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. 2021 Sep 16;12:5479. doi: 10.1038/s41467-021-25748-4

Fig. 3. XopC2 enhances JA signaling by promoting ubiquitination and degradation of JAZ proteins.

Fig. 3

a, b MeJA-induced expression of the JA-responsive genes OsLOX2 (a) and OsJAZ8 (b) was enhanced in XopC2-expressing rice seedlings. The wild-type, IE-17, IE-D391A-2, and IE-N396A-14 transgenic seedlings were treated with DEX or mock solution for 24 h followed by MeJA application (50 μM). Gene expression was detected by qRT-PCR using OsActin as an internal reference gene. Data are presented as means ± SE (n = 3 replicates per measurement). Asterisk (*) indicates a statistically significant difference in relative gene expression between mock and DEX treatments (two-sided t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). c The accumulation of OsJAZs-HA was greatly reduced in rice protoplasts conditionally expressing XopC2-FLAG. OsJAZ7-HA and OsJAZ9-HA were transiently expressed in IE-17 transgenic rice protoplasts after mock and DEX treatments for 12 h. Western blotting was performed to detect OsJAZs-HA, XopC2-FLAG, and β-OsActin (as a protein loading control). MG132, a proteasome inhibitor. d OsJAZ9-HA was rapidly degraded during Xoc infection but remained relatively stable during ΔxopC2 infection. Three-week-old transgenic seedlings expressing OsJAZ9-HA driven by its native promoter were sprayed with the wild-type, ΔxopC2, and ΔxopC2 strains complemented with a wild-type copy of xopC2 (C-ΔxopC2) or a kinase-defective copy (C-ΔxopC2D391A). OsJAZ9-HA was detected by immunoblotting at the indicated timepoints post-inoculation. e OsJAZ9 ubiquitination was enhanced in the presence of XopC2 in a semi-in vitro assay. After incubation with His6-OsJAZ9, NTA beads were divided equally and were mixed with GST-XopC2, HA-ubiquitin (Ub), and total rice protein extracts (TRPE) as indicated in the ubiquitination buffer. OsJAZ9 ubiquitination was detected at the indicated timepoints by immunoblotting with an anti-HA-HRP antibody. The protein loading was shown by CBB staining. f OsJAZ9 ubiquitination was enhanced in the presence of GST-XopC2 revealed by a refined semi-in vitro assay. The SCFOsCOI1b complex was immunoprecipitated with anti-FLAG M2 affinity beads from the extract of rice protoplasts co-expressing OsCOI1b-FLAG, OsCullin1a-HA, OSK1-HA, and OsRBX1-HA. The SCFOsCOI1b complex was then incubated with human UBE1 (E1), UBCH5α (E2), HA-ubiquitin (Ub), His6-OsJAZ9, coronatine (COR), and GST-XopC2/XopC2D391A/XopC2N396A in the ATP-containing reaction buffer. OsJAZ9 ubiquitination was detected by immunoblotting with anti-HA-HRP and anti-His-HRP antibodies. The experiments were independently repeated 3 times with similar results in the panels a–f.