An NLR pair in the Pm68 locus confers powdery mildew resistance in durum and common wheat

He, Huagang; Tang, Qiulian; Zhang, Qianyuan; Zhu, Shanying; Lv, Sijia; Bao, Yinguang; Liang, Jiabao; Wang, Jiale; Wang, Jin; Xu, Hongxing; Cavalet-Giorsa, Emile; Krattinger, Simon G.; Li, Hongjie; Wu, Chundu; Gao, Anli; Wang, Yajun

doi:10.1038/s41467-025-64048-z

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Published: 10 October 2025

An NLR pair in the Pm68 locus confers powdery mildew resistance in durum and common wheat

Nature Communications volume 16, Article number: 9039 (2025) Cite this article

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Abstract

Common wheat and durum wheat are widely cultivated cereal crops, both of which are infected by the obligate biotrophic pathogen Blumeria graminis f. sp. tritici (Bgt), causing powdery mildew. Recently, Pm68 gene was identified on the short arm of chromosome 2B in the Greek durum wheat line TRI 1796, conferring resistance to this disease. Here, we have cloned Pm68 from TRI 1796 using an integrated approach of genetic mapping, association analysis and PacBio sequencing. Transgenic assays demonstrate that Pm68 mediated resistance is controlled by a pair of genetically linked nucleotide-binding leucine-rich repeat (NLR)-encoding genes, Pm68-1 and Pm68-2. Transient expression assays in Nicotiana benthamiana leaves reveal that the activation of Pm68-1 is positively modulated by Pm68-2, likely through its N-terminal coiled-coil (CC)-like domain. Evolutionary analysis traces the origin of Pm68 to a specific wild emmer subpopulation. The introgression and transgenic wheat lines carrying this gene show no significant negative effects on major agronomic traits, highlighting the potential value of Pm68 for disease-resistant breeding programs of both durum wheat and common wheat.

Introduction

Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) and durum wheat (T. turgidum subsp. durum Desf. Husn., 2n = 4x = 28, AABB) are two related species with distinct grain properties and end-use purposes. Common wheat is globally cultivated and contributes approximately 18% of the calories consumed by humans¹. Durum wheat, on the other hand, is cultivated in a more limited range of agricultural regions and its grains are mainly used to make pasta². Both common and durum wheat productions are frequently challenged by various fungal diseases, including powdery mildew, rusts, and Fusarium head blight. Powdery mildew, a foliar disease caused by the obligate biotrophic pathogen Blumeria graminis f. sp. tritici (Bgt), is prevalent in most wheat-growing regions worldwide, resulting in significant yield losses and posing a threat to global food security^3,4.

The mining and utilization of powdery mildew resistance (Pm) genes are crucial for the management of powdery mildew of common wheat and durum wheat. More than 100 Pm genes/alleles have been characterized from wheat and its relatives^5,6. Up to now, 24 Pm genes have been cloned, including Pm1a⁷, Pm2⁸, Pm3⁹, Pm4¹⁰, Pm5e¹¹, Pm8¹², Pm12¹³, Pm13^4,14, Pm17¹⁵, Pm21¹⁶, Pm24¹⁷, Pm26¹⁸, Pm36¹⁹, Pm38/Yr18/Lr34/Sr57²⁰, Pm41²¹, Pm46/Yr46/Lr67/Sr55²², Pm55²³, Pm57²⁴, Pm60²⁵, Pm69²⁶, WTK4²⁷, PmTR1²⁸, Pm6Sl²⁹ and PmAeu1³⁰. Among them, 15 genes encode coiled coil nucleotide-binding leucine-rich-repeat (NLR) proteins, while the others encode kinase fusion proteins (KFPs) or multi-transmembrane transporters^18,31,32,33.

