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T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.

Kemppainen M, Duplessis S, Martin F, Pardo AG - Microb Biotechnol (2008)

Bottom Line: Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria.Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes.Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de MicologĂ­a Molecular, Departamento de Ciencia y TecnologĂ­a, Universidad Nacional de Quilmes, Roque SĂĄenz PeĂąa 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.

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pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus: glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus. hph: hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.
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f1: pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus: glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus. hph: hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.

Mentions: We had previously shown that Agrobacterium can be used for gene transfer in L. bicolor. The T‐DNA seemed to integrate mostly as a single copy and at different places in the fungal genome suggesting this method proper for non‐targeted mutagenesis of the fungus (Kemppainen et al., 2005). These conclusions were drown from Southern hybridization analysis knowing that it was possible that integration after all could had happened in sites sharing some common features or just in non‐coding sequences. When Laccaria genome sequencing was completed mapping of precise integration sites became possible and AMT of the fungus could be further evaluated. In order to gain more information regarding the Agrobacterium T‐DNA integration in L. bicolor we constructed the plasmid rescue binary vector pHg/pBks (Fig. 1). This plasmid does not carry homologous sequences with L. bicolor genome; its T‐DNA allows the selection of fungal transgenic strains by hygromycin resistance and the rescue of the RB–gDNA junction by cutting with SacI under ampicillin selection in E. coli.


T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.

Kemppainen M, Duplessis S, Martin F, Pardo AG - Microb Biotechnol (2008)

pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus: glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus. hph: hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.
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Related In: Results  -  Collection

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f1: pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus: glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus. hph: hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.
Mentions: We had previously shown that Agrobacterium can be used for gene transfer in L. bicolor. The T‐DNA seemed to integrate mostly as a single copy and at different places in the fungal genome suggesting this method proper for non‐targeted mutagenesis of the fungus (Kemppainen et al., 2005). These conclusions were drown from Southern hybridization analysis knowing that it was possible that integration after all could had happened in sites sharing some common features or just in non‐coding sequences. When Laccaria genome sequencing was completed mapping of precise integration sites became possible and AMT of the fungus could be further evaluated. In order to gain more information regarding the Agrobacterium T‐DNA integration in L. bicolor we constructed the plasmid rescue binary vector pHg/pBks (Fig. 1). This plasmid does not carry homologous sequences with L. bicolor genome; its T‐DNA allows the selection of fungal transgenic strains by hygromycin resistance and the rescue of the RB–gDNA junction by cutting with SacI under ampicillin selection in E. coli.

Bottom Line: Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria.Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes.Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de MicologĂ­a Molecular, Departamento de Ciencia y TecnologĂ­a, Universidad Nacional de Quilmes, Roque SĂĄenz PeĂąa 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.

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Related in: MedlinePlus