MOE Laboratory of Biosystems Homeostasis & Protection, Institute of Microbiology, College of Life Sciences, Zhejiang University , Hangzhou, Zhejiang, China.
College of Agricultural and Food Science, Zhejiang A&F University , Lin'an, Zhejiang, China.
Virulence. 2020 Dec;11(1):1415-1431. doi: 10.1080/21505594.2020.1836903.
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 , 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 or . 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 Δ and Δ mutants were less tolerant to metal cations (Na, K, Li, Zn, Mn and Fe), 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 . 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.
ENA1 和 ENA2 是 P 型 IID/ENA Na/K-ATPase，是酵母细胞内稳态所必需的，但在丝状真菌昆虫病原体中仍知之甚少。在这里，我们对编码五个 ENA1/2 同源物（ENA1a-c 和 ENA2a/b）和两个 P 型 IIC/NK Na/K-ATPase（NK1/2）的七个基因在 中进行了特征描述， 是一种昆虫病原真菌，是全球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 线索高度敏感。只有在没有 或 的情况下，细胞内的 Na、K 和 H 内稳态才会受到干扰。稳态失调的特征是加速液泡酸化，在 pH 5.0 到 9.0 时升高胞质内的 Na/K 水平，并在 5 天的浸没孵育过程中稳定胞外 H 水平至初始 pH7.5。尽管菌丝生长和无性发育几乎没有缺陷，但Δ 和 Δ 突变体对金属阳离子（Na、K、Li、Zn、Mn 和 Fe）、细胞壁扰动、氧化、非阳离子高渗和 UVB 照射的耐受性较低，通过正常的表皮感染，昆虫致病性严重受损，通过血腔注射，毒力减弱。其他五个 ENA 和 NK 基因的缺失突变体在液泡 pH 和所有检查的表型方面几乎没有变化。因此，只有 ENA1a 和 ENA2b 显然参与跨膜和液泡活动，对于细胞内阳离子内稳态、昆虫致病性和 对宿主昆虫和环境的多种胁迫耐受性是必需的。这些发现为 ENA1a 和 ENA2b 依赖的液泡 pH 稳定性、阳离子稳态过程和真菌适应宿主昆虫和环境的能力提供了新的见解。
