Establishing Gene Expression and Knockout Methods in Esteya vermicola CBS115803

Pine wilt disease, which is caused by the nematode Bursaphelenchus xylophilus, is one of the most destructive forest diseases worldwide. Esteya vermicola, a nematophagous fungus, has emerged as a promising biological control agent. However, the limited availability of gene function analysis techniques hinders further genetic modification of this fungus. In this study, we employed a combination of enzymes (driselase, snailase, and cellulase) to enzymatically degrade the cell wall of the fungus, resulting in a high yield of protoplasts. Furthermore, by utilizing 0.6 M sucrose as an osmotic pressure stabilizer, we achieved a significant protoplast regeneration rate of approximately 31%. Subsequently, we employed the polyethylene glycol-mediated protoplast transformation method to successfully establish a genetic transformation technique for E. vermicola CBS115803. Additionally, through our investigation, we identified the Olic promoter from Aspergillus nidulans, which effectively enhanced the expression of the DsRed gene encoding a red fluorescent protein in E. vermicola CBS115803. Moreover, we successfully implemented a split-marker strategy to delete the EvIPMD gene in E. vermicola CBS115803. In summary, our findings present valuable experimental methodologies for gene function analysis in E. vermicola CBS115803.