Virulence assay and trichothecene analysis.

<p>(A) Flowering wheat head were inoculated with conidia of the wild-type (WT) Z-3639, Δ<i>myt3</i> mutant, complemented strain Δ<i>myt3</i>-com, and overexpressed strain <i>MYT3</i>-oe, respectively. Mock indicates a negative control inoculated with 0.01% Tween 20. Wheat heads were photographed 14 days after inoculation. (B) Analysis of total trichothecenes (deoxynivalenole and 15-acetyl-deoxynivalenol) was performed on each strain grown in minimal liquid medium supplemented with 5 mM agmatine (MMA). The measured amount of trichothecenes was normalized to the biomass of each strain. (C) Expression of <i>TRI5</i> and <i>TRI6</i> in the wild type, Δ<i>myt3</i>, <i>MYT3</i>-oe strains. Transcript levels were analyzed by qRT-PCR from cultures grown in MMA for 4 days and presented as relative transcript level compared to a control gene, <i>CYP1</i>.</p

Transcriptional/translational expression of <i>MYT3</i>.

<p>(A) Transcript levels of <i>MYT3</i> in each strain were analyzed by qRT-PCR. Total RNA of each strain was extracted from vegetative cultures grown on carrot agar media, and from sexual cultures 3, 5, and 7 days after induction of sexual development. WT, wild-type strain Z-3639; Δ<i>myt3, MYT3</i> deletion mutant; <i>MYT3</i>-oe, over expression strain where <i>MYT3</i> expression is driven by the <i>EF1α</i> promoter. d, day; n.d., not detected. (B) Western blot analysis of <i>MYT3</i>-gfp stain. Lane 1, whole cell extracts were prepared from vegetative cultures grown on carrot agar media for 5 days; Lane 2–4, whole cell extracts were from sexual cultures 3, 5, and 7 days after induction of sexual development, respectively. Top, western blotting data; bottom, coomassie blue staining used as a loading control.</p

Nuclear localization of MYT3.

<p>Representative examples show co-localization of MYT3 fused with green fluorescent protein (GFP) and histone H1 fused with red fluorescent protein (RFP). A strain containing MYT3::GFP and hH1::RFP was grown on CM (hypha), YMA (conidia), and carrot agar media (ascospores) for the microscopic observation, respectively. DIC, differential interference contrast; scale bar = 20 μm.</p

The Δ<i>myt3</i> strain exhibits a lack of sexual development.

<p>(A) Strains grown on carrot agar media were self-fertilized. Photos were taken 9 days after sexual induction. Dissecting the perithecia showed the asci and ascospores of each strain (inset boxes). WT, wild-type strain Z-3639; Δ<i>myt3, MYT3</i> deletion mutant; Δ<i>myt3</i>-com, complemented strain of the Δ<i>myt3</i>; <i>MYT3</i>-oe, overexpressed strain of <i>MYT3</i>. Scale bar = 500 μm. (B) Heterozygous outcrosses. The Δ<i>myt3</i> strain, as either a female or male strain, was outcrossed with self-sterile Δ<i>mat2</i> strain. Photos were taken 9 days after outcrossing. Scale bar = 500 μm.</p

Conidiation, growth, and virulence of <i>Fusarium graminearum</i> strains.

a<p>(-N) indicates lacking of nitrogen source in MM.</p>b<p>Mean and standard deviation of diseased spikelets per wheat head.</p>c<p>Values with different letters within a column indicate statistical difference (<i>p</i><0.05) based on Tukey's test. All experiments were repeated three times with three replicates each.</p

<i>Fusarium graminearum</i> strains used in this study.

<p><i>Fusarium graminearum</i> strains used in this study.</p

Alignment of Myb-like DNA-binding domains of MYT3 homologs.

<p>(A) MYT3 homologs in <i>Fusarium oxysporum</i> (FOXG_00743), <i>Magnaporthe oryzae</i> (MGG_14558), <i>Neurospora crassa</i> (NCU_10346), <i>Botrytis cinerea</i> (BC1G_02829) were used for this analysis. Each Myb domain contains three helices, indicated with an underline, and residues that form their hydrophobic cores, indicated by black dots above the residue. The second domain contains residues that are implicated to have direct contact with the DNA, indicated by asterisks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094359#pone.0094359-Xiang1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094359#pone.0094359-Garzia1" target="_blank">[46]</a>. (B) Ribbon diagram of Myb-like DNA binding domains in MYT3.</p

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

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

The sexual development and virulence of Δ<i>Fgnot3</i> mutants.

<p>(A) Fertility tests. Each strain was inoculated on carrot agar and mock-fertilized (self-cross) or outcrossed with the corresponding male strains (WT, Δ<i>Fgnot3</i> and Δ<i>mat1</i>). The photographs were taken 7 days after sexual induction. Scale bar = 500 μm. (B) Asci of an outcross Δ<i>mat1</i> × Δ<i>Fgnot3</i>. Eight ascospores of an ascus from the Δ<i>mat1</i> × Δ<i>Fgnot3</i> outcross showing 1:1 segregation with and without Gfp-tagged histone H1. The photographs were taken 9 days after sexual induction. Scale bar = 10 μm. (C) Virulence on wheat heads. The center spikelet of each wheat head was injected with 10 μl of a conidial suspension. Pictures were taken 21 days after inoculation. Mock, negative control mock-inoculated with 0.01% of Tween 20; WT, <i>F</i>. <i>graminearum</i> wild-type strain Z-3639; Δ<i>Fgnot3</i>, <i>FgNOT3</i> deletion mutant; FgNot3c, Δ<i>Fgnot3</i>-derived strain complemented with <i>FgNOT3</i>.</p

The vegetative growth of Δ<i>Fgnot3</i> mutants.

<p>(A) Mycelial growth on complete medium (CM) and minimal medium (MM). The pictures were taken 5 days after inoculation. The pictures were taken from the upper (top) and the side (middle) of the plates. (B) Microscopic observation of hyphae. The differential interference contrast (DIC) images were taken 2 days after inoculation. Scale bar = 50 μm. (C) Swollen hyphae of Δ<i>Fgnot3</i> mutants on CM agar. Scale bar = 50 μm. WT, <i>F</i>. <i>graminearum</i> wild-type strain Z-3639; Δ<i>Fgnot3</i>, <i>FgNOT3</i> deletion mutant; FgNot3c, Δ<i>Fgnot3</i>-derived strain complemented with <i>FgNOT3</i>.</p