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MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae.

Dou X, Wang Q, Qi Z, Song W, Wang W, Guo M, Zhang H, Zhang Z, Wang P, Zheng X - PLoS ONE (2011)

Bottom Line: The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium.Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant.Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

View Article: PubMed Central - PubMed

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

ABSTRACT
Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

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MoVam7 has a role in vacuole morphogenesis and the formation of the Spitzenkörper and endocytosis.(A) Mycelia were stained with neutral red. At 14 hours, numerous small vacuoles were seen in the ΔMovam7 mutants (right panel), whereas large vacuoles of fewer numbers were present in the wild type strain. (B) Observation by transmission electron microscopy revealed numerous small, fragmented vacuoles in contrast to the large ones in the wild type hyphae. Bars represent 500 nm in the wild type strain or 800 nm in the ΔMovam7 mutant. (C) The wild type strain shows the presence of an intact Spitzenkörper (arrowheads) at the tips of the hyphae, which was missing in the ΔMovam7 mutants after exposure to FM4-64 staining for 10 min. Strains were grown for 2 days on CM-overlaid microscope slides before staining. (D) FM4-64 staining also revealed that the ΔMovam7 mutant was defective in endocytosis. Strains were grown for 2 days on the CM-overlaid microscope slides before adding FM4-64 and photographs were taken after 10 and 30 min exposure to FM4-64. Camera exposure is indicated in second.
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pone-0016439-g002: MoVam7 has a role in vacuole morphogenesis and the formation of the Spitzenkörper and endocytosis.(A) Mycelia were stained with neutral red. At 14 hours, numerous small vacuoles were seen in the ΔMovam7 mutants (right panel), whereas large vacuoles of fewer numbers were present in the wild type strain. (B) Observation by transmission electron microscopy revealed numerous small, fragmented vacuoles in contrast to the large ones in the wild type hyphae. Bars represent 500 nm in the wild type strain or 800 nm in the ΔMovam7 mutant. (C) The wild type strain shows the presence of an intact Spitzenkörper (arrowheads) at the tips of the hyphae, which was missing in the ΔMovam7 mutants after exposure to FM4-64 staining for 10 min. Strains were grown for 2 days on CM-overlaid microscope slides before staining. (D) FM4-64 staining also revealed that the ΔMovam7 mutant was defective in endocytosis. Strains were grown for 2 days on the CM-overlaid microscope slides before adding FM4-64 and photographs were taken after 10 and 30 min exposure to FM4-64. Camera exposure is indicated in second.

Mentions: As MoVam7 was identified based on its homology to S. cerevisiae Vam7, which is a SNARE protein involved in vacuole assembly and membrane fusion, we performed staining with vital dyes such as neutral red and FM4-64. In normal cells, neutral red is quickly internalized allowing staining of vesicles such as vacuoles. The ΔMovam7 mutants displayed vacuole formation by neutral red staining and transmission electron microscopy (Figure 2A and 2B). However, the vacuoles were smaller and more numerous (Figure 2A and 2B), which was similar to the observed phenotypes of the Δvam7 mutant of S. cerevisiae [34], [35], indicating a role for MoVam7 in vacuolar formation/assembly.


MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae.

Dou X, Wang Q, Qi Z, Song W, Wang W, Guo M, Zhang H, Zhang Z, Wang P, Zheng X - PLoS ONE (2011)

MoVam7 has a role in vacuole morphogenesis and the formation of the Spitzenkörper and endocytosis.(A) Mycelia were stained with neutral red. At 14 hours, numerous small vacuoles were seen in the ΔMovam7 mutants (right panel), whereas large vacuoles of fewer numbers were present in the wild type strain. (B) Observation by transmission electron microscopy revealed numerous small, fragmented vacuoles in contrast to the large ones in the wild type hyphae. Bars represent 500 nm in the wild type strain or 800 nm in the ΔMovam7 mutant. (C) The wild type strain shows the presence of an intact Spitzenkörper (arrowheads) at the tips of the hyphae, which was missing in the ΔMovam7 mutants after exposure to FM4-64 staining for 10 min. Strains were grown for 2 days on CM-overlaid microscope slides before staining. (D) FM4-64 staining also revealed that the ΔMovam7 mutant was defective in endocytosis. Strains were grown for 2 days on the CM-overlaid microscope slides before adding FM4-64 and photographs were taken after 10 and 30 min exposure to FM4-64. Camera exposure is indicated in second.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025985&req=5

pone-0016439-g002: MoVam7 has a role in vacuole morphogenesis and the formation of the Spitzenkörper and endocytosis.(A) Mycelia were stained with neutral red. At 14 hours, numerous small vacuoles were seen in the ΔMovam7 mutants (right panel), whereas large vacuoles of fewer numbers were present in the wild type strain. (B) Observation by transmission electron microscopy revealed numerous small, fragmented vacuoles in contrast to the large ones in the wild type hyphae. Bars represent 500 nm in the wild type strain or 800 nm in the ΔMovam7 mutant. (C) The wild type strain shows the presence of an intact Spitzenkörper (arrowheads) at the tips of the hyphae, which was missing in the ΔMovam7 mutants after exposure to FM4-64 staining for 10 min. Strains were grown for 2 days on CM-overlaid microscope slides before staining. (D) FM4-64 staining also revealed that the ΔMovam7 mutant was defective in endocytosis. Strains were grown for 2 days on the CM-overlaid microscope slides before adding FM4-64 and photographs were taken after 10 and 30 min exposure to FM4-64. Camera exposure is indicated in second.
Mentions: As MoVam7 was identified based on its homology to S. cerevisiae Vam7, which is a SNARE protein involved in vacuole assembly and membrane fusion, we performed staining with vital dyes such as neutral red and FM4-64. In normal cells, neutral red is quickly internalized allowing staining of vesicles such as vacuoles. The ΔMovam7 mutants displayed vacuole formation by neutral red staining and transmission electron microscopy (Figure 2A and 2B). However, the vacuoles were smaller and more numerous (Figure 2A and 2B), which was similar to the observed phenotypes of the Δvam7 mutant of S. cerevisiae [34], [35], indicating a role for MoVam7 in vacuolar formation/assembly.

Bottom Line: The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium.Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant.Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

View Article: PubMed Central - PubMed

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

ABSTRACT
Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

Show MeSH
Related in: MedlinePlus