Cloning And Functional Analysis Of LaeA Gene In The Mycoparasite Coniothyrium Minitans

Sclerotinia sclerotiorum(Lib.) de Bary is a widespread plant pathogenic fungus infecting more than 400 plant species. The mycoparasite Coniothyrium minitans has a excellent potential in control of the sclerotinia sclerotiorum stem rot caused by S. sclerotiorum. Mycoparasitism and production of antifungal substance(AFS) are the major mechanisms of C. minitans to control S. sclerotiorum, which is regulated by environmental p H value. Numerous reports indicated that Lae A regulates fungal secondary metabolism and development, such as in Aspergillus sp., Fusarium sp., Penicillium sp. and Trichoderma sp. However, information about Lae A gene in C. minitans remains unavailable in the literature.This study was conducted to clone Lae A homologous gene in C. minitans, to analyze the function of Cmlae A by protoplast mediated transformation and to understand the roles of Cmlae A gene in mycoparasitism and antibiosis between S. sclerotiorum and C. minitans interaction. Results are summarized below:Firstly, lae A gene and its promoter sequence of C. minitans were cloned in wild type Chy-1, named Cmlae A(Gen Bank Acc. No. KP998106). The Cmlae A coding sequence was 2017 bp with six exons and five introns. Cmlae A gene was predicted to encode a polypepetide contained 583 amino acid residues showing high homology to the S-adenosyl-methionine binding domain containing protein of Pyrenophora tritici-repentis with 80% identity. The expression pattern of Cmlae A gene under different light signal was analyzed by q RT-PCR. The transcript level of Cmlae A in wild type increased at 24 h dark treatment, and the transcript level was down regulated at 24 h light condition.Secondly, Cmlae A-disruption mutant named ΔCmlae A-2 and its corresponding complementary mutant ΔCmlae A-2C were obtained through the PEG-medium transformation of protoplast. Either in 24 h light or 24 h dark condition, mycelial growth of ΔCmlae A-2 mutant was no significant difference comparison with the wild type and the complementary mutant ΔCmlae A-2C on PDA cultures. However, the conidiation capacity and surface hydrophobicityof ΔCmlae A-2 were significantly affected. So, mutant lacking Cmlae Ahad reduced in conidial production and surface hydrophobicty as compared to the wild type and the complementary mutant ΔCmlae A-2C,and these biological characteristics are not regulated by light signal.Thirdly, the effect of Cmlae A on mycoparasitism of C. minitans was determined. Parasitism results showed that deletion of Cmlae A in C. minitans reduced its ability to infect S. sclerotiorum.Furthermore, production of parasite-related enzyme of two mutants and wild type of C. minitans were investigated. Activity of extracellular protease, chitinase and glucanase producted by ΔCmlae A-2 were significantly lower than the wild type and ΔCmlae A-2C. Therefore, Cmlae A is required for production of parasite-related enzyme(extracellular protease, chitinase and glucanase) in mycoparasitism of C. minitans on S. sclerotiorum.Fourthly, the cultural filtrates of the mutant ΔCmlae A-2 in PDB media showed enhanced antifungal activity. Meanwhile, the p H value of ΔCmlae A-2 culture filtratewas significantly lower than the wild type and the complementary mutant ΔCmlae A-2C. In the modified Czapek-Dox broth p H-buffered experiment, antifungal activity of the mutant ΔCmlae A-2 has no significant difference with the wild type and the complementary mutant ΔCmlae A-2C in p H 3.0, 4.0 and 6.0, and the antifungal activity was high of all C. minitans strains in a low level of p H ranging from 3.0 to 6.0. But in p H 5.0, the antifungal activity of ΔCmlae A-2 was markedly reduced. Thess results indicated that ΔCmlae A-2 enhanced antifungal activity in PDB likely because of decreased ambient p H.In conclusion, Cmlae A plays an important role in conidial production, surface hydrophobicity, andmycoparasitism and antibiosis in C. minitans against S. sclerotiorum.