Functional Characterization Of MCM1 Gene In Fusarium Graminearum

In eukaryotic cells MADS-box genes are known to play major regulatory roles in various biological processes by combinatorial interactions with other transcription factors.This class of proteins has been identified in yeasts, plants, insects, nematodes, lower vertebrates and mammals. To date, the plant databases reveal the existence of several hundreds of putative MADS box genes. These proteins contain a conserved DNA binding and dimerization domain named the MADS box after the five founding members of the family: Mcm1(yeast), Arg80(yeast) or Agamous(Arabidopsis), Deficiens and SRF(human).The F. graminearum genome has two putative MADS-box anscription factors.One of them is orthologous to Mcm1, which is an essential gene in S. cerevisiae, the other MADS-box protein in F. graminearum is orthologous to Rlm1 belongs to MEF2-like(type II) classes. Mcm1 plays an important role in various biological processes, including minichromosome maintenance, general metabolism, cell identity and pheromone responses.In this study we functionally characterized the Fg MCM1 MADS-box gene in Fusarium graminearum the causal agent of wheat and barley head blight.The number of conidia produced by Fgmcm1 mutant was over 2,000-fold less than that of PH-1 and failed to produce phialides.Conidia were directly formed on hyphal tips or branches.The Fgmcm1 was defective in conidiogenesis, approximately 77.2±5.6 % of Fgmcm1 mutant conidia were 1 to 3-celled and approximately 19.9±1.7% of Fgmcm1 conidia had a cell connected to the blunt end(DAPI/Calcoflour stained).Whereas wild-type conidia had uni-nucleate compartments, multiple nuclei were present in individual conidium compartments in Fgmcm1 mutant.These observations indicate that Fg MCM1 is required for phialide and conidiogenesis development in F. graminearum.Deletion of Fg MCM1 resulted in the loss of perithecium production.This suggests the Fg Mcm1 plays important roles in sexual reproduction regulation.The Fgmcm1 mutant had decreased sensitivities to oxidative and membrane stresses.In infection assays with flowering wheat heads and corn stalks, the Fgmcm1 mutant was significantly reduced in plant infection and DON production.The Fg MCM1-GFP fusion construct was generated by gap-repair and transformed into the Fgmcm1 mutant.The resulting transformant was normal in growth, conidiation, DON production, and virulence.GFP signals were observed in the nucleus in conidia and hyphae of the Fgmcm1/Fg MCM1 transformant.Half of the Fgmcm1 mutants were unstable and produced stunted sub-cultures.These stunted subcultures(such as MD1) were alive but produced colonies with limited growth on various media tested, including CM, PDA, 5x YEG, and MM.In addition, hyphae of the stunted subcultures tended to be irregular in the width.Stunted subcultures of the Fgmcm1 mutant also were unstable, some of the colonies of stunted cultures produced fast growing sectors When the fast growing sectors were transferred to fresh medium, these spontaneous suppressors had similar growth rate and colony morphology with the original Fgmcm1 mutant.This indicated that stunted subcultures were likely caused by changes in Fg Mcm1-interacting genes or related transcriptional controls.In the heterothallic F verticillioides, culture instability was not observed in the Fvmcm1 mutants, the phenotype of the Fvmcm1 mutants were similar with the Fgmcm1 mutant, include the phenotype of the conidiation(include macro- and micro-conidium) and the FB1 production, but we do not obserse the growth unstable phenotype.In comparison with the wild-type strains, the Fvmcm1 mutants produced longer and skinnier phialides.We also observe that microconidia germinated to produce both germ tubes and microconidia, which may be also related to cell identity defects resulted from Fv MCM1 deletion.In yeast two-hybrid assays Fg Mcm1 interacted with Mat1-1-1 and Fst12 two transcription factors important for sexual reproduction.Whereas Fgmcm1 mat1-1-1 but not Fgmcm1 fst12 double mutants were stable, the Fgmcm1 fst12 mutant was unstable and approximately 42.6% of its subcultures had the stunted growth phenotype, suggesting that the instability of the Fgmcm1 mutant may be related to the interactions of Fg Mcm1 with the mating type locus genes.To understand why the stunted subcultures were restricted in growth, we also examined its transcript profile. RNA-seq analysis indicated that a number of sexual reproduction-related genes were up-regulated in stunted subcultures compared to the Fgmcm1 mutant which was down-regulated in the expression of genes involved in pathogenesis secondary metabolism and conidiation.Overall our data indicate that Fg Mcm1 plays a critical role in the regulation of cell identity, sexual and asexual reproduction, secondary metabolism and pathogenesis in F graminearum...