An efficient targeted gene deletion approach for Trichoderma atroviride using Agrobacterium tumefaciens-mediated
Mengke Li1, Yihong Li1, Xiaoyu Zhang1
1Shenyang Agricultural University, College of Plant Protection, 110161 Shenyang, PR China.
None:
Trichoderma atroviride is well-known biocontrol fungus that plays a crucial role in controlling plant fungal diseases. In this study, the Serine Protease (T. atSp1) gene of T. atroviride was selected as the target gene to investigate the effects of Agrobacterium tumefaciens concentration, conidial concentration, mixing ratio of conidia and Agrobacterium cells, and induction time on transformation efficiency. The optimal knockout system was achieved under conditions that the density of A. tumefaciens was OD600 = 0.5, the concentration of conidia suspension was 106 conidia/mL, the mixture ratio of conidia suspension and A. tumefaciens AGL-1 was 1:1, and the induction time was 0.5 h. The transformation efficiency reached 28.33 to 61.67 transformants per 106 conidia under the optimized conditions. The ΔT. atSp1 was successfully validated by PCR analysis. Additionally, two genes of T. atEDG1 and T. atchi18 were also knocked out and verified, further demonstrating the robustness of this ATMT system. This study provides a stable and efficient genetic manipulation protocol for T. atroviride, facilitating further to understand genes function and biocontrol mechanisms.
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