Atypical NLR pairs in wheat: Sensor–helper NLRs conferring disease resistance

This commentary discusses new research showing that six structurally and functionally atypical NLR pairs in wheat confer disease resistance via a sensor NLR-helper NLR module. The functional mechanisms of some NLR pairs differ from the classical NLR pair model, revealing the complexity and diversity of the wheat immune system. The discovery and deployment of nucleotide-binding leucine-rich repeat receptors (NLRs) from wild relatives are critical strategies to broaden the disease resistance of wheat. Six papers have reported the discovery of several atypical NLR pairs from wild emmer wheat (WEW), demonstrating a sensor–helper mechanism. Wheat is the most widely cultivated crop in the world, providing approximately 20% of the global dietary calories and protein (The International Wheat Genome Sequencing Consortium IWGSC, 2014). The genetic bottleneck effect during polyploidization and domestication, coupled with the concentrated selection of a few superior genotypes in modern breeding, leads to the narrow genetic diversity of wheat (Zhou et al., 2020). The limited genetic variation renders wheat varieties particularly vulnerable to rapidly evolving fungal pathogens, leading to the rapid breakdown of resistance, especially powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and stripe rust (Puccinia striiformis f. sp. tritici, Pst). Most of the disease-resistant (R) proteins belong to the NLR protein family (Jones and Dangl, 2006). The typical characteristics of the NLR gene include an N-terminal variable domain containing either a coiled coil (CC) or Toll/ interleukin-1 receptor (TIR) domain, a central nucleotide-binding and Apaf-1, R proteins, and CED-4 (NB-ARC) domain, and a C-terminal leucine-rich repeat sequence (LRR) domain (Zhang et al., 2017). Some NLRs work in pairs or more complex networks: one is the sensor NLR (sNLR), which recognizes a pathogen effector, and the other is the helper NLR (hNLR), which induces downstream immune responses (Adachi et al., 2019). Typical sNLRs contain integrated domains (IDs), such as kinase, WRKY, and potassium-dependent sodium–calcium exchanger (NCKX). Genetically linked NLR pairs, such as RGA5/RGA4 and Pik-1/Pik-2 in rice and Arabidopsis RRS1/RPS4, function as sNLR and hNLR (Figure 1A) (Adachi et al., 2019). Six NLR pairs from wild emmer wheat confer rust and powdery mildew resistance in wheat (A) Mechanism model of regulating immune responses by RRS1/RPS4, RGA5/RGA4, and Pik-1/Pik-2 receptor complexes. (B) Gene loci, protein structures, and functions of six NLR pairs of wild emmer wheat. TdNLR1, CNL, TdCNL1, MlIW39-R2, PmWR183-NLR2, and Pm68-2 act as sensor NLR (sNLR), and TdNLR2, NL, TdCNL5, MlIW39-R1, PmWR183-NLR1, and Pm68-1 function as helper NLR (hNLR). A, Alanine; Bgt, Blumeria graminis f. sp. tritici; CC, coiled coil; CCl, coiled coil-like; E, Glutamic acid; HR, Hypersensitive response; K, Lysine; L, Leucine; LRR, leucine-rich repeat; NB-ARC, nucleotide-binding adaptor shared by Apaf-1, R proteins, and Ced-4; NCKX, potassium-dependent sodium–calcium exchanger; Pst, Puccinia striiformis f. sp. tritici; S, Serine; T, Threonine. In the latest issue of Nature Genetics, Hu et al. (2025) and Klymiuk et al. (2025) cloned and identified two pairs of NLR genes encoded by alleles of the same rust resistance locus (Figure 1B). Additionally, Zhu et al. (2025) have achieved successful cloning and identification of a pair of NLR genes that mediate resistance to powdery mildew (Figure 1B). Similarly, in Nature Communications, He et al. (2025), Yang et al. (2025), and Zhang et al. (2025) cloned three alleles encoding an NLR pair on a single powdery mildew resistance locus (Figure 1B). The genomic loci and domain structures of these NLR pairs are illustrated in Figure 1B. Hu et al. (2025) mapped the yellow rust R gene YrTD121 to a 1.1-Mb interval on chromosome 1BS and identified two head-to-head oriented NLR genes: TdNLR1 and TdNLR2 (an atypical