title: A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea.
abstract: Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen.
authors.affiliation: School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall Avenue, Bristol, BS8 1TQ, United Kingdom.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Ford
authors.affiliation: United States Department of Agriculture-Agricultural Research Service, 363 Hutchison Hall, University of California, One Shields Avenue, Davis, CA 95616, United States.
authors.forename: Kendra
authors.initials: K
authors.lastname: Baumgartner
authors.affiliation: The Royal Horticultural Society, Wisley, Woking, Surrey, GU23 6QB, United Kingdom.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Henricot
authors.affiliation: School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall Avenue, Bristol, BS8 1TQ, United Kingdom.
authors.forename: Andy M
authors.initials: AM
authors.lastname: Bailey
authors.affiliation: School of Biological Sciences, Life Sciences Building, University of Bristol, 24 Tyndall Avenue, Bristol, BS8 1TQ, United Kingdom.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Foster
mesh_terms: D000364:Agaricus / Q000235:genetics; D055392:Armillaria / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D002479:Inclusion Bodies / Q000235:genetics; D007438:Introns / Q000235:genetics*; D025282:Mycelium / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D020075:Phanerochaete / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins