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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

Expression of PL1332 proteins in Escherichia coli.A. Cloning of PL1332 cDNA and successful transformation of the expression vector. Lane 1: PL1332 cDNA pMAL-c2x expression vector after digestion with BamH1 and HindIII; Lane 2: empty pMAL-c2x vector; Lanes 3–8: recombinant plasmids purified from E. coli transformed with the expression construct. All plasmids in lanes 2–8 were digested with BamH1 while the plasmid in lane 1 was digested with BamH1 and HindIII. The clone shown in lane 3 was used for protein production. B. Expression of the maltose binding protein (MBP) and PL1332 fusion (MBP-PL1332) protein. Lane 1: protein markers; Lane 2: total crude extract before IPTG induction; Lane 3: total crude extract after IPTG induction; Lane 4: total crude extract; Lane 5: supernatant of the lysate; Lane 6: flow through; Lane 7: purified protein. Expected size of the MBP-PL1332 in lanes 2 through 7 was 68 KD and smaller bands marked with an asterisk are degraded fusion proteins; Lane 8: total crude extract of MBP protein; Lane 9: supernatant of the lysate; Lane 10: purified MBP. Expected size of MBP protein in lanes 8 through 10 was 42.5 KD.
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pone.0127140.g004: Expression of PL1332 proteins in Escherichia coli.A. Cloning of PL1332 cDNA and successful transformation of the expression vector. Lane 1: PL1332 cDNA pMAL-c2x expression vector after digestion with BamH1 and HindIII; Lane 2: empty pMAL-c2x vector; Lanes 3–8: recombinant plasmids purified from E. coli transformed with the expression construct. All plasmids in lanes 2–8 were digested with BamH1 while the plasmid in lane 1 was digested with BamH1 and HindIII. The clone shown in lane 3 was used for protein production. B. Expression of the maltose binding protein (MBP) and PL1332 fusion (MBP-PL1332) protein. Lane 1: protein markers; Lane 2: total crude extract before IPTG induction; Lane 3: total crude extract after IPTG induction; Lane 4: total crude extract; Lane 5: supernatant of the lysate; Lane 6: flow through; Lane 7: purified protein. Expected size of the MBP-PL1332 in lanes 2 through 7 was 68 KD and smaller bands marked with an asterisk are degraded fusion proteins; Lane 8: total crude extract of MBP protein; Lane 9: supernatant of the lysate; Lane 10: purified MBP. Expected size of MBP protein in lanes 8 through 10 was 42.5 KD.

Mentions: We failed to measure knockout effects of the PL1332 gene on the enzyme activity of pectate lyases secreted in the culture medium because the PL1332 gene was expressed at extremely low levels in the liquid medium, with or without pectin (Fig 1, GYEB and pectin). Further, it was not possible to measure enzyme activity in the inoculum collected from the infection sites when the PL1332 gene was highly induced because the fungal biomass was extremely small at 4 to 24 hours postinoculation. To verify its enzyme activity, we expressed the PL1332 protein by cloning the gene in a heterologous protein expression system (Fig 4A). PL1332 was expressed as a fusion protein by linking it to maltose binding protein (MBP) (Fig 4B). Two amino acids at the N-terminus were deleted during the cloning of PL1332 cDNA in the proper reading frame, following the MBP-coding region. After IPTG-was induced, all transformants abundantly expressed the ~68 KDa proteins expected from the fusion of MBP and PL1332 (MBP-PL1332). The fusion proteins were soluble and stayed in the cytosol of E. coli cells. However, the proteins were partially degraded after purification, unlike the intact MBP proteins (Fig 4B, compare lane 7 and lane 10). Treatment of the fusion protein with Factor Xa to remove the MBP domain caused complete degradation of the protein during overnight incubation at 4°C (data not shown). Thus, we were not able to perform enzyme assays using PL1332 after removing the MBP binding domain. Instead we performed enzymatic assays using the fusion proteins that were partially degraded. Enzyme activity was measured by the extent of enzymatic digestion of polygalacturonic acid to oligogalacturonic acid using a titrimetric stop-reaction method.


