Extended Data Fig. 8: Assays of the interaction between CaTCP21 and NSs, CaCOI1, CaTIR1, CaMAX2 or Tsw-PRL.

a, Y2H assay of the interactions between CaTCP21-1 to −7 and NSs. AD-CaTCP21-1 to AD-CaTCP21-7 were tested for the interaction with BD-NSs in yeast. The yeast co-transformed with BD and AD derivative constructs was plated and assayed on both SD/-L-T (left) and SD/-L-T-H-A (right) dropout media. b, Assays for the ability of NSs-RB and NSs-RB^Y30A mutants to suppress RNA silencing and to induce Tsw-mediated HR cell death. Wild-type (WT) NSs, NSs-RB, NSs-RB^Y30A mutant or pCambia2300S empty vector (EV) was co-expressed with GFP in 16c transgenic N. benthamiana plant leaves and assayed for the RNA silencing suppression activity (top). NSs-RB was from a resistance-breaking TSWV isolate 272. The eGFP fluorescence was examined under a handhold UV lamp at 5 dpi. Each of those NSs constructs was also co-expressed with Tsw in WT N. benthamiana plant leaves and assayed for HR induction activity (bottom). The HR phenotype was photographed at 5 dpi. c, BiFC analysis of the interaction between CaTCP21-1 and WT NSs or NSs-RB. cYFP-NSs or cYFP-NSs-RB was co-expressed with nYFP-CaTCP21-1 in N. benthamiana plant leaves and analysed by BiFC. The fluorescence signals were examined and photographed at 48 hpi. White arrows indicate the BiFC fluorescence signals. The numbers in each image are the number of scans displaying protein distribution equivalent to the image shown out of the total number of scans. Scale bar, 50 µm. Relative fluorescence signal intensity of each treatment in the left was quantified and shown in the right. Data are presented as mean values ± s.e.m.; n = 30 independent scans collected from three independent experiments. Data were analysed by two-sided Student’s t-test; P values are shown in the figure. d, Y2H assay of the interaction between CaTCP21-1 and CaCOI1, CaTIR1, CaMAX2, Tsw-PRL or NSs. AD-CaTCP21-1 was tested for the interaction with BD-CaCOI1, BD-CaTIR1, BD-CaMAX2, BD-Tsw-PRL and BD-NSs. BD, pGBKT7; AD; pGADT7. The results of the interaction between CaTCP21-1 and CaCOI1, CaTIR1, CaMAX2, Tsw-PRL or NSs on SD/-L-T dropout media plate is shown in this Figure and results on SD/-L-T-H-A dropout media plate is shown in Fig. 4a. e, BiFC analysis of the interaction between CaTCP21-1 and CaCOI1, CaTIR1 or CaMAX2 in planta. Agrobacterium (OD₆₀₀ = 1) individually carrying cYFP-CaCOI1, cYFP-CaTIR1, cYFP-CaMAX2, or cYFP was co-expressed with Agrobacterium (OD₆₀₀ = 1) carrying nYFP-CaTCP21-1 or nYFP in N. benthamiana plant leaves. The reconstituted YFP fluorescence signals were examined by confocal microscopy and photographed at 48 hpi. Scale bar, 50 µm. f, nYFP-CaTCP21-1 and cYFP-CaCOI1, cYFP-CaTIR1 or cYFP-CaMAX2 were co-expressed with H2B-mCherry, a nuclear marker, in N. benthamiana plant leaves. The white arrow indicates the co-localization of CaTCP21, CaCOI1/CaTIR1/CaMAX2 and H2B in the nucleus. Scale bar, 50 µm. g, h, GST pull-down analysis of the interaction between Tsw-PRL and NbTCP21-1 (g) or CaTCP21-1 (h). Purified GST-Tsw-PRL was used to pull-down purified HIS-FLAG-NbTCP21-1 (FLAG-NbTCP21-1) or HIS-FLAG-CaTCP21-1 (FLAG-CaTCP21-1). Blots were detected using GST and FLAG specific antibodies. Experiments were repeated at least three times with similar results.

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