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NLR surveillance of pathogen interference with hormone receptors induces immunity

Nature volume 613, pages 145–152 (2023)Cite this article

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An Author Correction to this article was published on 11 January 2023

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Abstract

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence3. However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor—TCP21—which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity.

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Fig. 1: TSWV NSs interferes with JA, AUX and SL phytohormone signalling pathways.
Fig. 2: TCP21 interacts with TSWV NSs and JA, AUX and SL receptors, and negatively regulates three phytohormone signalling pathways.
Fig. 3: NSs enhances the interaction of JA, AUX and SL receptors with TCP21 and blocks their association with transcription repressors and degradation.
Fig. 4: TCP21 promotes the interaction between NSs and Tsw and is required for Tsw-mediated cell death and resistance.

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Full versions of all gels and blots are provided in Supplementary Fig. 1.  Source data are provided with this paper.

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Acknowledgements

We thank J. Li for providing Arabidopsis mutant max2-1 seeds; D. Xie and S. Song for the coi1-1 seeds; D. Yang for the tir1/afb seeds; S. Cui for the tcp7/21 seeds; H. Zhao for B. cinerea strain B05.10; M. Turina for the C. chinense PI 152225 seeds and NSs-RB-272 construct; M. Deng for the C. annum fasciculatum seeds; S. Wang for the C. annum CM334 seeds; and Dr. Xin Shun Ding for critical reading of the manuscript. This work was supported by National Natural Science Foundation of China (31925032, 32220103008 and 31870143), the Youth Science and Technology Innovation Program, 333 Project and Funds from the Independent Innovation of Agricultural Science and Technology of Jiangsu Province (CX(22)2039) to X.T. NLR work in the S.P.D.-K. laboratory is supported by NSF-IOS-1354434 and NSF-IOS-1339185. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Jing Chen, Yanxiao Zhao

Authors and Affiliations

  1. Key Laboratory of Plant Immunity, Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China

    Jing Chen, Yanxiao Zhao, Xuanjie Luo, Hao Hong, Tongqing Yang, Shen Huang, Chunli Wang, Hongyu Chen, Xin Qian, Mingfeng Feng, Zhengqiang Chen, Yongxin Dong, Zhenchuan Ma, Jia Li, Min Zhu & Xiaorong Tao

  2. Howard Hughes Medical Institute, Department of Biology, Duke University, Durham, NC, USA

    Sheng Yang He

  3. Department of Plant Biology and The Genome Center, College of Biological Sciences, University of California, Davis, CA, USA

    Savithramma P. Dinesh-Kumar

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Contributions

J.C. and X.T. conceived and conceptualized the study. J.C., Y.Z., X.L. and H.H. generated materials used in this study. J.C. and X.T. performed structural modelling analysis. J.C. and Y.Z. performed Y2H screening. J.C. and X.L. performed RT–qPCR and analysed the data. J.C., Z.C. and Y.D. performed thrips preference assays. J.C., Y.Z., H.H. and X.L. performed co-immunoprecipitation, western blots and GST pull-down experiments. T.Y. and S.H. performed SLC assays and analysed the data. J.C., H.C. and X.L. performed the BiFC assays and confocal microscopy and analysed the data. J.C. and C.W. conducted protein degradation assays. Y.Z. and X.L. performed the jasmonic acid, auxin and strigolactone assays. H.C., X.Q. and M.F. contributed towards TSWV inoculation and accumulation assays. Y.Z. and X.L. performed VIGS assays. H.H. and X.L. performed the genetic analysis. J.C. performed statistical analysis. Z.M., J.L., M.Z., S.Y.H. and S.P.D.-K. analysed data, provided critical feedback and helped to shape the research. J.C. and X.T. wrote the original draft of the paper. S.P.D.-K. and X.T. reviewed and edited the paper with input from all authors.

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Correspondence to Xiaorong Tao.

