Nucleus-directed fluorescent reporter system for promoter studies in the ectomycorrhizal fungus Laccaria bicolor.

Currently ectomycorrhizal research suffers from a lack of molecular tools specifically adapted to study gene expression in fungal symbionts. Considering that, we designed pReNuK, a cloning vector for transcriptional promoter studies in the ectomycorrhizal basidiomycete Laccaria bicolor. The pReNuK vector offers the use of a nuclear localizing and chromatin incorporating histone H2B-mCherry fluorescent reporter protein and it is specifically optimized for efficient transgene expression in Laccaria. Moreover, pReNuK is designed to work in concert with Agrobacterium-mediated transformation under hygromycin B resistance selection. The functionality of the pReNuK reporter system was tested with the constitutive Laccaria glyceraldehyde 3-phosphate dehydrogenase gene promoter and further validated with the nitrogen source regulated nitrate reductase gene promoter. The expression of the nucleus-directed H2B-mCherry reporter is highly stable in time. Moreover, the transformation of Laccaria with pReNuK and the expression of the reporter do not have negative effects on the growth of the fungus. The pReNuK offers a novel tool for studying in vivo gene expression regulation in Laccaria, the leading fungal model for ectomycorrhizal research.

Improved GFP Variants to Study Gene Expression in Haloarchaea.

The study of promoter activities in haloarchaea is carried out exclusively using enzymes as reporters. An alternative reporter is the gene encoding the Green Fluorescent Protein (GFP), a simple and fast tool for investigating promoter strengths. However, the GFP variant smRS-GFP, used to analyze protein stabilities in haloarchaea, is not suitable to quantify weak promoter activities, since the fluorescence signal is too low. We enhanced the fluorescence of smRS-GFP 3.3-fold by introducing ten amino acid substitutions, resulting in mGFP6. Using mGFP6 as reporter, we studied six haloarchaeal promoters exhibiting different promoter strengths. The strongest activity was observed with the housekeeping promoters Pfdx of the ferredoxin gene and P2 of the ribosomal 16S rRNA gene. Much lower activities were determined for the promoters of the p-vac region driving the expression of gas vesicle protein (gvp) genes in Halobacterium salinarum PHH1. The basal promoter strength dropped in the order PpA , PpO > PpF , PpD . All promoters showed a growth-dependent activity pattern. The GvpE-induced activities of PpA and PpD were high, but lower compared to the Pfdx or P2 promoter activities. The mGFP6 reporter was also used to investigate the regulatory effects of 5'-untranslated regions (5'-UTRs) of three different gvp mRNAs. A deletion of the 5'-UTR always resulted in an increased expression, implying a negative effect of the 5'-UTRs on translation. Our experiments confirmed mGFP6 as simple, fast and sensitive reporter to study gene expression in haloarchaea.

Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.

Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina.