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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 is required for pathogenicity.(A) Pathogenicity test on rice (Oryza sativa cv. CO39). Rice leaves, unwounded (a) or wounded by abrasion (b), were inoculated with the wild-type strain, ΔMovam7 mutants, and complemented mutant (ΔMovam7R) strains, with water as a control. Mycelia cultured in CM for 2 days were harvested by centrifugation (10 min at 5000×g), washed twice with distilled water, and fragmented into 30-50 µm lengths by homogenization. The fragmented mycelia suspension was adjusted to 5×104 pieces/ml and inoculated onto intact or abraded leaves of susceptible rice. The experiments were repeated three times each with similar results. (B) Appressorium formation at the hyphal tip was blocked in ΔMovam7 mutants. The fragmented mycelia suspension was incubated on the surface of hydrophobic Gelbond film as described in Materials and Methods. The appressoria formation rates were obtained at 12 and 24 hours post incubation. (C) and (D) Penetration assays on onion and rice sheath epidermal cells. Fragmented mycelia suspensions (5×104 pieces/ml) of the wild-type and ΔMovam7 mutant strains were inoculated on strips of onion epidermis and rice sheath, appressoria onion (D) and infectious hyphae on rice sheath (E), all pointed by arrows, were photographed 1 day after inoculation.
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pone-0016439-g008: MoVam7 is required for pathogenicity.(A) Pathogenicity test on rice (Oryza sativa cv. CO39). Rice leaves, unwounded (a) or wounded by abrasion (b), were inoculated with the wild-type strain, ΔMovam7 mutants, and complemented mutant (ΔMovam7R) strains, with water as a control. Mycelia cultured in CM for 2 days were harvested by centrifugation (10 min at 5000×g), washed twice with distilled water, and fragmented into 30-50 µm lengths by homogenization. The fragmented mycelia suspension was adjusted to 5×104 pieces/ml and inoculated onto intact or abraded leaves of susceptible rice. The experiments were repeated three times each with similar results. (B) Appressorium formation at the hyphal tip was blocked in ΔMovam7 mutants. The fragmented mycelia suspension was incubated on the surface of hydrophobic Gelbond film as described in Materials and Methods. The appressoria formation rates were obtained at 12 and 24 hours post incubation. (C) and (D) Penetration assays on onion and rice sheath epidermal cells. Fragmented mycelia suspensions (5×104 pieces/ml) of the wild-type and ΔMovam7 mutant strains were inoculated on strips of onion epidermis and rice sheath, appressoria onion (D) and infectious hyphae on rice sheath (E), all pointed by arrows, were photographed 1 day after inoculation.

Mentions: Since we were not able to collect enough spores from the ΔMovam7 mutant for traditional assessment of pathogenicity, we collected mycelia and fragmented them before inoculation on the leaves of susceptible rice cultivar CO-39. The ΔMovam7 mutant caused no symptoms after 3–5 days, in contrast to the wild-type strain. The same result was obtained when the fragmented mycelia were sprayed on to abraded leaves, demonstrating that MoVam7 is essential for pathogenicity (Figure 8A).


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 is required for pathogenicity.(A) Pathogenicity test on rice (Oryza sativa cv. CO39). Rice leaves, unwounded (a) or wounded by abrasion (b), were inoculated with the wild-type strain, ΔMovam7 mutants, and complemented mutant (ΔMovam7R) strains, with water as a control. Mycelia cultured in CM for 2 days were harvested by centrifugation (10 min at 5000×g), washed twice with distilled water, and fragmented into 30-50 µm lengths by homogenization. The fragmented mycelia suspension was adjusted to 5×104 pieces/ml and inoculated onto intact or abraded leaves of susceptible rice. The experiments were repeated three times each with similar results. (B) Appressorium formation at the hyphal tip was blocked in ΔMovam7 mutants. The fragmented mycelia suspension was incubated on the surface of hydrophobic Gelbond film as described in Materials and Methods. The appressoria formation rates were obtained at 12 and 24 hours post incubation. (C) and (D) Penetration assays on onion and rice sheath epidermal cells. Fragmented mycelia suspensions (5×104 pieces/ml) of the wild-type and ΔMovam7 mutant strains were inoculated on strips of onion epidermis and rice sheath, appressoria onion (D) and infectious hyphae on rice sheath (E), all pointed by arrows, were photographed 1 day after inoculation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016439-g008: MoVam7 is required for pathogenicity.(A) Pathogenicity test on rice (Oryza sativa cv. CO39). Rice leaves, unwounded (a) or wounded by abrasion (b), were inoculated with the wild-type strain, ΔMovam7 mutants, and complemented mutant (ΔMovam7R) strains, with water as a control. Mycelia cultured in CM for 2 days were harvested by centrifugation (10 min at 5000×g), washed twice with distilled water, and fragmented into 30-50 µm lengths by homogenization. The fragmented mycelia suspension was adjusted to 5×104 pieces/ml and inoculated onto intact or abraded leaves of susceptible rice. The experiments were repeated three times each with similar results. (B) Appressorium formation at the hyphal tip was blocked in ΔMovam7 mutants. The fragmented mycelia suspension was incubated on the surface of hydrophobic Gelbond film as described in Materials and Methods. The appressoria formation rates were obtained at 12 and 24 hours post incubation. (C) and (D) Penetration assays on onion and rice sheath epidermal cells. Fragmented mycelia suspensions (5×104 pieces/ml) of the wild-type and ΔMovam7 mutant strains were inoculated on strips of onion epidermis and rice sheath, appressoria onion (D) and infectious hyphae on rice sheath (E), all pointed by arrows, were photographed 1 day after inoculation.
Mentions: Since we were not able to collect enough spores from the ΔMovam7 mutant for traditional assessment of pathogenicity, we collected mycelia and fragmented them before inoculation on the leaves of susceptible rice cultivar CO-39. The ΔMovam7 mutant caused no symptoms after 3–5 days, in contrast to the wild-type strain. The same result was obtained when the fragmented mycelia were sprayed on to abraded leaves, demonstrating that MoVam7 is essential for pathogenicity (Figure 8A).

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