Abstract
ENA1 and ENA2 are P-type IID/ENA Na(+)/K(+)-ATPases required for cellular homeostasis in yeasts but remain poorly understood in filamentous fungal insect pathogens. Here, we characterized seven genes encoding five ENA1/2 homologues (ENA1a-c and ENA2a/b) and two P-type IIC/NK Na(+)/K(+)-ATPases (NK1/2) in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of fungal insecticides worldwide. Most of these genes were highly responsive to alkaline pH and Na(+)/K(+) cues at transcription level. Cellular Na(+), K(+) and H(+) homeostasis was disturbed only in the absence of ena1a or ena2b. The disturbed homeostasis featured acceleration of vacuolar acidification, elevation of cytosolic Na(+)/K(+) level at pH 5.0 to 9.0, and stabilization of extracellular H(+) level to initial pH 7.5 during a 5-day period of submerged incubation. Despite little defect in hyphal growth and asexual development, the Δena1a and Δena2b mutants were less tolerant to metal cations (Na(+), K(+), Li(+), Zn(2+), Mn(2+) and Fe(3+)), cell wall perturbation, oxidation, non-cation hyperosmolarity and UVB irradiation, severely compromised in insect pathogenicity via normal cuticle infection, and attenuated in virulence via hemocoel injection. The deletion mutants of five other ENA and NK genes showed little change in vacuolar pH and all examined phenotypes. Therefore, only ENA1a and ENA2b evidently involved in both transmembrane and vacuolar activities are essential for cellular cation homeostasis, insect pathogenicity and multiple stress tolerance in B. bassiana. These findings provide a novel insight into ENA1a- and ENA2b-dependent vacuolar pH stability, cation-homeostatic process and fungal fitness to host insect and environment.