Extended Data Fig. 5: RHA3A/B ubiquitinate BIK1 in vivo.
From: Ligand-induced monoubiquitination of BIK1 regulates plant immunity
a, GST–RHA3ACD possesses E3 ligase activity in vitro. An in vitro ubiquitination assay was performed with GST–RHA3ACD followed by deubiquitination reactions with GST–USP2-cc. N-ethylmaleimide (NEM) (10 mM), an inhibitor of deubiquitinases, and heat-inactivated (HI, 95 °C for 5 min) USP2-cc are controls. Samples were analysed by SDS–PAGE and silver staining. b, GST–RHA3ACD possesses multi-monoubiquitination activity in vitro. A ubiquitination assay was done as in a but using the ubiquitin mutant with all lysine residues mutated to arginine (UBQ(K0)). Ubiquitinated proteins were detected by immunoblotting with anti-UBQ (left) or anti-RHA3A (right) antibodies. c, RHA3 expression in T-DNA insertion mutants. RHA3A expression in the T-DNA knockout line SALK_052714 and RHA3B expression in SALK_064303 were analysed as in Extended Data Fig. 4b. Mean ± s.e.m. fold change (WT set as 1.0); two-tailed Student’s t-test, n = 3. d, Screen for the optimal amiR-RHA3A and amiR-RHA3B. Protoplasts were transfected with RHA3A-HA or RHA3B-HA with control, amiR-RHA3A or amiR-RHA3B. RHA3A or RHA3B proteins were examined by immunoblotting with anti-HA antibody. e, RHA3A and RHA3B are required for BIK1 ubiquitination in vivo. A BIK1 ubiquitination assay was carried out by co-expressing control, artificial microRNA targeting RHA3A (amiR-RHA3A) or amiR-RHA3A together with microRNA targeting RHA3B (amiR-RHA3A amiR-RHA3B). f, RHA3A and RHA3B expression in amiR-RHA3A/B transgenic plants. qRT–PCR was carried out to detect RHA3A and RHA3B transcripts with ACTIN2 as a control. Mean ± s.e.m. fold change in gene expression from two independent transgenic lines (lines 1 and 2); one-way ANOVA, n = 5. g, RHA3A and RHA3B are required for BIK1 ubiquitination in transgenic plants. Protoplasts from amiR-RHA3A/B transgenic plants were transfected with BIK1–HA and FLAG–UBQ for ubiquitination assay. Bottom, quantification of BIK1 ubiquitination in amiR-RHA3A/B transgenic plants. Intensity of Ub-BIK1 or BIK1 bands was quantified with Image Lab (Bio-Rad). The amount of BIK1 ubiquitination is the relative intensity of the Ub-BIK1 band to the BIK1 band (no treatment in wild-type set as 1.0). Mean ± s.e.m.; different letters indicate significant difference with others (P < 0.05, one-way ANOVA, n = 3). h, Sequencing analysis of RHA3A and RHA3B genes in the CRISPR–Cas9 rha3a/b mutant. PCR fragments corresponding to RHA3A and RHA3B in rha3a/b were amplified, sequenced, and aligned to wild-type coding sequences. The reverse complement of the PAM sequence is underlined in red, and red arrowheads indicate the theoretical Cas9 cleavage sites. The experiments were repeated three times with similar results.
