Helber and Requena 2008

Expression of the flourescence markers DsRed and GFP fused to a nuclear localization signal in the arbuscular mycorrhizal fungas Glomus intraradices, New Phytologist. 177: 537-548.

Helber, N. and Requena N.

Abstract

Here, arbuscular mycorrhizal (AM) fungi were monitored in vivo introducing the fluorescent reporters DsRed and GFP (green fluorescent protein) in Glomus intraradices using a biolistic approach and Agrobacterium tumefaciens-mediated transformation. Both reporter genes were fused to the nuclear localization signal of the Aspergillus nidulans transcription factor StuA to target fluorescence to nuclei. Expression of DsRed was driven by two Glomus mosseae promoters highly expressed during early symbiosis, GmPMA1 and GmFOX2, while expression of GFP was driven by the A. nidulans gpd promoter. All promoters worked in G. intraradices as well as in A. nidulans. Red and green fluorescence was localized to nuclei of G. intraradices spores and hyphae 3 d after bombardment. However, expression was transient. The efficiency of the Agrobacterium-mediated transformation was very low. These results indicate that the biolistic method allows the expression of foreign DNA into G. intraradices with high frequency, but it is insufficient to render stable transformants. DsRed vs GFP is a more appropriate living reporter to be used in G. intraradices because of the lower autofluorescence in the red channel but targeted to the nucleus both marker genes can be visualized. This is the first report of fluorescent marker expression in an AM fungus driven by arbuscular mycorrhizal promoters.