T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.
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.
Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.
Minna
M
Kemppainen
Sebastien
S
Duplessis
Francis
F
Martin
Alejandro G
AG
Pardo
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D018014:Gene Transfer Techniques; D016681:Genome, Fungal; D055399:Laccaria / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D038821:Mycorrhizae / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012231:Rhizobium / Q000235:genetics
D004269:DNA, Bacterial; C036483:T-DNA
New Phytol. 2005 Feb;165(2):599-611
15720670
EMBO J. 2001 Nov 15;20(22):6550-8
11707425
Plant J. 2001 Jan;25(2):181-91
11169194
Curr Opin Plant Biol. 2007 Apr;10(2):204-10
17291823
Fungal Genet Biol. 2007 Feb;44(2):123-38
16979359
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1871-6
11172043
Plant Mol Biol. 2003 Jul;52(4):761-73
13677465
EMBO J. 1990 Oct;9(10):3077-84
2209538
Theor Appl Genet. 1991 Sep;82(3):257-63
24213168
Science. 2006 Sep 15;313(5793):1596-604
16973872
Nat Biotechnol. 1998 Sep;16(9):839-42
9743116
Mol Gen Genet. 2000 Sep;264(1-2):20-8
11016829
New Phytol. 2003 Jul;159(1):117-129
33873685
Fungal Genet Biol. 2007 Oct;44(10):1050-64
17544743
FEMS Microbiol Lett. 2004 Dec 15;241(2):207-14
15598534
Yeast. 2002 Apr;19(6):529-36
11921101
EMBO J. 1995 Jul 17;14(14):3585-95
7628458
Mol Plant Microbe Interact. 2006 Jun;19(6):588-96
16776292
EMBO Rep. 2004 Jun;5(6):632-7
15153934
Science. 2003 Aug 1;301(5633):653-7
12893945
Mol Plant Microbe Interact. 1999 Oct;12(10):862-71
10517026
Plant J. 1997 Jan;11(1):15-29
9025300
Plant J. 1994 Aug;6(2):271-82
7920717
Trends Genet. 2004 Aug;20(8):375-83
15262410
Nature. 2008 Mar 6;452(7183):88-92
18322534
EMBO J. 1991 Mar;10(3):697-704
2001683
Eukaryot Cell. 2004 Apr;3(2):420-9
15075272
Science. 1999 Aug 6;285(5429):901-6
10436161
EMBO J. 1995 Jul 3;14(13):3206-14
7621833
Mol Cells. 2001 Dec 31;12(3):407-11
11804343
EMBO Rep. 2002 Dec;3(12):1152-7
12446565
Curr Genet. 2005 Jul;48(1):1-17
15889258
FEMS Microbiol Lett. 2003 Mar 14;220(1):141-8
12644240
Appl Environ Microbiol. 2000 Oct;66(10):4510-3
11010906
Curr Genet. 2004 Feb;45(2):111-9
14634789
Mol Plant Microbe Interact. 2004 Feb;17(2):202-15
14964534
Mycorrhiza. 2005 Dec;16(1):19-22
16133248