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Plug-in strategy for resistance engineering inspired by potato NLRome

Nature volume 649, pages 396–405 (2026)Cite this article

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Abstract

Potato late blight, which is caused by Phytophthora infestans and was responsible for the Irish potato famine, remains a major threat to global food security1. Most late-blight resistance (R) genes encode nucleotide-binding leucine-rich repeat proteins (NLRs), but many have been overcome by the rapid evolution of P. infestans2. Deploying R genes through hybrid potato breeding3,4,5,6,7 offers a promising solution to manage late blight. Here we construct a section-wide NLRome comprising 39,211 NLR genes from 31 wild and 21 cultivated potato genomes, representing Solanum section Petota, the tuber-bearing clade. This includes newly sequenced genomes of seven wild species with strong resistance to late blight. Phylogenomic analyses reveal asymmetric patterns of evolution that distinguish sensor and helper NLRs. Mining of the NLRome enabled us to clone Rpi-cph1, a homologue of which has previously been identified only in American black nightshade, and Rpi-cjm1, a Toll/interleukin-1 receptor (TIR) domain-containing NLR against late blight. We show that non-canonical NLR integrated domains are widespread in the NLRome. Tracing their evolutionary trajectory enabled us to identify Rpi-brk1, an R gene that perceives a P. infestans effector through its heavy-metal-associated (HMA) domain. We find that incorporating the HMA domain into the potato NLR R1 broadens its resistance spectrum, suggesting that a domain ‘plug-in’ strategy could be used to engineer disease resistance. These findings provide a paradigm for R-gene discovery through comparative and evolutionary genomics, and a strategy for R-gene engineering.

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Fig. 1: Section-wide NLRome of Petota.
Fig. 2: Identification and functional characterization of Rpi-cph1.
Fig. 3: Identification and functional characterization of Rpi-cjm1.
Fig. 4: Rpi-brk1 is an HMA-domain-containing NLR that confers resistance to P. infestans.
Fig. 5: Plug-in strategy for engineering the potato R1 resistance gene.

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Data availability

All PacBio HiFi sequences and transcriptome data used in this study have been deposited at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject accession number PRJNA831581. Genome assemblies of the seven potato accessions have been deposited in the NCBI GenBank database under the accession number PRJNA831581. Genome assemblies and annotations for the seven potato accessions are available at GitHub (https://github.com/HongboDoll/PotatoNLRome/releases/tag/v1.2.1). NLR annotations for the 52 potato accessions are available at GitHub (https://github.com/HongboDoll/PotatoNLRome/releases/tag/v1.0). The genotypes of the 50 accessions used for GWAS are available at GitHub (https://github.com/HongboDoll/PotatoNLRome/releases/tag/v1.3). Previously released potato genome assemblies were downloaded from the Pan-Potato Database (http://solomics.agis.org.cn/potato/ftp/genome/) and Spud DB (http://spuddb.uga.edu/dm_v6_1_download.shtml). Publicly available potato transcriptomic data were downloaded from NCBI with BioProject accession number PRJNA754534. The embryophyta_odb10 database is available at https://busco-data.ezlab.org/v4/data/lineages/. The sequences of cloned R genes have been deposited in the NCBI GenBank database (Rpi-cjm1, BankIt2946483: PV474667; Rpi-cph1.1, BankIt2946490: PV474668; Rpi-cph1.2, BankIt2946493: PV474669; Rpi-brk1, BankIt2946498: PV474670). Source data are provided with this paper.

Code availability

All custom scripts used in this study are available at GitHub (https://github.com/HongboDoll/PotatoNLRome) and Zenodo (https://doi.org/10.5281/zenodo.14211048) (ref. 100).

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Acknowledgements

We thank S. Kamoun, J. D. G. Jones, Y. Wang, C. Zhang, Y. Zhang, J. Chen, Z. Shao, S. Li, L. Kong, R. van der Hoorn, I. Hein, K. Sohn, P. Birch, H. Wang, J. Huang, D. Yan and Y. Yang for project discussions and critical comments; T. Qi for providing N. benthamiana knockout lines; X. Liu, T. Wang and Q. Zhu for assistance with enzymatic analysis of Rpi-cjm1 and BN-PAGE experiments; Q. Fu, F. Zhang, X. Zhao and all other members of the S.D. and S.H. laboratories for assistance during this work; H. Luo for assistance with protein purification; and the AGIS CAAS-YNNU Joint Academy of Potato Sciences for assisting with greenhouse work. This work was supported by the National Natural Science Foundation of China (32488302 and 32130088), Guangdong Major Project of Basic and Applied Basic Research (2021B0301030004), the Agricultural Science and Technology Innovation Program (CAAS-ZDRW202101 and CAAS-ZDRW202404), the National Key Research and Development Program of China (2019YFA0906200 and 2019YFE0120500), the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District (RC201901-05), Shenzhen Outstanding Talents Training Fund, the Shenzhen Science and Technology Program (KQTD2016113010482651), the Natural Science Foundation of Shandong Province (ZR2025QC1405), Taishan Scholar Foundation of the People’s Government of Shandong Province (ts202507142) and Young Talent of Lifting Engineering for Science and Technology in Shandong, China (SDAST2024QTA030).

