Fig. 5: TaHAKAIR phosphorylation enhances its ubiquitination activity.
From: An m6A methyltransferase confers host resistance by degrading viral proteins through ubiquitination
A An LCI assay was used to confirm the interaction between TaHAKAIR, TaHAKAIS, and TaHAKAID with P2. P, positive control; N, negative control. B Relative fluorescence intensity of TaHAKAIR, TaHAKAIS, and TaHAKAID with P2. The p value is ‘0.7994’, ‘0.4266’, ‘0.9548’ and ‘<0.0001’, respectively. C An MST assay was used to determine the differences in the binding affinities of TaHAKAIR-mbp, TaHAKAIS-mbp and TaHAKAID-mbp for P2-GST. The MBP + P2-GST group was used as a negative control. The value is the mean ± SD, n = 3 biologically independent experiments. D Coinfiltration experiment of TaHAKAIR, TaHAKAIS, and TaHAKAID with P2. Ponceau staining was used to assess protein loading. E Ubiquitination assay of TaHAKAIR, TaHAKAIS, and TaHAKAID in vitro. Coomassie Brilliant Blue (CBB) assay was performed for protein loading. F Dot blot analysis of TaHAKAIR, TaHAKAIS, and TaHAKAID on the overall m6A modification levels in wheat. G Quantitative statistical analysis of the m6A level in (F). The p value in 500 ng: p = ‘0.0003’, p < ‘0.0001’, p = ‘0.0002’, p = ‘0.5346’, p = ‘0.9987’ p = ‘0.6177’. The p value in 250 ng: p = ‘0.0003’, p = ‘0.0002’, p = ‘0.0001’, p = ‘0.9979’, p = ‘0.7407’ p = ‘0.8307’. H Determination of viral RNA replication levels. 35S-GFP vs TaHAKAIR-GFP, p = ‘0.0053’; 35S-GFP vs TaHAKAIS-GFP, p = ‘0.0039’; TaHAKAIR-GFP vs TaHAKAIS-GFP, p = ‘0.9471’. I Determination of WYMV CP protein accumulation. Ponceau staining was used to assess protein loading. J The relative abundance of the CP protein in (I). 35S-GFP vs TaHAKAIR-GFP, p = ‘0.0421’; 35S-GFP vs TaHAKAIS-GFP, p = ‘0.0039’; TaHAKAIR-GFP vs TaHAKAIS-GFP, p = ‘0.0003’. K Dot blot analysis of TaHAKAIR/S on the overall m6A modification levels in WYMV genomic RNA. L Quantitative statistics on the m6A level in (K). The p value in 500 ng: p = ‘0.0016’, p < ‘0.0027’ and p = ‘8145’. The p value in 250 ng: p = ‘0.0001’, p = ‘0.0001 and p = ‘0.9919’. In B, G, H, J, L, the value is the mean ± SD (ANOVA with two-sided t-tests; n = 3 biologically independent experiments, ‘*’ p < 0.05, ‘**’ p < 0.01, ‘ns’ not significant).
