Functional Characterization Of MoMET13 Encoding Methylenetetrahydrofolate Reductasein Magnaporthe Oryzae

In the study, a nonpathogenic mutant Wh-672 was obtained using Agrobacterium tumefaciens-mediated transformation (ATMT) technique and the T-DNA tagged gene MoMET13 in the mutant was identified. Furthermore, the biological function of MoMET13 was analyzed by gene deletion and complementation strategy.Wh-672 mutant of M. oryzae was completely nonpathogenic to both susceptible barley and rice. Phenotypic analysis showed that the mutant did not produce conidia on complete medium. To identify the disrupted gene in Wh-672, the genorriic DNA flanking T-DNA was amplified using hiTAIL-PCR. The tertiary 610bp PCR product for the right border was purified and cloned into pGEM-T easy vector. DNA sequencing and BLAST analysis showed that the T-DNA inserted the site upsteam of a gene MGG₀1728.6 (chromosome I) with 1948bp containing 2 exons and 1 intron. The gene encodes 627 amino acids. With NCBI BLAST search, homologs of MGG₀1728.6 were found in the genomes of related fungal species. MGG₀1728.6 showed 46.9%,48.03%,similarity with Sc MET13and Sp MET9 respectively. These proteins belong to methylenetetrahydrofolate reductase (MTHFR) superfamily, which catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate,a co-substrate in the synthesis of methionine from homocysteine.Ninty six transformants were obtained by protoplast mediated transformation with MoMET13 genereplacement vector. Four AMomet13 mutants from these transformants were identified by PCR and southern blot. Phenotypic analysis showed that the AMometl3 mutants could not grow on MM plate without methionine and were completely nonpathogenic on leaves of barley and rice, even the leaves were wounded. Other phenotypes of the AMomet13 mutants were consistent with these of Wh-672. To determine whether the phenotypes of Wh-672 were caused by a T-DNA insertion, we constructed a complementation vector pMoMET13-GFP containing the MoMET13 and GFP fusion. The vector was used to transform into the Wh-672 mutant and 58 transformants were obtained. The phenotypes of Wh-672, including vegetative growth, conidiation, appressorium formation, mating and pathogenicity, were fully complemented by reintroduction of the gene. However, green fluorescence was not observed by laser scanning confocal microscope, indicating that MoMET13 expressed in a low level during mycelia growth, conidiation, conidia germination, and appressorium formation.Bioinformatics analysis found the other gene, MoMET12, encoding methylenetetrahydrofolate reductase in M. oryzae genome, which is homologus with MET12 of Saccharomyces cerevisiae. Two hundred and sixty-nine transformants were obtained by transforming MoMET12 knochout vector into protoplasts of Guy 11 and fourâ–³Momet12 mutants were identified by PCR. Theâ–³Mometl2 mutants could not grow well on MM plate without methionine, however, other biological characters are the same with the wild type Guy11.Taken together, MoMET13 is essential for vegetative growth, conidiation and pathogenicity in the rice blast fungus.