Enhancing Gene Knockout Efficiency in the Multinucleate Fungus <i>Botrytis cinerea</i> through High-Pressure Double Selection Combined with RNAi Triggering

Gene knockout is often challenging for the notorious crop killer <i>Botrytis cinerea</i> due to its multinucleate nature. Here, we established an efficient method for gene knockout in <i>B. cinerea</i> based on high-pressure positive and negative double selection (HPDS). For knockout of the toxin biosynthesis gene <i>BOA6</i>, positive selections in the high-pressure mode significantly increased the proportion of homokaryotic mutants. When an <i>HSVtk(Bc)</i>-associated negative selection system was simultaneously introduced, all obtained transformants were confirmed as homokaryotic knockout mutants. For highly expressed locus <i>oahA</i>, we employed a reverse replacement strategy to reduce the positive selection marker expression by triggering RNAi, facilitating efficient knockout via HPDS. This strategy is also efficiently applicable to the field strain CLS1. We established a high-throughput gene knockout system by which 69 of 71 predicted genes encoding secreted proteins were knocked out in CLS1, indicating its efficient uses for large-scale gene knockout in <i>B. cinerea</i>.