Identifition Of Conidiation And Growth Associated Genes In Coniothyrium Minitans

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a T-DNA insertional library with the wildtype strain ZS-1. A conidiation-deficient mutant ZS-1NT22803and a growth-deficient mutant ZS-1T25694.ZS-1NT22803mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the T-DNA flanking sequences revealed that a peroxisome biogenesis factor6(PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the T-DNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6was responsible for the phenotype of ZS-1NT22803. Further analysis showed that both ZS-1NT22803and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H2O2) than wild-type strain ZS-1. The conidiation of ZS-1TN22803was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans.The ROS produced by NADPH oxidases (Nox) play significant roles in animals, plants and fungi. The NADPH oxidases (Noxl/Nox2) genes were also found in Coniothyrium minitans. The gene knock-out and complementary experiments indicated that it is CmNoxl but not CmNox2which is necessary for conidium-producing and parasitic ability. Further more, qRT-PCR results indicated that high expression of CmNoxl was induced by S. sclerotiorum. NADPH oxidase complexes were also found cloned in C. minitans. CmRacl interacts with CmNoxR together to regulate CmNoxA and control the conidium-producing and parasitic ability. The yeast two-hybrid and bimolecular fluorescence complementation assays indicated that CmNoxl interacts with CmSlt2(homolog of Slt2encoding cell wall integrity-related MAP kinase). The phenotypes of knock-out transformant of CmNoxl and CmSlt2are very similar:they all lose the ability of conidium-producing and parasitic. GFP-CmSlt2fusion protein lost the ability to locate to the cell nucleus accurately in the△CmNoxlmutant. The sporulation capacity could be partially restored when the CmSlt2expressed in△CmRacl, indicating that the CmSlt2is the downstream regulatory factor of CmNoxl and is involved in sporulation and parasitism. The knock-out transformants of the genes in CmNox1signal pathways (CmRacl, CmNoxR, CmNoxl and CmSlt2) are all weaken in ability of melanin biosynthesis and parasitic, the qRT-PCR indicated that the expression of melanin biosynthesis associated polyketide synthase-encoding gene (CmPksl) and cell wall degrading enzyme-encoding gene (Cmgl and Chl) are all suppressed when these transformants are cultivated on PDA or on sclerotium. So, it infers that CmNOX1control CmPksl, Cmgl and CHI by regulating the MAPK signal pathways, thus to regulate ability of melanin biosynthesis and parasitic.Polar growth is a very significant biology characteristic. The abnormal polar growth mutant ZS-1T25694was isolated from the T-DNA insertion library of Coniothyrium minitans. The phenotype researches found that the growth rate of ZS-1T25694was obviously decreased, but the formation of conidia and the ability of parasitic on sclerotium were not affected. The Southern blot verified that the ZS-1T25694was single T-DNA insertion mutant. The i-PCR identified that the gene encode serine/threonine protein kinase in ZS-1T25694was disrupted by the T-DNA insertion, and this serine/threonine protein kinase of C. minitans share very high homology with Cla4in S. cerevisiae, so it was named CmCla4. The yeast two hybrid system verified that the CmCla4interact with upstream regulation factors CmRacl (Small GTP-binding proteins in C. minitans). The phenotype of CmRacl knockout mutant was similar to the ZS-1T25694, the growth rate obviously decreased, but CmRacl knockout mutant was lost the ability of conidia production and parasitic on sclerotium. It indicated that in C. minitans, CmRacl-CmCla4interaction is responsible for growth rate. In S. cerevisiae, the Cla4is the member of the p21-activated kinase (PAK) family, which activating the FUS3/KSS1MAP kinase pathway. In C. minitans, the homology genes of FUS3in FUS3/KSS1MAP kinase pathway were found, named CmFUS3. The gene functions researches found that CmFus3knockout mutants lose the ability of conidium-producing and parasitic, but not the growth rate. These results suggest that CmCla4could interact with CmRacl to regulate the polar growth of C. minitans, but CmCla4do not play critical role in activating the CmFus3MAP kinase pathway during polar growth. In summary, these results indicate that there are two regulation pathways of CmRAC1in C. minitans, the first one is that the CmRAC1interact with CmCla4to regular the growth; the other one is that the complex composed of CmRAC1and CmNOXR regular the CmNOX1, thus to regular the conidial production and parasitism, and the CmNOX1could also control the nuclear localization of CmSlt2, thus to regular the transcript accumulation of CmPksl, Ch1and Cmgl. Furthermore, CmPks1could catalyze the synthesis of melanin utilizing the Acetyl-CoA. These indicate that conidial production of C. minitans is a complicated process and regulated by multi-genes.