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A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host.

Chi MH, Park SY, Kim S, Lee YH - PLoS Pathog. (2009)

Bottom Line: For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant.Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration.These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University, Seoul, Korea.

ABSTRACT
For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant. We demonstrate that a novel pathogenicity gene, DES1, in Magnaporthe oryzae regulates counter-defenses against host basal resistance. The DES1 gene was identified by screening for pathogenicity-defective mutants in a T-DNA insertional mutant library. Bioinformatic analysis revealed that this gene encodes a serine-rich protein that has unknown biochemical properties, and its homologs are strictly conserved in filamentous Ascomycetes. Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration. Conidial size of the mutant became smaller than that of the wild type, but the mutant displayed no defects on cell wall integrity. The Deltades1 mutant was hypersensitive to exogenous oxidative stress and the activity and transcription level of extracellular enzymes including peroxidases and laccases were severely decreased in the mutant. In addition, ferrous ion leakage was observed in the Deltades1 mutant. In the interaction with a susceptible rice cultivar, rice cells inoculated with the Deltades1 mutant exhibited strong defense responses accompanied by brown granules in primary infected cells, the accumulation of reactive oxygen species (ROS), the generation of autofluorescent materials, and PR gene induction in neighboring tissues. The Deltades1 mutant displayed a significant reduction in infectious hyphal extension, which caused a decrease in pathogenicity. Notably, the suppression of ROS generation by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, resulted in a significant reduction in the defense responses in plant tissues challenged with the Deltades1 mutant. Furthermore, the Deltades1 mutant recovered its normal infectious growth in DPI-treated plant tissues. These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense.

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Expression Profiles of M. oryzae Peroxidases in the Δdes1 Mutant.A combination of the phylogenetic tree, expression characteristics, and domain architecture of 16 putative peroxidases in the M. oryzae genome were displayed. The phylogenetic tree was generated by ClustalW sequence alignment with 1000 bootstrappings and divided into three clades. The transcript levels of the the putative peroxidase encoding genes in the oxidative condition and/or in the Δdes1 mutant are indicated. Relative abundance of transcript compared with standard condition (wild type, normal condition) is displayed as a white triangle (up-regulated) or an inverted black triangle (down-regulated). Triangles indicating more than 2.0 (fold change) are displayed as trapezoids by cutting the top of the triangle. Fold changes of the standard condition (1.0) are not shown. Up-regulated genes in the Δdes1 mutant (more than 1.5 fold) were indicated in blue, and down-regulated genes in the Δdes1 mutant (less than 0.6 fold) were indicated in red. The InterPro terms and signal peptides are indicated (see legend).
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ppat-1000401-g008: Expression Profiles of M. oryzae Peroxidases in the Δdes1 Mutant.A combination of the phylogenetic tree, expression characteristics, and domain architecture of 16 putative peroxidases in the M. oryzae genome were displayed. The phylogenetic tree was generated by ClustalW sequence alignment with 1000 bootstrappings and divided into three clades. The transcript levels of the the putative peroxidase encoding genes in the oxidative condition and/or in the Δdes1 mutant are indicated. Relative abundance of transcript compared with standard condition (wild type, normal condition) is displayed as a white triangle (up-regulated) or an inverted black triangle (down-regulated). Triangles indicating more than 2.0 (fold change) are displayed as trapezoids by cutting the top of the triangle. Fold changes of the standard condition (1.0) are not shown. Up-regulated genes in the Δdes1 mutant (more than 1.5 fold) were indicated in blue, and down-regulated genes in the Δdes1 mutant (less than 0.6 fold) were indicated in red. The InterPro terms and signal peptides are indicated (see legend).

