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Remodelling autoactive NLRs for broad-spectrum immunity in plants

Nature volume 645, pages 737–745 (2025)Cite this article

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Abstract

Remodelling plant immune receptors has become a vital strategy for creating new disease resistance traits to combat the growing threat of plant pathogens to global food security and environmental sustainability1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17. However, current methods are constrained by the rapid evolution of plant pathogens and often lack broad-spectrum and durable protection. Here we report an innovative strategy to engineer broad-spectrum, durable and complete disease resistance in plants through expression of a chimeric protein containing a flexible polypeptide coupled with a single or dual conserved pathogen-originated protease cleavage site fused in frame to the N terminus of an autoactive nucleotide-binding and leucine-rich-repeat immune receptor (NLR) containing a coiled-coil or RESISTANCE TO POWDERY MILDEW 8-like coiled-coil domain. Following invasion, pathogen-originated specific proteases cleave the inactive chimeric protein to form free autoactive NLR, triggering broad-spectrum plant disease resistance. We demonstrate that a single engineered NLR can confer broad-spectrum and complete resistance against multiple potyviruses. Given that many pathogenic organisms across kingdoms encode proteases, this strategy has the potential to be exploited to control viruses, bacteria, oomycetes, fungi, nematodes and pests in plants.

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Fig. 1: HA–PCSPVY–aTm-22 confers complete resistance against multiple potyviruses.
Fig. 2: HA–PCSPVY–aAtNRG1.1 confers resistance against multiple potyviruses.
Fig. 3: HA–PCSTEV–PCSPVY–aAtNRG1.1 confers resistance against PVY and TEV.
Fig. 4: HA–PCSSMV–aAtNRG1.1 confers resistance in soybean against SMV.

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Data availability

Sequence data can be found in the GenBank/UniProtKB/SwissProt database under the following accession numbers: Tm-22 (AAQ10736.1), AtNRG1.1 (Q9FKZ1.1) and NbNRG1 (Q4TVR0). All data are available within the article and its Supplementary Information. Uncropped gel and immunoblot images are shown in Supplementary Fig. 1. Source data are provided with this paper.

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Acknowledgements

We thank F. Yan (Ningbo University) for providing ChiVMV and PepMoV, X. Zhang (Institute of Zoology, Chinese Academy of Sciences) for TuMV-GFP, X. Li (Shandong Agricultural University) for PVY-GFP, H. Guo (Institute of Microbiology, Chinese Academy of Sciences) for PPV-GFP and J.-M. Zhou (Yazhouwan National Laboratory) for the 35S::AvrRpt2 clone. This work was supported by the National Natural Science Foundation of China (32130086, 32430085, 32172360 and 32200118), a Biological Breeding-National Science and Technology Major Project (2024ZD04077) and the National Key Research and Development Program of China (2021YFD1400400 and 2022YFD1400800).

Author information

Authors and Affiliations

  1. MOE Key Laboratory of Bioinformatics and Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Junzhu Wang, Tianyuan Chen, Zhendong Zhang, Mengjie Song, Tianxin Shen, Xin Wang, Xiyin Zheng, Yan Wang, Ke Song, Tiancong Qi & Yule Liu

  2. Tsinghua–Peking Center for Life Sciences, Beijing, China

    Junzhu Wang, Tianyuan Chen, Zhendong Zhang, Mengjie Song, Tianxin Shen, Xin Wang, Xiyin Zheng, Yan Wang, Ke Song, Tiancong Qi & Yule Liu

  3. State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang, China

    Xiaoyang Ge & Fuguang Li

  4. Jiangsu Key Laboratory for Pathogens and Ecosystems, College of Life Sciences, Nanjing Normal University, Nanjing, China

    Kai Xu

  5. State Key Laboratory of North China Crop Improvement and Regulation and College of Horticulture, Hebei Agricultural University, Baoding, China

    Yiguo Hong

  6. School of Life Sciences, University of Warwick, Coventry, UK

    Yiguo Hong

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  1. Junzhu Wang
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Contributions

Y.L. conceived the research. Y.L. and J.W. designed the experiments. J.W. and T.C. constructed expression plasmids. J.W., Z.Z., M.S., T.S. and X.W. preformed N. benthamiana-related experiments including plant transformation and confirmation of transgenic resistance, with assistance from T.C., X.Z., K.S., Y.W. and T.Q. X.G. and F.L. generated transgenic soybean plants. J.W. tested viral resistance in transgenic soybean plants with assistance from K.X. The manuscript was written by Y.L., J.W. and Y.H. with contributions from all authors.

