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. 2018 Aug 16;30(9):2099–2115. doi: 10.1105/tpc.18.00036

Figure 7.

Figure 7.

SUMOylated JAZ6 Negatively Regulates COI1-JAZ6 Interaction.

(A) GST pull-down assays indicate that interaction between His:JAZ6 and GST:COI1 is weakened by the addition of increasing amounts of His:SUMO1 protein. His:JAZ6 protein mixed with different amounts of His:SUMO1 and pulled down with either GST:COI1 or GST alone in the presence or absence of coronatine (10 µM). The eluates were then probed with anti-His tag (αHis) or anti-GST (αGST) antibodies to detect His:JAZ6 or GST-tagged proteins, respectively.

(B) GST pull-down assays performed as above but with GST:COI1 replaced by the COI1 SIM mutant GST:COI1V553 indicate that mutation of Val to Ala rescues the interaction between GST:COI1V553A and His:JAZ6 even in the presence of His:SUMO1 protein. The eluates were probed with anti-His tag (αHis) or anti-GST (αGST) antibodies to detect His-JAZ6 or GST-tagged proteins, respectively.

(C) Coimmunoprecipitation of JAZ6:GFP with myc:COI1 in planta indicates that SUMOylated JAZ6:GFP binds to myc:COI1 even in the absence of JA mimic coronatine. Agrobacterium culture containing 35S::JAZ6:GFP was mixed with Agrobacterium cultures containing both 35S::myc:COI1 and 35S::HA:SUMO1 and transiently expressed in N. benthamiana. Total protein was extracted for immunoprecipitation with anti-myc antibodies (IP; αmyc) to pull down myc-COI1 and the immunoprecipitates were probed with anti-SUMO, anti-myc (IB: αmyc), and anti-GFP(IB: αGFP) antibodies to detect for the presence of SUMOylated and non-SUMOylated JAZ6:GFP and myc:COI1. Ponceau staining indicating Rubisco levels was employed to determine protein loading for the immunoprecipitation (IP:αGFP).

(D) In vivo degradation of JAZ6 was observed in coinfiltration experiments with increasing amounts of HA:COI1 or HA:COI1V553A in presence of 50 µM coronatine. The ratio of the relative concentration of agrobacteria used in the different coinfiltrations is indicated by numbers (top). Cell extracts were analyzed by immunoblot analysis with anti-GFP and anti-HA antibodies. Immunoblot analysis indicated that JAZ6:GFP was more unstable in plants transiently expressing HA:COI1V553A when compared with plants expressing HA:COI1. Ponceau red-stained Rubisco protein was used as a loading control.