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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

Selective deletion of the PL1332 gene without affecting the PL4813 gene.A. Schematic diagram of sequence comparisons between PL1332 and PL4813 loci. Three filled boxes at the 5’ side show blocks of similar sequences between the two genes. B. Schematic diagram of the wild-type locus, exogenus construct, and mutant locus. The mutant locus represents replacement of the PL1332 coding region with a single copy of a selectable marker, Hygromycin B (HygB) resistance cassette. C. Southern blots. The top panel shows a band shift from 6.7 Kb to 7.2 Kb by replacing 915 base pairs of the PL1332 coding and flanking region with 1436 base pairs of HygB cassette. The middle panel shows the PL4832 gene represented by a 10.9 kb band in all isolates, while the absence of PL1332 is represented by a 6.7 Kb band in all mutants compared to the wild type. This band does not appear in the top panel because sequence similarity was low at the probing region. The bottom panel shows the HygB cassette in all mutants except the wild type. Mutants represented by DNA lanes 1 and 2 were used for pathogenesis assays. The Δpl1332-1 mutant was complemented with a wild-type allele and mainly used for the virulence assays. P5’, P-PL1332, and P-Hyg indicate locations of the Southern probes. H indicates HindIII enzyme digestion sites.
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pone.0127140.g002: Selective deletion of the PL1332 gene without affecting the PL4813 gene.A. Schematic diagram of sequence comparisons between PL1332 and PL4813 loci. Three filled boxes at the 5’ side show blocks of similar sequences between the two genes. B. Schematic diagram of the wild-type locus, exogenus construct, and mutant locus. The mutant locus represents replacement of the PL1332 coding region with a single copy of a selectable marker, Hygromycin B (HygB) resistance cassette. C. Southern blots. The top panel shows a band shift from 6.7 Kb to 7.2 Kb by replacing 915 base pairs of the PL1332 coding and flanking region with 1436 base pairs of HygB cassette. The middle panel shows the PL4832 gene represented by a 10.9 kb band in all isolates, while the absence of PL1332 is represented by a 6.7 Kb band in all mutants compared to the wild type. This band does not appear in the top panel because sequence similarity was low at the probing region. The bottom panel shows the HygB cassette in all mutants except the wild type. Mutants represented by DNA lanes 1 and 2 were used for pathogenesis assays. The Δpl1332-1 mutant was complemented with a wild-type allele and mainly used for the virulence assays. P5’, P-PL1332, and P-Hyg indicate locations of the Southern probes. H indicates HindIII enzyme digestion sites.

Mentions: In addition to their similar expression pattern, the PL1332 and PL4813 genes shared three short blocks of similar sequences within a 1 kb sequence upstream from the start codon (Fig 2A) and an identical motif of a putative promoter [36]. The length of its genomic DNA was 835 nucleotides and contained one putative intron and two exons. We determined their cDNA sequence and defined the coding region for both genes (Fig 2A). There were three exons and two introns in each gene. The length of the coding regions and their nucleotide sequences were similar (Table 1 and S1 Fig). Their introns were also similar in their location, length, and sequence. Similarity in their expression profiles, gene structures, and gene sequences suggested that their functions were similar. For this reason, we decided to study one of the two genes instead of both.


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)

Selective deletion of the PL1332 gene without affecting the PL4813 gene.A. Schematic diagram of sequence comparisons between PL1332 and PL4813 loci. Three filled boxes at the 5’ side show blocks of similar sequences between the two genes. B. Schematic diagram of the wild-type locus, exogenus construct, and mutant locus. The mutant locus represents replacement of the PL1332 coding region with a single copy of a selectable marker, Hygromycin B (HygB) resistance cassette. C. Southern blots. The top panel shows a band shift from 6.7 Kb to 7.2 Kb by replacing 915 base pairs of the PL1332 coding and flanking region with 1436 base pairs of HygB cassette. The middle panel shows the PL4832 gene represented by a 10.9 kb band in all isolates, while the absence of PL1332 is represented by a 6.7 Kb band in all mutants compared to the wild type. This band does not appear in the top panel because sequence similarity was low at the probing region. The bottom panel shows the HygB cassette in all mutants except the wild type. Mutants represented by DNA lanes 1 and 2 were used for pathogenesis assays. The Δpl1332-1 mutant was complemented with a wild-type allele and mainly used for the virulence assays. P5’, P-PL1332, and P-Hyg indicate locations of the Southern probes. H indicates HindIII enzyme digestion sites.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127140.g002: Selective deletion of the PL1332 gene without affecting the PL4813 gene.A. Schematic diagram of sequence comparisons between PL1332 and PL4813 loci. Three filled boxes at the 5’ side show blocks of similar sequences between the two genes. B. Schematic diagram of the wild-type locus, exogenus construct, and mutant locus. The mutant locus represents replacement of the PL1332 coding region with a single copy of a selectable marker, Hygromycin B (HygB) resistance cassette. C. Southern blots. The top panel shows a band shift from 6.7 Kb to 7.2 Kb by replacing 915 base pairs of the PL1332 coding and flanking region with 1436 base pairs of HygB cassette. The middle panel shows the PL4832 gene represented by a 10.9 kb band in all isolates, while the absence of PL1332 is represented by a 6.7 Kb band in all mutants compared to the wild type. This band does not appear in the top panel because sequence similarity was low at the probing region. The bottom panel shows the HygB cassette in all mutants except the wild type. Mutants represented by DNA lanes 1 and 2 were used for pathogenesis assays. The Δpl1332-1 mutant was complemented with a wild-type allele and mainly used for the virulence assays. P5’, P-PL1332, and P-Hyg indicate locations of the Southern probes. H indicates HindIII enzyme digestion sites.
Mentions: In addition to their similar expression pattern, the PL1332 and PL4813 genes shared three short blocks of similar sequences within a 1 kb sequence upstream from the start codon (Fig 2A) and an identical motif of a putative promoter [36]. The length of its genomic DNA was 835 nucleotides and contained one putative intron and two exons. We determined their cDNA sequence and defined the coding region for both genes (Fig 2A). There were three exons and two introns in each gene. The length of the coding regions and their nucleotide sequences were similar (Table 1 and S1 Fig). Their introns were also similar in their location, length, and sequence. Similarity in their expression profiles, gene structures, and gene sequences suggested that their functions were similar. For this reason, we decided to study one of the two genes instead of both.

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