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Extended data figures and tables

Extended Data Fig. 1 Tsw contains an unusually large LRR domain that has structural similarity with the LRR domain of JA, AUX and SL phytohormone receptors/receptor-partner.

a, Pepper “PI152225” carrying Tsw (+Tsw) conferred resistance to TSWV infection, while “CM334” cultivar without Tsw (-Tsw) showed the typical symptoms including stunting and leaf curling. b, The RT-PCR of full-length cDNA of Tsw from two independent plant samples (lanes 1 and 2). M, the size marker. c, Schematic of Tsw protein containing a coiled-coil (CC), an NB-ARC and an extra-large LRR domain. As a comparison, CC-type NLR Rx is shown. The phytohormone receptor-like (Tsw-PRL) domain within LRR (896-2116 amino acid) is indicated by brackets. d, Protein Homology/analogY Recognition Engine (PHYRE) Version 2 detected that the extra-large LRR domain of Tsw has the amino acid sequence and structure similarity with LRR domain of JA receptor COI1, AUX receptor TIR1 and SL receptor-partner MAX2. e, Comparison of folding trends of 3D crystal structures of COI1 (PDB: 3OGM), TIR1 (PDB: 2P1N) and MAX2 (PDB: 5HYW). Depicted structure of COI1, TIR1 and MAX2 containing the F-box and LRR domain are shown on the left. The 3D crystal structures of three phytohormone receptors/receptor-partner that orientated in the same direction were displayed in the middle and the structures with 180º rotation were displayed in the right. f, Overlays of 3D structures of COI1, TIR1and MAX2 with the modeled structure of Tsw-PRL (1567-2050 a.a.) predicted by PHYRE 2. g, Co-expression of Tsw and NSs induces hypersensitive response (HR) cell death in Nicotiana benthamiana plant leaves. Tsw and pCambia2300S empty vector (EV) (i), NSs and EV (ii), EV (iii) or Tsw and NSs (iv) were co-expressed in N. benthamiana plant leaves via agro-infiltration. The HR cell death phenotype of infiltrated leaves was photographed at 3 dpi in the white light (WL; left) and Ultraviolet light (UV; right).

Extended Data Fig. 2

TSWV infection or overexpression of NSs in Arabidopsis affect JA, AUX and SL signalling pathways. a, Analysis of published transcriptome data50 showed that the majority of JA, AUX and SL responses genes are downregulated in Arabidopsis plants infected with TSWV at 9, 12 and 15 dpi. Gene expression up- or down-regulated (with at least a two-fold difference) are presented in red and green, respectively. b-d, Relative expression levels of JA, AUX and SL responses genes in Arabidopsis plants infected with TSWV at 12 dpi. Data are represented as means ± s.e.m.; n = 3 biologically independent samples. e, Amount of jasmonic acid (JA), indole-3-acetic acid (IAA) and 5-deoxystrigol (5-DS) in Arabidopsis plants infected with TSWV at 12 dpi. The Arabidopsis plants inoculated with 1×PBS buffer were used as Mock. Data are represented as mean values ± s.e.m.; n = 3 biologically independent samples. f, Transgenic Arabidopsis lines expressing NSs (1 to 8) were screened and examined by RT-PCR (top). Phenotypes of wild-type (WT) and NSs transgenic line #2 and line #3 Arabidopsis plants are shown at the bottom. g, The NSs transgenic plants are less sensitive to methyl jasmonate (MeJA)-induced root growth inhibition than WT plants. NSs transgenic line #2 (left) and #3 (right) were treated with MeJA. The photograph was taken 21 days post MeJA treatment. Scale bar, 1 cm. h, Relative expression levels of representative JA response genes in WT and NSs transgenic Arabidopsis plants in the presence of 0 or 100 µM MeJA. Data are represented as mean values ± s.e.m.; n = 3 biologically independent samples. i, Illustration of the experimental design for thrips attraction to WT and NSs transgenic plants. The NSs transgenic plants were placed at two corners across each other (i and iii) and WT plants placed in the other two corners (ii and iv) in an isolated box. The western thrips without carrying TSWV were placed in a petri dish in the center of the box. The thrips attracted onto WT and NSs transgenic plants were counted after the indicated time and shown in Fig. 1c. j, The NSs transgenic plants are less sensitive to auxin analog 2,4-D-induced root growth inhibition than WT plants. The photograph was taken at 14 days post 2,4-D treatment. Scale bar, 1 cm. k, Primary root lengths of 7-day-old WT and NSs transgenic Arabidopsis plants with or without 1 μM rac-GR24. Data are presented as mean values ± s.e.m.; n = 12 plants. Data in (b-e, h and k) 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.

