Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P. flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.

Pseudozyma flocculosa是一种附生酵母，对白粉病具有强大的拮抗活性。这种活性与一种罕见的抗真菌糖脂、絮凝素的生产有关。尽管发现了用于合成絮凝素的特定基因簇，但由于无法有效转化P. flocculosa，因此无法将功能作用归因于该分子。在这项研究中，利用同源重组 (HR) 和基于 CRISPR-Cas9 的基因组编辑两种不同的方法来破译絮凝素在P.  flocculosa生物防治活动中的作用。可以通过修改fat1来改变絮凝素的生产HR，但这些突变体表现出异常的表型和无法产生孢子。测序分析表明，HR 的转化导致基因组中的多次插入，解释了该方法的多营养效应。另一方面，CRISPR-Cas9 转化产生了一个突变体，该突变体在絮凝素的正确合成中发生了特异性改变。尽管失去了絮凝素的产生，但这种突变体在表型上与野生型相似，并且当测试其对白粉病的生物防治活性时，显示出相同的功效。这些结果提供了强有力的证据，即絮凝素介导的抗菌作用与P. flocculosa的作用方式无关并强调 CRISPR-Cas9 在其他难以转化的真菌（如P. flocculosa）的功能研究中的潜力。