Mascarin Gabriel Moura, Shrestha Somraj, de Carvalho Barros Cortes Marcio Vinícius, Ramirez Jose Luis, Dunlap Christopher A, Coleman Jeffrey J
Laboratory of Environmental Microbiology, Embrapa Environment, Brazilian Agricultural Research Corporation, Embrapa Environment, Rodovia SP 340, km 127.5, Jaguariúna, SP, 13918-110, Brazil.
Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, 36849, USA.
Fungal Biol Biotechnol. 2024 Nov 21;11(1):21. doi: 10.1186/s40694-024-00190-5.
Biocontrol agents play a pivotal role in managing pests and contribute to sustainable agriculture. Recent advancements in genetic engineering can facilitate the development of entomopathogenic fungi with desired traits to enhance biocontrol efficacy. In this study, a CRISPR-Cas9 ribonucleoprotein system was utilized to genetically improve the virulence of Beauveria bassiana, a broad-spectrum insect pathogen used in biocontrol of arthropod pests worldwide. CRISPR-Cas9-based disruption of the transcription factor-encoding gene Bbsmr1 led to derepression of the oosporein biosynthetic gene cluster resulting in overproduction of the red-pigmented dibenzoquinone oosporein involved in host immune evasion, thus increasing fungal virulence. Mutants defective for Bbsmr1 displayed a remarkable enhanced insecticidal activity by reducing lethal times and concentrations, while concomitantly presenting negligible or minor pleiotropic effects. In addition, these mutants displayed faster germination on the insect cuticle which correlated with higher density of free-floating blastospores in the hemolymph and accelerated mortality of the host. These findings emphasize the utility of genetic engineering in developing enhanced fungal biocontrol agents with customized phenotypic traits, and provide an efficient and versatile genetic transformation tool for application in other beneficial entomopathogenic fungi.
生物防治剂在害虫治理中发挥着关键作用，并有助于实现可持续农业。基因工程的最新进展能够推动具有所需性状的昆虫病原真菌的开发，以提高生物防治效果。在本研究中，利用CRISPR-Cas9核糖核蛋白系统对球孢白僵菌的毒力进行遗传改良，球孢白僵菌是一种广谱昆虫病原体，用于全球节肢动物害虫的生物防治。基于CRISPR-Cas9对转录因子编码基因Bbsmr1的破坏导致卵孢霉素生物合成基因簇的去抑制，从而导致参与宿主免疫逃避的红色色素二苯并醌卵孢霉素过量产生，进而提高了真菌的毒力。Bbsmr1缺陷型突变体通过缩短致死时间和降低致死浓度，显著增强了杀虫活性，同时伴随的多效性效应可忽略不计或较小。此外，这些突变体在昆虫表皮上萌发更快，这与血淋巴中游离芽生孢子的高密度以及宿主死亡率的加速相关。这些发现强调了基因工程在开发具有定制表型性状的增强型真菌生物防治剂方面的实用性，并为应用于其他有益昆虫病原真菌提供了一种高效且通用的遗传转化工具。
