is Required for β-1,3-glucan Synthesis, Cell Wall Integrity, and Full Virulence

Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the beta-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343-350). Here, we show that Rho4 is required for beta-1,3-glucan synthesis. Accordingly, Delta rho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Delta rho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Delta rho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Delta rho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.