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A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola.

Cho Y, Jang M, Srivastava A, Jang JH, Soung NK, Ko SK, Kang DO, Ahn JS, Kim BY - PLoS ONE (2015)

Bottom Line: When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue.The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2.It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola.

View Article: PubMed Central - PubMed

Affiliation: Incurable Diseases Research Center (WCI), Bio-Therapeutics Research Institute, Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk, 363-883, Republic of Korea.

ABSTRACT
Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development.

No MeSH data available.


Related in: MedlinePlus

Differential expression of eight pectate lyase-coding genes.Relative amount of transcripts for eight individual pectate lyase-coding genes in wild-type Alternaria brassicicola. The amount of transcripts for each gene is shown as a percentage of the amounts of Ef1-α transcripts at each stage. Y-axes indicate the relative quantity of the transcripts (RQT) of each gene compared to Ef1- α. Gene names are shown below the X-axes. Error bars indicate standard deviation (N = 3). hpi: hours postinoculation, when the fungal tissues were harvested; gyeb: fungal mycelium grown in glucose yeast extract broth; pectin: fungal mycelium grown in a minimal medium supplemented with pectin as a major carbon source.
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pone.0127140.g001: Differential expression of eight pectate lyase-coding genes.Relative amount of transcripts for eight individual pectate lyase-coding genes in wild-type Alternaria brassicicola. The amount of transcripts for each gene is shown as a percentage of the amounts of Ef1-α transcripts at each stage. Y-axes indicate the relative quantity of the transcripts (RQT) of each gene compared to Ef1- α. Gene names are shown below the X-axes. Error bars indicate standard deviation (N = 3). hpi: hours postinoculation, when the fungal tissues were harvested; gyeb: fungal mycelium grown in glucose yeast extract broth; pectin: fungal mycelium grown in a minimal medium supplemented with pectin as a major carbon source.

Mentions: Two putative pectate lyase-coding genes, PL1332 and PL4813, regulated by the transcription factor AbPf2, were dramatically induced soon after conidia were inoculated on leaves of host plants [36]. Expression levels of these genes and six other pectate lyase genes were further quantified and compared with transcripts of a gene encoding elongation factor 1-α (Ef1-α) (Fig 1). The expression levels of Ef1-α were more consistent than all other genes encoding housekeeping proteins [36]. The expression levels of all eight pectate lyase genes were less than 3% of the transcripts of Ef1-α at 4 hours postinoculation (hpi) (Fig 1A), but transcript levels of PL1332 and PL4831 were dramatically increased afterwards and reached levels comparable to Ef1-α by 12 hpi (Fig 1B). Subsequently, their expression levels decreased to less than 2% by 48 hpi when colonization was established. The expression levels of these genes remained low during saprophytic growth on both dead host tissue and axenic media (Fig 1D–1G). Notably, the presence of pectin as a major carbon source did not induce their expression (Fig 1G). The other six pectate lyase-coding genes (AB05514.1, AB00904.1, AB10322, AB06838.1, AB03608, AB10575.1) are putatively regulated by the AbVf19 transcription factor [34]. Although their expression was induced by AbVf19 during the late stage of infection, the magnitude of induction varied from less than 5% to over 200% compared to the expression levels of Ef1-α. Particularly, AB10322 and AB06838 among the six pectate lyase-coding genes were expressed at their highest levels during the late stages of infection. All eight of the pectate lyase-coding genes were expressed at low levels during saprophytic growth in a liquid culture medium and none were induced when pectin was a major carbon source in the medium.


A Pectate Lyase-Coding Gene Abundantly Expressed during Early Stages of Infection Is Required for Full Virulence in Alternaria brassicicola.

Cho Y, Jang M, Srivastava A, Jang JH, Soung NK, Ko SK, Kang DO, Ahn JS, Kim BY - PLoS ONE (2015)

Differential expression of eight pectate lyase-coding genes.Relative amount of transcripts for eight individual pectate lyase-coding genes in wild-type Alternaria brassicicola. The amount of transcripts for each gene is shown as a percentage of the amounts of Ef1-α transcripts at each stage. Y-axes indicate the relative quantity of the transcripts (RQT) of each gene compared to Ef1- α. Gene names are shown below the X-axes. Error bars indicate standard deviation (N = 3). hpi: hours postinoculation, when the fungal tissues were harvested; gyeb: fungal mycelium grown in glucose yeast extract broth; pectin: fungal mycelium grown in a minimal medium supplemented with pectin as a major carbon source.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4440746&req=5

pone.0127140.g001: Differential expression of eight pectate lyase-coding genes.Relative amount of transcripts for eight individual pectate lyase-coding genes in wild-type Alternaria brassicicola. The amount of transcripts for each gene is shown as a percentage of the amounts of Ef1-α transcripts at each stage. Y-axes indicate the relative quantity of the transcripts (RQT) of each gene compared to Ef1- α. Gene names are shown below the X-axes. Error bars indicate standard deviation (N = 3). hpi: hours postinoculation, when the fungal tissues were harvested; gyeb: fungal mycelium grown in glucose yeast extract broth; pectin: fungal mycelium grown in a minimal medium supplemented with pectin as a major carbon source.
Mentions: Two putative pectate lyase-coding genes, PL1332 and PL4813, regulated by the transcription factor AbPf2, were dramatically induced soon after conidia were inoculated on leaves of host plants [36]. Expression levels of these genes and six other pectate lyase genes were further quantified and compared with transcripts of a gene encoding elongation factor 1-α (Ef1-α) (Fig 1). The expression levels of Ef1-α were more consistent than all other genes encoding housekeeping proteins [36]. The expression levels of all eight pectate lyase genes were less than 3% of the transcripts of Ef1-α at 4 hours postinoculation (hpi) (Fig 1A), but transcript levels of PL1332 and PL4831 were dramatically increased afterwards and reached levels comparable to Ef1-α by 12 hpi (Fig 1B). Subsequently, their expression levels decreased to less than 2% by 48 hpi when colonization was established. The expression levels of these genes remained low during saprophytic growth on both dead host tissue and axenic media (Fig 1D–1G). Notably, the presence of pectin as a major carbon source did not induce their expression (Fig 1G). The other six pectate lyase-coding genes (AB05514.1, AB00904.1, AB10322, AB06838.1, AB03608, AB10575.1) are putatively regulated by the AbVf19 transcription factor [34]. Although their expression was induced by AbVf19 during the late stage of infection, the magnitude of induction varied from less than 5% to over 200% compared to the expression levels of Ef1-α. Particularly, AB10322 and AB06838 among the six pectate lyase-coding genes were expressed at their highest levels during the late stages of infection. All eight of the pectate lyase-coding genes were expressed at low levels during saprophytic growth in a liquid culture medium and none were induced when pectin was a major carbon source in the medium.

Bottom Line: When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue.The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2.It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola.

View Article: PubMed Central - PubMed

Affiliation: Incurable Diseases Research Center (WCI), Bio-Therapeutics Research Institute, Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk, 363-883, Republic of Korea.

ABSTRACT
Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development.

No MeSH data available.


Related in: MedlinePlus