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⁺, Zn²⁺, Mn²⁺ and Fe³⁺), 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.

摘要

ENA1 和 ENA2 是酵母细胞稳态所需的P 型 IID/ENA Na ⁺ /K ^{+ -ATP 酶，但在丝状真菌昆虫病原体中仍知之甚少。}在这里，我们鉴定了白僵菌（一种昆虫病原真菌）中编码 5 个 ENA1/2 同源物（ENA1a–c 和 ENA2a/b）和两个 P 型 IIC/NK Na ⁺ /K ⁺ -ATP 酶 (NK1/2) 的7 个基因。是全世界真菌杀虫剂的主要来源。^{这些基因中的大多数在转录水平上对碱性pH和Na +} /K ⁺信号高度敏感。仅当ena1a或ena2b缺失时，细胞的 Na ⁺、K ⁺和 H ⁺稳态才会受到干扰。在5天的水下培养期间，体内平衡紊乱的特点是液泡酸化加速、胞质Na ⁺ /K ^{+水平在pH 5.0至9.0升高，以及细胞外H +}水平稳定至初始pH 7.5。尽管菌丝生长和无性发育几乎没有缺陷，但Δena1a和Δena2b突变体对金属阳离子（Na ⁺，K ⁺，Li ⁺，Zn ²⁺，Mn ²⁺和Fe ³⁺）、细胞壁扰动的耐受性较差，氧化、非阳离子高渗透压和 UVB 照射，通过正常角质层感染严重损害昆虫的致病性，并通过血腔注射减弱毒力。其他五个 ENA 和 NK 基因的缺失突变体在液泡 pH 值和所有检查的表型方面几乎没有变化。因此，只有明显参与跨膜和液泡活动的ENA1a和ENA2b对于球孢白僵菌的细胞阳离子稳态、昆虫致病性和多重胁迫耐受性至关重要。这些发现为 ENA1a 和 ENA2b 依赖性液泡 pH 稳定性、阳离子稳态过程以及真菌对宿主昆虫和环境的适应性提供了新的见解。