Table 1.
G. zeae strains used in this study.
Figure 1.
Distribution of MYT2 homologs in fungi and genetic complementation.
(A) Distribution of MYT2 in representative fungal species. The distribution image was constructed using the BLASTMatrix tool that is available on the Comparative Fungal Genomics Platform (http://cfgp.riceblast.snu.ac.kr/) [72]. (B) Phylogenetic tree of MYT2 homologs in several fungal species. The alignment was performed with ClustalW, and the MEGA program, version 4.0, was used to perform a 1,000-bootstrap phylogenetic analysis using the neighbor-joining method. Pi, Phytophthora infestans; Pr, P. ramorum; Ps, P. sojae; Af, Aspergillus fumigatus; An, A. nidulans; Ao, A. oryzae; Hc, Histoplasma capsulatum; Bc, Botrytis cinerea; Fo, Fusarium oxysporum; Fv, F. verticillioides; Mg, Magnaporthe grisea; Nc, Neurospora crassa; Pa, Podospora anserine; Ca, Candida albicans; Kl, Kluyveromyces lactis; Sc, Saccharomyces cerevisiae; Cc, Coprinus cinereus; Cn, Cryptococcus neoformans; Pc, Phanerochaete chrysosporium; nd, not detected. (C) Targeted deletion and complementation of MYT2. WT, G. zeae wild-type strain Z-3639; myt2, MYT2 deletion mutant; MYT2com, myt2-derived strain complemented with MYT2-GFP; A, AvaI; gen, geneticin resistance gene cassette; hyg, hygromycin B resistance gene cassette. Lane 1, Z-3639; lanes 2 and 3, MYT2 mutants; lanes 4 and 5, MYT2com mutants. The sizes of the DNA standards (kb) are indicated to the left of the blot.
Figure 2.
(A) The MYT2 promoter region was replaced with the EF1α promoter. The left and right panels show the strategy of MYT2OE strain construction and Southern hybridization, respectively. In the blot, lane 1 and lanes 2–4 represent the wild-type strain and the MYT2-overexpressed mutants, respectively. (B) Expression of MYT2 in the wild-type, MYT2 deletion, and MYT2 overexpression strains. MYT2 transcript accumulation was analyzed by quantitative real time-PCR (qRT-PCR) during the vegetative and sexual induction stages. WT, wild-type strain Z-3639; MYT2OE, transgenic strain where the MYT2 promoter region was replaced with the EF1α promoter; P, PstI. The sizes of DNA standards (kb) are indicated to the left of the blot.
Figure 3.
Self-fertility and asci rosettes of the G. zeae strains.
(A) Perithecia of the G. zeae strains. Five-day old carrot agar culture was mock-fertilized to induce sexual reproduction and incubated for an additional 7 d. The upper and lower panels show the photographs of perithecia formed on a whole carrot agar plate and the photographs taken with a dissecting microscope, respectively. Scale bar = 200 µm. (B) Diameter of the perithecia of the G. zeae strains. The diameters of 300 perithecia were measured for each strain using a dissecting microscope. Values with different letters are significantly different (p<0.05) based on Tukey's test. (C) Asci rosettes of wild-type and MYT2 overexpression strains. Perithecia were dissected seven days after sexual induction. Scale bar = 20 µm. WT, G. zeae wild-type strain Z-3639; myt2, MYT2 deletion mutant; MYT2com, myt2-derived strain complemented with MYT2; MYT2OE, transgenic strain that has the EF1α promoter in place of the MYT2 promoter region.
Table 2.
Production and morphology of conidia and ascospores.
Figure 4.
Asci rosettes and ascospores per perithecium of the G. zeae strains.
Each strain was inoculated on carrot agar and was mock fertilized. (A) The perithecia from each strain were softly squeezed with cover slides to exude whole asci rosettes. The picture of each strain is representative of more than 20 repetitions. (B) All discharged ascospores were collected from the culture plate through 14 days after sexual induction. The number of ascospores per perithecia was obtained by dividing the number of perithecia by the number of discharged ascospores. Values with different letters are significantly different (p<0.05) based on Tukey's test. WT, G. zeae wild-type strain Z-3639; myt2, MYT2 deletion mutant; MYT2com, myt2-derived strain complemented with MYT2; MYT2OE, transgenic strain that has the EF1α promoter in place of the MYT2 promoter region.
Table 3.
Radial growth and germination rate.
Figure 5.
Mycelia growth and wheat head virulence of the MYT1 mutants.
(A) Mycelial growth on complete media (CM) and minimal media (MM) 5 d after inoculation. (B) A center spikelet of each wheat head was injected with 10 µl of conidia suspension. Values with different letters are significantly different (p<0.05) based on Tukey's test. Mock, negative control mock inoculated with 0.01 % Tween 20; WT, G. zeae wild-type strain Z-3639; myt2, MYT2 deletion mutant; MYT2com, myt2-derived strain complemented with MYT2; MYT2OE, transgenic strain that has the EF1α promoter in place of the MYT2 promoter region.
Figure 6.
Total trichothecene (deoxynivalenol and 15-acetyldeoxynivalenol) production and transcriptional analyses of trichothecene synthetic genes.
(A) Each strain was grown in minimal medium supplemented with 5 mM agmatine for 7 d. Trichothecenes were analyzed by GC-MS and quantified based on the biomass produced by each strain. Asterisks indicate data that significantly differed (p<0.05) based on Tukey's test (B) Expression of Tri5 and Tri6 in the wild-type, MYT2 deletion, and MYT2 overexpression strains. Gene transcription was analyzed by quantitative real time-PCR (qRT-PCR) 4 d after inoculation in MMA. WT, G. zeae wild-type strain Z-3639; Δmyt2, MYT2 deletion mutant; MYT2com, Δmyt2-derived strain complemented with MYT2; MYT2OE, transgenic strain that has the EF1α promoter inserted in place of the MYT2 promoter region.
Figure 7.
Cellular localization of MYT2.
MYT2 was fused with green fluorescent protein (GFP), and histone H1 was fused with red fluorescent protein (RFP). Co-localization of MYT2-GFP and hH1-RFP in spores (A) and germinated conidia 9 and 24 h after inoculation in complete medium (B). DIC, differential interference contrast Scale bar = 20 µm.