Extended Data Fig. 5: Effect of TCP21 on the expression of JA, AUX and SL response genes and the interaction between COI1/TIR1/MAX2 and JAZ1/IAA8/SMXL6.
From: NLR surveillance of pathogen interference with hormone receptors induces immunity
a, Generation of transgenic Arabidopsis overexpressing TCP21. Accumulation of TCP21 in 14 independent TCP21-transgenic lines was analysed by Western blot using HA specific antibodies (Top). Ponceau S staining was used to estimate sample loading. The phenotype of transgenic Arabidopsis overexpressing TCP21-3×HA photographed at 8-week-old stage was shown in the bottom. Lower left is the WT plant; lower middle and right are the TCP21 transgenic Arabidopsis lines #6 and #11. b, Relative expression levels of representative JA response genes in WT and AtTCP21 transgenic Arabidopsis plants in the presence of 0 or 100 µM MeJA. Data are presented as mean values ± s.e.m.; n = 3 biologically independent samples. c, Phenotype of tcp7/21 mutant and WT Arabidopsis plant. d-f, Relative expression levels of JA (d), AUX (e) and SL (f) response genes in tcp7/21 mutant. Data are presented as mean values ± s.e.m.; n = 3 biologically independent samples. g, Transient overexpression of AtTCP21 reduced the interaction between AtCOI1/AtTIR1/AtMAX2 and AtJAZ1/AtIAA8/AtSMXL6 assayed by SLC. The schematic diagram of the experiments is shown in the upper left. Co-expression of nLUC-A and cLUC-B produce luciferase activity (indicated by a thunder). A represents AtCOI1, AtTIR1 or AtMAX2 protein. B represents AtJAZ1, AtIAA8 or AtSMXL6 protein. nLUC-AtCOI1 and cLUC-AtJAZ1, nLUC-AtTIR1 and cLUC-AtIAA8, or nLUC-AtMAX2 and cLUC-AtSMXL6 were used to co-express with AtTCP21 or pCambia2300S empty vector (EV) in N. benthamiana plant leaves (lower left). The concentration of agrobacterium individually carrying those constructs were used at OD600 = 1.0. The luciferase activity was assayed at 48 hpi. The luciferase activity (Integrated Optical Density, IOD) in the treated leaves was quantified and shown in the lower right. Data are presented as mean values ± s.e.m.; n = 3 biologically independent samples. h, Western blot analysis of nLUC-AtCOI1 and cLUC-AtJAZ1, nLUC-AtTIR1 and cLUC-AtIAA8 in leaves shown in panel g using LUC and FLAG specific antibodies. Due to the protein level of cLUC-AtSMXL6 below detection, the analysis data of nLUC-AtMAX2 and cLUC-AtSMXL6 was not included here. Black, blue, grey and brown arrow indicates the band of nLUC-AtCOI1, cLUC-AtJAZ1, nLUC-AtTIR1 and cLUC-AtIAA8 protein, respectively. Ponceau S staining was used to estimate sample loading. i, Y2H analysis of the interaction between NbTCP21 and NbCOI1, NbTIR1, NbMAX2, Tsw-PRL or NSs. AD-NbTCP21-1 and AD-NbTCP21-2 were tested for the interaction with BD-NbCOI1, BD-NbTIR1, BD-NbMAX2, BD-Tsw-PRL and BD-NSs in yeast. Data in (b, d-g) were analysed by two-sided Student’s t-test; P values are shown in the figure. Experiments were repeated at least three times with similar results.
