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The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae.

Guo M, Chen Y, Du Y, Dong Y, Guo W, Zhai S, Zhang H, Dong S, Zhang Z, Wang Y, Wang P, Zheng X - PLoS Pathog. (2011)

Bottom Line: We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth.Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant.Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China.

ABSTRACT
Saccharomyces cerevisiae Yap1 protein is an AP1-like transcription factor involved in the regulation of the oxidative stress response. An ortholog of Yap1, MoAP1, was recently identified from the rice blast fungus Magnaporthe oryzae genome. We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth. The Moap1 mutant was sensitive to H₂O₂, similar to S. cerevisiae yap1 mutants, and MoAP1 complemented Yap1 function in resistance to H₂O₂, albeit partially. The Moap1 mutant also exhibited various defects in aerial hyphal growth, mycelial branching, conidia formation, the production of extracellular peroxidases and laccases, and melanin pigmentation. Consequently, the Moap1 mutant was unable to infect the host plant. The MoAP1-eGFP fusion protein is localized inside the nucleus upon exposure to H₂O₂, suggesting that MoAP1 also functions as a redox sensor. Moreover, through RNA sequence analysis, many MoAP1-regulated genes were identified, including several novel ones that were also involved in pathogenicity. Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant. Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

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The ROS accumulation is compromised in the Moap1 mutant during infection.(A) Detection of the superoxide by dihydrorhodamine 123 staining. Conidia were inoculated on coverslips and incubated in a moist chamber at 28°C for 0, 2 and 24 hrs before being stained for 2 hrs, rinsed twice with PBS and viewed by epifluorescence microscopy. Fluorescence images were captured using a 100-ms exposure for absorbed light using a GFP filter. Representative bright-field images at each time point are shown. (Scale bars  = 10 µm). (B) Detection of the superoxide by NBT staining. Conidia were prepared as above, stained with a 0.3 mM NBT aqueous solution for 1 hr and viewed by light microscopy. Multiple observations were made and the representative figures were presented (Scale bars  = 10 µm).
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ppat-1001302-g004: The ROS accumulation is compromised in the Moap1 mutant during infection.(A) Detection of the superoxide by dihydrorhodamine 123 staining. Conidia were inoculated on coverslips and incubated in a moist chamber at 28°C for 0, 2 and 24 hrs before being stained for 2 hrs, rinsed twice with PBS and viewed by epifluorescence microscopy. Fluorescence images were captured using a 100-ms exposure for absorbed light using a GFP filter. Representative bright-field images at each time point are shown. (Scale bars  = 10 µm). (B) Detection of the superoxide by NBT staining. Conidia were prepared as above, stained with a 0.3 mM NBT aqueous solution for 1 hr and viewed by light microscopy. Multiple observations were made and the representative figures were presented (Scale bars  = 10 µm).

Mentions: In fission yeast Schizosaccharomyces pombe, the transcription factor Pap1, together with another transcription factor SpAtf1, regulates the genes involved in ROS homeostasis and the response to the extrinsic oxidative stress [44], [45]. Intracellular ROS is known to have multiple functions in fungal pathogenicity [14], [15], [30], [32], [46]. In M. oryzae, intracellular ROS is the key to its virulence in rice seedlings, and the NADPH oxidase mutants lost virulence on the susceptible rice cultivar CO-39 because of their obstructed ROS production [46]. Thus, visualization of ROS accumulation was performed to investigate ROS metabolism during conidiation and germination in Moap1 mutants. We first investigated the production of ROS using dihydrorhodamine 123, which exhibits green fluorescence during reduction by superoxide radicals. Using this technique, it appeared that the Moap1 conidium accumulated higher amounts of superoxide than the wild-type (Figure 4A). Such increased accumulation of superoxide was also detected in the Moap1 mutants during conidia germination. Green fluorescence was typically more intense in the germ tubes and mature appressoria of the Moap1 mutants than the wild-type strain or the complemented Moap1/AP1 strain (Figure 4A). To further confirm enhanced accumulation of ROS in the Moap1 mutants, another kind of reactive oxygen species detection probe, nitroblue tetrazolium (NBT), which forms a dark-blue water-insoluble formazan precipitate on reduction by superoxide radicals, was used. Using this procedure, we found that the Moap1 mutants accumulated higher amounts of superoxide, with more intense formazan precipitates in the germ tubes and mature appressoria (Figure 4B). In contrast, the appressoria and infection hyphae of the wild type strain had less formazan precipitates than the Moap1 mutants (Figure 4B). Thus, both staining results indicated that MoAP1 disruption leads to excess oxidative bursts in conidiation and germination.


The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae.

