Figure 1.
Identification of the non-pathogenic mutant, WH672, and the T-DNA-tagged gene.
(A) Colonies of the wild type strain Guy11 and T-DNA insertional mutant WH672 on CM medium. (B) Barley and rice leaf segments were inoculated with the mycelium plugs from Guy11 and WH672 cultures. (C) Three round amplifications of 0.6 kb genomic DNA flanking right site of the integration T-DNA in WH672 using a hiTAIL PCR approach. (D) Position of T-DNA insertion in the WH672 mutant strain. Arrow indicates the T-DNA insertion positions in MGG_01728. Thick boxes represent the coding regions and the thin line joining these coding regions indicates position of the intron.
Figure 2.
Colony growth and pathogenicity assays of Δmet12, Δmet13 and Δmet13Δmet12 mutants.
(A) Colonies of different strains. (B) Barley and rice segments inoculated with mycelium plugs from the wild-type strain Guy11, Δmet13 (K56), ectopic transformant (ECT5), Δmet13Met13 (C3), Δmet12 (K12-7) and Δmet13Δmet12 (DK7). H2O was used as the negative control. (C) Rice root infection assays. Photographs were taken 5 days after inoculation.
Figure 3.
Phenotypic analysis of Δmet13 and Δmet12 mutants.
(A) Bar chart showing colony diameters of the wild-type strain Guy11, Δmet13 (K56), ectopic transformant (ECT5), Δmet13MET13 (C3), Δmet12 (K12-7) and Δmet13Δmet12 (DK7) on CM medium at 25°C for 10 days. Error bars represent the standard deviation. Lower case letters indicate significant differences at P = 0.05. (B) Microscopic observation of conidial development. Aerial hyphae were significantly reduced and conidia were not observed in the Δmet13 and Δmet13Δmet12 mutants. The wild-type strain Guy11 and the complementation strain (Δmet13MET13) formed normal conidiophores and numerous conidia. All the tested strains grown on CM medium for 4 days were examined by light microscopy. Bar = 20 μm. (C) Bar chart showing the conidial production. The WH672, Δmet13 and Δmet13Δmet12 mutants were unable to produce any conidia, while the Δmet12 muatnt produced significantly less conidia than Guy11, ECT5 and Δmet13MET13 on CM medium at 25°C for 12 days. Error bars represent standard deviation. (D) Appressoria were formed by the hyphal tips of Guy11, Δmet13, Δmet12 and Δmet13MET13 on hydrophobic surfaces. After 24 h incubation at 25°C in the dark, numerous appressoria were produced by Guy11, Δmet12 and Δmet13MET13, however, no appressoria were observed after inoculation of Δmet13. Bar = 20μm.
Figure 4.
Growth of Δmet13, Δmet13MET13 and Δmet12 on different medium.
The wild-type strain Guy11, Δmet13 (K56), Δmet13MET13 (C3) and Δmet12 (K12-7) were grown on MM, OMA and PDA media at 25°C for 10 days. The Δmet13 mutant was unable to grow and the growth of the Δmet12 mutant was significantly restricted on MM and OMA medium.
Figure 5.
Growth of the wild-type strain and the mutants on MM with methionine, homocysteine or ammonium sulfate.
The wild-type strain Guy11, WH672, Δmet13 (K56) and Δmet12 (K12-7) were grown on MM medium at 25°C for 10 days. Concentrations of methionine, homocysteine or ammonium sulfate are as indicated.
Figure 6.
Perithecium production of the Δmet13 and Δmet12 mutants on OMA medium with methionine.
Concentrations of methionine in OMA medium were indicated. Perithecium production of the Δmet13 (K56) and Δmet12 (K12-7) mutants was fully complemented by the supplement of 1 mM methionine.
Figure 7.
Complementation of appressorium formation, penetration and pathogenicity of the Δmet13 mutant by adding exogenous methionine.
(A) Appressoria of the wild-type strain Guy11, K56 (Δmet13) and K12-7(Δmet12) were induced on hydrophobic coverslips (HC) and onion epidermis (OE) surfaces at 25°C in darkness for 24 h. Bar represents 10 μm for top panels and 20 μm for bottom panels. (B) Rice infection assays. Conidial suspension of 1 × 105 conidia ml-1 were spray-inoculated onto rice seedlings. H2O was used as the negative control. Photographs were taken at 5 days after inoculation. For both A and B, conidia of Guy11, K56, C3 (Δmet13MET13), K12-7 and DK7 (Δmet13Δmet12) were harvested from the CM cultures with 2 mM methionine and then diluted in 1 mM methionine solution.
Figure 8.
Relative expression of MET13 and MET12 in Δmet13 and Δmet12 mutants.
Measurements of gene transcripts by quantitative RT-PCR analysis were normalized to β-tubulin (MGG_00604.6) and expressed as relative values, with 1 corresponding to Guy11. Error bars represent the standard deviation. Expression of MET12 in WH672 and K56 (Δmet13) and MET13 in K12-72 (Δmet12) was significantly elevated.
Figure 9.
The predicted pathway for methionine biosynthesis in M. oryzae and expression of several genes involved in the pathway in Δmet13, Δmet12 and Δmet13Δmet12 mutants.
(A) An outline of methionine metabolism in M. oryzae. The predicted genes and corresponding enzymes: 1. CGS1 (MGG_03583), CGS2 (MGG_09292) and CGS3 (MGG_04764) - cystathionine γ-synthase; 2. CGL1 (MGG_10380) - cystathionine γ-lyase; 3. CBL1/STR3 (MGG_07074) - cystathionine β-lyase; 4. CBS1 (MGG_07384) - cystathionine β-synthase; 5. MS1 (MGG_06712) - methionine synthase; 6. MET13 (MGG_01728) and MET12 (MGG_08171) - methylenetetrahydrofolate reductase (MTHFR); 7. SHM1 (MGG_13781) and SHM2 (MGG_00923) - serine hydroxymethyltransferase; 8. SAM1 (MGG_00383) - S-adenosyl-methionine synthase; 9. various methyltransferases; 10. SAH1 (MGG_05155) - S-adenosyl homocysteine hydrolase; 11. HCS1 (MGG_07195) - homocysteine synthase. SAM: S-adenosyl methionine; SAH: S-adenosyl homocysteine; OAH: O-acetylhomoserine; ADO: Adenosine; Ser: Serine; R: any methylacceptor substrate. (B) Expression of the predicted genes coding enzymes involved in methionine metabolism in the Δmet13, Δmet12 and Δmet13Δmet12 mutants measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The abundance of the gene transcripts was calculated relative to endogenous control (β-tubulin gene) using the 2–ΔΔCT method. Error bars represent the standard deviation.