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. 2019 Jun 20;116(28):14331–14338. doi: 10.1073/pnas.1905471116

Fig. 3.

Fig. 3.

HARP1 interacts with and stabilizes JAZ proteins. (A) Yeast two-hybrid assay. HARP1 was fused to GAL4 DNA-binding domain (BD), JAZ proteins of Arabidopsis (A. thaliana), cotton (G. hirsutum), and tobacco (N. benthamiana) were fused to GAL4 activation domain (AD), respectively. Interactions were examined with 1 mM 3-amino-1,2,4-triazole. (B and C) HARP1 reduces COI1-JAZ3 coprecipitation. Recombinant proteins of HIS-JAZ3 (B) and HIS-JAZ3δN (C) were incubated with total leaf proteins of the wild-type (WT) and 35S:6MYC-HARP1-1 (HARP1) Arabidopsis in the presence of 50 μM Coronatine. Anti-COI1 antibody was used to detect COI1 level before (Crude) or after (Pull down) immunoprecipitation. Anti-MYC antibody was used to detect the 6MYC-HARP1 (HARP1), and Anti-HIS antibody was used to detect HIS-JAZ3 and HIS-JAZ3δN. Band intensity was quantified by ImageJ and was shown under each blot. The intensity of the WT was set to 1. The relative COI1/HIS-JAZ3 ratios were listed in the bottom. The coimmunoprecipitation of COI1 with HIS-JAZ3 but not HIS-JAZ3δN was inhibited in the presence of HARP1. (D and E) HARP1 increases JAZ3 accumulation. The JAZ3-HA level is more stable in 35S:JAZ3-HA 35S:6MYC-HARP1 than in 35S:JAZ3-HA after 50 μM MeJA (D) or wounding (E) treatment. The plant leaves were collected 45 min after MeJA treatment or at the indicated time after wounding. Anti-HA antibody was used to detect JAZ3-HA (JAZ3). Band intensity was quantified by ImageJ and was shown under each blot. The intensity of the untreated 35S:JAZ3 –HA sample was set to 1. The relative JAZ3/COI1 ratios were listed in the bottom. The amount of total proteins in each loading was quantified by Bradford assay and visualized by CBB staining.