Abstract：
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.
摘要：
尽管越来越多的真菌microRNA样小RNA（milRNAs）被鉴定和报道，生物防治真菌中milRNAs的分析及其在病原真菌的霉菌寄生中的作用仍然有限。因此,在这项研究中，我们构建了一个GFP荧光菌株，以评估T.breveT069与灰霉病菌相互作用系统中霉菌寄生的关键时期。结果表明，木霉毛霉的早期发生在菌丝接触后12h，具有菌丝平行性的特征，而中期持续36小时的特征是包裹。在72小时发生的霉菌寄生虫病的晚期以灰霉病菌菌丝体的降解为特征。随后，我们使用高通量测序鉴定了短毛虫T069和灰霉病芽孢杆菌的sRNA。在ltR1中，为243个基因鉴定了45个潜在的milRNA靶标，和73个milRNAs靶向ltR3中的733个基因。此外,为了鉴定短小T.breveT069中潜在的跨界miRNA,我们筛选了在短小T.breveT069基因组中唯一表达且具有前体结构但在灰白B.cinerea基因组中不存在的miRNA。接下来,我们预测了灰霉病的靶基因。我们的发现表明，来自短小T.breveT069的14个潜在的跨界milRNAs靶向灰霉病中的41个基因。值得注意的是,cme-MIR164a-p5_1ss17CT可以靶向15个基因，包括Rim15(BCIN_15g00280)，NOP53(BCIN_12g03770),Skn7(BCIN_02g08650),和Vel3(BCIN_03g06410)，而ppe-MIR477b-p3_1ss11TC靶向聚酮合成酶(BCIN_03g04360,PKS3)。PC-5p-27397_41的靶基因是非核糖体肽合成酶(BCIN_01g03730,Bcnrps6)。PC-3p-0029(Tri-milR29)靶向的几丁质合成酶7.这些基因在灰霉病菌的正常菌丝生长和致病性中起着至关重要的作用。总之,这项研究强调了milRNAs在灰霉病木霉病中的意义。这一发现为miRNAs在真菌病原体的预防和治疗中的应用提供了新的策略。
