Fig. 5: NIa interacts with TaRD21A^S to affect its protease activity.

a The interaction between NIa and TaRD21A^S was confirmed by Y2H assays. b LCI assay was used to confirm the interaction between NIa-nLuc and TaRD21A^S-cluc. Leaf cells co-expressing sGF-nLuc and cLuc-SAR or nLuc and TaRD21A^S-cluc were used as positive or negative controls, respectively. c GST pull-down assay was used to detect the interaction between NIa-His and TaRD21A^S-GST. d Subcellular distribution of NIa: GFP, TaRD21A^S-ΔSP: RFP and TaRD21A^S: RFP in N. benthamiana epidermal cells. Three times each experiment was repeated independently with similar results. Bar, 50 μm. e Accumulation of 35 S: NIa: GFP, RD21Apro: TaRD21A^S-ΔSP: RFP and RD21Apro: TaRD21A^S: RFP in AF from assayed plants in (d) was determined by western blot assays using GFP or RFP antibody. f Mild chlorotic mosaic symptoms were observed at 14 dpi in the fourth leaves of the plants inoculated with Fes buffer (Mock), WYMV, BSMV, BSMV: TaRD21A^S, BSMV + WYMV and BSMV: TaRD21A^S + WYMV, respectively. g Accumulation of TaRD21A and BSMV RNA in the leaves of YM158(S) co-infected with BSMV + WYMV or BSMV: TaRD21A^S + WYMV. Values of qRT-PCR is the mean ± SD (two-sided t-test, n = 4 biologically independent experiments, ***P < 0.001, n.s., no significant). h AF was extracted from the leaves of YM158(S) infected with BSMV + WYMV and BSMV: TaRD21A^S + WYMV for western blot assay using NIa specific antibody. H3, PEPC and H⁺-ATPase antibody was used to verify the absence of nuclear, cytoplasmic, or plasma membrane components. i The effect of NIa on TaRD21A^S activity. The AF from wheat leaves with or without NIb expression were used as control. j, k Effect of NIa-GST on the cysteine proteases activity in AF from N. benthamiana leave expressing RD21Apro: TaRD21A^S: RFP or RD21Apro: RFP. l The effect of WYMV infection on TaRD21A^S activity. (−)DCG-04 indicated AF without DCG-04 labelling and used for distinguish from background signals. EV in (i–k) is representative of expressing vector which containing His tag. Coomassie Blue staining in (e, h–l) shows the assayed protein loaded. The data in (c, e, h–l) are representative of n = 3 independent experiments. Source data are provided as a Source Data file.