Mentions: We examined the transcriptional regulation of genes encoding peroxidases. Putative peroxidase-encoding genes were identified from the annotated M. oryzae genome database. There were 19 such genes that had peroxidase-related InterPro domains, including IPR010255 (haem peroxidase), IPR000889 (glutathione peroxidase), and IPR000028 (chloroperoxidase). Three of these 19 genes were excluded because their transcripts were not detected under the given experimental conditions. Sixteen putative peroxidase genes in M. oryzae could be classified into three clades by phylogenetic analysis, and most of them possessed a signal peptide when assessed using the SignalP program (Fig. 8). Differences in the transcriptional expression of the peroxidase genes between the wild type and the Δdes1 mutant under oxidative (1 mM H2O2) or normal (no H2O2) conditions were examined using quantitative RT-PCR, and fold changes were calculated using wild type under normal condition as a standard condition. The expression level of some peroxidase genes in clade 1 (plant ascorbate peroxidases: MGG04545, MGG10368, MGG08200, and MGG09398; fungal lignin peroxidase: MGG07790) was up-regulated under the oxidative condition in the wild type (Fig. 8). The transcription of MGG07790, MGG08200, and MGG09398 was down-regulated in the Δdes1 mutant, and the reduced transcription was not recovered by treatment with H2O2. The expression of MGG10368 and MGG04545 was also repressed in the Δdes1 mutant, but the reduced transcription was partially recovered by treatment of H2O2 (Fig. 8). The expression level of the other peroxidase genes in clade 1 (catalase peroxidases: MGG04337 and MGG09834; haem peroxidases: MGGG00461 and MGG10877) was not significantly altered by the deletion of the DES1 gene. Peroxidase genes in clade 2 (chloroperoxidases: MGG07574, MGG11849, MGG07871, and MGG07574) were responsive to H2O2 treatment, but their expression was not down-regulated in the Δdes1 mutant. Peroxidase genes in clade 3 (cytochrome P450 peroxidases: MGG10859 and MGG13239; glutathione peroxidase: MGG07460) were not responsive to H2O2 under the experimental conditions, but their expression was down-regulated in Δdes1 (Fig. 8). The expression of putative laccase-encoding genes was also examined in the Δdes1 mutant. Seventeen genes having two or three multicopper oxidase domains (IPR001117, IPR011706, or IPR011707) were identified from the M. oryzae genome database. We excluded one of them (MGG09102) from the analysis because the transcript was not detected in the given experimental conditions. Expression levels of laccase genes in the oxidative condition (1 mM H2O2) were rather reduced or similar in wild type. Transcription of all laccase genes except one (MGG07500) was also down-regulated in the Δdes1 mutant, even differences in the transcription level of the laccase genes between wild type and the Δdes1 mutant were more severe than those of the peroxidase genes (Fig. S8).


A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host.

Chi MH, Park SY, Kim S, Lee YH - PLoS Pathog. (2009)

Expression Profiles of M. oryzae Peroxidases in the Δdes1 Mutant.A combination of the phylogenetic tree, expression characteristics, and domain architecture of 16 putative peroxidases in the M. oryzae genome were displayed. The phylogenetic tree was generated by ClustalW sequence alignment with 1000 bootstrappings and divided into three clades. The transcript levels of the the putative peroxidase encoding genes in the oxidative condition and/or in the Δdes1 mutant are indicated. Relative abundance of transcript compared with standard condition (wild type, normal condition) is displayed as a white triangle (up-regulated) or an inverted black triangle (down-regulated). Triangles indicating more than 2.0 (fold change) are displayed as trapezoids by cutting the top of the triangle. Fold changes of the standard condition (1.0) are not shown. Up-regulated genes in the Δdes1 mutant (more than 1.5 fold) were indicated in blue, and down-regulated genes in the Δdes1 mutant (less than 0.6 fold) were indicated in red. The InterPro terms and signal peptides are indicated (see legend).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2668191&req=5

