CgCFEM1 and CgCFEM2 modulate virulence in Colletotrichum gloeosporioides by integrated regulation of TOR and cAMP-PKA signaling pathways

Colletotrichum gloeosporioides causes anthracnose in multiple plants via specialized infection structures, which are regulated by conserved pathways including the Target of rapamycin (TOR) and cyclic adenosine monophosphate-protein kinase A (cAMP-PKA). Effectors with Common in Fungal Extracellular Membranes (CFEM) domain are critical virulence factors. Although CgCFEM1 has been characterized, the role of its closest homolog CgCFEM2 and their potential interplay remains unknown. This study demonstrates that CgCFEM2 is a crucial pathogenicity effector, with its gene expression highly induced during infection. Pathogenicity assays on rubber tree leaves revealed that the ΔCgCFEM2 mutant was severely attenuated in virulence, and the double mutant ΔCgCFEM1/2 exhibited an even more severe defect, indicating CgCFEM1 and CgCFEM2 play complementary yet distinct roles in pathogenicity. Both effectors were essential for normal conidia morphology, conidiation, and the critical transition from appressoria to invasive hyphae. Mechanistically, we discovered that loss of CgCFEM1 and CgCFEM2 leads to deregulated cell cycle progression during infection-related development, resulting in aberrant germ tube elongation and impaired appressorium formation. Additionally, the mutants exhibited hyperactive phosphorylation of p70-S6 kinase (p70 S6K) and insensitivity to rapamycin, indicating the involvement of TOR signaling. A key finding is that both CgCFEM1 and CgCFEM2 positively regulate intracellular cAMP levels, but through fundamentally distinct targets: CgCFEM1 promotes cAMP synthesis by upregulating the adenylate cyclase gene CgMac1, whereas CgCFEM2 inhibits cAMP degradation by repressing the phosphodiesterase gene CgPdeH.​ Consequently, the loss of these effectors led to reduced cAMP levels and impaired phosphorylation of MAP kinase Pmk1. Our findings establish that CgCFEM1 and CgCFEM2 as pivotal, synergistic regulators of the C. gloeosporioides infection cycle. They converge on elevating cAMP levels via different mechanisms: synthesis versus degradation. They also influence TOR signaling. Together, they ensure proper fungal development and pathogenicity. This study provides novel insights into the sophisticated effector network employed by phytopathogenic fungi to achieve successful infection.