AbaA Regulates Conidiogenesis in the Ascomycete Fungus <i>Fusarium graminearum</i>

<div><p><i>Fusarium graminearum</i> (teleomorph <i>Gibberella zeae</i>) is a prominent pathogen that infects major cereal crops such as wheat, barley, and maize. Both sexual (ascospores) and asexual (conidia) spores are produced in <i>F. graminearum</i>. Since conidia are responsible for secondary infection in disease development, our objective of the present study was to reveal the molecular mechanisms underlying conidiogenesis in <i>F. graminearum</i> based on the framework previously described in <i>Aspergillus nidulans</i>. In this study, we firstly identified and functionally characterized the ortholog of AbaA, which is involved in differentiation from vegetative hyphae to conidia and known to be absent in <i>F. graminearum</i>. Deletion of <i>abaA</i> did not affect vegetative growth, sexual development, or virulence, but conidium production was completely abolished and thin hyphae grew from abnormally shaped phialides in <i>abaA</i> deletion mutants. Overexpression of <i>abaA</i> resulted in pleiotropic defects such as impaired sexual and asexual development, retarded conidium germination, and reduced trichothecene production. AbaA localized to the nuclei of phialides and terminal cells of mature conidia. Successful interspecies complementation using <i>A. nidulans</i> AbaA and the conserved AbaA-WetA pathway demonstrated that the molecular mechanisms responsible for AbaA activity are conserved in <i>F. graminearum</i> as they are in <i>A. nidulans</i>. Results from RNA-sequencing analysis suggest that AbaA plays a pivotal role in conidiation by regulating cell cycle pathways and other conidiation-related genes. Thus, the conserved roles of the AbaA ortholog in both <i>A. nidulans</i> and <i>F. graminearum</i> give new insight into the genetics of conidiation in filamentous fungi.</p></div

Germination rate of conidia.

<p>One ml of conidium suspension of each strain was incubated in 10 ml of complete medium (CM) at 25°C on a rotary shaker (150 rpm). One hundred spores were observed in each examination with light microscopy, and the number of conidia that germinated was counted. All data were obtained from three biological replicates. Asterisks indicate data that significantly differed (<i>p</i><0.05) based on Tukey's test. WT, <i>F. graminearum</i> wild-type strain Z-3639; AbaA-OE, a transgenic strain with the <i>abaA</i> promoter replaced with the <i>ef1α</i> promoter.</p

Identification and deletion of <i>abaA</i> in <i>F.</i><i>graminearum</i>.

<p>(A) Re-annotation of the <i>abaA</i> ortholog gene in <i>F. graminearum</i>. cDNA sequencing analysis revealed an exact <i>abaA</i> open reading frame with an intron. (B) Comparison of AbaA orthologs between <i>A. nidulans</i> and <i>F. graminearum</i>. A schematic representation of the protein orthologs shows the location and alignment of conserved domains. The TAE and TEA/ATTS domains (IPR000818) predicted with InterProScan; NLS, nuclear localization signal predicted by using NLStradamus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072915#pone.0072915-NguyenBa1" target="_blank">[62]</a>. (C) Distribution of AbaA homologs 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.0072915#pone.0072915-Park2" target="_blank">[74]</a>. 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>; Pm, <i>Penicillium marneffei</i>, Hc, <i>Histoplasma capsulatum</i>; Bd, <i>Blastomyces dermatitidis</i>; Pb, <i>Paracoccidioides brasiliensis</i>; Fo, <i>Fusarium oxysporum</i>; Fv, <i>F. verticillioides</i>; Mo, <i>Magnaporthe oryzae</i>; Pa, <i>Podospora anserine</i>; Nc, <i>Neurospora crassa</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. (D) Targeted deletion of the <i>abaA</i> gene. The <i>abaA</i> gene was deleted from the <i>F. graminearum</i> wild-type genome. Left panel, schematic representation of the homologous gene recombination strategy used to generate the <i>abaA</i> deletion mutants. Right panel, Southern blot analysis. Sizes of the DNA standards used are indicated in kilobases to the left of the blot.</p

Phenotypic analyses of <i>abaA</i> deletion mutants.

<p>(A) Mycelial growth of <i>F. graminearum</i> strains on complete media (CM). Pictures were taken 5 days after inoculation on CM. (B) Perithecium formation of <i>F. graminearum</i> strains on carrot agar. Pictures were taken 7 days after sexual induction from carrot agar. Scale bar = 500 µm. (C) Asci rosettes of <i>F. graminearum</i> strains. Imaging was performed 7 days after sexual induction. (D) Virulence on wheat heads. A center spikelet of each wheat head was injected with 10 µl of a conidium suspension. Pictures were taken 21 days after inoculation. Values with different letters are significantly different (<i>p</i><0.05) based on Tukey's test. Scale bar = 20 µm. WT, <i>F. graminearum</i> wild-type strain Z-3639; Δ<i>abaA</i>, <i>abaA</i> deletion mutant; AbaA-OE, transgenic strain with the abaA promoter replaced with the <i>ef1α</i> promoter.</p

Percentage of DEGs among AbaA binding motif-containing genes.

<p>All information on <i>F. graminearum</i> sequences and annotation was obtained from the <i>F. graminearum</i> Database (<a href="http://mips.helmholtz-muenchen.de/genre/proj/FGDB/" target="_blank">http://mips.helmholtz-muenchen.de/genre/proj/FGDB/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072915#pone.0072915-Wong1" target="_blank">[51]</a>. From this genome sequence and gene annotation, 500-bp upstream sequences were collected from all predicted genes and surveyed for finding CATTCY motifs in these regions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072915#pone.0072915-Andrianopoulos1" target="_blank">[60]</a>. DEGs between <i>F. graminearum</i> wild-type strain Z-3639 and <i>abaA</i> deletion mutant were compared with genes having CATTCY motifs within 500-bp upstream sequences to calculate percentages of DEGs among CATTCY motifs -containing genes. DEG, differentially expressed genes; RPKM, reads per kilobase of exon per million.</p

Overexpression of <i>abaA</i>.

<p>(A) The <i>abaA</i> promoter was replaced with the <i>ef1α</i> promoter. The left and right panels show the strategy of AbaA-OE strain construction and Southern hybridization, respectively. The sizes of DNA standards (kb) are indicated on the left of the blot. (B) Relative transcript accumulation of <i>abaA</i> in wild-type, <i>abaA</i>-deleted, and <i>abaA</i>-overexpressed strains. The transcript level of <i>abaA</i> was analyzed by quantitative real time-PCR (qRT-PCR) during the conidium induction stage. WT, <i>F. graminearum</i> wild-type strain Z-3639; Δ<i>abaA</i>, <i>abaA</i> deletion mutant; AbaA-OE, a transgenic strain with the <i>abaA</i> promoter replaced with the <i>ef1α</i> promoter.</p

Morphology of conidiophores of <i>F.</i><i>graminearum</i> strains.

<p>Morphology of conidiophores of <i>F. graminearum</i> strains in carboxymethyl cellulose (CMC) medium. Pictures were taken 1 to 3 days after conidium induction. Scale bar = 10 µm. WT, <i>F. graminearum</i> wild-type strain Z-3639; Δ<i>abaA</i>, <i>abaA</i> deletion mutant; AbaA-OE, transgenic strain with the abaA promoter replaced with the <i>ef1α</i> promoter.</p

Cellular localization of AbaA.

<p>AbaA was fused with green fluorescent protein (GFP), and histone H1 was fused with red fluorescent protein (RFP). (A) The GFP signals were highly fluorescent in the phialides (indicated with arrows) and maturing conidia (indicated with arrow heads). The GFP signals became blurred after germination. (B) The GFP signals were undetectable in mature ascospores. Scale bar = 10 µm.</p

Mycelial growth and trichothecene production of <i>F. graminearum</i> strains.

a<p>Radial growth was measured after a 4-day incubation on complete media (CM).</p>b<p>Biomass was measured after a 3-day incubation in complete media (CM).</p>c<p>DON and 15-AcDON concentrations were measured after a 7-day incubation in a minimal medium containing 5 mM agmatine.</p>**<p>Asterisk indicates data differed significantly (<i>p</i><0.01) based on Tukey's test.</p

Relative transcript accumulation of <i>wetA</i>.

<p>The transcript levels of <i>wetA</i> (FGSG_17727) were analyzed by quantitative real time-PCR (qRT-PCR) during the conidium induction stage in the wild-type, <i>abaA</i> deletion mutant, and <i>abaA</i> overexpression mutant strains. WT, <i>F. graminearum</i> wild-type strain Z-3639; Δ<i>abaA</i>, <i>abaA</i> deletion mutant; AbaA-OE, a transgenic strain with the <i>abaA</i> promoter replaced with the <i>ef1α</i> promoter.</p