StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum

doi:10.1016/S2095-3119(20)63233-6

摘要/Abstract

摘要：

同源重组（homologous recombination, HR）和非同源末端连接（nonhomologous end joining, NHEJ）是真核生物两种主要的双链断裂（DSB）修复方法。通常抑制NHEJ中关键组分的活性能够提高靶基因敲除的效率或者影响真核生物的生长和发育。然而，在玉米大斑病菌（Exserohilum turcicum）有关NHEJ途径的作用了解甚少。为了研究玉米大斑病菌中编码NHEJ途径关键组分蛋白Ku80基因的功能，我们在玉米大斑病菌鉴定并分析了该基因对病菌生长发育及致病性调控作用。方法：本研究通过利用同源比对的方法，在玉米大斑病菌中鉴定到与酵母Ku80同源的基因，命名为StKU80，并对该基因进行了相关生物信息分析；利用农杆菌介导的遗传转化技术（ATMT）获得了两株稳定的StKU80基因敲除突变体，并对基因的功能进行了分析。结果：保守结构域分析表明，StKu80包含真核生物的KU70p / KU80p蛋白典型的结构域VWA，Ku78和Ku-PK-bind；进一步的系统发育分析表明，StKu80与来自小麦颖枯病菌（Parastagonospora nodorum）的Ku80（XP_001802136.1）亲缘关系较近，其次是来自红曲霉（Monascus ruber）的Ku80（AGF90044.1）；突变体与野生型（WT）菌株相比，突变体的菌丝间隔变的更长，细胞壁较薄，在细胞壁表面的物质变的变少以及细胞中线粒体的含量变多；对突变体致病相关的结构进行分析表明，突变体不产生分生孢子和成熟的附着胞，但是突变体的HT毒素活性与WT类似，表明StKU80影响了病菌了侵染过程，但并未影响病菌的致病力；对StKU80能否参与调控胁迫响应反应分析发现，突变体对由H₂O₂产生的氧化反应高度敏感，但是对紫外不敏感。结论：StKU80在调控玉米大斑病菌的生长发育、致病性及胁迫响应过程中发挥着重要的作用。

Abstract:

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are considered the two main double-strand break (DSB) repair approaches in eukaryotes. Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes. However, little is known about the roles of the NHEJ pathway in foliar pathogens. Here we identified a gene designated StKU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum. Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in StKu80. Phylogenetic analysis indicated that StKu80 is most closely related to Ku80 (XP_001802136.1) from Parastagonospora nodorum, followed by Ku80 (AGF90044.1) from Monascus ruber. Furthermore, the gene knockout mutants ΔStKU80-1 and ΔStKU80-2 were obtained. These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type (WT) strain but similar HT-toxin activity. The mutants did not produce conidia and mature appressoria. On the other hand, the mutants were highly sensitive to H₂O₂, but not to ultraviolet radiation. In summary, the StKU80 plays devious roles in regulating the development of E.?turcicum.

Key words: Exserohilum turcicum , pathogenicity , gene knockout , growth and development

. [J]. Journal of Integrative Agriculture, 2021, 20(1): 147-158.

GONG Xiao-dong, LIU Yu-wei, BI Huan-huan, YANG Xiao-rong, HAN Jian-min, DONG Jin-gao, GU Shou-qin. StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum[J]. Journal of Integrative Agriculture, 2021, 20(1): 147-158.

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