ppat-1000401-g008: Expression Profiles of M. oryzae Peroxidases in the Δdes1 Mutant.A combination of the phylogenetic tree, expression characteristics, and domain architecture of 16 putative peroxidases in the M. oryzae genome were displayed. The phylogenetic tree was generated by ClustalW sequence alignment with 1000 bootstrappings and divided into three clades. The transcript levels of the the putative peroxidase encoding genes in the oxidative condition and/or in the Δdes1 mutant are indicated. Relative abundance of transcript compared with standard condition (wild type, normal condition) is displayed as a white triangle (up-regulated) or an inverted black triangle (down-regulated). Triangles indicating more than 2.0 (fold change) are displayed as trapezoids by cutting the top of the triangle. Fold changes of the standard condition (1.0) are not shown. Up-regulated genes in the Δdes1 mutant (more than 1.5 fold) were indicated in blue, and down-regulated genes in the Δdes1 mutant (less than 0.6 fold) were indicated in red. The InterPro terms and signal peptides are indicated (see legend).
Mentions: We examined the transcriptional regulation of genes encoding peroxidases. Putative peroxidase-encoding genes were identified from the annotated M. oryzae genome database. There were 19 such genes that had peroxidase-related InterPro domains, including IPR010255 (haem peroxidase), IPR000889 (glutathione peroxidase), and IPR000028 (chloroperoxidase). Three of these 19 genes were excluded because their transcripts were not detected under the given experimental conditions. Sixteen putative peroxidase genes in M. oryzae could be classified into three clades by phylogenetic analysis, and most of them possessed a signal peptide when assessed using the SignalP program (Fig. 8). Differences in the transcriptional expression of the peroxidase genes between the wild type and the Δdes1 mutant under oxidative (1 mM H2O2) or normal (no H2O2) conditions were examined using quantitative RT-PCR, and fold changes were calculated using wild type under normal condition as a standard condition. The expression level of some peroxidase genes in clade 1 (plant ascorbate peroxidases: MGG04545, MGG10368, MGG08200, and MGG09398; fungal lignin peroxidase: MGG07790) was up-regulated under the oxidative condition in the wild type (Fig. 8). The transcription of MGG07790, MGG08200, and MGG09398 was down-regulated in the Δdes1 mutant, and the reduced transcription was not recovered by treatment with H2O2. The expression of MGG10368 and MGG04545 was also repressed in the Δdes1 mutant, but the reduced transcription was partially recovered by treatment of H2O2 (Fig. 8). The expression level of the other peroxidase genes in clade 1 (catalase peroxidases: MGG04337 and MGG09834; haem peroxidases: MGGG00461 and MGG10877) was not significantly altered by the deletion of the DES1 gene. Peroxidase genes in clade 2 (chloroperoxidases: MGG07574, MGG11849, MGG07871, and MGG07574) were responsive to H2O2 treatment, but their expression was not down-regulated in the Δdes1 mutant. Peroxidase genes in clade 3 (cytochrome P450 peroxidases: MGG10859 and MGG13239; glutathione peroxidase: MGG07460) were not responsive to H2O2 under the experimental conditions, but their expression was down-regulated in Δdes1 (Fig. 8). The expression of putative laccase-encoding genes was also examined in the Δdes1 mutant. Seventeen genes having two or three multicopper oxidase domains (IPR001117, IPR011706, or IPR011707) were identified from the M. oryzae genome database. We excluded one of them (MGG09102) from the analysis because the transcript was not detected in the given experimental conditions. Expression levels of laccase genes in the oxidative condition (1 mM H2O2) were rather reduced or similar in wild type. Transcription of all laccase genes except one (MGG07500) was also down-regulated in the Δdes1 mutant, even differences in the transcription level of the laccase genes between wild type and the Δdes1 mutant were more severe than those of the peroxidase genes (Fig. S8).

Bottom Line: For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant.Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration.These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Fungal Pathogenesis, Seoul National University, Seoul, Korea.

ABSTRACT
For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant. We demonstrate that a novel pathogenicity gene, DES1, in Magnaporthe oryzae regulates counter-defenses against host basal resistance. The DES1 gene was identified by screening for pathogenicity-defective mutants in a T-DNA insertional mutant library. Bioinformatic analysis revealed that this gene encodes a serine-rich protein that has unknown biochemical properties, and its homologs are strictly conserved in filamentous Ascomycetes. Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration. Conidial size of the mutant became smaller than that of the wild type, but the mutant displayed no defects on cell wall integrity. The Deltades1 mutant was hypersensitive to exogenous oxidative stress and the activity and transcription level of extracellular enzymes including peroxidases and laccases were severely decreased in the mutant. In addition, ferrous ion leakage was observed in the Deltades1 mutant. In the interaction with a susceptible rice cultivar, rice cells inoculated with the Deltades1 mutant exhibited strong defense responses accompanied by brown granules in primary infected cells, the accumulation of reactive oxygen species (ROS), the generation of autofluorescent materials, and PR gene induction in neighboring tissues. The Deltades1 mutant displayed a significant reduction in infectious hyphal extension, which caused a decrease in pathogenicity. Notably, the suppression of ROS generation by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, resulted in a significant reduction in the defense responses in plant tissues challenged with the Deltades1 mutant. Furthermore, the Deltades1 mutant recovered its normal infectious growth in DPI-treated plant tissues. These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense.

Show MeSH
Related in: MedlinePlus