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Author notes

  1. These authors contributed equally: Luyao Wang, Hongbo Li, Yuhang Ke

Authors and Affiliations

  1. National Key Laboratory of Tropical Crop Breeding, Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Luyao Wang, Hongbo Li, Ping Zhu, Yuying Li, Pei Wang, Shaoqun Zhou & Sanwen Huang

  2. State Key Laboratory of Agricultural and Forestry Biosecurity, College of Plant Protection, Nanjing Agricultural University, Nanjing, China

    Luyao Wang, Yuhang Ke, Ping Zhu, Haohao Liu, Yajie Li & Suomeng Dong

  3. College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, China

    Hongbo Li

  4. School of Life Sciences, Yunnan Key Laboratory of Potato Biology, Yunnan Normal University, Southwest United Graduate School, Kunming, China

    Pei Wang

  5. State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Jingqi Chen & Li Wan

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

    Savithramma P. Dinesh-Kumar

  7. National Key Laboratory of Tropical Crop Breeding, Chinese Academy of Tropical Agricultural Sciences, Haikou, China

    Sanwen Huang

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  3. Yuhang Ke
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  4. Ping Zhu
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  5. Yuying Li
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  11. Li Wan
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  12. Savithramma P. Dinesh-Kumar
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  13. Suomeng Dong
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Contributions

S.H. and S.D. conceived and designed the project. L. Wang, Y.K., Yuying Li, H. Liu, Yajie Li and J.C. performed experiments. H. Li., L. Wang and P.Z. performed bioinformatic analyses. P.W., L. Wang and Yuying Li contributed to greenhouse work. H. Li., L. Wang, S.D. and S.H. wrote the manuscript. S.H., S.D., S.P.D.-K., L. Wan and S.Z. revised the manuscript. S.H. and S.D. coordinated the project. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Suomeng Dong or Sanwen Huang.

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Competing interests

The authors declare no competing interests.

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Nature thanks Doil Choi, Zuhua He and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Overview of NLRs in Petota.

a, Phylogenetic relationship among the 52 wild and cultivated potato accessions and one tomato. Species names marked using asterisks denote the seven wild potatoes whose genomes are sequenced and assembled in this study. S. stenotomum, S. tuberosum Group Stenotomum; S. phrueja, S. tuberosum Group Phrueja. b, NLR copy number in potato. CNL: CC-NB-LRR, TNL (TIR-NLR): TIR-NB-LRR, NL: NB-LRR, RNL: RPW8-NB-LRR. c, Correlation between assembled monoploid genome size and number of predicted NLRs. The linear regression line is represented in blue. The 95% confidence interval is illustrated using grey-shaded regions. A two-tailed Pearson’s correlation test was used to calculate the correlation coefficient and P value. d, Violin plot shows the number of CNL, TNL, NL, and RNL in the 52 potato accessions. There are 52 samples in each category. The upper and lower dotted lines denote 75% and 25% quartiles, and the central dotted line indicates the median. The density curves show the data distribution.

Source Data

Extended Data Fig. 2 Characterization of type I and type II NLRs.

a, Phylogenetic relationships among type I and type II NLR clades. NLR clades whose mean branch length relative to their leaves is less than 1 are collapsed, marked using triangles in the phylogeny. b, Number of type I NLRs clades sister to type II or type I NLR clades. c, Number of previously reported sensor and helper NLRs belonging to Type I and Type II NLR clades. d, Proportions of type I and type II NLRs distributed in NLR gene clusters. e, Number of previously reported sensors, NLRs, which recognize pathogen effectors from five types of pathogens, are distributed in type I and type II NLR clades. f, Evolutionary features of type I and type II NLRs, using NRC4, R1 (type I NLRs) and NRC0, NRCx, NRC1, NRC2, NRC3, Prf (type II NLRs) as examples. Bootstrap values > 75 are displayed for the corresponding nodes.

