The Vacuolar Ca2+/H+ Exchanger AaVCX Regulates Calcium Homeostasis, the Differentiation of Infection Structures and Pathogenicity in Alternaria alternata Under the Transcriptional Control of AaCrz1.

Calcium ion (Ca2+) signalling is crucial for multiple biological processes in fungi, including growth, stress adaptation and pathogenic behaviour. The vacuolar Ca2+/H+ exchanger (VCX) is vital for maintaining calcium homeostasis within fungal cells, yet its specific biological functions in phytopathogenic fungi remain poorly understood. This study focused on the role and transcriptional regulation of AaVCX in Alternaria alternata, the organism responsible for pear black spot disease. By employing a split-marker strategy, we generated AaVCX knockout (ΔAaVCX) and complemented (ΔAaVCX-C) strains. Phenotypic assessments revealed that ΔAaVCX exhibited reduced mycelial growth, abnormal accumulation of intracellular Ca2+ and heightened sensitivity to various stressors. Deletion of AaVCX severely hindered spore germination, differentiation of appressoria and invasive hyphae in A. alternata triggered by pear peel wax. The ΔAaVCX nearly lost its ability to penetrate cellophane, and its virulence on pear fruits and leaves was markedly diminished. Molecular docking analysis indicated that AaCrz1, a key transcription factor in the calcium signalling pathway, can bind to the promoter of AaVCX. Dual-luciferase and yeast one-hybrid assays demonstrated that AaCrz1 binds to the AaVCX promoter in vitro and positively regulates its expression, with a predicted GCC-core motif identified in the promoter region. Furthermore, transcript levels of AaVCX were significantly reduced in the ΔAaCrz1 background, indicating that AaVCX is positioned downstream of AaCrz1 in a regulatory cascade. Our findings enhance the understanding of calcium signalling networks in phytopathogenic fungi and propose that components involved in vacuolar calcium storage may serve as potential targets for disease management.