Analysis of the mode of action of fungicides and drug design based on structural analysis of pathogenicity related genes products from plant pathogenic fungi

点击翻译按钮获取中文摘要

Melanin biosynthesis is essential for appressorial penetration of Colletotrichum lagenarium. Three major melanin biosynthetic enzymes, polyketide synthase PKS1, scytalone dehydratase SCD1 and 1,3,8-trihydroxynaphthalene reductase THR1 were expressed in E.coli and purified. We used either pMAL and pET system for the heterologous expression of proteins and purified by affinity chromatography. And in the case of PKS1, we constructed an expression vector for Aspergillus oryzae and identified the products by PKS1. We analyzed the mode of action of an novel anti-rice blast fungicide carpropamid by using purified enzymes. First, we established in vitro reaction system and then analyzed the inhibitory activity against the dehydration reaction by carpropamid. The result obtained was that carpropamid directly act to the enzyme and thus bringing about inhibition of melanin biosynthesis. Further we analyzed multimer formation of SCD1 by yeast two-hybrid system. The result obtained indicated that SCD1 enzyme associates to each other and would form multimer. Lastly we prepared polyclonal antibody against purified PKS1, SCD1 and THR1 and immuno-blot analysis of those melanin biosynthetic enzymes indicated that there would be a kind of post-translational regulation for the enzyme activity.