Source Data

Extended Data Fig. 3 Rpi-cph1.1 and Rpi-cph1.2 confer resistance to P. infestans.

a, Phylogenetic relationships among Rpi-cph1, Rpi-amr3 and Rpi-amr3 homologues in Clade006. The phylogeny was constructed using alignments of amino acid sequences from the NB-ARC domain. ZAR1 was used as an outgroup. b, Two alleles of Rpi-cph1 conferred resistance against P. infestans JH19 when transiently expressed in N. benthamiana leaves. Late-blight resistance was not observed in two Rpi-amr3 homologues in S. hintonii and S. cajamarquense. Images were taken 5 days post-agro-infiltration. Scale bar = 1.5 cm. c, Western blot analysis of tested Rpi-amr3 homologues with c-terminus 10×myc tag. d, Resistance of Rpi-cph1.1 and Rpi-cph1.2 relied on NRC helpers in N. benthamiana. Images were taken 5 days post-agro-infiltration and inoculated with zoospore suspension of the P. infestans isolate JH19. Scale bar = 1 cm. e, Three independent transgenic potato lines of the Rpi-cph1.2 (#7, #16, #22) allele showed resistance against the P. infestans isolate 88069. Images were taken 5 days post-zoospore inoculation. Scale bar = 1.5 cm. f,g, Rpi-cph1.1 (#10, #13, #17) (f) and Rpi-cph1.2 (#7, #16, #22) (g) transgenic potato lines exhibited high resistance against 5 selected P. infestans isolates. WT, wild type. All transgenic potato lines were generated in the biological background of S. tuberosum cv. Désirée. h, Self-activation induced by two MHD mutants of Rpi-cph1.1 and Rpi-cph1.2 required native NRC helpers in potato. MHD mutants and NRC helpers from S. cardiophyllum were co-expressed in 4-week-old N. benthamiana leaves by agro-infiltration in the same final concentration (OD600 = 0.3). Sc, S. cardiophyllum. The numbers on the right bottom of the photographs indicate the number of leaves showing HR cell death out of the total number of leaves infiltrated. i, Rpi-cph1.1 and Rpi-cph1.2 required native NRC helpers for sensing AVRamr3 in N. benthamiana. Rpi-cph1.1 and Rpi-cph1.2 were co-expressed with AVRamr3 and the selected NRC helpers. Sc, S. cardiophyllum. St, S. tuberosum. Nb, N. benthamiana. All used genes were driven by the CaMV 35S promoter. Images were taken 5 days post-agro-infiltration. The numbers on the right bottom of the photographs indicate the number of leaves showing HR cell death out of the total number of leaves infiltrated. j, Western blot analysis of NRC helpers from S. cardiophyllum (ScNRC2, ScNRC3, ScNRC4), from S. tuberosum cv. Désirée (StNRC4) and from N. benthamiana (NbNRC4) with c-terminus 10×myc tag. Data in b,d,f–h are presented as mean + s.d. Different lower-case letters indicate significant differences among treatments (P ≤ 0.01, one-way ANOVA–Tukey’s HSD test, n  =  9 independent experiments in b, n  =  12 independent experiments in d, n  =  9 independent experiments in f–h, detailed P values between each two comparisons are provided in the Source Data file). The experiments in c,j were performed three times independently with similar results. For gel source data, see Source Data Extended Data Fig. 3.

Source Data

Extended Data Fig. 4 Transient expression of Rpi-cjm1 in N. benthamiana confers resistance to P. infestans.

a, Transient expression of CaMV 35S-driven Rpi-cjm1 in N. benthamiana leaves conferred resistance to the P. infestans isolate JH19. Data are presented as mean + s.d. The three asterisks indicate significant differences between infected area in leaves expressed Rpi-cjm1 or gfp control (P = 0.00000016, Student’s t-test, n  =  10 independent experiments). Scale bar =1.5 cm. b, Rpi-cjm1 induced HR cell death in N. benthamiana leaves when co-expressed with the P. infestans effector PITG_04085. c, HR symptoms when co-expressing different NLR with AVR proteins in wild-type (WT) and nrc2/3-KO, nrc4-KO, nrc2/3/4-KO, eds1-KO, nrg1-KO and adr1-KO N. benthamiana plants. KO, knockout. The numbers on the bottom right of the photographs indicate the number of HR symptom sites and the number of experiments. d, Resistance conferred by Rpi-cjm1 against P. infestans required NbEDS1 and NbNRG1. Images were taken 5 days post-agro-infiltration and inoculated with zoospore suspension of the P. infestans isolate JH19. Scale bar = 1 cm. Data are presented as mean ± s.d. Different lower-case letters indicate significant differences among treatments (P ≤ 0.01, one-way ANOVA–Tukey’s HSD test, n  =  9 independent experiments, detailed P values between each two comparisons are provided in the Source Data file).

