Abstract
Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.
Highlights
Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyzes the reduction of 5, 10-methylenetetrahydrofolate (CH2-THF) to 5-methyltetrahydrofolate (CH3-THF), which is required for methionine biosynthesis
MTHFR is a critical enzyme for converting 5,10-CH2-THF to 5-CH3-THF, and the latter serves as a major methyl donor in the re-methylation of homocysteine to methionine
We identified a non-pathogenic mutant WH672 of M. oryzae using T-DNA insertional mutagenesis, and identified MET13, which putatively encodes a homologue of S. cerevisiae Met13 (MTHFR1)
Summary
Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) catalyzes the reduction of 5, 10-methylenetetrahydrofolate (CH2-THF) to 5-methyltetrahydrofolate (CH3-THF), which is required for methionine biosynthesis. Genes encoding MTHFRs of many species, including bacteria, fungi, plants and mammals, have previously been identified and characterized [1,3,4,5]. Most fungal species appear to have two MTHFR encoding genes, such as MET12 and MET13 in Saccharomyces cerevisiae [1], MET11 and MET9 in Schizosaccharomyces pombe [6], META and METF in Aspergillus nidulans [7], and FgMET13 and FgMET12 in Fusarium graminearum [8]. In A. nidulans, disruption of either METF (MET13 homologue) or META (MET12 homologue) results in methionine auxotrophy [7]. Targeted gene replacement of either F. graminearum FgMET13 or FgMET12 leads to methionine auxotrophy and affects pigment biosynthesis [8]. The role of MTHFRs in plant infection by pathogenic fungi has not been explored in detail
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