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

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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.