NB-ARC-LRR, lacking a canonical N-terminal signaling domain) (Figure 1B). Functional validation through ethyl methanesulfonate (EMS) mutagenesis, virus-induced gene silencing (VIGS), genome editing, and transgenic assays confirmed their interdependence in YrTD121-mediated stripe rust resistance. Molecular interaction and cell death assays further demonstrated that TdNLR1CC self-associates and triggers cell death in N. benthamiana leaves, while TdNLR2 likely functions as the hNLR. To more precisely identify the dominant Yr84 resistance locus, Klymiuk et al. (2025) localized it to a 0.5 cM interval on chromosome 1BS, revealing two head-to-head NLR genes: CNL and NL (an atypical NB-ARC-LRR, lacking a canonical N-terminal signaling domain) (Figure 1B). EMS and transgenic assays confirmed their co-dependency, with CNL likely acting as an sNLR and NL acting as an hNLR. Their LRR domains interacted in vivo, despite no full-length protein interaction being detected. The Yr84 and YrTD121 alleles were detected in 21% (38/177) and 17% (62/363) of WEW accessions, respectively. Among WEW populations, 10 CNL and 12 NL protein haplotypes were identified for the Yr84 locus, and 16 TdNLR1 and 11 TdNLR2 protein variants were identified for the YrTD121 locus, forming 20 haplotype combinations. Among these, Yr84 and its allele YrTD121 were functional haplotypes. TdNLR1 differs from the CNL reported here by two amino acid substitutions (S520L and A866T), whereas TdNLR2 corresponds to NL but carries a single amino acid substitution (E110K). The introgression of Yr84 or YrTD121 into several durum and bread wheat cultivars conferred robust resistance without compromising agronomic traits, as evidenced by field trials. Zhu et al. (2025) precisely located the powdery mildew resistance site MLIW170/PM26 to the range of 0.07 cM on chromosome 2BS, identifying an atypical TdCNL1 characterized by the incorporation of an NCKX domain into the NB-ARC domain and a typical TdCNL5 (Figure 1B). Notably, the TdCNL1/TdCNL5 pair is not in a head-to-head orientation. Both genes proved essential for resistance through mutagenesis and VIGS experiments, with loss of function of either TdCNL1 or TdCNL5 leading to susceptibility in the original host backgrounds. TdCNL1 likely acts as an sNLR, whereas TdCNL5 may correspond to an hNLR. Interestingly, transgenic experiments in the Fielder wheat background revealed that resistance required either TdCNL1 alone or both genes, but not TdCNL5 alone. The functional pair was detected in just 8.8% of WEW, with three haplotypes showing distinct geographic distributions. He et al. (2025), Yang et al. (2025), and Zhang et al. (2025), localized the powdery mildew resistance alleles PmWR183, MlIW39, and Pm68, respectively, to the same locus on chromosome 2BS (Figure 1B). Transgenic experiments proved that resistance for each allele requires paired NLR proteins: A canonical CC-type hNLR (PmWR183-NLR1, MlIW39-R1, and Pm68-1) and an atypical sNLR (PmWR183-NLR2, MlIW39-R2, and Pm68-2) with a CC-like (CCl) domain (Figure 1B). Transient expression in N. benthamiana and protein interaction experiments showed that the activity of each hNLR depends on its corresponding sNLR, with physical interaction occurring between the two partners within each pair. These hNLRs and sNLRs originate from different Triticeae chromosomes and became genetically linked in WEW. While not yet fully deployed in cultivated wheat, their introgression does not appear to adversely affect key agronomic traits. In the well-studied NLR pairs RGA5/RGA4, Pik-1/Pik-2, and RRS1/RPS4, the hNLRs are also known as the executors (Adachi et al., 2019). In contrast, the sNLRs TdNLR1 and TdCNL1 respectively show cell death execution capabilities and autoactivation characteristics, indicating that they are more like executors rather than mere pathogen sensors. Supporting this view, the presumed hNLR TdNLR2 lacks the pore-forming CC domain, while both TdNLR2 and TdCNL5 may function as regulatory subunits or scaffolds. This functional