Programa de Pós-Graduação em Proteção de Plantas, Instituto Federal Goiano, Rodovia Geraldo Silva Nascimento, Km 2,5, Urutaí, GO, CEP: 75790-000, Brazil.
Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte, Brasília, DF, CEP: 70770-900, Brazil.
Sci Rep. 2021 Jan 13;11(1):1085. doi: 10.1038/s41598-020-80186-4.
Trichoderma harzianum is a filamentous fungus used as a biological control agent for agricultural pests. Genes of this microorganism have been studied, and their applications are patented for use in biofungicides and plant breeding strategies. Gene editing technologies would be of great importance for genetic characterization of this species, but have not yet been reported. This work describes mutants obtained with an auxotrophic marker in this species using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas (CRISPR-associated) system. For this, sequences for a guide RNA and Cas9 overexpression were inserted via biolistics, and the sequencing approach confirmed deletions and insertions at the pyr4 gene. Phenotypic characterization demonstrated a reduction in the growth of mutants in the absence of uridine, as well as resistance to 5-fluorotic acid. In addition, the gene disruption did not reduce mycoparasitc activity against phytopathogens. Thus, target disruption of the pyr4 gene in T. harzianum using the CRISPR/Cas9 system was demonstrated, and it was also shown that endogenous expression of the system did not interfere with the biological control activity of pathogens. This work is the first report of CRISPR Cas9-based editing in this biocontrol species, and the mutants expressing Cas9 have potential for the generation of useful technologies in agricultural biotechnology.
哈茨木霉是一种丝状真菌，可用作农业害虫的生物防治剂。该微生物的基因已被研究，并已为其在生物杀菌剂和植物育种策略中的应用申请了专利。基因编辑技术对于该物种的遗传特征具有重要意义，但尚未有报道。本工作使用 CRISPR（成簇的规律间隔短回文重复序列）/ Cas（CRISPR 相关）系统，在该物种中用营养缺陷型标记获得了突变体。为此，通过弹道法插入了向导 RNA 和 Cas9 过表达的序列，并通过测序方法证实了 pyr4 基因的缺失和插入。表型特征表明突变体在缺乏尿嘧啶的情况下生长减少，并且对 5-氟尿嘧啶具有抗性。此外，基因破坏并未降低针对植物病原菌的真菌寄生活性。因此，证明了使用 CRISPR/Cas9 系统靶向破坏哈茨木霉中的 pyr4 基因，并且该系统的内源性表达也不会干扰病原体的生物防治活性。这是该生物防治物种中首次报道基于 CRISPR Cas9 的编辑，表达 Cas9 的突变体有望在农业生物技术中生成有用的技术。