Corresponding author

Correspondence to Yule Liu.

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Competing interests

Y.L. and J.W. have filed a patent application covering the entire content of this study (patent applicant: Tsinghua University; inventors: Yule Liu, Junzhu Wang; application no.: PCT/CN2025/075120). The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Expression and traits of transgenic plants of HA-PCSPVY-aTm-22.

a, Transient expression of either NIaPVY-Myc or NIa-ProTuMV-Myc caused cell death in T0 transgenic HA-PCSPVY-aTm-22 plants (lines 3 and 5), but not in wild-type (WT) plants at 2 days post agroinfiltration. Bar is 2 cm. b, Successful expression of Myc-tagged proteins was confirmed by immunoblotting. Loading of total protein samples for analyses are shown (lower panel). Sizes and positions of protein markers are indicated. c, T1 transgenic HA-PCSPVY-aTm-22 plants of two independent lines showed no developmental defects. Bar is 5 cm. d, No significant trait difference between wild-type and transgenic plants. Height and fresh weight were measured when the plants are 3 months old, and seed setting indicated by numbers of seed pods were measured when the plants are 4 months old. “ns” means no significance as determined by two-sided Student’s t tests (n = 6 biologically independent samples). Data are represented as mean ± s.d. e, Transgene and protein expression of the HA-PCSPVY-aTm-22 chimeric protein were detected by genomic PCR (upper panel) and immunoblotting (middle panel) in three independent individuals of T1 plants from two independent transgenic lines 3 and 5. Loading of total protein samples for analyses are shown (lower panel). Sizes and positions of DNA (upper panel) and protein markers (middle and lower panels) are indicated. f, Cleavage of HA-PCSPVY-aTm-22 by Myc-NIa-ProPVY in T1 transgenic plants. Myc-NIa-ProPVY and AvrRpt2-Myc were expressed at the left or right halves of the same leaves from the transgenic plants. Wide-type leaf tissues without agroinfiltration served as negative controls. Total proteins were extracted and analyzed. NLR proteins and proteases were detected by anti-HA and anti-Myc antibodies, respectively. Experiments were repeated at least three times with similar results.

Source data

Extended Data Fig. 2 HA-PCSPVY-aTm-22 confers resistance against PVY-GFP.

a, Inoculated leaves under normal light (left panel) and ultra-violet light (right panel) at 7 dpi by PVY-GFP. Leaves of WT, 3-T1 and 5-T1 came from the same plants showed in Fig. 1g. HR lesions are indicated (arrows). Bar is 2 cm. b, c, Inoculated leaves showed HR lesions at 7 dpi (b), but whole plant showed systemic hypersensitive response (SHR) at 21 dpi (c) for a few T1 transgenic HA-PCSPVY-aTm-22 plant infected by PVY-GFP. Wild-type plant was used as control. The bar is 2 cm and 5 cm in panels b and c, respectively.

Extended Data Fig. 3 HA-PCSPVY-aTm-22 confers resistance against potyviruses.

a-d, Leaves of T1 transgenic HA-PCSPVY-aTm-22 plants were inoculated with TuMV-GFP (a), PPV-GFP (b), PepMoV (c) and ChiVMV (d), respectively, and photographed at 7 dpi. HR lesions are indicated (arrows) in T1 transgenic HA-PCSPVY-aTm-22 plants inoculated with PepMoV (c). Bar is 2 cm.

Extended Data Fig. 4 Confirmation of expression in transgenic plants of HA-PCSPVY-aAtNRG1.1.

a, NIaPVY-Myc caused cell death in the leaf of T0 transgenic HA-PCSPVY-aAtNRG1.1 plants. b, T1 transgenic plants of line 1 grew and developed normally. c, Transgene and HA-PCSPVY-aAtNRG1.1 protein were detected by genomic PCR and immunoblotting in three independent individuals of T1 plants of transgenic line 1.

Extended Data Fig. 5 HA-PCSPVY-aAtNRG1.1 confers resistance against multiple potyviruses.

a, Inoculated leaves of wild-type and T1 transgenic HA-PCSPVY-aAtNRG1.1 plants challenged with PVY-GFP. Photographs were taken under normal light and UV light at 7 dpi. HR lesions are indicated (arrows). Bar is 2 cm. b, Systemic tissues of PPV-inoculated WT and transgenic plants at 7 dpi. Bar = 5 cm. c, Inoculated leaves of T1 transgenic HA-PCSPVY-aAtNRG1.1 plants co-infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV and wild-type plants infected with each of the four individual potyviruses. Two halves of one leaf of the transgenic plant were infected with TuMV-GFP and PPV-GFP, respectively. Two halves of another leaf of the same transgenic plant were infected with PepMoV and ChiVMV, respectively. One half leaf from different wild-type plants was infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV, separately. Leaf photographs were taken at 7 dpi. HR lesions are indicated (arrows). Bar = 2 cm.

