Fig. 2: Resistance conferred by TaHAKAI^R is dependent on ubiquitination activity.

A Phylogenetic analysis of TaHAKAIs from different species. HAKAI protein sequences from Hordeum vulgare (Hv), Zea mays (Zm), Oryza sativa (Os), and Arabidopsis thaliana (At) were identified. B Conserved RING ___domain analysis of TaHAKAI-A, TaHAKAI-B and TaHAKAI-D among different plant species. The asterisk indicates the conserved RING ___domain amino acid residue. C In vitro ubiquitination assay of TaHAKAI^R. n = 3 biologically independent experiments. The TaHAKAI^R-mbp protein purified from E. coli was incubated with E1, E2, Ub, and ATP in the reactions. Immunoblot analysis was performed with anti-MBP and anti-ubiquitin antibodies. The asterisks represent the positions of the MBP and TaHAKAI^R-MBP size. D Determination of viral RNA replication levels via qRT‒PCR at different times. The value is the mean ± SD (ANOVA with two-sided t-test; n = 3 biologically independent experiments, ‘**’ p < 0.01, ‘ns’ not significant). The p value are as follows. ‘0.0606’, ‘0.0551’ and ‘0.9968’ in 6 h; ‘0.0014’, ‘0.0022’ and ‘0.8653’ in 12 h; ‘0.0043’, ‘0.0019’ and ‘0.6328’ in 24 h; ‘0.0003’, ‘0.0007’ and ‘0.5142’ in 36 h; ‘<0.0001’, ‘<0.0001’ and ‘0.8435’ in 48 h. E Detection of WYMV protein accumulation using WB. The value is the mean ± SD (n = 3 biologically independent experiments). Ponceau staining was used to assess protein loading. F The relative abundance of the CP protein in (E). The value is the mean ± SD (ANOVA with two-sided t-tests; n = 3 biologically independent experiments, ‘*’ p < 0.05, ‘**’ p < 0.01). 35S-GFP vs TaHAKAI^R-GFP, p = ‘0.0070’; 35S-GFP vs TaHAKAI^R(H111Y)-GFP, p = ‘0.0384’; TaHAKAI^R-GFP vs TaHAKAI^R(H111Y)-GFP, p = ‘0.0006’. G, I Dot blotting was used to determine the m⁶A modification levels of host RNA and viral RNA using an anti-m⁶A antibody. MB staining of total RNA and viral RNA served as equal loading controls. H, J Quantitative statistics were performed on the m⁶A level in (G, I). The value is the mean ± SD (ANOVA with two-sided t-test; n = 3 biologically independent experiments, ‘**’ p < 0.01, ‘ns’ not significant). The p value in H is as follows. ‘<0.0001’, ‘<0.0001’and ‘0.9954’ in 500 ng; ‘0.0002’, ‘0.0003’ and 0.8885 in 250 ng. The p value in J is as follows. ‘0.0015’, ‘0.0019’ and ‘0.9695’ in 500 ng; ‘0.0013’, ‘0.0011’ and ‘0.9819’ in 250 ng.