| Summary: | 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 <i>Colletotrichum graminicola</i>, formerly erroneously annotated as a Rho1 protein, physically interacts with the β-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343–350). Here, we show that Rho4 is required for β-1,3-glucan synthesis. Accordingly, Δ<i>rho4</i> 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 Δ<i>rho4</i> strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δ<i>rho4</i> strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δ<i>rho4</i> 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 <i>C. graminicola</i>.
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