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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

Enzyme activity of pectate lyase measured by a titrimetric stop reaction method.A. Enzyme activity calculated by free iodines not covalently bound to oligogalacturonic acids that originated in polygalacturonic acids. MBP: maltose binding protein, MBP-PL1332: fusion protein, GST: glutathione-S-transferase, GST-PL1332: fusion protein. B. Visual comparison of the relative amounts of free iodine. Intensity of dark brown color indicates relative amounts of free iodine, which inversely correlates with enzyme activity. ** p < 0.01.
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pone.0127140.g005: Enzyme activity of pectate lyase measured by a titrimetric stop reaction method.A. Enzyme activity calculated by free iodines not covalently bound to oligogalacturonic acids that originated in polygalacturonic acids. MBP: maltose binding protein, MBP-PL1332: fusion protein, GST: glutathione-S-transferase, GST-PL1332: fusion protein. B. Visual comparison of the relative amounts of free iodine. Intensity of dark brown color indicates relative amounts of free iodine, which inversely correlates with enzyme activity. ** p < 0.01.

Mentions: Purified MBP-PL1332 fusion proteins did not show enzyme activity under any of the test conditions (S2 Fig). We speculated that the fusion proteins were either degraded during protein purification and subsequent enzyme-assay conditions, or the enzyme required unknown co-factors for its activity. To circumvent possible problems caused by protein degradation, the absence of unknown cofactors in the reaction mixture, or both, we measured enzyme activity in the soluble fraction of whole lysates of E. coli that expressed MBP-PL1332 fusion proteins. Soluble lysate of E. coli expressing MBP was used as a control and it showed weak enzyme activity, as expected (Fig 5 MBP-PL1332). The soluble lysate of E. coli expressing MBP-PL1332 fusion proteins, however, showed significantly stronger enzyme activity (p < 0.01) than the control. We performed similar experiments using PL1332 proteins fused to glutathione-S-transferase (GST). The GST-PL1332 fusion proteins in soluble bacterial lysate also showed stronger enzyme activity than GST in soluble bacterial lysate (Fig 5 GST-PL1332).


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)

Enzyme activity of pectate lyase measured by a titrimetric stop reaction method.A. Enzyme activity calculated by free iodines not covalently bound to oligogalacturonic acids that originated in polygalacturonic acids. MBP: maltose binding protein, MBP-PL1332: fusion protein, GST: glutathione-S-transferase, GST-PL1332: fusion protein. B. Visual comparison of the relative amounts of free iodine. Intensity of dark brown color indicates relative amounts of free iodine, which inversely correlates with enzyme activity. ** p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440746&req=5

pone.0127140.g005: Enzyme activity of pectate lyase measured by a titrimetric stop reaction method.A. Enzyme activity calculated by free iodines not covalently bound to oligogalacturonic acids that originated in polygalacturonic acids. MBP: maltose binding protein, MBP-PL1332: fusion protein, GST: glutathione-S-transferase, GST-PL1332: fusion protein. B. Visual comparison of the relative amounts of free iodine. Intensity of dark brown color indicates relative amounts of free iodine, which inversely correlates with enzyme activity. ** p < 0.01.
Mentions: Purified MBP-PL1332 fusion proteins did not show enzyme activity under any of the test conditions (S2 Fig). We speculated that the fusion proteins were either degraded during protein purification and subsequent enzyme-assay conditions, or the enzyme required unknown co-factors for its activity. To circumvent possible problems caused by protein degradation, the absence of unknown cofactors in the reaction mixture, or both, we measured enzyme activity in the soluble fraction of whole lysates of E. coli that expressed MBP-PL1332 fusion proteins. Soluble lysate of E. coli expressing MBP was used as a control and it showed weak enzyme activity, as expected (Fig 5 MBP-PL1332). The soluble lysate of E. coli expressing MBP-PL1332 fusion proteins, however, showed significantly stronger enzyme activity (p < 0.01) than the control. We performed similar experiments using PL1332 proteins fused to glutathione-S-transferase (GST). The GST-PL1332 fusion proteins in soluble bacterial lysate also showed stronger enzyme activity than GST in soluble bacterial lysate (Fig 5 GST-PL1332).

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