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Title:   Author  item
                                            Baumgartner, Kendraitem
                                            FOSTER, GARY - University Of Bristolitem
                                            BAILEY, ANDY - University Of Bristol
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/30/2010
                                    Publication  Date: 12/1/2010
                                    Citation: Baumgartner, K., Foster, G.D., Bailey, A.M. 2010. Agrobacterium-mediated transformation for the investigation of somatic recombination in the fungal pathogen Armillaria. Applied and Environmental Microbiology. 76:7990-7996.Interpretive Summary: The mushroom Armillaria mellea is a destructive soil-borne pathogen that infects over 300 plant species, and is of increasing interest due to its ability to aid in the process of biofuel production.  Here we report the study tool of genetic transformation of this species.  A range of techniques was evaluated, and Agrobacterium-mediated transformation was successful.  This was accomplished with A. tumefaciens carrying plasmid pBGgHg, which contains hygromycin phosphotransferase (hph) and intronless enhanced green fluorescent protein (eGFP) cassettes.  Success was dependent on testing both mycelium and basidiospores, the latter of which are obtained only from field-collected mushrooms.  In six independent transformation attempts of spores and mycelium of two Armillaria species (A. mellea, A. gallica), hygromycin-resistant colonies were obtained only with spores of A. mellea.  Hph was integrated into all transformants, as indicated by serial transfer to selection media, PCR, and RT-PCR.  eGFP was not viusalized, but was integrated and expressed at the RNA level, as indicated by PCR.  Southern hybridization confirmed random, mainly single copy integrations of the transforming DNA.  Microsatellite markers identified some diploid transformants, suggesting that either haploid spores mated during co-cultivation, or that some spores were diploid.  Transformants showed normal virulence against a susceptible host, indicating that transformation per se does not deter colonization, and so may be a useful tool to examine infection mechanisms.  Matings between haploid wild-types and haploid transformants created hygromycin-resistant diploids.  This genetic transformation system will advance research aimed at identification of Armillaria-tolerant crops and biofuel production by this important species.
                                Technical Abstract: The honey fungus Armillaria mellea is a destructive soil-borne pathogen that affects over 300 plant species, and is of increasing interest due to its ability to decompose lignin.  Here we report the transformation of this fungus.  A range of techniques was evaluated, and Agrobacterium-mediated transformation was successful.  This was accomplished with A. tumefaciens carrying plasmid pBGgHg, which contains hygromycin phosphotransferase (hph) and intronless enhanced green fluorescent protein (eGFP) cassettes.  Success was dependent on testing both mycelium and basidiospores, the latter of which are obtained only from field-collected mushrooms.  In six independent transformation attempts of spores and mycelium of two Armillaria species (A. mellea, A. gallica), hygromycin-resistant colonies were obtained only with spores of A. mellea.  Hph was integrated into all transformants, as indicated by serial transfer to selection media, PCR, and RT-PCR.  eGFP was not viusalized, but was integrated and expressed at the RNA level, as indicated by PCR.  Southern hybridization confirmed random, mainly single copy integrations of the transforming DNA.  Microsatellite markers identified some diploid transformants, suggesting that either haploid spores mated during co-cultivation, or that some spores were diploid.  Transformants showed normal virulence against a susceptible host, indicating that transformation per se does not deter colonization, and so may be a useful tool to examine infection mechanisms.  Matings between haploid wild-types and haploid transformants created hygromycin-resistant diploids.  This genetic transformation system will advance research aimed at pathogenesis and lignin degradation of this important species.


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