Source Data

Extended Data Fig. 3 The interaction between NSs and AtCOI1, AtTIR1, AtMAX2 or Tsw-PRL.

a, Co-immunoprecipitation (Co-IP) analysis of the interaction between NSs and AtCOI1/AtTIR1/AtMAX2. YFP-AtCOI1, YFP-AtTIR1 and YFP-AtMAX2 were used to Co-IP the NSs-FLAG in N. benthamiana plant. YFP was used as a control. The upper arrow indicates the band of YFP-AtCOI1, YFP-AtTIR1 and YFP-AtMAX2 protein; the lower arrow indicates the band of YFP protein. Blots were detected using YFP and FLAG specific antibodies. Ponceau S staining was used to estimate sample loading. b, SLC analysis of the interaction between NSs and AtCOI1/AtTIR1/AtMAX2 in planta. cLUC-AtCOI1, cLUC-AtTIR1, cLUC-AtMAX2 or cLUC control vector were co-expressed with nLUC-NSs or nLUC control vector in N. benthamiana plant leaves. Luciferase activity was detected at 48 hpi. c, BiFC analysis of the interaction between NSs and AtCOI1/AtTIR1/AtMAX2 or Tsw-PRL in planta. nYFP-AtCOI1, nYFP-AtTIR1, nYFP-AtMAX2, nYFP-Tsw-PRL or nYFP control vector was co-expressed with cYFP-NSs or cYFP control vector in N. benthamiana plant leaves. The reconstituted YFP fluorescence signals were examined by confocal microscopy and photographed at 48 hpi. Scale bar, 50 µm. d, NSs interacts with AtCOI1/AtTIR1/AtMAX2 in the nucleus assayed by BiFC. cYFP-NSs and nYFP-AtCOI1, nYFP-AtTIR1 or nYFP-AtMAX2 were co-expressed with H2B-mCherry, a nuclear marker, in N. benthamiana plant leaves. The white arrow indicates the co-localization of NSs, AtCOI1/AtTIR1/AtMAX2 and H2B in the nucleus. Scale bar, 50 µm. e, Co-IP analysis of the interaction between NSs and Tsw-PRL in planta. YFP-Tsw-PRL was used to Co-IP NSs-FLAG in N. benthamiana plant. YFP was used as a control. Blots were detected using YFP and FLAG specific antibodies. Ponceau S staining is used to estimate sample loading. f, SLC analysis of the interaction between NSs and Tsw-PRL in planta. cLUC-Tsw-PRL or cLUC control vector was co-expressed with nLUC-NSs or nLUC control vector in N. benthamiana plant leaves. Luciferase activity was detected at 48 hpi. g, Y2H analysis of the interaction between NSs and AtCOI1, AtTIR1, AtMAX2 or Tsw-PRL. pGBKT7 empty vector containing a DNA binding domain (BD) and pGADT7 empty vector containing an activation domain (AD) were used as negative controls. BD-NSs and AD-NSs were used as a positive control. 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. Experiments were repeated at least three times with similar results.

Extended Data Fig. 4 Yeast two hybrid screening identified AtTCP21 as a common interactor for NSs, AtCOI1, AtTIR1, AtMAX2 and Tsw-PRL.