Four designated Bgt resistance loci have been identified in durum wheat. Mld is a recessive gene on chromosome 4B that has lost its effectiveness³⁴. Pm3h is a dominant gene on chromosome 1AS of an Ethiopian durum wheat accession³⁵, whose sequence is identical to Pm3d³⁶. PmDR147 is a dominant gene on 2AL of durum wheat accession DR147³⁷. Pm68 was identified in the Greek durum wheat accession TRI 1796. Through bulked segregant RNA-Seq (BSR-Seq) analysis combined with genetic mapping using a bi-parent population, Pm68 was mapped to a 0.44-cM genetic interval on the terminal part of chromosome arm 2BS, corresponding to a 1.78-Mb physical region of the reference genome of durum wheat cv. Svevo³⁸. Durum wheat accession TRI 1796 is highly resistant to all 22 tested Bgt isolates, suggesting that this gene has potential for powdery mildew resistance breeding in both durum wheat and common wheat.

In this study, we report the cloning of Pm68 from durum wheat using a strategy that combines genetic fine mapping, association analysis and PacBio sequencing. Pm68 resistance is mediated by the NLR pair Pm68-1 and Pm68-2. Pm68-1 encodes a canonical NLR containing a coiled-coil (CC) domain, a nucleotide-binding site (NBS) domain and a leucine-rich repeat (LRR) domain, whereas Pm68-2-encoded NLR lacks a typical CC domain but possesses a four-helix bundle structurally analogous to a CC domain, designated here as a CC-like (CC_l) domain. Transgenic plants expressing either Pm68-1 or Pm68-2 are susceptible to powdery mildew, while hybrid F₁ and F₄ carrying the two NLRs are all resistant. Transient over-expression analyses in Nicotiana benthamiana leaves indicate that Pm68-1 plays a critical role in triggering cell death, which is regulated by Pm68-2, likely through its N-terminal CC_l domain. The Pm68-carrying chromosome segment could be traced to a genetically distinct and geographically restricted wild emmer subpopulation. We have transferred Pm68 into the elite common wheat cultivar Yangmai 158 (YM158) by interspecific hybridization. In both common wheat introgression lines and transgenic lines expressing Pm68, no significant deleterious effects on major agronomic traits were observed, suggesting that Pm68 has a potential value for common wheat and durum wheat breeding.

Results

Pm68 is delimited to 266-kb and 297-kb regions corresponding to Svevo and Zavitan reference genomes, respectively

For genetic fine mapping of Pm68, the previously identified flanking markers Xdw03 and Xdw15 were used to screen 1382 F₂ individuals derived from the cross between durum wheat accessions TRI 1796 (resistant) and PI 584832 (susceptible). A total of 41 recombinants were obtained and then further genotyped using another 8 co-dominant markers (Supplementary Data 1 and 2). As a result, Pm68 was mapped to a 0.21-cM genetic interval flanked by markers Xdw05/Xdw06 and Xdw09/Xdw10 corresponding to a 434-kb region in the reference genome of durum wheat cv. Svevo³⁹. Three markers Xdw07, Xdw08 and Xdw08.9 co-segregated with Pm68 (Fig. 1a).

Fig. 1: Map-based cloning and functional validation of *Pm68.*

To further narrow the Pm68 locus, we inoculated 120 durum wheat accessions with Bgt iaolate BgtYZ01 at the one-leaf stage. As a result, 114 accessions displayed highly susceptible phenotypes (IT 4), while 6 accessions (including TRI 1796) showed resistance with hypersensitive reactions (HR) (IT 0;) which resembles Pm68-mediated resistance (Supplementary Fig. 1; Supplementary Data 3). We then genotyped the above six resistant and 85 susceptible durum wheat accessions using 7 co-dominant markers (Xdw05 − Xdw10 and Xdw8.9) spanning the Pm68 locus. Xdw07 and Xdw08 showed segregation with Pm68, left Xdw08.9 was the only marker co-segregated with Pm68 both in F₂ and association mapping populations (Fig.1a, b; Supplementary Data 2 and 4). Therefore, Pm68 was mapped to region flanked by markers Xdw08 and Xdw09, corresponding to a 266-kb and a 297-kb physical intervals of durum wheat Svevo³⁹ and wild emmer Zavitan⁴⁰ reference genomes, respectively (Fig.1c).

