Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens

Pardo, Alejandro Guillermo; Hanif, Mubashir; Raudaskoski, Marjatta; Gorfer, Markus

doi:10.1017/s0953756201005378

Cited by 63 publications

(26 citation statements)

References 23 publications

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“…Although ble-containing plasmids have been reported to generate phleomycin-resistant transformants of H. cylindrosporum (40), Paxillus involutus (40), P. chrysosporium (15), S. commune (52), Suillus bovinus (40), and T. versicolor (1), the same ble gene was not able to give rise to phleomycin-resistant transformants of C. passeckerianus. Insertion of an intron at the 5Ј end of the gene led to the production of phleomycin-resistant transformants of C. passeckerianus.…”

Section: Vol 75 2009 Genetic Transformation Of Clitopilus Passeckermentioning

confidence: 99%

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Kilaru

Collins

Hartley

et al. 2009

Appl Environ Microbiol

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We describe efficient polyethylene glycol (PEG)-mediated and Agrobacterium-mediated transformation systems for a pharmaceutically important basidiomycete fungus, Clitopilus passeckerianus, which produces pleuromutilin, a diterpene antibiotic. Three dominant selectable marker systems based on hygromycin, phleomycin, and carboxin selection were used to study the feasibility of PEG-mediated transformation of C. passeckerianus. The PEG-mediated transformation of C. passeckerianus protoplasts was successful and generated hygromycinresistant transformants more efficiently than either phleomycin or carboxin resistance. Agrobacterium-mediated transformation with plasmid pBGgHg containing hph gene under the control of the Agaricus bisporus gpdII promoter led to hygromycin-resistant colonies and was successful when homogenized mycelium and fruiting body gill tissue were used as starting material. Southern blot analysis of transformants revealed the apparently random integration of the transforming DNA to be predominantly multiple copies for the PEG-mediated system and a single copy for the Agrobacterium-mediated system within the genome. C. passeckerianus actin and tubulin promoters were amplified from genomic DNA and proved successful in driving green fluorescent protein and DsRed expression in C. passeckerianus, but only when constructs contained a 5 intron, demonstrating that the presence of an intron is prerequisite for efficient transgene expression. The feasibility of RNA interference-mediated gene silencing was investigated using gfp as a target gene easily scored in C. passeckerianus. Upon transformation of gfp antisense constructs into a highly fluorescent strain, transformants were recovered that exhibited either reduced or undetectable fluorescence. This was confirmed by Northern blotting showing depletion of the target mRNA levels. This demonstrated that gene silencing is a suitable tool for modulating gene expression in C. passeckerianus. The molecular tools developed in this study should facilitate studies aimed at gene isolation or characterization in this pharmaceutically important species.

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Section: Vol 75 2009 Genetic Transformation Of Clitopilus Passeckermentioning

confidence: 99%

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Kilaru

Collins

Hartley

et al. 2009

Appl Environ Microbiol

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“…Agrobacteriummediated transformation is not restricted to eukaryotes as Agrobacterium is also able to transform the gram positive bacterium Streptomyces lividans. (Tsuji et al, 2003) Colletotrichum trifolii (Takahara et al, 2004) Coniothyrium minitans (Rogers et al, 2004;Li et al, 2005) Cryphonectria parasitica (Park and Kim, 2004) Fusarium circinatum (Covert et al, 2001) Fusarium culmorum (Michielse et al, 2005) Fusarium oxysporum (Mullins et al, 2001;Takken et al, 2004;Khang et al, 2005 (Michielse et al, 2005) Ophiostoma piceae (Tanguay and Breuil, 2003) Ophiostoma piliferum (Hoffman and Breuil, 2004) Paecilomyces fumosoroseus (Michielse et al, 2005) Paracoccidioides brasiliensis (Leal et al, 2004) Penicillium chrysogenum (Sun et al, 2002) Saccharomyces cerevisiae (Bundock et al, 1995;Piers et al, 1996;Risseeuw et al, 1996;Bundock et al, 2002) Trichoderma asperellum (Michielse et al, 2005) Trichoderma atroviride (Zeilinger, 2004) Pardo et al, 2002 ) …”

Section: Non-plant Organisms Transformed By Agrobacteriummentioning

confidence: 99%

Agrobacterium-Mediated Transformation of Non-Plant Organisms

Soltani

Heusden

Hooykaas

2008

Agrobacterium: From Biology to Biotechnology

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Abstract. During the last decade it became clear that the ability of Agrobacterium to transform host organisms is not restricted to plants, but that numerous other organisms are transformable by Agrobacterium under laboratory conditions. It has been shown that Agrobacterium-mediated transformation is possible for at least 80 different non-plant species. Most of these organisms are fungi including yeasts, but also mammalian cells and algae can be transformed. Agrobacterium-mediated transformation is not restricted to eukaryotes as Agrobacterium is also able to act on the gram positive bacterium Streptomyces lividans. In general, the procedures for the transformation of different organisms are similar, but each organism has its own conditions for optimal transformation efficiency. Nowadays Agrobacterium-mediated transformation is the method of choice for the transformation of various fungi as transformation efficiencies are much higher than with other methods and the transformation protocols are relatively facile. Agrobacterium can transfer not only DNA but also proteins to the host organisms through its type four secretion system. This protein transfer has been shown for both plants and the yeast Saccharomyces cerevisiae. A major issue in

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“…Such a tool is necessary to clarify the role of those different genes in the symbiosis (e.g., are new transport events switched on as a result of interactions between the symbiotic partners?). Recently, a transformation protocol for ectomycorrhizal fungi based on the capacity of Agrobacterium tumefaciens to transfer its T-DNA to fungal cells as it does to plant cells (Bundock et al 1995) was developed by Pardo et al (2002). This method represents an interesting alternative to the traditional protoplast-mediated transformation, as this protocol can be implemented on intact fungal hyphae, thus circumventing the need for making protoplasts, which is tedious, time-consuming, and restricted to a limited number of ectomycorrhizal fungal species.…”

Section: Introductionmentioning

confidence: 99%

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

et al. 2006

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Hebeloma cylindrosporum is a model fungus for mycorrhizal studies because of its fast growth rate, simple nutritional requirements, and completion of its life cycle in vitro, and because it is amenable to transformation. To advance cell biological research during establishment of symbiosis, a tool that would enable the direct visualisation of fusion proteins in the different symbiotic tissues [namely, the expression of reporter genes such as Green Fluorescent Protein (GFP)] was still a missing tool. In the present study, H. cylindrosporum was transformed using Agrobacterium carrying the binary plasmid pBGgHg containing the Escherichia coli hygromycin B phosphotransferase (hph) and the EGFP genes, both under the control of the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter. EGFP expression was successfully detected in transformants. The fluorescence was uniformly distributed in the hyphae, while no significant background signal was detected in control hyphae. The suitability of EGFP for reporter gene studies in Hebeloma cylindrosporum was demonstrated opening up new perspectives in the Hebeloma genetics.

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Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens

Cited by 63 publications

References 23 publications

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianus

Agrobacterium-Mediated Transformation of Non-Plant Organisms

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

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