a, Interaction of full-length AtTCP7 and AtTCP14 with NSs in yeast. AD, pGADT7 vector. BD, pGBKT7 vector. The yeast co-transformed with BD and AD derivative constructs was plated on both SD/-L-T (left) and SD/-L-T-H-A (right) dropout media. b, Number and frequency of AtTCP7 and AtTCP14 identified in Y2H library screening using NSs as bait. c, Y2H analysis of the interaction between NSs and 24 TCP family members. The full-length coding sequence of 24 TCP family members from Arabidopsis plant was constructed into AD vector and co-transformed with BD-NSs or BD control vector into yeast and their growth and interaction were assayed on both SD/-L-T and SD/-L-T-H-A dropout media. d, Phylogenetic tree analysis of 24 TCP family members from Arabidopsis. The phylogenetic tree was constructed using MEGA 7.0 by the neighbor-joining method and bootstrap values (1000 replicates) are shown on the branches. e, Y2H assay showing direct interaction between AtTCP7/14/17/21 and AtCOI1/AtTIR1/AtMAX2 or between AtTCP7/14/17/21 and NSs. The results for SD/-L-T dropout media are shown in this Figure and results for SD/-L-T-H-A dropout media are shown in Fig. 2a. f, BD-AtCOI1, BD-F-box (AtCOI1), BD-LRR (AtCOI1) or BD vector control were tested for the interaction with AD-AtTCP21 or AD vector control in yeast. g, AD-AtTCP21, AD-AtTCP21 (1-103), AD-AtTCP21 (104-240), AD-AtTCP21 (30-103), AD-AtTCP21 (30-136), AD-AtTCP21 (137-240) or AD vector control were tested for the interaction with BD-AtCOI1 or BD vector control in yeast. The schematic diagram of truncation mutants of AtTCP21 was shown above the Y2H assays. h, SLC analysis of the interaction between NSs and AtTCP21 in planta. cLUC-AtTCP21 or cLUC was co-expressed with nLUC-NSs or nLUC in N. benthamiana plant leaves. Luciferase activity was detected at 48 hpi. i, Y2H analysis of the interaction between AtTCP21 and LRR domain of different proteins. BD-FBL6-LRR, BD-FBL17-LRR, BD-RPM1-LRR, BD-HRT-LRR, BD-FLS2-LRR, BD-EFR-LRR or BD vector control were tested for the interaction with AD-AtTCP21 or AD vector control in yeast. AD, pGADT7 vector. BD, pGBKT7 vector. FBL6 and FBL17 are two F-box family proteins; RPM1 and HRT are two CC-NLRs; FLS2 and EFR are two PRRs. The yeast cotransformed with BD and AD derivative constructs were plated and assayed on both synthetic double dropout media -Leu-Trp (SD/-L-T) and synthetic quadruple dropout media -Leu-Trp-HIS-Ade (SD/-L-T-H-A).

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.

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.

Source Data

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.

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.

Source Data

Extended Data Fig. 7 The effects of JA/AUX/SL phytohormone, overexpressing or knock-down/-out TCP21 on TSWV accumulation in Nicotiana benthamiana, pepper and Arabidopsis plant leaves.

a, Phenotype of TSWV-inoculated N. benthamiana plants treated with DMSO, MeJA, 2,4-D or rac-GR24. N. benthamiana plants were sprayed with DMSO, 100 µM MeJA, 100 µM 2,4-D and 100 µM rac-GR24, respectively. At 3 d post treatment, the fresh sap from TSWV infected tissues was mechanically inoculated onto phytohormone treated leaves. The phenotype of TSWV-inoculated plants was photographed at 9 d post inoculation. b, TSWV accumulation was analysed in systemic infected leaves of N. benthamiana plants treated with DMSO, MeJA, 2,4-D and rac-GR24, respectively, at 9 dpi by Western blot using TSWV N specific antibodies. Ponceau S staining was used to estimate sample loadings. c, Phenotype of TSWV-inoculated pepper plant (-Tsw) treated with DMSO, MeJA, 2,4-D or rac-GR24. C. annum fasciculatum (-Tsw) were sprayed with DMSO, 100 µM MeJA, 100 µM 2,4-D and 100 µM rac-GR24, respectively. At 3 d post treatment, the fresh sap from TSWV infected tissues was mechanically inoculated onto phytohormone treated leaves. The phenotype of TSWV-inoculated plants was photographed at 6 d post inoculation. d, TSWV accumulation was analysed in systemic infected leaves of pepper plant (-Tsw) treated with DMSO, MeJA, 2,4-D and rac-GR24, respectively, at 6 dpi by Western blot using TSWV N specific antibodies. Immunoblot analysis of actin is used to estimate the sample loadings. **, p < 0.01; ns, no significance. Data were analysed by two-sided Student’s t-test; exact P values are shown in the Source Data. e, TSWV infection phenotype on wild-type (WT) and AtTCP21 transgenic Arabidopsis plants. The photos of infected plants were taken at 10 dpi. f, TSWV infection phenotype on WT and tcp7/21 mutant Arabidopsis plants. The photos of infected plants were taken at 12 dpi. g, Silencing of TCP21 in pepper plants (-Tsw) enhances resistance to TSWV. The fusion of 200 bp cDNA fragments each of CaTCP21-1, 3, 4 and 7 (TRV-CaTCP21-1/3/4/7) was used to infect C. annum fasciculatum (-Tsw). Phenotype of TSWV-inoculated C. annum fasciculatum silenced for CaTCP21-1/3/4/7 was photographed at 7 dpi (left). TSWV accumulation in systemic infected leaves of C. annum fasciculatum silenced for CaTCP21-1/3/4/7 at 7 dpi was analysed by immunoblot analysis using TSWV N specific antibodies (right). Immunoblot analysis of actin is used to estimate sample loadings. **, p < 0.01. Data were analysed by two-sided Student’s t-test; the exact P values are shown in the Source Data. h, Phenotype of Botrytis cinerea infection lesions on detached leaves of WT (left), TCP21 transgenic (middle) and tcp7/21 mutant (right) Arabidopsis plant leaves at 48 h post inoculation. B. cinerea lesions on leaves were quantified and shown in the right. Data are presented as mean values ± s.e.m.; n = 6 biologically independent samples. Lowercase letters a-c represent statistically different groups (one-way ANOVA with Tukey’s test, p < 0.05); exact P values great than 0.0001 (adjusted) are shown in Source Data. In box plots the center line represents the median, box edges delimit lower and upper quartiles and whiskers show the highest and lowest data points. Experiments were repeated at least three times with similar results.

