Five Vacuolar Ca²⁺ Exchangers In Beauveria Bassiana Mediate Differentially Calcineurin- Dependent Ca²⁺/Mn²⁺ Tolerance, Multistress Responses And Virulence

Beauveria bassiana is one of typical filamentous entomopathogenic fungi that take important part in biological control of insect pests. The biocontrol potential and hence field control efficacy of a fungal formulation are largely dependent upon the fungal adaptability to target insect pests and their diverse habitats. Such adaptability is tightly associated with cellular responses to environmental and host stresses. Thus, it is important to understand how cellular multistress responses are linked to the fungal biocontrol potential.Multiple Vcx1 (vacuolar calcium exchanger) paralogues exist in many filamentous fungi but are functionally unexplored unlike a single Vcx1 orthologue well characterized in yeasts. B. bassiana harbors five Vcx1 proteins (Vcx1 A to VcxE) homologous to the yeast Vcx1. All the deduced Vcx1 proteins are featured with two conserved Na_Ca_Ex cores typical for the superfamily of Na+/Ca2+exchangers. This study sought to characterize the fungctions of Vcx1 in B. bassiana by generation and analyses of five single vcxl deletion mutants and their complementary rescued mutants.The five Vcxl paralogues were found functioning conditionally in intracellular Ca2+ homeostasis and contributed differentially to multistress tolerance and virulence in B. bassiana. Each vcxl deletion drastically upregulated transcriptional expressions of four other partners and six P-type Ca2+-ATPases, resulting in elevated or lowered intracellular Ca2+concentration in some deletion mutants treated with Ca2+ stress or untreated at 25 and 30?. When calcineurin was inactivated by cyclosporine A, Ca2+ tolerance decreased by 11-17% in five Avcxl mutants, but Mn2+ sensitivity increased only in ?vcxlA and ?vcx1D, at optimal 25?. These two mutants were also more sensitive to Ca2+ stress at 30? when calcineurin was active, and showed minor growth defect at 25 and 30? when calcineurin was inactive. Moreover, all the Avcxl 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 Avcxl mutants excluding Avcx1E, which showed unique defect in conidial thermotolerance. All the changes were restored by each vcxl complementation.In conclusion, all the five Vcxl paralogues in B. bassiana are important not only for intracellular Ca2+ homeostasis but also for various biological aspects due to their vital roles in the Ca2+-CN signaling pathway. The differential effects of single vcxl deletions on the transcriptional expressions of four other Vcxl partners and six Ca2+ ATPases and on multistress tolerance and virulence recall special attention to the functions of all possible Vcxl paralogues in many filamentous fungi although some of them are seemingly redundant under certain conditions. Our results demonstrate that the Vcxl paralogues may function much more than what has been learned from a single orthologue in model yeasts.