Source Data

Extended Data Fig. 5 Diversity and evolution of NLR IDs in Petota.

a, Number of distinct IDs in the four phylogenetic groups of Petota. The sample sizes are 6, 20, 5, and 21, respectively. P value = 0.002291 in Kruskal–Wallis non-parametric (two-tailed) test. Fisher’s least significant difference was used for multiple comparisons. CND, S. candolleanum. b, The top 15 most prevalent IDs in the NLRome. c, Phylogenetic relationships among representative IDs and NLRs in the S. cardiophyllum genome. The phylogeny was constructed based on the alignment of NB-ARC domain amino acid sequences. Branches that do not contain NLRs with IDs are collapsed. IDs with a presence frequency ≥ 10 are indicated by text. d, Proportions of IDs present exclusively in NLRs and those also found in other non-NLR genes. e, Ka/Ks values of pairs of IDs present in NLR and non-NLR genes. f, Distribution of Tajima’s D values. NLR-ID refers to IDs present in NLRs. Non-NLR-ID refers to IDs present in non-NLR genes. For e,f, the number of samples in each category is 52. Grey lines connect values of the same ID in NLRs and non-NLR genes. Red lines highlight the HMA domain. In a,e,f, the upper and lower edges of boxes represent the 75th and 25th percentiles, respectively, and the central line indicates the median. The whiskers extend to the minimum and maximum data points. * P < 0.05 and *** P < 0.001 in the two-tailed Wilcoxon rank sum test. g, Distribution of NLR-HMA, total NLRs and non-NLR HMA genes across 94 Solanaceae species. Branch length indicates divergence time (million years ago) between species. NLR-HMA refers to NLRs with HMA domains.

Source Data

Extended Data Fig. 6 IDs in wild and cultivated potato.

a, Intersection of identified IDs in species from the four phylogenetic taxa of Petota. CND is the proposed progenitor of cultivated potatoes Solanum candolleanum. b, Phylogenetic relationships among NLRs carrying the HMA domain (NLR-HMA) and the R1 resistance gene. The phylogeny was constructed based on the alignment of NB-ARC domain amino acid sequences.

Source Data

Extended Data Fig. 7 Phylogenetic relationships among NLRs with HMA in Solanaceae.

The phylogeny was constructed using alignments of amino acid sequences from the NB-ARC domain.

Source Data

Extended Data Fig. 8 Phylogenetic analyses of HMA-containing genes in Solanaceae species.

a, Phylogenetic relationships across 3,521 genes carrying HMA domains in 42 Solanaceae species. The NLR-HMA clade is depicted using purple lines. b, A zoom-in view of cladogram of the NLR-HMA clade and its sister clade. Domain architecture for each clade is also shown. In a,b, the phylogeny was constructed using alignments of amino acid sequences from the HMA domain.

Source Data

Extended Data Fig. 9 Genomic distribution of NLR-HMA and related genes.

a, Distribution of NLRs in the R1 gene cluster and NLR-HMA in the potato DM reference genome, with a window length of 500 kb. b, Genomic coordinates of genes surrounding the R1 gene cluster in 8 wild and cultivated potato species and 2 Solanum wild species.

Source Data

Extended Data Fig. 10 Protein complex of HMAbrk1 and AVRbrk1.

a, NRC dependency analysis of Rpi-brk1. HR index data was recorded 5 days post-agro-infiltration. b, NRC dependency analysis of Rpi-brk1. Images were taken 5 days post-zoospore inoculation of P. infestans isolate JH19. Scale bar =1 cm. c, Prediction of protein complex of HMAbrk1 and AVRbrk1. The protein complex structures were generated using ClusPro and visualized with PyMOL. d, Screening of key amino acid residues that regulate the interaction of the HMAbrk1-AVRbrk1 protein complex using split-luciferase assay. D1124 was used as a control. e, Three independent transgenic potato lines expressing Rpi-brk1 (#1, #3 and #9), but not Rpi-brk1-m7 mutant (#2, #6 and #11), conferred resistance against five P. infestans isolates. Images were taken 5 days post-zoospore inoculation. f, NRC dependency analysis of R1-HMAbrk1 and R1-HMAbrk1-m7. Resistant gene R1 fused with HMAbrk1 or derivate mutant HMAbrk1-m7 were co-expressed with AVRbrk1 or AVR1 in wild-type N. benthamiana or nrc2/3, nrc4, nrc2/3/4 knockout mutants. HR index data was recorded 5 days post-agro-infiltration. Data in a,b,e,f are presented as mean + s.d. Different lower-case letters indicate significant differences among treatments (P ≤ 0.01, one-way ANOVA–Tukey’s HSD test, n  =  12 independent experiments in a,b,e,f, detailed P values between each two comparisons are provided in the Source Data file).

Source Data

Extended Data Table 1 Summary of the 52 potato genomes used in this study
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Supplementary information

Supplementary Information

Supplementary Notes 1–7, Discussion, Figs. 1–16 and references.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–19.

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Source Data Supplementary Figs. 8, 10, 13, 14 and 16

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Wang, L., Li, H., Ke, Y. et al. Plug-in strategy for resistance engineering inspired by potato NLRome. Nature 649, 396–405 (2026). https://doi.org/10.1038/s41586-025-09678-5

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