The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

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.

MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae

“…Similarly, for Moarg1, Moarg5,6, and Moarg7, when conidia had been pre-incubated in arginine solution before the plant inoculation assays, exogenous arginine could partially restore the infection defects in appressorial penetration and invasive hyphae growth. The Moarg1, Moarg5,6, and Moarg7 mutants were able to penetrate plant leaf cuticles, but the penetration rate and invasive growth were obviously reduced compared with the wild type, which just like Momet13 and Molys2 mutants of M. oryzae (Yan et al, 2013;Chen et al, 2014). However, studies in str3 (methionine auxotrophy) and Moilv1 (isoleucine auxotrophy) showed that there was no statistical difference in penetration rate between mutants and wild type, but the growth of invasive hyphae was restricted in host cells (Wilson et al, 2012;Du et al, 2014).…”

“…Similarly, the Moarg1 and Moarg5,6 mutants were unable to produce perithecia, and Moarg7 produced few perithecia. Like met13 mutant (methionine auxotrophy) of M. oryzae (Yan et al, 2013), the perithecium production of the Arg − mutants was partially restored via supplementation of oatmeal agar medium with exogenous arginine, which indicated that arginine is required for the sexual reproduction of M. oryzae. Unexpectedly, perithecium production was abolished in the Arg − mutants and wild type, when medium was amended with 2.5 mM arginine.…”

“…Mating tests were carried out by pairing the wild type strain Guy11 (MAT1-2) and Arg − mutants with a standard tester strain TH3 (MAT1-1) on oatmeal agar (OMA) plates, as described previously (Yan et al, 2013). The plates were incubated at 25 • C until the colony margins contacted each other, and then placed under continuous white fluorescent light at 18 • C for 4 weeks.…”

“…In M. oryzae, two REMI transformants pth3 and met1 have been identified, which exhibit histidine auxotrophy and methionine auxotrophy, respectively (Sweigard et al, 1998;Balhadère et al, 1999). Recently, the functions of several synthetic enzyme genes involved in methionine (MET12, MET13 and STR3), leucineisoleucine-valine (ILV1, ILV2 and ILV6), lysine (LYS2), and purine (ADE1) biosynthesis were investigated in M. oryzae (Yan et al, 2013;Wilson et al, 2012;Du et al, 2013Du et al, , 2014Chen et al, 2014;Fernandez et al, 2013). A link between auxotrophy and the loss of, or a reduction in pathogenicity was found in most of these cases.…”

Carbamoyl Phosphate Synthetase Subunit MoCpa2 Affects Development and Pathogenicity by Modulating Arginine Biosynthesis in Magnaporthe oryzae

“…Previous studies have shown that several genes involved in fungal metabolism are important to the infection-related morphogenesis and pathogenicity of M. oryzae . Cystathionine beta-lyase (MoStr3) and methylene tetrahydrofolate reductase (MoMet13) affect growth and development processes by regulating methionine biosynthesis (Wilson et al, 2012; Yan et al, 2013). SAICAR synthetase (MoAde1) is required for de novo adenine biosynthesis and pathogenicity (Fernandez et al, 2013).…”