T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.
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.
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
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Mentions: We set up a plasmid rescue protocol for L. bicolor using 1–3 µg of completely SacI‐digested and self‐ligated gDNA which proved to be enough for producing ampicillin‐resistant E. coli colonies after electroporation. The number of colonies depended largely on the rescued fungal transgenic strain and normally ranged from 10 to 100 per electroporation. We noticed that a cleaning‐up of the ligation reaction with phenol‐chloroform before electroporation was not necessary while using the enzymes and buffers detailed in Experimental procedures. Of the 47 processed fungal transgenic strains 40 produced ampicillin‐resistant bacteria and up to eight plasmids per rescued strain were analysed by linearization with SacI for a minimum size of 3 kb corresponding to pBluescript KS+. Obtained rescued plasmids ranged from 3 to 10 kb in size indicating the incorporation of genomic DNA of Laccaria (a representative set of rescued plasmids is presented in Fig. 3). Transgenic strains showing abnormalities in any of the mentioned features were excluded from further analysis. Plasmids from 35 fungal transgenic strains were subjected to sequencing of which 29 were able to be sequenced with the Post‐RB primer that binds within the T‐DNA at 43–62 bases upstream of the RB nick site. A blastnsearch was performed with the obtained sequences on the JGILaccaria genome portal (http://genome.jgi‐psf.org/Lacbi1/Lacbi1.home.html) in order to identify the T‐DNA–gDNA junctions. The exact integration sites were mapped in the genome and the RB conservation was compared between 25 rescued sequences (Fig. 4). The RB was well conserved during the integration of the T‐DNAs in Laccaria genome and 15 out of 25 sequences at their maximum possible length reached up to the original adenine RB nick site, while the rest of the sequences showed deletions of one to five bases. The samples failing in the sequencing with the primer used in this work most likely have had larger RB truncations and lack the primer binding site. Altogether 24 sequences were located in the Laccaria genome (version 1.0 as of December 2007) while one rescued sequence did not carry genomic DNA flank long enough for mapping. According to the automatized gene‐prediction models integrations had happened in both coding and non‐coding regions of the fungal genome and we were able to locate them into exons, introns, upstream and downstream elements of predicted genes or in intergenic regions. Two of the sequences could not be located in Laccaria genome and were compared with the blastn algorithm to GenBank databases to identify possible DNA contaminations. No homologies were found proposing that these sequences most probably belong to the fungus and correspond to sequencing gaps in the Laccaria genome sequence. Eighteen T‐DNA integration sites were precisely located in ORFs and their upstream or downstream elements (Table 1). Thirteen ORF interruptions were found, 11 of which were in exons and two in predicted intronic sequences. All of these interrupted ORFs have homologues in other organisms and eight have a proposed protein function (Table 1). Three integrations fall in proposed promoters and three in downstream ORFs (Table 1). Two sequences showed signs of genetic rearrangements or tandem T‐DNA integrations. One of these last sequences was obtained from the rescued fungal transgenic strain 15 that already showed a double hybridization signal in the preliminary Southern blot analysis (Fig. 2), the lower signal being rather small for representing a full‐length integration of a second T‐DNA. In this transgenic strain the RB was first followed by the inner sequence of the T‐DNA and then by a Laccaria sequence indicating a genomic reorganization during or after T‐DNA integration. Also in another rescued fungal transgenic strain the primer sequence was immediately followed by the T‐DNA LB showing a possible incomplete tandem integration (data not shown).
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.