Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus
Fig 5
Microscopic analysis of CaMV11P6-infected A. thaliana.
(A) Arabidopsis thaliana GFP1-10 leaves were analyzed by confocal fluorescence microscopy at 3 and 40 days after inoculation (dpi) with CaMV11P6. GFP fluorescence and chloroplast autofluorescence are presented in green and grey, respectively. The greenish spots in the leaf tissue at 3 dpi are not due to GFP since they also fluoresced in blue when excited at 405 nm. The inset in the third panel shows details of an 11P6 inclusion in which darker circular spots are visible (yellow arrows). (B) CaMVwt-infected tissue sections were immunolabeled (magenta) at 28 dpi using the antisera (αP2 or αP6) as indicated. The yellow arrows point to the stronger stained cortex of immunostained VFs, the inset presents details of a VF. (C) CaMV11P6-infected tissue was immunolabeled at 40 dpi using P2 antiserum (magenta) and 11P6 was visualized by fluorescence of the reconstituted split GFP (green). The inset shows a confocal single section of such a green fluorescing inclusion labeled with P6 antiserum (magenta). Note that only the cortex of the inclusion is labeled. The yellow arrows point to putative VF lacunae. Cell walls in (B) and (C) were stained with Fluorescent Brightener 28 and are presented in blue. All confocal images are maximum projections except where indicated. (D) and (E) Transmission electron micrographs presenting (D) a CaMVwt and (E) a CaMV11P6-infected cell, both fixed at 33 dpi. VF and TB designate virus factories and transmission bodies, respectively; yellow lines (labeled “VP”) and blue arrows point to virus particles and lacunae, respectively. Different microscopes were used for image acquisition in (D) and (E). Brightness and contrast were corrected to allow better comparison of the micrographs. Scale bars in (A) 50 μm for the overviews and 5 μm for the inset, in (B) and (C) 10 μm and in (D) and (E) 1 μm.