Solanum bulbocastanum nucleotide-binding leucine-rich repeat receptor evolution reveals functional variants and critical residues in Rpi-blb1/RB

doi:10.1111/jipb.13950

• Research Article •

Solanum bulbocastanum nucleotide-binding leucine-rich repeat receptor evolution reveals functional variants and critical residues in Rpi-blb1/RB

Jie Li^1†, Sophie Mantelin^2†,‡,*, Miles Armstrong³, Amanpreet Kaur^2,3, Sonia Gomez^3,4, Jiahan Ying¹, Xiuli Qin¹, Kathryn M. Wright², Brian Harrower², Paolo Ribeca², Théo Chaumet², Gaynor McKenzie², Huanting Liu⁵, Malcolm F White⁵, Thomas Adams², Stuart Ronan Fisher³, Daolong Dou^1,6, Xiaodan Wang^1* and Ingo Hein^1,2,3*

1. State Key Laboratory of Agricultural and Forestry Biosecurity, MOA Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China
2. James Hutton Institute, Invergowrie, Dundee DD25DA, UK
3. University of Dundee, Dundee DD14HN, UK
4. National University of Colombia, Medellín 111321, Colombia
5. School of Biology, University of St Andrews, St Andrews KY16 9ST, UK
6. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

^†These authors contributed equally to this research.
^‡Present address: INRAE UMR 1355 Institute Sophia Agrobiotech, 400 route des Chappes, BP 167 Sophia Antipolis‐Cedex06903 France.
^*Correspondences: Xiaodan Wang (xdwang@cau.edu.cn); Ingo Hein (Ingo.Hein@hutton.ac.uk, Dr. Hein is fully responsible for the distribution of all the materials associated with this article)

Received:2025-03-06 Accepted:2025-05-15 Online:2025-06-17
Supported by:
This work was supported by the National Natural Science Foundation of China (32372558), National Natural Science Foundation of China‐The Royal Society (32061130211), the Rural and Environment Science and Analytical Services Division of the Scottish Government through project JHI‐B1‐1,the Biotechnology and Biological Sciences Research Council(BBSRC) through award BB/S015663/1, and the Royal Society through award NAF\R1\201061.

Abstract

Abstract: Host–pathogen co-evolution shapes resistance (R) proteins and their recognition of pathogen avirulence factors. However, little attention has been paid to naturally occurring genetic diversity in R genes. In this study, 12 Solanum bulbocastanum accessions from the Commonwealth Potato Collection were screened for resistance to Phytophthora infestans, identifying 11 resistant and one susceptible accession. Targeted enrichment sequencing of nucleotide-binding leucine-rich repeat (NLR) genes using RenSeq, followed by diagnostic RenSeq (dRenSeq) analysis, revealed that all accessions except 7650 contained Rpi-blb1/RB variants. Variants in accessions 7641 and 7648 were non-functional, while three novel functional variants were identified. Cloning and functional analysis of Rpi-blb1/RB variants assessed their recognition of the avirulence factor IPI-O1. Three variants were functional, conferring resistance to P. infestans. Variants in accessions 7644 and 7647 also recognized IPI-O4, confirmed in transgenic potatoes. Analysis of a non-functional variant in S. bulbocastanum accession 7648 identified amino acid Ser347 in the nucleotide-binding (NB-ARC) domain as critical for cell-death initiation following IPI-O1 recognition. Predictions from the FunFOLD2 protein–ligand interaction model suggested that Ser347 is essential for ATP binding, suggesting potential inhibition on pentameric resistosome assembly. Western blot analysis revealed that the mutation of Ser347 to Asn markedly compromises the Rpi-blb1/RB protein stability, and co-immunoprecipitation assay further confirmed that this mutation severely disrupts the self-association of CCNB, thereby preventing Rpi-blb1/RB activation. Consistently, substituting Asn347 with serine restored function, underscoring its key role in Rpi-blb1/RB activity. Cell biology experiments demonstrated that Rpi-blb1/RB relocalize to the plasma membrane in response to IPI-O1. This relocalization depends on Ser347, further supporting the idea that its mutation affects resistosome formation, impairing resistance. This study provides an in-depth functional analysis of natural Rpi-blb1/RB diversity, offering insights into NLR protein evolution and resistance mechanisms in potatoes.

Key words: enrichment sequencing, host–pathogen co‐evolution, nucleotide‐binding leucine‐rich repeat (NLR) gene, plant immunity, Solanum bulbocastanum

Jie Li, Sophie Mantelin, Miles Armstrong, Amanpreet Kaur, Sonia Gomez, Jiahan Ying, Xiuli Qin, Kathryn M. Wright, Brian Harrower, Paolo Ribeca, Théo Chaumet, Gaynor McKenzie, Huanting Liu, Malcolm F White, Thomas Adams, Stuart Ronan Fisher, Daolong Dou, Xiaodan Wang, Ingo Hein. Solanum bulbocastanum nucleotide-binding leucine-rich repeat receptor evolution reveals functional variants and critical residues in Rpi-blb1/RB[J]. J Integr Plant Biol., DOI: 10.1111/jipb.13950.

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Solanum bulbocastanum nucleotide-binding leucine-rich repeat receptor evolution reveals functional variants and critical residues in Rpi-blb1/RB

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