Extended Data Fig. 6: NSs enhances the interaction of COI1/TIR1/MAX2 receptors/receptor-partner with TCP21 and blocks degradation of JAZ1/IAA8/SMXL6 transcriptional repressors.
From: NLR surveillance of pathogen interference with hormone receptors induces immunity
a, NSsY30A that lacked RNA silencing suppression activity was able to induce the HR cell death in N. benthamiana plant leaves co-expressing Tsw. Wild-type (WT) NSs, NSsY30A 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). The eGFP fluorescence was examined under a handhold UV lamp at 5 dpi. WT NSs, NSsY30A mutant, or EV 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. b, NSsY30A enhances the interaction between AtCOI1/AtTIR1/AtMAX2 and TCP21 assayed by SLC in planta. In one half leaf of N. benthamiana plant, nLUC-AtCOI1, nLUC-AtTIR1 or nLUC-MAX2 was coexpressed with cLUC-TCP21 in the presence of a EV control, and in another half leaf of N. benthamiana plant, nLUC-AtCOI1, nLUC-AtTIR1 or nLUC-MAX2 was coexpressed with cLUC-TCP21 in the presence of NSs. Luciferase activity was assayed and photographed at 48 hpi. c, NSsY30A stabilizes the interaction between AtCOI1 and AtTCP21 in the presence of coronatine (COR) in planta. The schematic diagram of the experimental design is shown in the left. nLUC-AtCOI1 was co-expressed with cLUC-TCP21 in the presence of DMSO, 0.1 µM COR, DMSO + NSs and 0.1 µM COR + NSs in plant leaves of N. benthamiana. Luciferase signal was assayed and photographed at 48 hpi (middle). Quantification of the luciferase activity of each treatment was shown in the right. Data are presented as mean values ± s.e.m.; n = 3 biologically independent samples. Lowercase letters a-d represent statistically different groups (one-way ANOVA with Tukey’s test, p < 0.05); the exact P values greater than 0.0001 are shown in the Source Data. d, Combined effect of AtTCP21 and NSsY30A on the interaction between AtCOI1/AtTIR1 and AtJAZ1/AtIAA8 assayed by SLC in planta. nLUC-AtCOI1 (OD600 = 1.0), cLUC-AtJAZ1 (OD600 = 1.0) and AtTCP21 (OD600 = 0.1) or nLUC-AtTIR1 (OD600 = 1.0), cLUC-AtIAA8 (OD600 = 1.0) and AtTCP21 (OD600 = 0.1) were co-expressed with NSsY30A (OD600 = 1.0) or pCambia2300S empty vector (EV; OD600 = 1.0) in N. benthamiana plant leaves. The luciferase activity in the leaves was assayed at 48 hpi. The luciferase activity was quantified and shown in the right of each treatment. Data are presented as mean values ± s.e.m.; n = 3 biologically independent samples. Data were analysed by two-sided Student’s t-test; the exact P values are shown in the figure. e, NSs fails to enhance the interaction of AtCOI1 (left) /AtTIR1 (middle) /AtMAX2 (right) receptors/receptor-partner with AtJAZ1/AtIAA8/AtSMXL6 transcription repressors in the absence of TCP21. The purified HIS-FLAG-AtCOI1/HIS-FLAG-AtTIR1 /HIS-FLAG-AtMAX2 (FLAG-AtCOI1/FLAG-AtTIR1/FLAG-AtMAX2) was used to pull-down purified HIS-MBP-AtJAZ1/HIS-MBP-AtIAA8/HIS-FLAG-AtSMXL6 (MBP-AtJAZ1/MBP-AtIAA8/FLAG-AtSMXL6) in the presence of 0.1 µM COR, 0.1 µM 2,4-D or equivalent DMSO. Increasing amount of HIS-NSs (NSs) was added to the reaction. GST or HIS-YFP (YFP) was added as a control. Blots were detected using MBP, FLAG, NSs, GST and YFP specific antibodies. The band intensity of the pulled down HIS-MBP-JAZ1, HIS-MBP-IAA8 or HIS-FLAG-AtSMXL6 in the first lane was set as 1 and the degradation of repressors in other lanes was quantified. Arrow indicates the band of HIS-MBP-AtJAZ1 protein (≈ 68 kDa). f, TSWV NSs inhibition on the degradation of transcription repressors NbJAZ1 (left), NbIAA8 (middle) and NbSMXL6 (right) in NbTCP21-1 and NbTCP21-2 (NbTCP21-1/2) silenced N. benthamiana plants. NSs was transiently overexpressed in N. benthamiana plant leaves silenced for NbTCP21-1/2 or TRV-GUS control plant leaves via agroinfiltration. The protein extract of infiltrated leaves for each treatment was mixed with purified HIS-MBP-NbJAZ1, HIS-MBP-NbIAA8 or HIS-NbSMXL6 proteins in the presence of 0.1 µM COR, 0.1 µM 2, 4-D, or 0.1 µM rac-GR24 and assayed for in vitro degradation from 0-60 min at 24 °C. The blots were detected using HIS specific monoclonal antibodies. The amount of repressor proteins at 0 min was set as 1 and the degradation of repressors in other lanes was quantified. Experiments were repeated at least three times with similar results.
