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ARS Home » Pacific West Area » Davis, California » Crops Pathology and Genetics Research » Research » Publications at this Location » Publication #329593
Title: A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

Author
item FORD, KATHRYN - University Of Bristol
item Baumgartner, Kendra
item HENRICOT, BEATRICE - The Royal Horticultural Society, Uk
item BAILEY, ANDREW - University Of Bristol
item FOSTER, GARY - University Of Bristol

Submitted to: Nature Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2016
Publication Date: 7/7/2016
Citation: Ford, K.L., Baumgartner, K., Henricot, B., Bailey, A.M., Foster, G.D. 2016. A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea. Nature Scientific Reports. 6: 29226. Doi: 10.1038/srep29226.

Interpretive Summary: Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. We previously developed a method to genetically transform the fungus, but the technique was not very efficient; we had to transform tens of thousands of spores, just to be successful with a few dozen. To improve efficiency of the method, we used a yeast-adapted pCAMBIA0380 Agrobacterium vector, to allow easy exchange of promoters and inclusion of introns within the transgene. 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 successfully used 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 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.

Technical 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 successfully used 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 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.