Gene Expression Profiling during Conidiation in the Rice Blast Pathogen <em>Magnaporthe oryzae</em>

<div><p>Conidiation of phytopathogenic fungi is a key developmental process that plays a central role in their life cycles and in epidemics. However, there is little information on conidiation-induced molecular changes in the rice blast fungus <em>Magnaporthe oryzae</em>. As a first step to understand conidiogenesis in this fungus, we measured genome-wide gene expression profiles during conidiation using a whole genome oligonucleotide microarray. At a two-fold expression difference, approximately 4.42% and 4.08% of genes were upregulated and downregulated, respectively, during conidiation. The differentially expressed genes were functionally categorized by gene ontology (GO) term analysis, which demonstrated that the gene set encoded proteins that function in metabolism, cell wall biosynthesis, transcription, and molecule transport. To define the events of the complicated process of conidiogenesis, another set of microarray experiments was performed using a deletion mutant for <em>MoHOX2</em>, a stage-specific transcriptional regulator essential for conidial formation, which was expressed <em>de novo</em> in a conidiation-specific manner in <em>M. oryzae</em>. Gene expression profiles were compared between the wild-type and the Δ<em>Mohox2</em> mutant during conidiation. This analysis defined a common gene set that was upregulated in the wild-type and downregulated in the Δ<em>Mohox2</em> mutant during conidiation; this gene set is expected to include conidiation-related downstream genes of <em>MoHOX2</em>. We identified several hundred genes that are differentially-expressed during conidiation; our results serve as an important resource for understanding the conidiation, a process in <em>M. oryzae,</em> which is critical for disease development.</p> </div

Measurements of the transcripts obtained by qRT-PCR in the wild-type (open) and in the Δ<i>Mohox2</i> mutant (gray) during conidiation.

<p>The MGG locus number and description of the genes are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043202#pone-0043202-t001" target="_blank">Table 1</a>.</p

Molecular functions of the genes induced and repressed during conidiation of <i>M. oryzae</i> at a two-fold expression threshold based on the Gene Ontology (GO) terms.

<p>Molecular functions of the genes induced and repressed during conidiation of <i>M. oryzae</i> at a two-fold expression threshold based on the Gene Ontology (GO) terms.</p

The genome-wide analysis of changes in mRNA abundance in the Δ<i>Mohox2</i> mutant during conidiation.

<p>(<b>A</b>). The number of genes induced (left) or repressed (right) with fold change values by comparing RNA levels in wild type with those in the Δ<i>Mohox2</i> mutant during conidiation. (<b>B</b>). The number of genes induced in wild-type and repressed in the Δ<i>Mohox2</i> mutant during conidiation. (<b>C</b>). Validation of microarray data by qRT-PCR. Graph shows the transcript levels of each gene on the same x-axis in the wild-type (white squares) and in the Δ<i>Mohox2</i> mutant (black squares) during conidiation.</p

<i>Magnaporthe oryzae</i> genes used in qRT-PCR.

<p><i>Magnaporthe oryzae</i> genes used in qRT-PCR.</p

The genome-wide analysis of changes in mRNA abundance during conidiation.

<p>(<b>A</b>). Measurement of the conidial density of <i>M. oryzae</i> on polycarbonate membrane-laid OMA plates at the indicated times. (<b>B</b>). The number of genes induced (left) or repressed (right) during conidiation based on the comparison of RNA levels of non-conidiating mycelia (NCMY) with conidiating mycelia (CNMY) of <i>M. oryzae</i>. (<b>C</b>). The number of genes induced (right) or repressed (left) with fold change values during conidiation of <i>M. oryzae</i>. (<b>D</b>). Validation of the microarray data by qRT-PCR. Transcript levels of each gene in CNMY were normalized to <i>β</i>-tubulin and expressed as relative values, with 1 corresponding to the NCMY. Each of five-digit number on the x-axis indicates the MGG locus number.</p

Experimental Evolution Reveals Genome-Wide Spectrum and Dynamics of Mutations in the Rice Blast Fungus, <i>Magnaporthe oryzae</i>

<div><p>Knowledge on mutation processes is central to interpreting genetic analysis data as well as understanding the underlying nature of almost all evolutionary phenomena. However, studies on genome-wide mutational spectrum and dynamics in fungal pathogens are scarce, hindering our understanding of their evolution and biology. Here, we explored changes in the phenotypes and genome sequences of the rice blast fungus <i>Magnaporthe oryzae</i> during the forced <i>in vitro</i> evolution by weekly transfer of cultures on artificial media. Through combination of experimental evolution with high throughput sequencing technology, we found that mutations accumulate rapidly prior to visible phenotypic changes and that both genetic drift and selection seem to contribute to shaping mutational landscape, suggesting the buffering capacity of fungal genome against mutations. Inference of mutational effects on phenotypes through the use of T-DNA insertion mutants suggested that at least some of the DNA sequence mutations are likely associated with the observed phenotypic changes. Furthermore, our data suggest oxidative damages and UV as major sources of mutation during subcultures. Taken together, our work revealed important properties of original source of variation in the genome of the rice blast fungus. We believe that these results provide not only insights into stability of pathogenicity and genome evolution in plant pathogenic fungi but also a model in which evolution of fungal pathogens <i>in natura</i> can be comparatively investigated.</p></div

Induction of cell death in HeLa cells by the PFKFB3 inhibitors, N4A and YN1.

<p>The cells were treated with two different concentrations of inhibitors, 25 µM and 50 µM. (<b>A</b>) Induced cell death at two different concentrations of N4A was measured by flow cytometry after double staining with Annexin V and PI. (<b>B</b>) Quantitation of the flow-cytometric data (mean ± SD) showing a dose-related effect of N4A. (<b>C</b>) Cytograms of YN- induced cell death and (<b>D</b>) quantitation of the flow-cytometric data.</p

Treatment of HeLa cells with N4A and YN1 inhibits soft agar colony formation.

<p>(<b>A</b>) Anchorage-independent cell growth in soft agar. HeLa cells were grown in soft agar for 21 days in the presence of the indicated concentrations of N4A and YN1 respectively (20×). (<b>B</b>) Statistical analysis of the experiment. Columns, mean (n = 5); bars, SD.</p

Structure of PFKFB3 in complex with inhibitors.

<p>(<b>A</b>) Ribbon diagram of the crystal structure of the PFKFB3•N4A complex. N4A bound to the PFKFB3 Fru-6-P site in the 2-Kase catalytic pocket is shown with a concomitant |F_o|−|F_c| omit map at a 2.5 level. The Fru-6-P bound to the 2-Pase domain is also shown in gray for comparison. (<b>B</b>)The interactions between N4A and PFKFB3 are shown. Hydrogen bonds are shown as yellow dotted lines and a Cation-π interaction is represented by a red broken line. (<b>C</b>) The water-mediated hydrogen bonds between PFKFB3 and N4A are shown in yellow dotted lines. (<b>D</b>)Inhibitor-induced local conformational changes around the N4A binding groove. Comparison of the structures of the PFKFB3•AMPPCP•Fru-6-P complex (dark gray) and the PFKFB3•N4A complex (color) was made. (<b>E</b>)YN1 bound to the same pocket with a |F_o|−|F_c| omit map at 2.5 level is shown. Hydrogen bonds between YN1 and PFKFB3 are shown as yellow dotted lines and Cation-π interactions as red broken lines.</p