Fig. 2: TaRD21A is a member of the papain-like cysteine proteases (PLCPs) family.

a Subcellular localization of TaRD21A^R in N. benthamiana leaves. RFP under TaRD21A^R native promoter was used as negative control. RD21Apro: AtPIP1: RFP, RD21Apro: TaRD21A^R: RFP or RD21Apro: RFP was co-expressed with plasma membrane (PM) marker 35 S: AtPIP2A: RFP in N. benthamiana leaves. Yellow arrows indicate apoplastic spaces and white arrows indicate PM. Bar, 10 µm. Three times each experiment was repeated independently with similar results. b The AF and total protein of the N. benthamiana leaves transiently expressing RD21Apro: AtPIP1: RFP, RD21Apro: TaRD21A^R: RFP or RD21Apro: RFP were analyzed by western blot assay using an anti-RFP antibody. c AF were prepared from N. benthamiana leaves transiently expressing RD21Apro: TaRD21A^R: RFP or RD21Apro: RFP and then labeled with DCG-04 in the presence or absence of E-64. d TaRD21A^R-GST was expressed and purified from E. coli (BL21) and then co-incubated with DCG-04. The purified GST was used as control. e TaRD21A protease activity is involved in WYMV resistance in N. benthamiana. The conserved catalytic triad Cys-His-Asn in TaRD21A^R was substituted with Glycine (TaRD21A^R-M) and then fused to C-terminus of RFP. AF from these assayed leaves was extracted for analysis protease activity. (−)DCG-04 in (c–e) indicated that the AF or GST fusion protein without DCG-04 labelling and used for distinguish from background signals. Coomassie Blue staining in (b–e) shows the assayed protein loaded. The data in (b–e) are representative of n = 3 independent experiments. Source data are provided as a Source Data file.