Rho1-GTPase Activating Protein, Bem2 is Required for Antifungal Action of Fludioxonil

A. Sharma, Alok K Mondal


Background: Fludioxonil is a member of phenylpyrrole group of fungicides which is widely used in agriculture for crop management. It is known to act through group III HHKs (NIK1/OS-1 orthologs) in fungal pathogens. Exposure of fungi to fludioxonil affects Nik1 ortholog which activates HOG pathway through Hog1p phosphorylation. However the details of the molecular events leading to HOG pathway activation, growth arrest or cell death remained unclear Methods: Mutagenized DNA from the mTn-lacZ-LEU2 genomic library was transformed into S. cerevisiae strain BY4742 harboring pRS-ClNIK1. Screening was done for resistant transformants on 50ppm concentration of fludioxonil media. Confocal microscopy was done to check the localization of Hog1p- GFP and analyze the budding pattern on drug treatment. Results: We find that heterologous expression of ClNik1 (Nik1 ortholog from Candida lusitaniae) renders S. cerevisiae cells - which are otherwise resistant - susceptible to fludioxonil. Next we checked the localization of Hog1p-GFP and observed that drug induced phosphorylation did not change the cytoplasm localization of Hog1p Moreover, both [[unable to display character: ∆]]gpd1[[unable to display character: ∆]]gpd2 mutant and [[unable to display character: ∆]]nmd5 mutant remained sensitive to fludioxonil and thereby confirming that the intracellular accumulation of glycerol or the nuclear translocation of Hog1p was not essential. Thus it appeared that the antifungal action of fludioxonil was mediated by cytoplasmic action of Hog1p. We further checked the effect of fludioxonil on cell cycle and budding pattern of yeast. We found that it causes G1 delay and cytokinesis defect leading to cell growth inhibition. By a genome wide screening Bem2p, a known Rho1 GAP, was identified to be involved in the antifungal action of fludioxonil and the deletion of bem2 resulted in fludioxonil resistance. Over expression of RHO1, RHO2, RHO5 and CDC42 in the wild type background did not lead to fludioxonil resistance. None of the other Rho1-GAP proteins have role in antifungal action. Mutational study revealed that both GAP dependent and independent role of Bem2p was crucial for drug action. Genetic data suggested that deletion of MPK1, SKN7, SWE1, SWI6, CRZ1 in [[unable to display character: ∆]]bem2 genetic background suppressed the resistance to drug. Conclusions: Study provides evidence that more than one signalling cascade regulate antifungal action of fludioxonil


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American Society for Microbiology General Meeting
    • ASM 113th (2013)