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

Abstract

Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium-mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T-DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T-DNA right border (RB)-genomic DNA junctions in Escherichia coli. Fifty-one Agrobacterium-transformed fungal strains, picked up at random from a larger collection of T-DNA tagged strains (about 500), were analysed. Sixty-nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. 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.

Figure 1

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.

Figure 2

Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λBstE II, L. bicolor wild type (Wt) and transgenic strains.

Figure 3

Set of rescued plasmids linearized with SacI. Genomic DNA isolated after L. bicolor transformation was self‐ligated and electroporated into E. coli. Plasmids isolated from bacterial clones were linearized with SacI and separated in an 1% agarose gel. From left to right: molecular size marker λBstE II, representative set of plasmids rescued from independent L. bicolor transgenic strains.

Figure 4

Right border conservation of Agrobacterium‐transformed Laccaria transgenic strains. The pCAMBIA 1300 sequence is presented till the RB nick site. Letters in italics represent bases that could originate from both RB or genomic DNA.

Figure 5

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).

Figure 6

Laccaria bicolor transgenic strain 24 and wild type grown on P5 medium supplemented with 4 mM KNO₃ as the sole N source.