Fluorescent labeling of peroxisome and nucleus in Botrytis cinerea

Abstract:

[Objective] To label the nuclei and peroxisomes of Botrytis cinerea with fluorescent proteins, as a tool for further investigation on biogenesis and dynamics of the cellular structures of the fungus. [Methods] The green fluorescent protein (GFP) and the red fluorescent proteins (DsRed and mCherry) were used as reporter proteins to label the nuclei and peroxisomes in B. cinerea. Three fluorescent labeling vectors were separately introduced into the B. cinerea strain B05.10 via Agrobacterium tumefaciens-mediated transformation. The resulting transformants were selected and confirmed by PCR, and then analyzed with the confocal fluorescent microscope. [Results] The single-spore purified recombinant strains expressing red or green fluorescence were obtained. The PCR amplification and fluorescence detection indicated that the fluorescent genes were integrated into the genome of the transformants. Round fluorescent spots were detected in mycelia and conidia of the strains with nuclear labeling, overlapping well with DAPI staining. In the strains with peroxisomes labeling, small green or red fluorescent dots were present in mycelia and conidia. Upon induction on lipids, the number of the dots was significantly increased, corresponding well with the described distribution profile of peroxisomes. In addition, the blue fluorescence produced by Calcofluor white staining did not interfere with the fluorescence of red or green fluorescent proteins, capable of forming well-integrated multi-fluorescent images. [Conclusion] We obtained the B. cinerea strains with fluorescent labeled nuclei or peroxisomes, which are maybe ideal tools for studying the biogenesis and dynamics of cellular organelles, the developments and even the pathogenesis of the fungus.