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

Figure 5.

Figure 5.

SUMOylation of JAZ6 Is Enhanced during B. cinerea Infection.

(A) Immunoblots indicating significantly increased SUMOylation and protein abundance of GFP-tagged JAZ6 from 4-week-old (35S:JAZ6:GFP transgenics in wild-type background) plants infected with B. cinerea. Samples were collected at different time points after infection and mock-treated samples were used for immunoprecipitation with anti-GFP antibodies (IP: αGFP). Immunoblots (IB) were probed with GFP (IB:αGFP) or AtSUMO1/2 antibodies (IB:αSUMO1). Ponceau staining indicating Rubisco levels was employed to determine protein loading for the immunoprecipitation (IP:αGFP).

(B) Immunoblots probed with anti-HA antibodies showing HA-OTS1 levels in 35S::OTS1-HA transgenic Arabidopsis lines infected with B. cinerea. Four-week-old 35S::OTS1-HA transgenic Arabidopsis leaves were pressure infiltrated with B. cinerea and mock treated with magnesium chloride solution. Protein extracts were harvested from leaf samples collected at different time points after infection. Ponceau red stained Rubisco protein was used to indicate total protein levels.

(C) Immunoblots indicating greatly reduced SUMOylation and protein abundance of GFP-tagged JAZ6 from 15-d-old seedlings (35S:JAZ6:GFP transgenics in wild-type background) treated with 100 µM JA for 30 min. Protein samples were collected for immunoprecipitation with anti-GFP antibodies (IP: αGFP) at 0 and 30 min after treatment. Immunoblots (IB) were probed with GFP (αGFP) or AtSUMO1/2 antibodies (αSUMO1). Ponceau staining indicating Rubisco levels was employed to determine protein loading for the immunoprecipitation (IP:αGFP).

(D) Coimmunoprecipitation of HA-OTS1 with JAZ6:GFP in planta. Agrobacterium cultures containing 35S::HA-OTS1 were mixed with Agrobacterium cultures containing either 35S::GFP or 35S::JAZ6:GFP and transiently expressed in N. benthamiana. Total protein was extracted for immunoprecipitation with anti-GFP beads. Immunoprecipitates were analyzed by immunoblotting using anti-HA and anti-GFP antibodies to detect for the presence of OTS1-HA or JAZ6:GFP, respectively. Ponceau red stained Rubisco protein was used to indicate total protein levels used in the immunoprecipitation in the time points