Guo M, Chen Y, Du Y, Dong Y, Guo W, Zhai S, Zhang H, Dong S, Zhang Z, Wang Y, Wang P, Zheng X - PLoS Pathog. (2011)

The ROS accumulation is compromised in the Moap1 mutant during infection.(A) Detection of the superoxide by dihydrorhodamine 123 staining. Conidia were inoculated on coverslips and incubated in a moist chamber at 28°C for 0, 2 and 24 hrs before being stained for 2 hrs, rinsed twice with PBS and viewed by epifluorescence microscopy. Fluorescence images were captured using a 100-ms exposure for absorbed light using a GFP filter. Representative bright-field images at each time point are shown. (Scale bars  = 10 µm). (B) Detection of the superoxide by NBT staining. Conidia were prepared as above, stained with a 0.3 mM NBT aqueous solution for 1 hr and viewed by light microscopy. Multiple observations were made and the representative figures were presented (Scale bars  = 10 µm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044703&req=5

ppat-1001302-g004: The ROS accumulation is compromised in the Moap1 mutant during infection.(A) Detection of the superoxide by dihydrorhodamine 123 staining. Conidia were inoculated on coverslips and incubated in a moist chamber at 28°C for 0, 2 and 24 hrs before being stained for 2 hrs, rinsed twice with PBS and viewed by epifluorescence microscopy. Fluorescence images were captured using a 100-ms exposure for absorbed light using a GFP filter. Representative bright-field images at each time point are shown. (Scale bars  = 10 µm). (B) Detection of the superoxide by NBT staining. Conidia were prepared as above, stained with a 0.3 mM NBT aqueous solution for 1 hr and viewed by light microscopy. Multiple observations were made and the representative figures were presented (Scale bars  = 10 µm).
Mentions: In fission yeast Schizosaccharomyces pombe, the transcription factor Pap1, together with another transcription factor SpAtf1, regulates the genes involved in ROS homeostasis and the response to the extrinsic oxidative stress [44], [45]. Intracellular ROS is known to have multiple functions in fungal pathogenicity [14], [15], [30], [32], [46]. In M. oryzae, intracellular ROS is the key to its virulence in rice seedlings, and the NADPH oxidase mutants lost virulence on the susceptible rice cultivar CO-39 because of their obstructed ROS production [46]. Thus, visualization of ROS accumulation was performed to investigate ROS metabolism during conidiation and germination in Moap1 mutants. We first investigated the production of ROS using dihydrorhodamine 123, which exhibits green fluorescence during reduction by superoxide radicals. Using this technique, it appeared that the Moap1 conidium accumulated higher amounts of superoxide than the wild-type (Figure 4A). Such increased accumulation of superoxide was also detected in the Moap1 mutants during conidia germination. Green fluorescence was typically more intense in the germ tubes and mature appressoria of the Moap1 mutants than the wild-type strain or the complemented Moap1/AP1 strain (Figure 4A). To further confirm enhanced accumulation of ROS in the Moap1 mutants, another kind of reactive oxygen species detection probe, nitroblue tetrazolium (NBT), which forms a dark-blue water-insoluble formazan precipitate on reduction by superoxide radicals, was used. Using this procedure, we found that the Moap1 mutants accumulated higher amounts of superoxide, with more intense formazan precipitates in the germ tubes and mature appressoria (Figure 4B). In contrast, the appressoria and infection hyphae of the wild type strain had less formazan precipitates than the Moap1 mutants (Figure 4B). Thus, both staining results indicated that MoAP1 disruption leads to excess oxidative bursts in conidiation and germination.

Bottom Line: We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth.Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant.Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China.

ABSTRACT
Saccharomyces cerevisiae Yap1 protein is an AP1-like transcription factor involved in the regulation of the oxidative stress response. An ortholog of Yap1, MoAP1, was recently identified from the rice blast fungus Magnaporthe oryzae genome. We found that MoAP1 is highly expressed in conidia and during invasive hyphal growth. The Moap1 mutant was sensitive to H₂O₂, similar to S. cerevisiae yap1 mutants, and MoAP1 complemented Yap1 function in resistance to H₂O₂, albeit partially. The Moap1 mutant also exhibited various defects in aerial hyphal growth, mycelial branching, conidia formation, the production of extracellular peroxidases and laccases, and melanin pigmentation. Consequently, the Moap1 mutant was unable to infect the host plant. The MoAP1-eGFP fusion protein is localized inside the nucleus upon exposure to H₂O₂, suggesting that MoAP1 also functions as a redox sensor. Moreover, through RNA sequence analysis, many MoAP1-regulated genes were identified, including several novel ones that were also involved in pathogenicity. Disruption of respective MGG_01662 (MoAAT) and MGG_02531 (encoding hypothetical protein) genes did not result in any detectable changes in conidial germination and appressorium formation but reduced pathogenicity, whereas the mutant strains of MGG_01230 (MoSSADH) and MGG_15157 (MoACT) showed marketed reductions in aerial hyphal growth, mycelial branching, and loss of conidiation as well as pathogenicity, similar to the Moap1 mutant. Taken together, our studies identify MoAP1 as a positive transcription factor that regulates transcriptions of MGG_01662, MGG_02531, MGG_01230, and MGG_15157 that are important in the growth, development, and pathogenicity of M. oryzae.

Show MeSH
Related in: MedlinePlus