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

Fig 6. APC10 negatively regulates DRB4 levels.

Fig 6

(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.