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

粗糙脉孢菌是一种丝状真菌，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，这表明真菌在环境响应和分化中使用了多种组氨酸激酶。我们利用RIP突变技术对粗毛拟青霉进行了基因破坏，分离并鉴定了8种组氨酸激酶基因干扰物。然而，除os-1基因外，所有的干扰物在菌丝生长、渗透胁迫或氧化胁迫、杀菌剂敏感性和育性方面都没有表现出异常。这些结果提示真菌组氨酸激酶可能是冗余的。为了通过基因交叉筛选具有多个组氨酸激酶基因突变的菌株，我们建立了检测每个组氨酸激酶基因的RIP突变的聚合酶链式反应方法。我们鉴定了os-4(MAPKK激酶)和os-5(MAPK激酶)基因，它们是由os-1组氨酸激酶下游调控的。此外，我们还发现NcCTT1和NcGPD1基因的转录被杀菌剂二碳六亚胺激活，它们的激活依赖于os-1途径。基于上述结果，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.