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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 Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.

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Reverse transcription polymerase chain reaction (RT‐PCR) expression patterns of the genes coding for Laccaria bicolor Zn finger protein and Ser/Threo protein kinase (protein ID 318751 and 379393 respectively). Total RNA from mycelium of L. bicolor wild‐type strain S238N and transgenic fungal strains 4, 24 and 36 where the gene models coding proteins 318751, 379393 and 318555 (transporter) respectively were interrupted by T‐DNA insertion was isolated and aliquots of 1 µl were used for first‐strand cDNA synthesis. A PCR was performed with 2 µl of first‐strand cDNA and between 15 and 30 cycles of amplification. The picture shows fragments amplified after 28 cycles. Laccaria glucokinase (protein ID 312018) specific primers were used as a reference to check for equal transcripts amplification in the wild‐type and the different transgenic strains. Lines 1 and 2 in each panel are L. bicolor wild‐type S238N stored at INRA‐Nancy and a subculture of it stored at University of Quilmes respectively. All the transformants were obtained using the last one as recipient strain for transformation. Lines 3–5 in each panel are L. bicolor transgenic strains 4, 24 and 36 respectively (T‐DNA insertion in protein ID 318751, Zn finger; 379393, Ser/Threo protein kinase; and 318555, transporter). No transcript could be detected for the gene interrupted in the transgenic strain 36 and coding for a putative transporter neither in the transgenic strains or in the wild type (not shown in this figure). A control with no RT in the first‐strand cDNA synthesis reaction mix was included for each strain and set of specific primers (not shown).
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f5: Reverse transcription polymerase chain reaction (RT‐PCR) expression patterns of the genes coding for Laccaria bicolor Zn finger protein and Ser/Threo protein kinase (protein ID 318751 and 379393 respectively). Total RNA from mycelium of L. bicolor wild‐type strain S238N and transgenic fungal strains 4, 24 and 36 where the gene models coding proteins 318751, 379393 and 318555 (transporter) respectively were interrupted by T‐DNA insertion was isolated and aliquots of 1 µl were used for first‐strand cDNA synthesis. A PCR was performed with 2 µl of first‐strand cDNA and between 15 and 30 cycles of amplification. The picture shows fragments amplified after 28 cycles. Laccaria glucokinase (protein ID 312018) specific primers were used as a reference to check for equal transcripts amplification in the wild‐type and the different transgenic strains. Lines 1 and 2 in each panel are L. bicolor wild‐type S238N stored at INRA‐Nancy and a subculture of it stored at University of Quilmes respectively. All the transformants were obtained using the last one as recipient strain for transformation. Lines 3–5 in each panel are L. bicolor transgenic strains 4, 24 and 36 respectively (T‐DNA insertion in protein ID 318751, Zn finger; 379393, Ser/Threo protein kinase; and 318555, transporter). No transcript could be detected for the gene interrupted in the transgenic strain 36 and coding for a putative transporter neither in the transgenic strains or in the wild type (not shown in this figure). A control with no RT in the first‐strand cDNA synthesis reaction mix was included for each strain and set of specific primers (not shown).

Mentions: None of the analysed dikaryotic fungal transgenic strains with a T‐DNA insertion showed an obvious altered phenotype such as hyphal morphology or growth rate when cultivated on P5 media. Three transgenic strains (4, 24 and 36, Table 1) with an interruption in a predicted gene were selected in order to test by reverse transcription polymerase chain reaction (RT‐PCR) whether a single integration in one of the gene copies of the dikaryotic strain can cause a detectable reduction on the gene expression level or not (Fig. 5).


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)

Reverse transcription polymerase chain reaction (RT‐PCR) expression patterns of the genes coding for Laccaria bicolor Zn finger protein and Ser/Threo protein kinase (protein ID 318751 and 379393 respectively). Total RNA from mycelium of L. bicolor wild‐type strain S238N and transgenic fungal strains 4, 24 and 36 where the gene models coding proteins 318751, 379393 and 318555 (transporter) respectively were interrupted by T‐DNA insertion was isolated and aliquots of 1 µl were used for first‐strand cDNA synthesis. A PCR was performed with 2 µl of first‐strand cDNA and between 15 and 30 cycles of amplification. The picture shows fragments amplified after 28 cycles. Laccaria glucokinase (protein ID 312018) specific primers were used as a reference to check for equal transcripts amplification in the wild‐type and the different transgenic strains. Lines 1 and 2 in each panel are L. bicolor wild‐type S238N stored at INRA‐Nancy and a subculture of it stored at University of Quilmes respectively. All the transformants were obtained using the last one as recipient strain for transformation. Lines 3–5 in each panel are L. bicolor transgenic strains 4, 24 and 36 respectively (T‐DNA insertion in protein ID 318751, Zn finger; 379393, Ser/Threo protein kinase; and 318555, transporter). No transcript could be detected for the gene interrupted in the transgenic strain 36 and coding for a putative transporter neither in the transgenic strains or in the wild type (not shown in this figure). A control with no RT in the first‐strand cDNA synthesis reaction mix was included for each strain and set of specific primers (not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815887&req=5

f5: Reverse transcription polymerase chain reaction (RT‐PCR) expression patterns of the genes coding for Laccaria bicolor Zn finger protein and Ser/Threo protein kinase (protein ID 318751 and 379393 respectively). Total RNA from mycelium of L. bicolor wild‐type strain S238N and transgenic fungal strains 4, 24 and 36 where the gene models coding proteins 318751, 379393 and 318555 (transporter) respectively were interrupted by T‐DNA insertion was isolated and aliquots of 1 µl were used for first‐strand cDNA synthesis. A PCR was performed with 2 µl of first‐strand cDNA and between 15 and 30 cycles of amplification. The picture shows fragments amplified after 28 cycles. Laccaria glucokinase (protein ID 312018) specific primers were used as a reference to check for equal transcripts amplification in the wild‐type and the different transgenic strains. Lines 1 and 2 in each panel are L. bicolor wild‐type S238N stored at INRA‐Nancy and a subculture of it stored at University of Quilmes respectively. All the transformants were obtained using the last one as recipient strain for transformation. Lines 3–5 in each panel are L. bicolor transgenic strains 4, 24 and 36 respectively (T‐DNA insertion in protein ID 318751, Zn finger; 379393, Ser/Threo protein kinase; and 318555, transporter). No transcript could be detected for the gene interrupted in the transgenic strain 36 and coding for a putative transporter neither in the transgenic strains or in the wild type (not shown in this figure). A control with no RT in the first‐strand cDNA synthesis reaction mix was included for each strain and set of specific primers (not shown).
Mentions: None of the analysed dikaryotic fungal transgenic strains with a T‐DNA insertion showed an obvious altered phenotype such as hyphal morphology or growth rate when cultivated on P5 media. Three transgenic strains (4, 24 and 36, Table 1) with an interruption in a predicted gene were selected in order to test by reverse transcription polymerase chain reaction (RT‐PCR) whether a single integration in one of the gene copies of the dikaryotic strain can cause a detectable reduction on the gene expression level or not (Fig. 5).

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 Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.

Show MeSH
Related in: MedlinePlus