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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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Deletion of Movam7 resulted in altered distribution of chitins.(A) The ΔMovam7 mutants (#1 and #10), complemented mutant (ΔMovam7R), and wild type strains were incubated on CM plates supplemented with various stress inducers at 28°C for 6 days. Growth of the ΔMovam7 mutant in media supplemented with SDS (0.01%; vs wild type P<0.01), lysing enzymes (2 mg/ml), and Congo Red (20 µg/ml). (B) Deletion of Movam7 altered the distribution of chitin within the cell. In the wild-type strain Guy11, CFW fluorescence was mainly distributed at the hyphal and septal apices, whereas in the ΔMovam7 mutant, fluorescence was not restricted to growing apices and found also on the lateral walls along the hyphal axe. This abnormal distribution of cell wall components was restored by the introduction of the wild-type MoVAM7 gene. (C) Reduced expression was found in six out of seven genes that encode chitin synthases in the ΔMovam7 mutants of M. oryzae. RNA was extracted from mycelia that were grown for 3 days in liquid CM. Error bars represent the standard deviation and asterisks represent significant different among stains tested. All of the reductions are significant (P = 0.01 or P = 0.05) according to Duncan's multiple range test.
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pone-0016439-g005: Deletion of Movam7 resulted in altered distribution of chitins.(A) The ΔMovam7 mutants (#1 and #10), complemented mutant (ΔMovam7R), and wild type strains were incubated on CM plates supplemented with various stress inducers at 28°C for 6 days. Growth of the ΔMovam7 mutant in media supplemented with SDS (0.01%; vs wild type P<0.01), lysing enzymes (2 mg/ml), and Congo Red (20 µg/ml). (B) Deletion of Movam7 altered the distribution of chitin within the cell. In the wild-type strain Guy11, CFW fluorescence was mainly distributed at the hyphal and septal apices, whereas in the ΔMovam7 mutant, fluorescence was not restricted to growing apices and found also on the lateral walls along the hyphal axe. This abnormal distribution of cell wall components was restored by the introduction of the wild-type MoVAM7 gene. (C) Reduced expression was found in six out of seven genes that encode chitin synthases in the ΔMovam7 mutants of M. oryzae. RNA was extracted from mycelia that were grown for 3 days in liquid CM. Error bars represent the standard deviation and asterisks represent significant different among stains tested. All of the reductions are significant (P = 0.01 or P = 0.05) according to Duncan's multiple range test.

Mentions: To examine the role of MoVam7 in cell wall integrity, we investigated the effects of various cell-wall perturbing agents on the ΔMovam7 mutant. The sensitivity of the ΔMovam7 mutant to lytic enzymes (LE), and Congo Red (CR) is not significantly different from that of the wild–type strain and the complemented mutant strains, but it was more sensitive to SDS (Figure 5A). No significant difference in the rate of protoplast release was found at 30 min and 90 min (Figure S3). CFW staining was also used to probe the distribution of chitin on the cell wall. In the wild type strain, CFW fluorescence was mostly distributed at the septa and tips where chitin, one of main components of the fungal cell wall, was actively synthesized, while in the mutants, patches of bright fluorescence were observed on the lateral wall of hyphae in addition to septa and hyphal tips (Figure 5B). Since CFW is a fluorescent dye that intercalates with nascent chitin chains, this result might suggest the altered distribution of chitin on cell wall in the ΔMovam7 mutant was due to the aberrant cell wall synthesis activity. Chitin contents analysis showed the chitin contents were reduced by 10% in the ΔMovam7 mutant compared to the wild-type strain (Figure S4). We also examined the expression of several genes encoding the putative chitin synthases in M. oryzae by quantitative RT-PCR assay. Interestingly, the result showed that with the exception of MGG_06064, the expression for six out of seven genes was reduced in the ΔMovam7 mutant in comparison to the wild strain (Figure 5C). These findings indicated that the aberrant chitin distribution, combining with reduced chitin production due to reduced gene expression, contributed together to weakened altered cell wall integrity in ΔMovam7 mutants.


