Functional Analysis Of Gene Of Small RNA Pathways In Coniothyrium Minitans

Coniothyrium minitans is an important biocontrol agent of plant pathogens Sclerotinia sclerotiorum. Small RNA is widespread from prokaryotes to eukaryotes. The Dicer plays an important role in the splicing of hairpin-like RNA and double-stranded RNA, while the Argonaute proteins are essential components of the microRNA-induced silencing complex (RISC), and both of them play important roles in microRNA biogenesis. The function of AGO, DCL1and DCL2have been well studied in animals, plants and some filamentous fungus, while the roles of AGO, DCL1and DCL2in C. minitans are still not clear. Herein, the function of AGO, DCL1and DCL2were studied in C. minitans.Firstly, the transcription of CmAGO, CmDCL1and CmDCL2were detected by RT-PCR, and the results showed that these genes express constructively in C. minitans. Strains containing null-mutations of CmAGO, CmDCL1and CmDCL2were constructed by homologous recombination. Gene knock-out vectors were transformed into conidia of C. minitans with Agrobactirium tumfaciens-mediated transformation. Three replacement mutants of CmAGO, four replacement mutants of CmDCLl and eight replacement mutants of CmDCL2were obtained. These mutants were verified by PCR and Southern blot. Complementary vector of CmAGO, CmDCLl and CmDCL2were constructed, and three complementary mutants of CmDCL2were obtained. Further studies showed that there were no significant difference in the morphology of colony, mycelium and conidium between knock-out mutants and wild-type strain ZS-1, however, the growth rate and rot of sclerotial parasitization of CmAGO, CmDCLl and CmDCL2knock-out mutants were noticeable decreased compared to ZS-1. The production of conidia and AFS in the knock-out mutants were increased in the antagonized area when dual cultured with S. sclerotiorum Ep-1PNA367. The conidial germination rate of CmAGO replacement mutants were significantly higher than ZS-1. Viruses could be detected in CmAGO and CmDCL2replacement mutants after antagonized with the Botrytis cinerea10HN454which contain virus BcHV/10HN454and BcTV/10HN454, and the phenotype of the virus infected strains were altered remarkably, including the reduction of conidiation in CmDCL2replacement mutants and lost the conidiation in CmAGO replacement mutants completely. The phenotypes of CmDCL2replacement mutants were fully complemented by re-introduction of the gene CmDCL2. These results suggest that CmAGO, CmDCLl and CmDCL2play critical roles in the growth development and sclerotial parasitism in C. minitans.