Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

Lipid composition of Botrytis cinerea and inhibition of its radiolabelling by the fungicide iprodione.

• Botrytis cinerea is an important plant pathogen that causes grey mould in over 200 hosts. It is often controlled by dicarboximides, which have various proposed mechanisms of action, including effects on lipids. Here we have examined the effect of one dicarboximide, iprodione, on lipid metabolism. • B. cinerea, cultured in malt extract media, was challenged with iprodione and its lipids extracted, separated by TLC, and analysed by GLC. Lipid metabolism was followed using [1-14 C]acetate. • Triacylglycerol was the major nonpolar and phosphatidylcholine the main polar lipid in B. cinerea. Linoleate, followed by α-linolenate, were the major fatty acids and most lipid classes had compositions broadly similar to the total fatty acid pattern. Iprodione, at concentrations causing a cessation of growth (5 µM) caused a decrease in polar lipid but not total nonpolar lipid labelling. Within the nonpolar lipids, DAG was better labelled. • The data show that iprodione had a selective effect on lipid metabolism. The altered pattern of labelling suggested that choline (ethanolamine) phosphotransferase would be worth investigating as a primary site of action.