Fig 1.
COP1 is a positive regulator of HRT-mediated defense against TCV.
(A) HR formation in TCV-inoculated HRT COP1 and HRT cop1 genotypes at 3 dpi. The HR phenotype was evaluated in ~30 plants that were analyzed in three separate experiments. (B) Trypan blue stained leaves showing microscopic cell death phenotype at 3 dpi with TCV. Scale bars, 270 microns. At least six independent leaves were analyzed with similar results. (C) Real-time quantitative RT-PCR analysis showing relative expression levels of PR-1 in mock- and pathogen-inoculated plants. Leaves were sampled 24 h post treatments. The error bars indicate SD (n = 3). Asterisk denotes significant differences from mock-treated leaves (t test, P<0.003). Results are representative of two independent experiments. (D) Western blot showing relative CP levels in indicated genotypes inoculated with TCV. Leaves were sampled at 3 dpi. Ponceau-S staining of Rubisco was used as the loading control. This experiment was repeated two times with similar results. (E) Western blots showing relative levels of HRT-Flag in indicated genotypes expressing HRT-Flag transgene. Ponceau-S staining of the Western blots was used as the loading control. This experiment was repeated three times with similar results. (F) Real-time quantitative RT-PCR analysis showing relative expression levels of HRT in indicated genotypes. The error bars indicate SD (n = 3). Results are representative of two independent experiments. (G) Typical morphological phenotypes of TCV inoculated HRT COP1 (Di-17 ecotype), HRT cop1 and hrt (Col-0 ecotype) plants. Plants were photographed at 18 dpi. (H) Western blot showing relative CP levels in distal bolt tissues of indicated genotypes. Plants were inoculated with TCV and the distal uninoculated tissues were sampled at 3 dpi. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated two times with similar results. (I) Western blot showing relative CP levels in mock- and TCV-inoculated genotypes. Plants were inoculated with buffer or TCV and the inoculated tissues were sampled at 3 dpi. Ponceau-S staining of the western blot was used as the loading control. This experiment was repeated two times with similar results.
Fig 2.
COP1 is a positive regulator of RPM1-mediated defense against Pst.
(A) Western blot showing relative levels of RPM1-Myc in wild-type and cop1 plants. Ponceau-S staining of the western blots was used as the loading control. Arrow indicates the target protein corresponding to the indicated antibody. This experiment was repeated three times with similar results. (B) Growth of Pst avrRpm1 on cop1. Error bars indicate SD. Asterisks indicate data statistically significant from that of control (Col-0) (P<0.05, n = 4). (C) IP of COP1-Myc with RPM1-Flag. N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-Myc and α-Flag. This experiment was repeated twice with similar results. (D) IP of COP1-Flag with RPM1-Myc. RPM1-Myc and COP1-Flag were expressed under their native or 35S promoters, respectively. Arrows indicate the target protein corresponding to the indicated antibody. The immunoprecipitated proteins were analyzed with α-Myc and α-Flag and this experiment was repeated twice with similar results.
Fig 3.
COP1 regulates DRB1 and DRB4 levels and thereby TCV resistance.
(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.
Fig 4.
DRB proteins are required for the stability of HRT.
(A) HR formation in TCV-inoculated Di-17, Col-0 and HRT drb genotypes at 10 dpi. The HR phenotype was evaluated in ~20–30 plants that were analyzed in four separate experiments. (B) Western blots showing relative levels of HRT-Flag in Di-17 and drb genotypes expressing HRT-Flag transgene. Ponceau-S staining of the Western blots was used as the loading control. This experiment was repeated three times with similar results. (C and D) IP of DRB1-Myc (C) and DRB2-Myc (D) with HRT-Flag. All proteins were expressed under their respective native promoters in Arabidopsis. The immunoprecipitated proteins were analyzed with α-Myc and α-Flag and this experiment was repeated twice with similar results. (E) Visual phenotype of Nicotiana benthamiana leaves expressing indicated proteins. Agroinfiltration was used to express HRT, CP, EDS1 (E90-At3g48090), and DRB1, DRB3 or DRB4 proteins. The leaf was photographed at 4 days post treatment. (F) Trypan blue stained leaves of Di-17 and transgenic plants overexpressing DRB1, DRB3 and DRB4 in Di-17 background. The plants were inoculated with TCV and the inoculated leaves were sampled at 36 h post inoculation. Scale bars, 270 microns. At least four independent leaves were analyzed with similar results. (G) Electrolyte leakage in genotypes shown in F. The leaves were sampled at 0 and 24 h post TCV inoculation. Error bars represent SD. Asterisks indicate data statistically significant from that of control (Col-0) (P<0.05, n = 4).
