Fig. 5: TaMTB positively regulates m6A level of WYMV R1.
From: N6-methyladenosine RNA modification promotes viral genomic RNA stability and infection
a RNA immunoprecipitation (RIP) of WYMV RNAs with a specific anti-m6A antibody. Total RNA extracted from WYMV-infected wheat plants was incubated with anti-m6A plus IgA or IgA alone. Dilutions of the immunoprecipitated RNAs were blotted on Hybond-N+ membrane and detected with Dig-WYMV. b m6A peaks in WYMV RNA1 and RNA2. The detail peaks and viral gene annotation are shown in Supplementary Table 2. Top numbers show the full length of the analyzed RNA segments, bp, base-pair. Green color lines are the m6A region. Red, IP peak. Gray, Input peak. c Real-time fluorescence amplification curves and bar plot of the threshold cycle (CT) of qPCR showing SELECT result for detecting the m6A site in peak3 of RNA 1. Rn is the raw fluorescence for the associated well normalized to the fluorescence of the passive reference dye (ROX). Values are means ± SD (two-sided t test, n  =  3, P  <  0.0001). **P  <  0.01. d m6A-IP-qPCR assay of peak3 in WYMV-infected wheat. Total RNA extracted from WYMV-infected wheat plants incubated with anti-m6A or IgG (negative control) for IP. Values are means ± SD (two-sided t test, n  =  3, P  <  0.0001). **P  <  0.01. e RIP-qPCR assay revealing the binding of MTB to peak3 region. The protein-RNA complexes were extracted from WYMV-infected TaMTB-Flag-OE wheat plants and subjected to immunoprecipitation with anti-Flag or mouse IgG (negative control). Values are means ± SD (two-sided t test, n  =  3, P  <  0.0001). f In vitro interaction of TaMTB-His(E. coli) with probes 1-3 detected by MST experiment. TaMTB protein+ probe 3 was used as a negative control. Each binding assay was repeated three times independently, and error bars represent SD. Data in (f) are represented as means ± SD. m6A-IP-qPCR assay of peak3 in WYMV-infected BSMV:00 / BSMV:TaMTB (g) or wild type (WT) / TaMTB-OE (h) plants. BSMV:00 and WT were used as negative controls, respectively. Values are means ± SD (two-sided t test, n  =  3, (g) P  = 0.0074, (h) 0.0021). i SELECT analysis for detecting m6A6800 site in RNA 1 with or without TaMTB treatment. Values are means ± SD (two-sided t test, n  =  3, P  <  0.0001). Source data are provided as a Source Data file.