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)

Deletion of Movam7 resulted in altered distribution of chitins.(A) The ΔMovam7 mutants (#1 and #10), complemented mutant (ΔMovam7R), and wild type strains were incubated on CM plates supplemented with various stress inducers at 28°C for 6 days. Growth of the ΔMovam7 mutant in media supplemented with SDS (0.01%; vs wild type P<0.01), lysing enzymes (2 mg/ml), and Congo Red (20 µg/ml). (B) Deletion of Movam7 altered the distribution of chitin within the cell. In the wild-type strain Guy11, CFW fluorescence was mainly distributed at the hyphal and septal apices, whereas in the ΔMovam7 mutant, fluorescence was not restricted to growing apices and found also on the lateral walls along the hyphal axe. This abnormal distribution of cell wall components was restored by the introduction of the wild-type MoVAM7 gene. (C) Reduced expression was found in six out of seven genes that encode chitin synthases in the ΔMovam7 mutants of M. oryzae. RNA was extracted from mycelia that were grown for 3 days in liquid CM. Error bars represent the standard deviation and asterisks represent significant different among stains tested. All of the reductions are significant (P = 0.01 or P = 0.05) according to Duncan's multiple range test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016439-g005: Deletion of Movam7 resulted in altered distribution of chitins.(A) The ΔMovam7 mutants (#1 and #10), complemented mutant (ΔMovam7R), and wild type strains were incubated on CM plates supplemented with various stress inducers at 28°C for 6 days. Growth of the ΔMovam7 mutant in media supplemented with SDS (0.01%; vs wild type P<0.01), lysing enzymes (2 mg/ml), and Congo Red (20 µg/ml). (B) Deletion of Movam7 altered the distribution of chitin within the cell. In the wild-type strain Guy11, CFW fluorescence was mainly distributed at the hyphal and septal apices, whereas in the ΔMovam7 mutant, fluorescence was not restricted to growing apices and found also on the lateral walls along the hyphal axe. This abnormal distribution of cell wall components was restored by the introduction of the wild-type MoVAM7 gene. (C) Reduced expression was found in six out of seven genes that encode chitin synthases in the ΔMovam7 mutants of M. oryzae. RNA was extracted from mycelia that were grown for 3 days in liquid CM. Error bars represent the standard deviation and asterisks represent significant different among stains tested. All of the reductions are significant (P = 0.01 or P = 0.05) according to Duncan's multiple range test.
Mentions: To examine the role of MoVam7 in cell wall integrity, we investigated the effects of various cell-wall perturbing agents on the ΔMovam7 mutant. The sensitivity of the ΔMovam7 mutant to lytic enzymes (LE), and Congo Red (CR) is not significantly different from that of the wild–type strain and the complemented mutant strains, but it was more sensitive to SDS (Figure 5A). No significant difference in the rate of protoplast release was found at 30 min and 90 min (Figure S3). CFW staining was also used to probe the distribution of chitin on the cell wall. In the wild type strain, CFW fluorescence was mostly distributed at the septa and tips where chitin, one of main components of the fungal cell wall, was actively synthesized, while in the mutants, patches of bright fluorescence were observed on the lateral wall of hyphae in addition to septa and hyphal tips (Figure 5B). Since CFW is a fluorescent dye that intercalates with nascent chitin chains, this result might suggest the altered distribution of chitin on cell wall in the ΔMovam7 mutant was due to the aberrant cell wall synthesis activity. Chitin contents analysis showed the chitin contents were reduced by 10% in the ΔMovam7 mutant compared to the wild-type strain (Figure S4). We also examined the expression of several genes encoding the putative chitin synthases in M. oryzae by quantitative RT-PCR assay. Interestingly, the result showed that with the exception of MGG_06064, the expression for six out of seven genes was reduced in the ΔMovam7 mutant in comparison to the wild strain (Figure 5C). These findings indicated that the aberrant chitin distribution, combining with reduced chitin production due to reduced gene expression, contributed together to weakened altered cell wall integrity in ΔMovam7 mutants.

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