Functional analysis on fungal histidine kinases based on genome information and its application on fungicide screening

粗糙脉孢菌（Neurospora crassa）是一种丝状真菌，其基因组中有11个组氨酸激酶基因，而酿酒酵母（Saccharomyces cerevisiae）只有一个组氨酸激酶基因。大多数组氨酸激酶在包括稻瘟病菌在内的丝状真菌中高度保守，表明真菌利用多种组氨酸激酶进行环境响应和分化。我们在N.通过RIP突变的方法，分离并鉴定了8种组氨酸激酶基因的破坏物。然而，除了os-1基因外，所有干扰物在菌丝生长、渗透或氧化胁迫、杀菌剂敏感性和育性方面均未表现出异常。这些结果表明真菌组氨酸激酶可能存在冗余。为了通过遗传杂交筛选具有多个组氨酸激酶基因缺失的菌株，我们建立了PCR方法检测每个组氨酸激酶基因的RIP突变。我们鉴定了os-4（MAPK激酶）和os-5（MAPK激酶）基因，它们在os-1组氨酸激酶的下游受到调节。此外，我们发现，NcCTT 1和NcGPD 1基因的转录被二甲酰亚胺类杀菌剂激活，其激活依赖于os-1途径。在此基础上，组氨酸激酶抑制剂筛选体系的构建正在进行中。

Neurospora crassa, a filamentous fungus, has eleven histidine kinase genes in its genome, while Saccharomyces cerevisiae has only one histidine kinase gene. Most of the histidine kinases were highly conserved in filamentous fungi including Magnaporthe girisea, suggesting that fungi use many kinds of histidine kinases for environmental response and differentiation. We carried out gene disruption in N. crassa by RIP mutation, and isolated and characterized eight kinds of histidine kinase gene disruptants. However, all disruptants, except os-1 gene, did not show abnormality in mycelial growth, osmotic or oxidative stress, fungicide sensitivity, and fertility. These results suggested the possibility of redundancy of fungal histidine kinase. To screen the isolates with several histidine kinase gene disruption by genetic cross, we constructed the PCR method to detect RIP mutations for each histidine kinase gene.Os-1 histidine kinase is responsible for osmotic stress response and dicarboximide-resistance. We identified os-4 (MAPKK kinase) and os-5 (MAPK kinase) genes, which are regulated down stream of os-1 histidine kinase. Furthermore, we revealed that transcription of NcCTT1 and NcGPD1 genes was activated by dicarb6ximide fungicides and their activation was dependent on os-1 pathway. Based on above results, construction of the screening system for histidine kinase inhibitors is undergoing.