Helper NLR immune protein NRC3 evolved to evade inhibition by a cyst nematode virulence effector
Yu Sugihara, Jiorgos Kourelis, Mauricio P. Contreras, Hsuan Pai, Adeline Harant, Muniyandi Selvaraj, AmirAli Toghani, Claudia Martínez-Anaya, Sophien Kamoun
Abstract
Parasites can counteract host immunity by suppressing nucleotide binding and leucine-rich repeat (NLR) proteins that function as immune receptors. We previously showed that a cyst nematode virulence effector SPRYSEC15 (SS15) binds and inhibits oligomerisation of helper NLR proteins in the expanded NRC1/2/3 clade by preventing intramolecular rearrangements required for NRC oligomerisation into an activated resistosome. Here we examined the degree to which NRC proteins from multiple Solanaceae species are sensitive to suppression by SS15 and tested hypotheses about adaptive evolution of the binding interface between the SS15 inhibitor and NRC proteins. Whereas all tested orthologs of NRC2 were inhibited by SS15, some natural variants of NRC1 and NRC3 are insensitive to SS15 suppression.
Introduction
Molecular interactions between plant immune receptors and pathogen effectors drive their coevolution, shaping the dynamics of host-pathogen relationships [1–4]. Harold H. Flor, in 1942, proposed the hypothesis that single genes in plants and pathogens determine the outcome of their interactions; that is, a plant carrying a certain gene displays resistance against a pathogen carrying a corresponding gene [5,6]. In plant-pathogen interactions, Flor’s model is generally interpreted as the recognition of pathogen effectors (known as AVR effectors) by plant immune receptors (encoded by disease resistance genes or R genes), triggering a robust resistance mechanism. This gene-for-gene model has provided an invaluable conceptual framework and has significantly influenced both applied and basic research in disease resistance [7].
Materials and method
Plant growth conditions
N. benthamiana triple nrc2/3/4 CRISPR knock-out (KO) mutant line ‘210.5.5.1’ was grown in a controlled environment growth chamber with a temperature range of 22–25°C, 45–65% humidity, and a 16/8 h light/dark cycle.
Results
Natural variants of NRC1 and NRC3 but not NRC2 are insensitive to SS15 inhibition
We previously showed that SS15 inhibits NRC1, NRC2 and NRC3 but not NRC4 [35,41]. Given that the homologous NRC1, NRC2, NRC3, along with the NLR modulator NRCX, form a well-supported clade in the Solanaceae NRC tree (Figs 1A and S2) [24,37], we investigated the degree to which NRC orthologs from multiple Solanaceae species are inhibited by SS15. For this purpose, we cloned 11 representative NRC sequences from Nicotiana benthamiana (NbNRC2 and NbNRC3), Capsicum annuum (pepper; CaNRC1, CaNRC2 and CaNRC3), Solanum tuberosum (potato; StNRC1, StNRC2 and StNRC3) and Solanum lycopersicum (tomato; SlNRC1, SlNRC2 and SlNRC3) from the phylogenetically related NRC1/2/3 clade (Figs 1 and S2).
Discussion
Plant pathogen effectors can function as suppressors of NLR-mediated immunity [15]. However, the degree to which NLRs have evolved to evade pathogen immunosuppressors has remained unknown. In this study, we discovered that some natural variants of helper NLRs in the NRC family are insensitive to suppression by the cyst nematode effector SS15. We found that some natural variants of NRC1 and NRC3 are insensitive to SS15 suppression, while all tested NRC2s are suppressed by SS15 (Fig 1).
Acknowledgments
C.M.-A. is grateful to the DGAPA-PASPA UNAM Program for financing a sabbatical year at TSL. We thank all members of the TSL Support Services for their invaluable assistance.
Citation: Sugihara Y, Kourelis J, Contreras MP, Pai H, Harant A, Selvaraj M, et al. (2025) Helper NLR immune protein NRC3 evolved to evade inhibition by a cyst nematode virulence effector. PLoS Genet 21(4): e1011653. https://doi.org/10.1371/journal.pgen.1011653
Editor: Tiancong Qi,, Tsinghua University, CHINA
Received: September 10, 2024; Accepted: March 9, 2025; Published: April 9, 2025
Copyright: © 2025 Sugihara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The datasets used in this study are archived in Zenodo (https://doi.org/10.5281/zenodo.14584438). The ancestral sequence reconstruction pipeline “anceseq” is available on Github (https://github.com/YuSugihara/ancseq) and archived in Zenodo (https://doi.org/10.5281/zenodo.10808871).
Funding: 1. This study was supported by The Gatsby Charitable Foundation (YS, JK, MPC, HP, AH, MS, AT, CMA, SK), Biotechnology and Biological Sciences Research Council (BBSRC) BB/P012574 (Plant Health ISP) (YS, JK, MPC, HP, AH, MS, AT, CMA, SK), BBSRC BBS/E/J/000PR9795 (Plant Health ISP - Recognition) (YS, JK, MPC, HP, AH, MS, AT, CMA, SK), BBSRC BBS/E/J/000PR9796 (Plant Health ISP - Response) (YS, JK, MPC, HP, AH, MS, AT, CMA, SK), BBSRC BBS/E/J/000PR9797 (Plant Health ISP – Susceptibility) (YS, JK, MPC, HP, AH, MS, AT, CMA, SK), BBSRC BBS/E/J/000PR9798 (Plant Health ISP – Evolution) (YS, JK, MPC, HP, AH, MS, AT, CMA, SK), BBSRC BB/V002937/1 (SK, MPC), European Research Council (ERC) 743165 (SK). More information about the funding sources can be found at the following websites: the Gatsby Charitable Foundation (https://www.gatsby.org.uk/), BBSRC (https://www.ukri.org/councils/bbsrc/), ERC (https://erc.europa.eu) The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: S.K. receives funding from industry on NLR biology and has cofounded a start-up company (Resurrect Bio Ltd.) related to NLR biology. J.K., M.P.C. and S.K. have filed patents on NLR biology. M.P.C. has received fees from Resurrect Bio Ltd.
