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Identification and analysis of in planta expressed genes of Magnaporthe oryzae.

ABSTRACT: BACKGROUND: Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction. RESULTS: A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence. CONCLUSION: This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.

SUBMITTER: Kim S

PROVIDER: S-EPMC2832786 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Identification and analysis of in planta expressed genes of Magnaporthe oryzae.

Kim Soonok S Park Jongsun J Park Sook-Young SY Mitchell Thomas K TK Lee Yong-Hwan YH

BMC genomics 20100210

<h4>Background</h4>Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryz ...[more]

PMID: 20146797

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Project description:BACKGROUND: Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc). RESULTS: Changes in gene expression of the African Xoo strain MAI1 in the susceptible rice cultivar Nipponbare were profiled, using an SSH Xoo DNA microarray. Microarray hybridization was performed comparing bacteria recovered from plant tissues at 1, 3, and 6 days after inoculation (dai) with bacteria grown in vitro. A total of 710 bacterial genes were found to be differentially expressed, with 407 up-regulated and 303 down-regulated. Expression profiling indicated that less than 20% of the 710 bacterial transcripts were induced in the first 24 h after inoculation, whereas 63% were differentially expressed at 6 dai. The 710 differentially expressed genes were one-end sequenced. 535 sequences were obtained from which 147 non-redundant sequences were identified. Differentially expressed genes were related to metabolism, secretion and transport, pathogen adherence to plant tissues, plant cell-wall degradation, IS elements, and virulence. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. The Xoo MAI1 non-redundant set of sequences was compared against several X. oryzae genomes, revealing a specific group of genes that was present only in MAI1. Numerous IS elements were also found to be differentially expressed. Quantitative real-time PCR confirmed 86% of the identified profile on a set of 14 genes selected according to the microarray analysis. CONCLUSIONS: This is the first report to compare the expression of Xoo genes in planta across different time points during infection. This work shows that as-yet-unidentified and potentially new virulence factors are appearing in an emerging African pathogen. It also confirms that African Xoo strains do differ from their Asian counterparts, even at the transcriptional level.

Project description:Manganese is a crucial trace element that constitutes the cofactors of many enzymes. However, excessive Mn2+ can be toxic for both prokaryotes and eukaryotes. The mechanism of fungal genetics and metabolism in response to Mn2+ stress remains understudied, warranting further studies. Magnaporthe oryzae is well-established as the most destructive pathogen of rice. A field strain, YN2046, more sensitive to Mn2+ toxicity than other strains, was obtained from a previous study. Herein, we explored the genetic mechanisms of Mn2+ sensitivity in YN2046 through comparative transcriptomic analyses. We found that many genes previously reported to participate in Mn2+ stress were not regulated in YN2046. These non-responsive genes might cause Mn2+ sensitivity in YN2046. Weight gene correlation network analysis (WGCNA) was performed to characterize the expression profile in YN2046. Some overexpressed genes were only found in the Mn2+ tolerant isolate YN125. Among these, many single nucleotide polymorphism (SNP) were identified between YN125 and YN2046, which might disrupt the expression levels of Mn responsive genes. We cloned two uncharacterized genes, MGG_13347 and MGG_16609, from YN125 and transformed them to YN2046 with a strong promoter. Our results showed that the heterologous overexpression of two genes in YN2046 restored its sensitivity. Transcriptomic and biochemical analyses were performed to understand Mn tolerance mechanisms mediated by the two heterologous overexpressed genes. Our results showed that heterologous overexpression of these two genes activated downstream gene expression and metabolite production to restore M. oryzae sensitivity to Mn, implying that SNPs in responsive genes account for different phenotypes of the two strains under Mn stress. IMPORTANCE Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. The comparative transcriptome analysis showed that fewer genes were regulated in the Mn-sensitive strain. WGCNA and SNP analyses found that mutations in promoter and coding sequence regions might disrupt the expression of genes involved in Mn detoxification in the sensitive strain. We transferred two unannotated genes that were cloned from the Mn-tolerant strain into a sensitive strain with strong promoters, and the transformants exhibited an enhanced tolerance to Mn2+ toxicity. Transcriptome and biochemistry results indicated that heterologous overexpression of the two genes enhanced the tolerance to Mn toxicity by reactivation of downstream genes in M. oryzae.

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Identification and analysis of in planta expressed genes of Magnaporthe oryzae.

Publications

Identification and analysis of in planta expressed genes of Magnaporthe oryzae.

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