Extended Data Fig. 6 HA-PCSPVY-aNLR failed to confer resistance against TEV.

a, Co-expression of HA-PCSPVY-aNLR with PVY NIa-Myc, but not TEV NIa-Myc, caused cell death. Photograph was taken at 3 days post agroinfiltration. b, TEV-GFP spread over WT and T1 transgenic HA-PCSPVY-aAtNRG1.1 plants of line 1 at 7 dpi. c, RT-PCR detected the presence of TEV RNA in WT and transgenic HA-PCSPVY-aAtNRG1.1 plants at 7 dpi by TEV-GFP. Experiments were repeated at least three times with similar results.

Extended Data Fig. 7 Confirmation of expression and resistance in transgenic plants of HA-PCSTEV-PCSPVY-aAtNRG1.1.

a, T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants from line 3 showed no abnormal development. b, Transgene and its protein expression were detected by genomic PCR and immunoblotting in three independent individual T1 plants of line 3. c, d, The inoculated leaves of T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants were photographed under normal light and UV light at 7 days post infection with PVY-GFP (c) and TEV-GFP (d). Bar is 2 cm.

Extended Data Fig. 8 HA-PCSTEV-PCSPVY-aAtNRG1.1 confers resistance against multiple potyviruses.

a, The inoculated leaves of T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. HR lesions are indicated with arrows. Photographs were taken at 7 dpi. Bar is 2 cm. b, T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants showed resistance against co-infection of TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. T1 transgenic plants were co-infected with all four potyviruses TuMV-GFP, PPV-GFP, PepMoV and ChiVMV. Two halves of one leaf of the transgenic plant were infected with TuMV-GFP and PPV-GFP, respectively. Two halves of another leaf of the same transgenic plant were infected with PepMoV and ChiVMV, respectively. One half leaf from different wild-type plants was infected with TuMV-GFP, PPV-GFP, PepMoV and ChiVMV, separately. Photographs were taken at 21 dpi. Bar is 5 cm. c, RT-PCR showed that no viral RNA was detected in the systemic leaves of T1 transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants co-infected by TuMV-GFP, PPV-GFP, PepMoV and ChiVMV.

Extended Data Fig. 9 Some transgenic HA-PCSTEV-PCSPVY-aAtNRG1.1 plants showed systemic hypersensitive response (SHR) upon TEV-GFP infection.

a, The inoculated leaves and whole plants were photographed under normal light and UV light at 7 and 21 dpi respectively. b, The enlarged view of top tissues accompanied with SHR at 21 dpi. c, RT-PCR showed that small amount of TEV RNA was detected in the systemic leaves of SHR plants.

Extended Data Fig. 10 HA-PCSSMV-aAtNRG1.1 confers resistance against SMV in T1 transgenic soybean plants.

a, Expression of HA-PCSSMV-aAtNRG1.1 was confirmed in T1 transgenic soybean plants. b, The top view of uninoculated WT, inoculated T1 and WT plants at 66 days post infection of SMV-eGFP. Bar is 5 cm. c, Inoculated leaves of SMV-eGFP were shown. Bar = 2 cm. d, Agronomic traits of wild-type and T1 transgenic soybean plants. Plant height, seed setting (seedpod number and seed number) and yield (seed weight) per plant are shown. Two-sided Student’s t-tests (n = 4 biologically independent samples) were performed. Data are represented as mean ± s.d. “ns” denotes no statistically significant difference, while *** (p < 0.001) indicates a highly significant difference as assessed by Student’s t-tests. P values are shown in the Source Data.

Source data

Supplementary information

Supplementary Fig. 1

This file contains all uncropped blots and gel images.

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1. NIa protease cleavage sites between NIb and CP among 199 versus 110 analysed potyviruses follow XXVXXQ↓A(G/S) versus XXVXHQ↓A(G/S). Supplementary Table 2. NIa protease cleavage sites in polyproteins encoded by seven potyviruses in this paper. Supplementary Table 3. Primers used in this study.

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Wang, J., Chen, T., Zhang, Z. et al. Remodelling autoactive NLRs for broad-spectrum immunity in plants. Nature 645, 737–745 (2025). https://doi.org/10.1038/s41586-025-09252-z

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