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

The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum.

BACKGROUND: Small non-coding RNAs (sRNA) are emerging as major components of the cells regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in Gram- prokaryotes, but hardly any in Gram+ prokaryotes. Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress (or heat-shock protein, HSP) response as well as specialized metabolic programs. RESULTS: Using RNA deep sequencing (RNA-seq) we examined the sRNome of C. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional (6S, S-box &solB) and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq. CONCLUSIONS: Our results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications

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

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

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

First report of Fusarium equiseti-the incitant of post flowering stalkrot of maize (Zea mays L.) in India

Maize (Zea mays L.) is one of the important cereal crops in the world and is the third largest grown cereal crop in India. Field surveys conducted in 2013-15 recorded stalk rot incidence of 28-35% in southern states of India. The typical symptoms were observed after pollination with the drying of the lower leaves and eventually entire plant wilted prematurely, lower internodes turned in to grey-green color and stalks are hollow and weak leading to the lodging of the plant. Stalk rot associated pathogen was isolated on PDA medium. Out of 219 Fusarium isolates, 19 were distinct and the fungal colonies on PDA medium showed the development of pale brown to dark brown pigment. Macro conidia were produced in orange sporodochia from monophialides on branched conidiophores with apical cells tapered and elongated. Chlamydospores were solitary and intercalary. All 19 isolates were morphologically identical, and a representative isolate was used for molecular identification. The ITS rDNA and TEF gene were amplified and sequenced using ITS1/ITS4, TEF1/TEF2primer pairs. The nBLAST search and phylogenetic analysis confirmed that the pathogen was Fusarium equiseti. Pathogenicity tests conducted on 50-day-old maize plants by injecting conidial suspension of F. equiseti produced typical stalk rot symptoms after 15 days of post-inoculation and the pathogen's identity was confirmed by cultural and morphological features after re-isolation. Association of F. equiseti as the causal agent of sheath rot of maize was reported from China. The association of F. equiseti with stalk rot of maize is the first report in India

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