Studies On The Screening Of Phytopathogenic Fungal Inhibitors Using Glucan Synthase As A Target

Phytopathogenic fungus are responded to most of the plant diseases, and this causes a heavy loss in the crops. Because of the unique structure of fungal cell wall, it is considered to be an excellent target of antifungal inhibitor.β-1,3-glucan is one of the compounds in cell wall, the synthesis of which is catalyzed byβ-1,3-glucan synthase. The amino acid sequence of this enzyme is highly conserved in fungus. So it is possible to discover glucan synthase inhibitor with broad-spectrum capacity. There are two homologous genes FKS1 and FKS2 encode the catalytic subunit of glucan synthase in Saccharomyces cerevisiae. TheΔfks1 strain is hypersensitive to glucan synthase inhibitor compare with the wild-type strain. This pair of strains can be used as a target-based whole-cell model for screening glucan synthase inhibitors. In this experiment, multidrug resistance gene pdr1 null mutant of S. cerevisea was chosen to construct fks1 null mutant strain using PCR-based one step gene disruption. The target of the inhibitor can be originally confirmed by comparing the different sensitivities betweenΔfks1 strain and the wild-type strain to the inhibitor. The double-deck platform method and strain-mixed group screening method were established to screening microorganisms that inhibit the yeast from soil samples. We obtained 173 strains with antifungal activity. Especially, strain ZJA-65 inhibited theΔfks1 pdr1 strain more obviously comparing with the pdr1 strain. The ZJA-65 strain is being further studied.Botrytis cinerea is a widespread phytopathogenic fungus that affects a lot of vegetable and fruit crops. In order to study glucan synthase and its inhibiters in B. cinerea, a putative FKS gene of B. cinerea was cloned using RT-PCR. TheΔfks1Δfks2 pFKS1 yeast strain was constructed by transforming the plasmid harboring FKS1 intoΔfks1 strain, and then FKS2 of this strain was disrupted. ThisΔfks1Δfks2 pFKS1 strain is being used to study the putative FKS gene of B. cinerea by complementation.