Transcriptomics of the rice blast fungus Magnaporthe oryzae in response to the bacterial antagonist Lysobacter enzymogenes reveals candidate fungal defense response genes.

Plants and animals have evolved a first line of defense response to pathogens called innate or basal immunity. While basal defenses in these organisms are well studied, there is almost a complete lack of understanding of such systems in fungal species, and more specifically, how they are able to detect and mount a defense response upon pathogen attack. Hence, the goal of the present study was to understand how fungi respond to biotic stress by assessing the transcriptional profile of the rice blast pathogen, Magnaporthe oryzae, when challenged with the bacterial antagonist Lysobacter enzymogenes. Based on microscopic observations of interactions between M. oryzae and wild-type L. enzymogenes strain C3, we selected early and intermediate stages represented by time-points of 3 and 9 hours post-inoculation, respectively, to evaluate the fungal transcriptome using RNA-seq. For comparative purposes, we also challenged the fungus with L. enzymogenes mutant strain DCA, previously demonstrated to be devoid of antifungal activity. A comparison of transcriptional data from fungal interactions with the wild-type bacterial strain C3 and the mutant strain DCA revealed 463 fungal genes that were down-regulated during attack by C3; of these genes, 100 were also found to be up-regulated during the interaction with DCA. Functional categorization of genes in this suite included those with roles in carbohydrate metabolism, cellular transport and stress response. One gene in this suite belongs to the CFEM-domain class of fungal proteins. Another CFEM class protein called PTH11 has been previously characterized, and we found that a deletion in this gene caused advanced lesion development by C3 compared to its growth on the wild-type fungus. We discuss the characterization of this suite of 100 genes with respect to their role in the fungal defense response

Confocal images of the interaction assay of <i>M. oryzae</i> and <i>L. enzymogenes</i> wild-type strain C3.

<p><i>M. oryzae</i> expressing a green fluorescent protein and <i>L. enzymogenes</i> expressing a dsRed fluorescent protein at 3hpi (A) and 9 hpi (B), and a mock inoculated sample (C). The long, thin structures are hyphae, whereas the tear-drop shaped structures are conidia. The smaller red rod shapes are bacteria. <i>M. oryzae</i> conidium size ranges from 20 to 30 µm. Scale bar: 20µm.</p

Promoter element motifs genes repressed by <i>L. enzymogenes</i> wild-type C3 and induced by mutant DCA.

<p>Promoters for the 100 fungal genes repressed by wild-type C3 and induced by mutant DCA were analyzed for common motifs using the MEME suite. Thirty-three genes had promoter elements with similarity to the binding site for the AZF1transcription factor (A) and 23 genes had promoter elements with similarity to the binding site for the STP2 transcription factor in <i>S. cerevisiae</i> (B).</p

Protein motifs for genes repressed by <i>L. enzymogenes</i> wild-type C3, and induced by mutant DCA.

<p>Amino acid sequences of 100 genes were analyzed using the motif finding program MEME, revealing five significant classes. Motifs 1 and 2 were found in MGG_08623.6, MGG_10662.6, and MGG_09601.6. Motif 3 was found in MGG_09857.6, MGG_01231.6, and MGG_00220.6. Motif 4 was found in MGG_14292.6, MGG_01863.6, and MGG_06587.6. Motif 5 was found in MGG_12589.6, MGG_00689.6, MGG_03201.6, and MGG_05025.6.</p

Venn diagrams reveal number of overlapping genes in <i>M. oryzae</i> challenged with <i>L. enzymogenes</i> wild-type C3 and mutant DCA.

<p>The area proportional Venn diagram shows numbers of fungal genes differentially expressed when challenged with <i>L. enzymogenes</i> wild-type strain C3 (red circles) and with mutant DCA (green circles) at 3 and 9 hpi. The induced genes by the C3 treatment were compared to the repressed genes by the DCA treatment and no overlapping genes were found at 3hpi, but 3 genes overlapped at 9 hpi. One hundred genes, which were repressed by C3 treatment and induced by DCA, overlapped at 3 hpi, whereas no overlapping genes were found at 9 hpi.</p

Functional categorization of <i>M. oryzae</i> genes repressed by <i>L. enzymogenes</i> wild-type C3 and induced by the mutant DCA.

<p>The graph shows functional categorization of 100 genes repressed by the wild-type bacterial strain C3 and induced by the mutant bacterial strain DCA. Numbers of genes in each functional category (y-axis) are shown across the x-axis. Genes were categorized using the Universal Protein Resource, Uniprot.</p

Fungal viability and bacterial load.

<p>Fungal killing assay using the MTT staining protocol to determine % viability of fungal cells, after treatment with either <i>Lysobacter enzymogenes</i> strains C3 or DCA (A). By 24 hours post-inoculation (hpi), the C3-treated sample is only 25% viable compared with untreated fungal cells, whereas the DCA-treated sample retained approximately 85% viability. Bars represent the average of 3 replicates and lines represent the standard error. Tukey-Kramer test was performed on the wild-type and mutant bacterium at each time-point (3 hr: p-value > 0.46; 9 hr: p-value > 0.76; 24 hr: p-value > 0.2). Bacterial burden assay showing no significant differences in bacterial numbers between C3 and DCA –treated samples at 0, 3 and 9 hours post-inoculation (B). Each plotted value indicates the population (cfu-colony forming units) of <i>L. enzymogenes</i> that colonized fungal cells per 100 µl inoculation. Bars represent the average of three replicates, lines represent standard error and capital letters over each bar represent lack of significance between pairs (C3 and DCA) at each time-point. Statistics were performed with the Tukey-Kramer test.</p