discrepancy challenges the rigid sensor–helper (executor) binary classification. Therefore, adopting a more nuanced terminology, such as executor–enhancer or executor–regulator, would more accurately reflect the atypical relationship observed in these NLR pairs. Among paired NLRs RRS1/RPS4, RGA5/RGA4, and Pik-1/Pik-2, sNLRs and hNLRs form heterogeneous complexes in both the resting and activated states (Figure 1A) (Adachi et al., 2019). In contrast, no direct physical interaction was detected between full-length TdNLR1 and TdNLR2, or between CNL and NL. This discrepancy hints at a transient activation mode reminiscent of the Solanaceous NRC network, or alternatively, a wheat-specific membrane-centric assembly that evades conventional cytosolic interaction screens. Although interactions were observed between CNLLRR and NLLRR, this may represent only a snapshot of a larger membrane-anchored complex. To clarify the activation mechanism, an integrated experimental approach is recommended. Future work should combine (1) sub-cellular fractionation with membrane-protected co-immunoprecipitation or split-luciferase assays in N. benthamiana protoplasts with defined lipid perturbations; (2) total internal reflection fluorescent microscope study of fluorophore-tagged full-length proteins to visualize transient membrane micro-domains; and (3) AlphaFold3 multimer predictions constrained to membrane-embedded states to identify lipid-binding surfaces that stabilize the interfaces between TdNLR1 and TdNLR2 or between CNL and NL. For these TdNLR pairs in WEW, key questions remain unresolved regarding the identification of their corresponding effector proteins, which probably directly interact with NLRLRR or indirectly associate through host guardees/decoys or integrated decoy proteins (Figure 1B). During pathogen perception, oligomeric resistosomes are formed by NLRs. The structures of the resistosomes formed by these NLR pairs from WEW also need to be further resolved. Is sNLR alone, hNLR alone, or the sNLR–hNLR complex assembled into a higher-order oligomer structure to give rise to calcium (Ca2+) permeable channels (Figure 1B)? Notably, TdNLR2 and NL lack the pore-forming CC domain. The CC domain in hNLRs (e.g., ADR1s, NRG1s, and NRCs) is critical for oligomerization, membrane association, and formation of Ca2+-permeable pores. However, the sequence and architecture of TdNLR2 and NL preclude this executive function. What is the structural role of this type of hNLR in the resistosomes? Does it act as a “Plug” or a “Scaffold”? The relatively low frequency of these NLR pairs in WEW accessions highlights the need for focused mining of its diversity. Furthermore, their successful cloning of these NLR pairs in WEW inspires us to systematically explore more NLR pairs from more wild relatives of wheat, providing more resources for breeding. Alternatively, pyramiding of the R gene is a promising strategy to achieve sufficient and sustainable disease resistance in plants. Importantly, the introgression of these NLR pairs into cultivated wheat has been achieved without a yield penalty, supporting their breeding potential. Therefore, combining these different NLR pairs may develop a more complex immune network, thereby enhancing the multi-disease resistance of wheat without reducing its yield. This work was supported by grants from the National Key R&D Program of China (2023YFE0123400), the National Natural Science Foundation of China NSFC (32472527, 32470342), the China Postdoctoral Science Foundation (2024M751986), the Natural Science Foundation of Shanghai (24ZR1430100), the Taishan Scholars Program (tsqn202306306), and the Guizhou Plant Bacteria and Biological Control Science and Technology Innovation Talent Team Development (Qian Ke He Talent-BQW [2025]003). The authors declare no conflicts of interest. Y.G., F.L., and H.C. conceived the manuscript. Y.G. drafted the manuscript. X.Z., Z.K.S., J.C., and G.Q. revised the manuscript. All authors have read and approved the contents of this paper.