Source Data

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-RBY30A mutants to suppress RNA silencing and to induce Tsw-mediated HR cell death. Wild-type (WT) NSs, NSs-RB, NSs-RBY30A 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.

Source Data

Extended Data Fig. 9 Effect of TSWV infection or NSs on the interactions between CaCO1/CaTIR1/CaMAX2 and CaTCP21 or Tsw-PRL and CaTCP21.

a, BiFC analysis of the interaction between CaTCP21-1 and CaCOI1 (upper left), CaTIR1 (upper right), CaMAX2 (lower left) or Tsw-PRL (lower right) in Mock-inoculated or TSWV infected N. benthamiana plant. Agrobacterium (indicated OD600) individually carrying cYFP-CaCOI1, cYFP-CaTIR1, cYFP-CaMAX2 or cYFP-Tsw-PRL was co-expressing with Agrobacterium carrying nYFP-CaTCP21-1 (indicated OD600) in N. benthamiana leaves that pre-infected with TSWV or inoculated with 1×PBS buffer (Mock) 3 days earlier. BiFC fluorescence signals were examined by Zeiss 710 confocal microscope and photographed at 2 days post infiltration (dpi). b, BiFC analysis of the interaction between Tsw-PRL and CaTCP21-1 in the presence of pCambia2300S empty vector (EV), NSsY30A or NSs-RBY30A. c, BiFC analysis of the interaction between tsw-PRL and CaTCP21-1 in the presence of EV or NSsY30A. d, e, Competitive analysis of the interactions between CaTCP21-1 and Tsw-PRL, CaTCP21-1 and CaCOI1/CaTIR1 with or without NSsY30A. BiFC analysis of the interaction between CaTCP21-1 and CaCOI1/CaTIR1 in the presence of pCambia2300S empty vector (EV), Tsw-PRL, Tsw-PRL+EV, or Tsw-PRL+NSsY30A was shown in panel d. BiFC analysis of the interaction between CaTCP21-1 and Tsw-PRL in the presence of EV, CaCOI1, CaCOI1 + EV, CaCOI1 + NSsY30A or EV, CaTIR1, CaTIR1 + EV, CaTIR1 + NSsY30A was shown in panel e. The fluorescence signals were examined and photographed at 48 hpi. For a-e, 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. Data in (a) were analysed by two-sided Student’s t-test; P values are shown in the figure. Data in (b-e) were analysed by one-way ANOVA with Tukey’s test; lowercase letters a, b and c represent statistically different groups (p < 0.05); exact P values greater than 0.0001 (adjusted) are shown in the Source Data. Experiments were repeated three times with similar results.

