Département de Phytologie, Université Laval, Québec, QC, Canada.
Département de Phytologie, Université Laval, Québec, QC, Canada; Universidade Federal de Viçosa, Departamento de Bioquímica e Biologia Molecular, Viçosa, MG, Brazil.
Fungal Genet Biol. 2021 Aug;153:103573. doi: 10.1016/j.fgb.2021.103573. Epub 2021 May 21.
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.
出芽短梗霉是一种具有强大抗白粉病能力的附生酵母。这种活性与一种罕见的抗真菌糖脂——絮凝素的产生有关。尽管已经发现了絮凝素合成的特定基因簇，但由于无法有效地转化出芽短梗霉，因此试图赋予该分子功能一直受到阻碍。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方法，来解析絮凝素在出芽短梗霉生物防治活性中的作用。通过 HR 进行 fat1 的编辑可以改变絮凝素的产生，但这种突变体表现出异常表型和无法产生分生孢子。测序分析表明，HR 转化导致基因组中发生多次插入，解释了该方法的多效性效应。另一方面，CRISPR-Cas9 转化产生了一个在絮凝素正确合成中发生改变的突变体。尽管丧失了絮凝素的产生，但这种突变体在表型上与野生型相似，并且在测试其对白粉病的生物防治活性时，表现出相同的功效。这些结果有力地证明了絮凝素介导的抑菌作用不是出芽短梗霉作用模式的原因，并突出了 CRISPR-Cas9 用于功能研究的潜力，特别是对于其他难以转化的真菌，如出芽短梗霉。
