Two fluffy family proteins, CgrFlbA and CgrFlbC, coordinately regulate hyphal growth, oxidative stress, asexual development, and pathogenicity in Colletotrichum graminicola.

Colletotrichum graminicola, the causal agent of maize anthracnose, is responsible for substantial global crop losses. While the fluffy family proteins (FlbA-FlbE, FluG) are known as key upstream activators of asexual development in Aspergillus nidulans, their functions in C. graminicola remain largely unexplored. Here, we systematically characterized the biological roles of the fluffy gene homologs in this plant pathogen. Deletion of CgrflbA, which encodes a regulator of G-protein signaling (RGS) protein, resulted in a fluffy phenotype, reduced oval conidiation, and a complete block in falcate conidium formation. The ΔCgrflbA mutant also demonstrated enhanced melanin deposition, increased hyphopodium formation, improved oxidative stress tolerance, and consequently, elevated virulence. In contrast, deletion of CgrflbC (encoding a C2H2 transcription factor) led to slowed hyphal growth, increased H2O2 sensitivity, and a sharp decrease in falcate conidium production. This mutant also failed to produce hyphopodia and was significantly attenuated in pathogenicity. Furthermore, disruption of CgrflbD impaired falcate conidiation, whereas deletions of CgrflbB, CgrflbE, and CgrfluG had no significant impact on fungal growth and development. Collectively, our findings demonstrate that CgrFlbA and CgrflbC are key regulators that coordinately govern vegetative growth, oxidative stress response, asexual development, and pathogenicity in C. graminicola.