Source Data

Extended Data Fig. 10 TCP21 is required for the HR induction by Tsw and NSs in N. benthamiana plant and required for Tsw-mediated resistance in pepper against TSWV infection.

a, Effect of CaTCP21-1 on the HR cell death induced by co-expression of Tsw and NSs. CaTCP21-1 (i), Tsw (ii), Tsw+CaTCP21-1 (iii), Tsw+NSs+EV (iv) or Tsw+NSs+CaTCP21-1 (v) was/were (co)expressed in N. benthamiana leaves via agroinfiltration. The HR was monitored from 0-8 dpi and photographed at 5 dpi. b, Effect of CaTCP3 and CaTCP17 on the HR cell death induced by co-expression of Tsw and NSs. Data are represented as mean values ± s. e. m.; n = 6 biologically independent samples. c, Effect of CaTCP21-1 on the HR cell death induced by co-expression of Sw-5b NLR and NSm effector. HR phenotype was photographed at 5 dpi. The ion leakage of infiltrated leaves was measured at 2 dpi and shown in the right. Data are represented as mean values ± s. e. m.; n = 6 biologically independent samples. d, Schematic diagram of TRV-mediated gene silencing of NbTCP21 in N. benthamiana plant. TRV carrying a 300 bp cDNA fragment each of NbTCP21-1 and NbTCP21-2 (TRV-NbTCP21-1/2) was used to infect N. benthamiana plant. After 20 days post TRV treatment, the upper new leaves that silenced with TCP21 were co-expressed with Tsw and NSsY30A. The onset of HR was monitored from 0-8 dpi. e, The phenotype of N. benthamiana plant treated with TRV-GUS and TRV-NbTCP21-1/2 at 20 dpi. f, Relative RNA expression of NbTCP21-1 and NbTCP21-2 in N. benthamiana plant treated with TRV-GUS and TRV-NbTCP21-1/2. Data are presented as mean values ± s.e.m.; n = 3 independent plant samples. g, BiFC analysis of the interaction between NSs and Tsw-PRL in TRV-GUS treated control plant leaves or NbTCP21-1/2 silenced N. benthamiana plant leaves. The BiFC fluorescence signals were examined and photographed at 39 h post agroinfiltration. h, Silencing of NbTCP21-1/2 did not affect the HR induced by Sw-5b NLR and NSm effector. Sw-5b and NSm were coexpressed by agro-infiltration in N. benthamiana plant treated with TRV-GUS or TRV-NbTCP21-1/2. HR phenotype of infiltrated leaves was monitored and photographed at 5 dpi. The ion leakage of infiltrated leaves was measured at 2 dpi and shown in the right. Data are presented as mean values ± s.e.m.; n = 6 biologically independent samples. i, Phenotype of pepper “PI152225” silenced for CaTCP21-1/3/4/7 and Tsw. TRV carrying a 300 bp cDNA fragment of GUS (TRV-GUS), Tsw (TRV-Tsw) or the fusion of 200 bp cDNA fragment each of CaTCP21-1, 3, 4 and 7 (TRV-CaTCP21-1/3/4/7) was used to infect pepper “PI152225” carrying Tsw. The phenotype of pepper treated with various TRV constructs were photographed at 30 dpi. TRV-CaPDS served as a positive control for silencing (right). j-l, Relative expression levels of CaTCP21-1, -3, -4, -7, Tsw and CaPDS genes in pepper treated with TRV-GUS, TRV-CaTCP21-1/3/4/7 (j), TRV-Tsw (k) or TRV-CaPDS (l). Data are represented as mean values ± s. e. m.; n = 3 biologically independent samples. Data in (b, c) were analysed by one-way ANOVA with Tukey’s test; lowercase letters a and b represent statistically different groups (p < 0.05). Data in (f, h, j-l) were analysed by two-sided Student’s t-test. P values in (b, c, j) are shown in the Source Data; P values in (f, h, k, i) are shown in the figure. In box plots of (b, c, h), the center line represents the median, box edges delimit lower and upper quartiles and whiskers show the highest and lowest data points.

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Chen, J., Zhao, Y., Luo, X. et al. NLR surveillance of pathogen interference with hormone receptors induces immunity. Nature 613, 145–152 (2023). https://doi.org/10.1038/s41586-022-05529-9

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