Five vacuolar Ca(2+) exchangers play different roles in calcineurin-dependent Ca(2+)/Mn(2+) tolerance, multistress responses and virulence of a filamentous entomopathogen.

Multiple Vcx1 (vacuolar calcium exchanger) paralogues exist in many filamentous fungi but are functionally unexplored unlike a single Vcx1 ortholog well characterized in yeasts. Here we show that five Vcx1 paralogues (Vcx1A-E) in Beauveria bassiana are conditionally functional for intracellular Ca(2+) homeostasis and contribute differentially to multistress tolerance and virulence in the filamentous entomopathogen. Each vcx1 deletion drastically upregulated transcriptional expressions of four other partners and six P-type Ca(2+)-ATPases, resulting in elevated or lowered intracellular Ca(2+) concentration in some deletion mutants treated with Ca(2+) stress or untreated at 25 and 30 °C. When calcineurin was inactivated by cyclosporine A, Ca(2+) tolerance decreased by 11-17% in five Δvcx1 mutants, but Mn(2+) sensitivity increased only in Δvcx1A and Δvcx1D, at optimal 25 °C. These two mutants were also more sensitive to Ca(2+) stress at 30 °C when calcineurin was active, and showed minor growth defect at 25 and 30 °C when calcineurin was inactive. Moreover, all the Δvcx1 mutants were more sensitive to dithiothreitol (stress-response trigger to endoplasmic reticulum) and Congo red (cell wall stressor); three of them were consistently less tolerant to the oxidants menadione and H2O2. The fungal virulence to Galleria mellonella larvae decreased by 15-40% in four Δvcx1 mutants excluding Δvcx1E, which was uniquely defective in conidial thermotolerance. All the changes were restored by each vcx1 complementation. Our findings indicate that the five Vcx1 paralogues in B. bassiana contribute differentially to calcineurin-dependent Ca(2+)/Mn(2+) tolerance, multistress responses and virulence, and recall attention to multifunctional Vcx1 paralogues in filamentous fungi.