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

Pathogenicity of wild-type, Δ<i>Moadh1</i> and Δ<i>Mosre1</i>.

<p>(A) The pathogenicity assay was performed by spraying a conidia suspension (5 × 10⁴ conidia/ml) of each strain onto susceptible rice seedlings. Photographs were taken 7 days after inoculation. (B) The pathogenicity assay via drop inoculation was examined. Conidia suspension (10⁵ conidia/ml) was dropped onto detached rice leaves and incubated at room temperature. dpi, days post inoculation.</p

Genome-Wide Analysis of Hypoxia-Responsive Genes in the Rice Blast Fungus, <i>Magnaporthe oryzae</i>

<div><p>Rice blast fungus, <i>Magnaporthe oryzae</i>, is the most destructive pathogen in the rice-growing area. This fungus has a biotrophic phase early in infection and later switches to a necrotrophic lifestyle. During the biotrophic phase, the fungus competes with its host for nutrients and oxygen. Continuous uptake of oxygen is essential for successful establishment of blast disease of this pathogen. Here, we report transcriptional responses of the fungus to oxygen limitation. Transcriptome analysis using RNA-Seq identified that 1,047 genes were up-regulated in response to hypoxia. Those genes are involved in mycelial development, sterol biosynthesis, and metal ion transport based on hierarchical GO terms, and are well-conserved among three fungal species. In addition, null mutants of two hypoxia-responsive genes were generated and their roles in fungal development and pathogenicity tested. The mutant for the sterol regulatory element-binding protein gene, <i>MoSRE1</i>, exhibited increased sensitivity to a hypoxia-mimicking agent, increased conidiation, and delayed invasive growth within host cells, which is suggestive of important roles in fungal development. However, such defects did not cause any significant decrease in disease severity. The other null mutant, for the alcohol dehydrogenase gene <i>MoADH1</i>, showed no defect in the hypoxia-mimicking condition (using cobalt chloride) and fungal development. Taken together, this comprehensive transcriptional profiling in response to a hypoxic condition with experimental validations would provide new insights into fungal development and pathogenicity in plant pathogenic fungi.</p></div

Transcriptional response of <i>M</i>. <i>oryzae</i> to hypoxic conditions.

<p>(A) Expression of annotated transcripts in hypoxia. A total of 7,720 transcripts were selected by the FDR correction (<i>P</i> < 0.05). A twofold threshold was applied for significantly regulated genes. (B) Enrichment of specific gene ontology terms under hypoxia. Numbers represent the counts of transcripts supporting the GO terms.</p

Conidiation of the wild-type, Δ<i>Moadh1</i> and Δ<i>Mosre1</i>.

<p>(A) Conidia were collected from 7-day-old cultures on V8 juice agar plates with 5-ml water. The number of conidia was counted using a hemocytometer under the microscope. (B) Conidiogenesis was monitored under a microscope 16 hours after incubation. Strains were grown on oatmeal agar plates and scraped for inducing conidiogenesis at the same time. Scale bar indicates 100 μm.</p

Pathogenicity of wild-type, Δ<i>Moadh1</i> and Δ<i>Mosre1</i>.

<p>(A) The pathogenicity assay was performed by spraying a conidia suspension (5 × 10⁴ conidia/ml) of each strain onto susceptible rice seedlings. Photographs were taken 7 days after inoculation. (B) The pathogenicity assay via drop inoculation was examined. Conidia suspension (10⁵ conidia/ml) was dropped onto detached rice leaves and incubated at room temperature. dpi, days post inoculation.</p

The steroid biosynthesis pathway in <i>M</i>. <i>oryzae</i>.

<p>Intermediates are boxed in white and final products are boxed in black. Genes involved in the pathway are circled. Numbers in the gene names are gene IDs that start with ‘MGG.’ Up-regulated genes are colored in red and down-regulated genes in blue. ‘No significant regulation’ is colored in white.</p

The <i>in planta</i> expressed genes in the hypoxia-induced transcriptome.

<p>The <i>in planta</i> expressed genes in the hypoxia-induced transcriptome.</p

Schematic diagram of SREBP regulation for adaptation to hypoxia.

<p>Sterols control activation of SREBP: it is inactive in the presence of and active in the absence of sterols. Growth in limited oxygen conditions inevitably resulted in a lack of sterols, activating SREBP. Increases in enzymes for sterol biosynthesis and other oxygen-dependent pathways produce more sterols, which can be used for mycelial growth.</p

Invasive growth, oxidative stress sensitivity and enzyme activity of the wild-type, Δ<i>Moadh1</i> and Δ<i>Mosre1</i>.

<p>(A) Infectious growth was observed in rice sheath cells. A conidial suspension (2 × 10⁴ conidia/ml) was inoculated into the excised rice sheath. Photographs were taken 48 hours after incubation. Scale bar indicates 20 μm. (B) Frequency of infected rice cells was determined by counting at least 100 appressorium-mediated penetration pegs with three replicates. Invasive growth was observed as described above. 1, Move to adjacent cell; 2, One cell filled; 3, Primary hyphae; 4, No penetration. (C) Extracellular oxidative stress sensitivity of the wild-type and two deletion mutants were examined. Wild-type and two deletion mutants were inoculated on CM and CM including 2.5 or 5 mM H₂O₂ and 3 mM methyl viologen (MV). (D) Wild-type, Δ<i>Mosre1</i> and <i>Mosre1c</i> were inoculated on CM containing 200 ppm Congo Red. Discoloration (halo) of Congo Red was observed at 9 days after incubation.</p