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)

Expression of PL1332 proteins in Escherichia coli.A. Cloning of PL1332 cDNA and successful transformation of the expression vector. Lane 1: PL1332 cDNA pMAL-c2x expression vector after digestion with BamH1 and HindIII; Lane 2: empty pMAL-c2x vector; Lanes 3–8: recombinant plasmids purified from E. coli transformed with the expression construct. All plasmids in lanes 2–8 were digested with BamH1 while the plasmid in lane 1 was digested with BamH1 and HindIII. The clone shown in lane 3 was used for protein production. B. Expression of the maltose binding protein (MBP) and PL1332 fusion (MBP-PL1332) protein. Lane 1: protein markers; Lane 2: total crude extract before IPTG induction; Lane 3: total crude extract after IPTG induction; Lane 4: total crude extract; Lane 5: supernatant of the lysate; Lane 6: flow through; Lane 7: purified protein. Expected size of the MBP-PL1332 in lanes 2 through 7 was 68 KD and smaller bands marked with an asterisk are degraded fusion proteins; Lane 8: total crude extract of MBP protein; Lane 9: supernatant of the lysate; Lane 10: purified MBP. Expected size of MBP protein in lanes 8 through 10 was 42.5 KD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440746&req=5

pone.0127140.g004: Expression of PL1332 proteins in Escherichia coli.A. Cloning of PL1332 cDNA and successful transformation of the expression vector. Lane 1: PL1332 cDNA pMAL-c2x expression vector after digestion with BamH1 and HindIII; Lane 2: empty pMAL-c2x vector; Lanes 3–8: recombinant plasmids purified from E. coli transformed with the expression construct. All plasmids in lanes 2–8 were digested with BamH1 while the plasmid in lane 1 was digested with BamH1 and HindIII. The clone shown in lane 3 was used for protein production. B. Expression of the maltose binding protein (MBP) and PL1332 fusion (MBP-PL1332) protein. Lane 1: protein markers; Lane 2: total crude extract before IPTG induction; Lane 3: total crude extract after IPTG induction; Lane 4: total crude extract; Lane 5: supernatant of the lysate; Lane 6: flow through; Lane 7: purified protein. Expected size of the MBP-PL1332 in lanes 2 through 7 was 68 KD and smaller bands marked with an asterisk are degraded fusion proteins; Lane 8: total crude extract of MBP protein; Lane 9: supernatant of the lysate; Lane 10: purified MBP. Expected size of MBP protein in lanes 8 through 10 was 42.5 KD.
Mentions: We failed to measure knockout effects of the PL1332 gene on the enzyme activity of pectate lyases secreted in the culture medium because the PL1332 gene was expressed at extremely low levels in the liquid medium, with or without pectin (Fig 1, GYEB and pectin). Further, it was not possible to measure enzyme activity in the inoculum collected from the infection sites when the PL1332 gene was highly induced because the fungal biomass was extremely small at 4 to 24 hours postinoculation. To verify its enzyme activity, we expressed the PL1332 protein by cloning the gene in a heterologous protein expression system (Fig 4A). PL1332 was expressed as a fusion protein by linking it to maltose binding protein (MBP) (Fig 4B). Two amino acids at the N-terminus were deleted during the cloning of PL1332 cDNA in the proper reading frame, following the MBP-coding region. After IPTG-was induced, all transformants abundantly expressed the ~68 KDa proteins expected from the fusion of MBP and PL1332 (MBP-PL1332). The fusion proteins were soluble and stayed in the cytosol of E. coli cells. However, the proteins were partially degraded after purification, unlike the intact MBP proteins (Fig 4B, compare lane 7 and lane 10). Treatment of the fusion protein with Factor Xa to remove the MBP domain caused complete degradation of the protein during overnight incubation at 4°C (data not shown). Thus, we were not able to perform enzyme assays using PL1332 after removing the MBP binding domain. Instead we performed enzymatic assays using the fusion proteins that were partially degraded. Enzyme activity was measured by the extent of enzymatic digestion of polygalacturonic acid to oligogalacturonic acid using a titrimetric stop-reaction method.

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