Fig 5.
DRB proteins interact with CP.
(A) Relative levels of DRB1 in nucleus and cytosolic fractions of Arabidopsis plants expressing DRB1-Myc under its self promoter. The blot was sequentially probed with indicated antibodies. Ponceau-S staining of the Western blot was used as the loading control. This experiment was repeated two times with similar results. Fold change, normalized with Rubisco, Actin or H3 proteins, in western blots was quantified using Image Quant software. (B-D) Co-IP of DRB1-Flag, (B) DRB2-Flag (C) and DRB5-Flag (D) in the presence of TCV. The transgenic Arabidopsis plants expressing DRB1-Flag, DRB2-Flag, and DRB5-Flag under their respective native promoters (NP) were inoculated with TCV and leaves sampled at 3 dpi were processed for Co-IP. The TCV inoculated Col-0 plants were used as a negative control. These experiments were repeated twice with similar results. (E and F) Co-IP of DRB1-Myc or DRB4-Myc with HRT-Flag in the presence or absence of TCV (E) or CP (F). Arabidopsis expressing DRB1 and HRT under their native promoters were used in E. For transient assays shown in F, N. benthamiana plants were agroinfiltrated and immunoprecipitated proteins were analyzed with α-Myc and α-Flag. The experiments shown in E and F were repeated two times with similar results.
Fig 6.
APC10 negatively regulates DRB4 levels.
(A) Western blot showing DRB4 levels in cop1 plants infiltrated with 10 or 50 μM MG132. The control (cnt) plants were infiltrated with DMSO and the leaves were sampled 24 h post infiltration. The Col-0 and drb4 plants were used as additional controls. (B) Western blots showing relative levels of DRB4 (upper panel), CP (middle panel) or DRB1 (lower panel) in APC10 overexpressing (OE) plants. Ponceau-S staining of the Western blots was used as the loading control. This experiment was repeated three times with similar results. (C) Quantitative RT-PCR analysis showing relative levels of APC10, DRB1 and DRB4 transcripts in wild-type (Col-0) and APC10 overexpressing (OE) plants. This experiment was repeated twice using two or more independent cDNA preparations as templates. (D) Proposed model of regulation of HRT levels by COP1, DRB1 and DRB4 proteins. HRT interact with DRB1 (D1), DRB4 (D4) and COP1 proteins. COP1 interacts with DRB1 [24], but not with DRB4. Moreover, DRB1 and DRB4 do not interact with each other. Although a mutation in either DRB1 or DRB4 results in degradation of HRT, only a mutation in DRB1 abolishes HR to TCV. These observations suggest that DRB1 and DRB4 might form separate complexes with HRT. COP1 was recently shown to stabilize DRB1 by negatively regulating an unknown protease [24]. Likewise, DRB4 was previously shown to interact with APC10 E3 ligase [30], which negatively regulates DRB4 levels. Thus, COP1 might be protecting DRB1 and DRB4 proteins by negatively regulating a putative protease or APC10, respectively. A loss of COP1 will therefore result in the activation of protease or E3 ligase, which in turn will degrade DRB1 and DRB4 proteins, respectively. Alternately, COP1- and APC10-mediated regulation of DRB4 could be independent processes that rely on the relative levels of APC10 in the cell. Together, these results show that components of the RNA silencing pathway and photomorphogenesis are intricately associated with the stability/activation of the R proteins.