is the causal agent of sclerotinia stem rot in over 400 plant species. In a previous study, the group III histidine kinase gene of () revealed its involvement in iprodione and fludioxonil sensitivity and osmotic stress. To further investigate the fungicide sensitivity associated with the high-osmolarity glycerol (HOG) pathway, we functionally characterized , which is the downstream kinase of . To generate knockout mutants, split marker transformation combined with a newly developed repeated protoplasting method and CRISPR/Cas9 ribonucleoprotein (RNP) delivery approach were used. The pure and knockout mutants showed reduced sensitivity to fungicides and increased sensitivity to osmotic stress. In addition, the knockout mutants demonstrated reduced virulence compared to knockout mutants and wild-type. Our results indicate that the repeated protoplasting method and RNP approach can generate genetically pure homokaryotic mutants and is involved in osmotic adaptation, fungicide sensitivity, and virulence in .
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