Correlation analyses of transcriptomes depending on expression levels.

Genes with more than 0 (A), 1 (B), 10 (C), and 100 (D) RPKM values 12 h after conidia induction were selected for the analysis.</p

Induction of asexual sporulation during perithecia production in <i>F</i>. <i>graminearum</i>.

(A) Conidia production after sexual induction. The number of conidia (conidia/mm2) was counted 0–5 days after sexual induction in the wild-type strain. (B) ABAA and WETA transcript levels during asexual and sexual reproduction. The transcript levels were quantified in reads per kilobase of exon per million mapped sequence reads (RPKM). (C) Mycelia and perithecia produced from the wild-type and abaA mutant strains. Scale bars represent 50 μm and 500 μm for the upper and below pictures, respectively. Arrows indicate mature conidia.</p

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

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

Phenotypic effects of <i>ABAA</i> deletion on representative transcription factor mutants.

<p>(A) Conidia production. The number of conidia was measured 1 day after sexual induction. The number of conidia in the wild-type strain was arbitrarily set to one. (B) Mycelia and conidia of representative transcription factor mutants. Pictures were taken 1 day after sexual induction. Scale bar = 50 μm. (C) Sexual development of the transcription factor mutant with and without the <i>ABAA</i> deletion. Pictures were taken 7 days after sexual induction. Scale bar = 500 μm.</p

Induction of conidiation-related genes during the initial stage of sexual development.

<p>(A) Venn diagrams illustrating the overlap between upregulated (A) or downregulated (B) wild-type-specific genes (blue circle, WT-1 d/<i>abaA</i>-1 d) and differentially expressed genes after sexual induction in the wild-type strain (green circle for downregulated genes, WT-24 h/WT-2 h < 2; orange circle for upregulated genes, WT-24 h/WT-2 h > 2). (C) Expression profiles of clustered groups including the conidiation-specific genes <i>WETA</i> and <i>ABAA</i>. Fuzzy clustering categorized 933 upregulated wild-type-specific genes into 10 groups. The genes included in groups 3 and 9 showed similar expression patterns during conidiation with <i>WETA</i> and <i>ABAA</i>, respectively. (D) The Venn diagram illustrating the overlap between the <i>WETA</i>- and <i>ABAA</i>-related genes (blue circle) and the upregulated genes after sexual induction in the wild-type strain (orange circle). Wild-type-specific genes (WT-1 d/<i>abaA</i>-1 d) represent differentially expressed genes in the wild-type strain compared to the <i>abaA</i> mutant 1 day after sexual induction. Genes that were differentially expressed 24 h after sexual induction compared to the uninduced condition (2 h) represent differentially expressed genes after sexual induction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155671#pone.0155671.ref030" target="_blank">30</a>].</p

Utilization of a Conidia-Deficient Mutant to Study Sexual Development in <i>Fusarium graminearum</i>

<div><p>Transcriptome analysis is a widely used approach to study the molecular mechanisms underlying development and the responses of fungi to environmental cues. However, it is difficult to obtain cells with a homogeneous status from the sexually-induced culture of the plant pathogenic fungus <i>Fusarium graminearum</i>. In this study, we provided phenotypic and genetic evidence to show that the current conditions applied for perithecia induction inevitably highly induced asexual sporulation in this fungus. We also found that hundreds of genes under the control of the conidiation-specific gene <i>ABAA</i> were unnecessarily upregulated after perithecia induction. Deletion of <i>ABAA</i> specifically blocked conidia production in both the wild-type strain and sexually-defective mutants during sexual development. Taken together, our results suggest that the <i>abaA</i> strain could be used as a background strain for studies of the initial stages of perithecia production in <i>F</i>. <i>graminearum</i>. Further comparative transcriptome analysis between the <i>abaA</i> mutant and the sexually-defective transcription factor mutant carrying the <i>ABAA</i> deletion would contribute to the construction of the genetic networks involved in perithecia development in <i>F</i>. <i>graminearum</i>.</p></div

Percentages of genes included in clusters 3 and 9 based on their expression levels.

<p>Genes with more than 1, 10, and 100 RPKM values 1 d after sexual induction were selected for the analysis.</p

Representative IGV images of genes possibly regulated by ex-siRNA.

<p>(A) Aligned sRNA-seq and mRNA-seq results of <i>F</i>. <i>graminearum</i> strains were visualized using IGV. Red stars indicate selected ex-siRNA for RT-PCR. (B) Relative transcript abundances of genes in the <i>F</i>. <i>graminearum</i> strains during sexual development. Transcript levels were analyzed via qRT-PCR 5 days after sexual induction. The transcript level of the wild type was arbitrarily set to 1.</p

Characterization of DEGs in RNAi-deficient mutants.

<p>(A) Expression profiles of clustered groups including the mating-type genes. Fuzzy clustering categorized total genes into 10 groups depending on their expression profiles during sexual development (0–5 day after sexual induction). The genes included in groups 2 and 9 showed similar expression patterns as those of the mating-type genes (<i>MAT1-1-1</i>, <i>MAT1-1-2</i>, <i>MAT1-1-3</i>, and <i>MAT1-2-1</i>) during sexual reproduction. Transcriptome data during sexual development were obtained from a previous study [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006595#pgen.1006595.ref008" target="_blank">8</a>] and re-analyzed for this study. (B) Gene Ontology (GO) enrichment network of the upregulated genes in the RNAi-deficient mutants. (C) GO enrichment network of the downregulated genes in the RNAi-deficient mutants. GO terms were statistically analyzed using GOstats [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006595#pgen.1006595.ref040" target="_blank">40</a>] and visualized using REVIGO [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006595#pgen.1006595.ref041" target="_blank">41</a>].</p

Characterization of sRNA-producing genes.

<p>(A) Number of genes that produce sRNAs depending on <i>F</i>. <i>graminearum</i> strains. (B) Number of genes that produce sRNAs with 5′-U depending on <i>F</i>. <i>graminearum</i> strains. (C) Correlation analyses of transcriptomes of <i>Fgdicer1 Fgdicer2</i> and <i>Fgago1 Fgago2</i> compared to that of the wild type depending on sRNA counts. The log₂ ratio of transcript abundance in <i>Fgdicer1 Fgdicer2</i> versus wild-type (x axis) and <i>Fgago1 Fgago2</i> versus wild-type (y axis) is plotted. Colors indicate the sRNA density (reads per kilobase). (D) Correlation analyses of sRNA counts and transcript abundance. Gene numbers with corresponding log₂ ratio of transcript abundance in <i>Fgdicer1 Fgdicer2</i> or <i>Fgago1 Fgago2</i> versus the wild-type strain Z-3639 were counted. Most genes producing antisense sRNAs more than 1000 counts per kilobase (red graphs) were positively regulated in <i>Fgdicer1 Fgdicer2</i> and <i>Fgago1 Fgago2</i> compared to those in the wild type. Colors indicate the sRNA density denoted in Fig 5C.</p