Asci rosettes and ascospores per perithecium of the <i>G. zeae</i> strains.

<p>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 (<i>p</i><0.05) based on Tukey's test. WT, <i>G. zeae</i> wild-type strain Z-3639; myt2, <i>MYT2</i> deletion mutant; MYT2com, myt2<i>-</i>derived strain complemented with <i>MYT2</i>; MYT2OE, transgenic strain that has the <i>EF1α</i> promoter in place of the <i>MYT2</i> promoter region.</p

Self-fertility and asci rosettes of the <i>G. zeae</i> strains.

<p>(A) Perithecia of the <i>G. zeae</i> 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 <i>G. zeae</i> strains. The diameters of 300 perithecia were measured for each strain using a dissecting microscope. Values with different letters are significantly different (<i>p</i><0.05) based on Tukey's test. (C) Asci rosettes of wild-type and <i>MYT2</i> overexpression strains. Perithecia were dissected seven days after sexual induction. Scale bar  = 20 µm. WT, <i>G. zeae</i> wild-type strain Z-3639; myt2, <i>MYT2</i> deletion mutant; MYT2com, myt2<i>-</i>derived strain complemented with <i>MYT2</i>; MYT2OE, transgenic strain that has the <i>EF1α</i> promoter in place of the <i>MYT2</i> promoter region.</p

Production and morphology of conidia and ascospores.

a<p>Conidiation was measured by counting the number of conidia produced after a 3-d incubation in CMC.</p>b<p>Macroconidia were produced on YMA. A total of 100 macroconidia were observed for each examination.</p>c<p>Ascospores were collected from culture plate lids 10 d after sexual induction. A total of 300 ascospores were observed for each examination.</p>d<p>All experiments were repeated three times with three replicates each. Values within a column with different letters are significantly different (<i>p</i><0.01) based on Tukey's test.</p

<i>MYT2</i> overexpression.

<p>(A) The <i>MYT2</i> promoter region was replaced with the <i>EF1α</i> 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 <i>MYT2</i>-overexpressed mutants, respectively. (B) Expression of <i>MYT2</i> in the wild-type, <i>MYT2</i> deletion, and <i>MYT2</i> overexpression strains. <i>MYT2</i> 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 <i>MYT2</i> promoter region was replaced with the <i>EF1α</i> promoter; P, <i>Pst</i>I. The sizes of DNA standards (kb) are indicated to the left of the blot.</p

Radial growth and germination rate.

a<p>Radial growth was measured after a 5-d incubation.</p>b<p>The germination percentage was measured after a 6-h incubation.</p>c<p>CM, complete medium; MM, minimal medium.</p>d<p>All experiments were repeated three times with three replicates each. Values within a column with different letters are significantly different (<i>p</i><0.05) based on Tukey's test.</p

<i>G. zeae</i> strains used in this study.

<p><i>G. zeae</i> strains used in this study.</p

Distribution of MYT2 homologs in fungi and genetic complementation.

<p>(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 (<a href="http://cfgp.riceblast.snu.ac.kr/" target="_blank">http://cfgp.riceblast.snu.ac.kr/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037859#pone.0037859-Park1" target="_blank">[72]</a>. (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, <i>Phytophthora infestans</i>; Pr, <i>P. ramorum</i>; Ps, <i>P. sojae</i>; Af, <i>Aspergillus fumigatus</i>; An, <i>A. nidulans</i>; Ao, <i>A. oryzae</i>; Hc, <i>Histoplasma capsulatum</i>; Bc, <i>Botrytis cinerea</i>; Fo, <i>Fusarium oxysporum</i>; Fv, <i>F. verticillioides</i>; Mg, <i>Magnaporthe grisea</i>; Nc, <i>Neurospora crassa</i>; Pa, <i>Podospora anserine</i>; Ca, <i>Candida albicans</i>; Kl, <i>Kluyveromyces lactis</i>; Sc, <i>Saccharomyces cerevisiae</i>; Cc, <i>Coprinus cinereus</i>; Cn, <i>Cryptococcus neoformans</i>; Pc, <i>Phanerochaete chrysosporium</i>; nd, not detected. (C) Targeted deletion and complementation of <i>MYT2</i>. WT, <i>G. zeae</i> wild-type strain Z-3639; myt2, <i>MYT2</i> deletion mutant; MYT2com, myt2<i>-</i>derived strain complemented with <i>MYT2-GFP</i>; A, <i>Ava</i>I; <i>gen</i>, geneticin resistance gene cassette; <i>hyg</i>, hygromycin B resistance gene cassette. Lane 1, Z-3639; lanes 2 and 3, <i>MYT2</i> mutants; lanes 4 and 5, MYT2com mutants. The sizes of the DNA standards (kb) are indicated to the left of the blot.</p

Mycelia growth and wheat head virulence of the <i>MYT1</i> mutants.

<p>(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 (<i>p</i><0.05) based on Tukey's test. Mock, negative control mock inoculated with 0.01 % Tween 20; WT, <i>G. zeae</i> wild-type strain Z-3639; myt2, <i>MYT2</i> deletion mutant; MYT2com, myt2<i>-</i>derived strain complemented with <i>MYT2</i>; MYT2OE, transgenic strain that has the <i>EF1α</i> promoter in place of the <i>MYT2</i> promoter region.</p

A Comparison of Transcriptional Patterns and Mycological Phenotypes following Infection of <i>Fusarium graminearum</i> by Four Mycoviruses

<div><p>Many fungi-infecting viruses, which are termed mycoviruses, have been identified, and most do not cause any visible symptoms. Some mycoviruses, however, can attenuate the virulence of the infected fungi, a phenomenon referred to as hypovirulence. To study fungus responses to virus infection, we established a model system composed of <i>Fusarium graminearum</i> and four mycoviruses including FgV1 (Fusarium graminearum virus 1), FgV2, FgV3, and FgV4. FgV1 and FgV2 infections caused several phenotypic alterations in <i>F. graminearum</i> including abnormal colony morphology, defects in perithecium development, and reductions in growth rate, conidiation, and virulence. In contrast, FgV3 and FgV4 infections did not cause any phenotypic change. An RNA-Seq-based analysis of the host transcriptome identified four unique <i>Fusarium</i> transcriptomes, one for each of the four mycoviruses. Unexpectedly, the fungal host transcriptome was more affected by FgV1 and FgV4 infections than by FgV2 and FgV3 infections. Gene ontology (GO) enrichment analysis revealed that FgV1 and FgV3 infections resulted in down-regulation of host genes required for cellular transport systems. FgV4 infection reduced the expression of genes involved in RNA processing and ribosome assembly. We also found 12 genes that were differentially expressed in response to all four mycovirus infections. Unfortunately, functions of most of these genes are still unknown. Taken together, our analysis provides further detailed insights into the interactions between mycoviruses and <i>F. graminearum</i>.</p></div

The 12 DEGs commonly identified in response to four different mycovirus infections by RNA-Seq.

<p>The 12 DEGs commonly identified in response to four different mycovirus infections by RNA-Seq.</p