Horizontal gene transfer from trees to ectomycorrhizal fungi: Lessons from laboratory and host plant liberation experiments Dr. Uwe Nehls 1,2, Dr. Chi Zhang 1, Dr. Mika Tarkka 1, Andrea Bock 1 1: University of Tübingen, Microbiological Institute, Physiological Ecology of Plants, Tübingen, Germany 2: University of Bremen, Faculty for Biology and Chemistry, Botany, Bremen, Germany
Longitudinal section of an ectomycorrhiza
Due to root cell degeneration plant DNA is released and can due to the intimate contact between both partners, be taken up by the fungus. Horizontal gene transfer from plant to fungi?
Scheme of an ectomycorrhizal fungal colony
Experimental system to investigate horizontal gene transfer Transgenic polar plants containing a small gene that will confer fungal Basta resistance after a horizontal transfer towards ectomycorrhizal fungi
Mycorrhized poplar plant Amanita muscaria Poplar plants in a Petri dish system A = non-mycorrhized root B = Mycorrhiza
Streptomyces hygroscopicus bar gene (800 bp) under the control of the Cochliobolus heterostrophus glyceraldehyde- 3-phosphate dehydrogenase (GPD) promoter (Zhang et al., 2005 Env Biosafety Res 4: 1-8)
Transformation of A. muscaria with the GPD/bar construct (Zhang et al., 2005 Env Biosafety Res 4: 1-8)
Detection of the bar gene in the genome of Basta resistant A. muscaria colonies. Southern blot of genomic DNA of Basta-resistant fungal colonies digested with HindIII and hybridized with labeled bar gene. (Lane 1) Wild-type DNA, (Lanes 2-10) DNA isolated from different transformants. (Zhang et al., 2005 Env Biosafety Res 4: 1-8)
Next Step: Generation of transgenic poplar plants containing the Basta-resistance cassette
Presence of the bar gene in the genomic DNA of kanamycin-resistant poplar transformants. PCR amplification of a 550 bp bar gene fragment from genomic poplar DNA. (Lanes 1-20) DNA of putative transformants, (P) diluted plasmid DNA containing the bar gene (C) DNA of a non-transformed poplar. (Zhang et al., 2005 Env Biosafety Res 4: 1-8)
Large scale mycorrhization of poplar plants under controlled conditions
From a total of 35.000 investigated mykorrhizas 102 slow growing fungal colonies were obtained on selection medium. (Zhang et al., 2005 Env Biosafety Res 4: 1-8)
However: None of these colonies was able to grow after a transfer onto fresh selection medium
No indication for the presence of the bar gene in genomic DNA isolated from these fungal colonies. A B PCR-amplification from genomic DNA using primers for: (A)the bar gene or (B) (B) a fungal gene C respective positive controls (Zhang et al., 2005 Env Biosafety Res 4: 1-8)
Conclusion: Under (well defined) lab conditions no hints for a horizontal gene transfer from poplar roots to A. muscaria hyphae were found in ectomycorrhizas
Estimation of horizontal gene transfer events that could be expected A. muscaria transformation efficiency: about 7.3 x 10 6 DNA molecules necessary to generate one transformant. 35,000 mycorrhizas contained 2.45 x 10 7 plant cells in direct and 1 x 10 8 plant cells not in direct contact with fungal hyphae. => 35,000 mycorrhizas represent about 3.4 to 17 times the number of plant genomes necessary to obtain one fungal transformant.
Horizontal gene transfer from poplar to ectomycorrhizal fungi under field conditions
Five independent poplar transformants (each about 30 plants) in plantation
Digging the root system 1-3 years)
Isolation of mycorrhizas, surface sterilization, distribution onto selection agar plates
Frequent mykorrhiza morphotypes
Large fluctuation of fungal partners Ectomycorrhiza morphotype frequency in spring/autumn (Nehls et al., 2006 In: Recent developments in tree transgenesis. Springer Verlag, 323-334)
Limitation 1: Certain fungal isolates do not grow on synthetic media
Limitation 2: From 120,000 investigated mycorrhizas, about 40,000 were naturally BASTA-tolerant. 60 50 40 30 20 10 0 Surface of fungal colony [mm 2 ] 2 4 5 6 B02 C02 D02 E02 47-29 hbel 2 48-38 SL 48-38 SL! 60-20 hbel 2 s1 s2 Isolate without Basta 200µg/ml Basta
BASTA-tolerant fungal isolates could be divided into two groups: Majority (95%): fast-growing fungi (colonized a Petri dish within a few days). Anatomical features and ITS-sequencing : fungal isolates are saprophytic ascomycotic fungi (mainly Aspergillus) Survived surface sterilization and growth suppression by benomyl and BASTA.
BASTA-tolerant fungal isolates could be divided into two groups: Smaller portion (5%) much slower growing fungi. 14 different isolates according to growth properties and anatomical features. Sequencing of genomic rrna fragments revealed them as ascomycetes, too. However, they included previously characterized root endophytes (but not ECM fungi). this second class of Poplar fine roots fungi are colonized by endophytic fungi in addition to mycorrhizal fungi.
PCR amplification to investigate the presence of the bar gene in slow growing isolates. No bar gene signal was ever obtained from any of the slow growing BASTA-tolerant isolates These fungal isolates are naturally herbicide resistant.
Summary Neither under laboratory conditions nor under field conditions indications for a horizontal gene transfer from tree roots to ectomycorrhizal fungi was obtained