Functional Characterization of CfRgs2 Reveals Its Critical Role in Growth, Conidiation, Stress Response, and Virulence of Colletotrichum fructicola

Colletotrichum fructicola is the predominant pathogenic agent responsible for anthracnose in Camellia oleifera. RGS2 is a GTPase-activating protein that negatively regulates G-protein signaling by inactivating Gα subunits. In this study, we characterized the ortholog of CfRGS2 in C. fructicola to explore its pathogenic roles. Seven canonical RGS genes were identified through BLASTp and keyword searches. Conserved domains and subcellular localizations were predicted bioinformatically. A CfRGS2 knockout mutant was generated via overlap-PCR and PEG-mediated transformation, verified by PCR, and complemented by reintroducing the wild-type gene. Phenotypic characterization showed that the growth rates of mutants ΔCfrgs2-1 and ΔCfrgs2-2 were significantly reduced compared with those of the wild-type and complemented strains. On both PDA and minimal medium, the mutant strains exhibited significantly smaller colony diameters of 3.3 cm and 3.1 cm, respectively, relative to the control strains. Moreover, conidiation in the mutants was only 4% of that in the wild-type and complemented strains, and appressorium formation was reduced to 6%, with statistical analyses confirming high significance. Under cell wall stress induced by 400 μg/mL Congo red, the growth inhibition rates of ΔCfrgs2-1 and ΔCfrgs2-2 were 44% and 48%, respectively, significantly higher than those of the control strains. Pathogenicity assays demonstrated that the mutants failed to induce lesions on unwounded leaves and caused 47% and 30% smaller lesion areas on wounded apple fruits, respectively. In summary, C. fructicola possesses seven canonical RGS proteins that regulate G-protein signaling, among which CfRgs2 is implicated in growth, conidiation, the stress response to cell wall perturbation, and virulence.