Transboundary milRNAs: Indispensable molecules in the process of Trichoderma breve T069 mycoparasitism of Botrytis cinerea

Despite the increasing number of fungal microRNA-like small RNAs (milRNAs) being identified and reported, profiling of milRNAs in biocontrol fungi and their roles in the mycoparasitism of pathogenic fungi remains limited. Therefore, in this study, we constructed a GFP fluorescence strain to evaluate the critical period of mycoparasitism in the interaction system between T. breve T069 and B. cinerea. The results showed that the early stage of Trichoderma mycoparasitism occurred 12 h after hyphal contact and was characterized by hyphal parallelism, whereas the middle stage lasted 36 h was characterized by wrapping. The late stage of mycoparasitism occurred at 72 h was characterized by the degradation of B. cinerea mycelia. We subsequently identified the sRNAs of T. breve T069 and B. cinerea during the critical period of mycoparasitism using high-throughput sequencing. In ltR1, 45 potential milRNA targets were identified for 243 genes, and 73 milRNAs targeted 733 genes in ltR3. Additionally, to identify potential transboundary miRNAs in T. breve T069, we screened for miRNAs that were exclusively expressed and had precursor structures in the T. breve T069 genome but were absent in the B. cinerea genome. Next, we predicted the target genes of B. cinerea. Our findings showed that 14 potential transboundary milRNAs from T. breve T069 targeted 41 genes in B. cinerea. Notably, cme-MIR164a-p5_1ss17CT can target 15 genes, including Rim15 (BCIN_15g00280), Nop53 (BCIN_12g03770), Skn7 (BCIN_02g08650), and Vel3 (BCIN_03g06410), while ppe-MIR477b-p3_1ss11TC targeted polyketide synthase (BCIN_03g04360, PKS3). The target gene of PC-5p-27397_41 was a non-ribosomal peptide synthetase (BCIN_01g03730, Bcnrps6). PC-3p-0029 (Tri-milR29) targeted chitin synthetase 7. These genes play crucial roles in normal mycelial growth and pathogenicity of B. cinerea. In conclusion, this study highlights the significance of milRNAs in Trichoderma mycoparasitism of B. cinerea. This discovery provides a new strategy for the application of miRNAs in the prevention and treatment of fungal pathogens.