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. 2018 Mar 7;14(3):e1006894. doi: 10.1371/journal.ppat.1006894

Fig 3. COP1 regulates DRB1 and DRB4 levels and thereby TCV resistance.

Fig 3

(A-B) Western blots showing relative levels of DRB1 (A), and DRB4 (B) in indicated genotypes. Ponceau-S staining of the Western blots was used as the loading control. Arrows indicate the target protein corresponding to the indicated antibody. This experiment was repeated three times with similar results. (C) Co-immunoprecipitation (IP) assay carried out between DRB4-Myc and COP1-Flag proteins. DRB4 and COP1 were expressed under their native or 35S promoters, respectively, and the transgenic plants were crossed to create a line co-expressing both the proteins. The immunoprecipitated proteins were analyzed with α-Myc and α-Flag and this experiment was repeated twice with similar results. (D) IP assay carried out between DRB1-Myc and DRB4 proteins. DRB1-Myc was expressed under its native promoter and the immunoprecipitated proteins were analyzed with α-Myc and α-DRB4 and this experiment was repeated twice with similar results. (E) HR formation in TCV-inoculated Di-17, Col-0, Col-0 containing an introgressed copy of HRT and HRT drb genotypes at 3 dpi. The HR phenotype of HRT drb plants was evaluated in ~40–50 plants per genotype that were analyzed in five to seven separate experiments. (F) Trypan blue stained leaves showing microscopic cell death phenotype at 3 dpi with TCV. Scale bars, 270 microns. At least five independent leaves were analyzed with similar results. (G) RNA gel blot analysis showing expression of PR-1 in indicated genotypes after inoculation with TCV. Total RNA was extracted from inoculated leaves at 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. H/H and H/h indicate plants homozygous or heterozygous for HRT, respectively. The experiment was repeated twice with similar results. (H) RNA gel blot analysis showing relative levels of genomic CP RNA in indicated genotypes inoculated with TCV. Leaves were sampled at 3 dpi. Ethidium bromide staining of rRNA was used as the loading control. This experiment was repeated three times with similar results.