A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea.

Kathryn L Ford,Gary D Foster,Béatrice Henricot,Andy M Bailey,Kendra Baumgartner

doi:10.1038/srep29226

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.

Highlights

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide
An additional molecular tool that has proven to be useful in other fungi, but is currently unavailable for Armillaria, is the expression of reporter genes
Earlier transformation work with A. mellea[15] used a vector previously developed for Agaricus bisporus, pBGgHg30, containing intronless hygromycin resistance and GFP cassettes driven by the A. bisporus

Summary

Introduction

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. An additional molecular tool that has proven to be useful in other fungi, but is currently unavailable for Armillaria, is the expression of reporter genes Reporter genes such as luciferase (LUC), (β-glucuronidase (GUS), green fluorescent protein (GFP) and red fluorescent protein (DsRed) and their derivatives have been used to analyse pathogen-host interactions in planta[17,18,19], facilitate early detection of infection in pathogenicity assays[20], permit promoter:reporter gene fusions to study gene expression patterns and localisation[21,22] and to assess requirements for heterologous gene expression[23,24,25,26]. Correspondence and requests for materials should be addressed to www.nature.com/scientificreports/

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