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。

The mitogen-activated protein kinase (MAPK), a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes.  However, little is known of the role MAPK plays in phytopathogenic fungi.  In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol (HOG) MAPK gene HOG1 of Saccharomyces cerevisiae.  In addition, gene knockout technology was employed to investigate the function of STK1.  Gene knockout mutants (KOs) were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain (WT).  Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface.  STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum (HT)-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43.  Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress.  These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis.  Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica.

Setosphaeria turcica, an essential phytopathogenic fungus, is the primary cause of serious yield losses in corn; however, its pathogenic mechanism is poorly understood. We cloned STK2, a newly discovered mitogen-activated protein kinase gene with a deduced amino acid sequence that is 96% identical to MAK2 from Phaeosphaeria nodorum, 56% identical to KSS1 and 57% identical to FUS3 from Saccharomyces cerevisiae. To deduce Stk2 function in S. turcica and to identify the genetic relationship between STK2 and KSS1/FUS3 from S. cerevisiae, a restructured vector containing the open reading frame of STK2 was transformed into a fus3/kss1 double deletion mutant of S. cerevisiae. The results show that the STK2 complementary strain clearly formed pseudohyphae and ascospores, and the strain grew on the surface of the medium after rinsing with sterile water and the characteristics of the complementary strain was the same as the wild-type strain. Moreover, STK2 complemented the function of KSS1 in filamentation and invasive growth, as well as the mating behavior of FUS3 in S. cerevisiae, however, its exact functions in S. turcica will be studied in the future research.