High efficient genome-editing system for a valuable plant-beneficial strain, Trichoderma harzianum NJAU4742

Youzhi Miao 1 Yanqiong Kong 1 Jian Zhang 1 Dongyang Liu 1 Feng Cai 1 Irina S. Druzhinina 3 Qirong Shen 1 Ruifu Zhang 1,2
1Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing, China
2Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China
3Research Area Biochemical Technology, TU Wien, Vienna, Austria

The specie of Trichoderma harzianum has valuable functions in agricultural applications, such as plant growth-promotion and anti-pathogens of plant. However, the deeper studies were strictly limited by genetic manipulation, especially in multiple genes editing. In this study, we developed a high efficient marker-free recycling gene-deletion system in T. harzianum strain NJAU4742, based on a strategy of two stepped homologous recombination (HR). Firstly, hygromycin B resistance gene, encoding aminoglycoside phosphotransferase, was used as a marker but to construct a marker-free auxotrophic mutant, NJAU4742Δura3, based on which the native ura3 gene was then used to screen the mutants of target genes with a destroyed expression box after the first HR. The final mutant, just deleting the whole sequence of target gene, would be screened out after the second HR using the plate with uridine and 5-Fluoroorotic acid (5-FOA). Meanwhile, screening steps were optimized to be able to keep the rate of HR in 10%-20% for strain NJAU4742 even without the contribution of ku70-deleted mutant, which was largely increased than the general value of 1% in filamentous fungi. Surely, no quantitatively restricted target genes could be knocked out one by one through this system in a quite short period. It is the first time to report the method of multiple gene editing in plant-beneficial T. harzianum strain, and this study will largely promote the scientific researches involved in the mechanisms of interactions among Trichoderma, plant and plant pathogens.