The Pm68-mediated resistance to powdery mildew is controlled by a pair of NLR genes

The 266-kb Pm68 interval of Svevo reference genome contains 15 annotated genes, while the corresponding 297-kb Zavitan interval contains 7 annotated genes (Fig.1c). Eight gene-derived markers (Xdw08.1 − Xdw08.8) were developed from five Svevo genes and three Zavitan genes, which were used to genotype the two parents, TRI 1796 and PI 584832. We found that five Svevo gene-derived markers (Xdw08.1 − Xdw08.5) only amplified from the susceptible parent PI 584832, while three Zavitan gene-derived markers (Xdw08.6 − Xdw08.8) only amplified from resistant parent TRI 1796 (Supplementary Fig. 2a), suggesting that TRI 1796 harbors a haplotype similar to wild emmer Zavitan, rather than durum wheat Svevo (Fig. 1c).

Since Zavitan exhibited a susceptible phenotype (IT 4) to Bgt isolate BgtYZ01 (Supplementary Fig. 3), we sequenced the genome of the Pm68 donor accession TRI 1796 using the PacBio circular consensus sequencing (CCS) platform and obtained 5,571,107 HiFi reads consisting of ~98 Gb genomic sequence data, approximately 9.4-fold coverage of Svevo genome (10.45 Gb) and 9.7-fold coverage of Zavitan genome (10.1 Gb). After sequence assembling, 11,467 contigs were obtained, with a contig N50 of 1.78 Mb and N90 of 0.52 Mb, and a total length of the assembly of 10.41 Gb. The flanking markers Xdw08 and Xdw09 were positioned on a single 4.46-Mb contig ptg004078l. Dot plot analyses revealed that the Pm68 interval of TRI 1716 is highly similar to Zavitan and diverged from Svevo (Supplementary Fig. 2b). The Xdw08 and Xdw09 flanked 305-kb TRI 1716 interval corresponds to 297-kb region of Zavitan genome, and the seven annotated genes from Zavitan interval also align to seven corresponding alleles from TRI 1716 (Fig. 1d).

To identify causal gene(s) of Pm68, we mapped RNA-seq reads from BgtYZ01-infected TRI 1716 leaf tissues to the TRI 1716 genome assembly. Four out of the seven annotated genes, including TRIDC2BG003960, TRIDC2BG003970, TRIDC2BG003990 and TRIDC2BG004000, were found to have relatively high read-mapping depth, while no reads mapped to the rest three genes, TRIDC2BG003940, TRIDC2BG003950 and TRIDC2BG003980 (Fig. 1d). This indicated that only four genes out the seven were expressed in BgtYZ01- infected TRI 1716 leaf tissues. Among these four expressed genes, TRIDC2BG003970 and TRIDC2BG003990 encode two NLR receptors, TRIDC2BG004000 encodes a U4/U6 small nuclear ribonucleoprotein Prp31, and TRIDC2BG003960 encodes an unknown function protein with no annotated conserved domain. Given that NLR genes are often responsible for disease resistance, the TRI 1796 genes corresponding to TRIDC2BG003970 and TRIDC2BG003990 in Zavitian were considered as the most likely the candidates for Pm68, and referred as Pm68-1 and Pm68-2 hereafter (Fig. 1d). Both NLR genes contain no introns and are arranged in a head-to-head orientation, 202 kb apart in the TRI 1796 genome. The qRT-PCR assay validated the expression of Pm68-1 and Pm68-2, and their transcript levels were not induced by Bgt infection (Supplementary Fig. 4). Sequence comparison of these two NLRs demonstrated that both resistant TRI 1796 and susceptible Zavitan have an identical Pm68-1 coding sequence (CDS) (2742 bp), but differ from one nucleotide in their Pm68-2 CDS (G1111T), leading to a premature stop codon in the Pm68-2 allele from Zavitan (Fig. 1d). As Zavitan is susceptible to BgtYZ01, it was suggested that a functional Pm68-2 is necessary for Pm68 resistance.

We amplified the full-length coding regions of Pm68-1 and Pm68-2, and separately ligated them into the vector pLGY02 with an expression cassette driven by the maize ubiquitin promoter to generate the constructs pLGY02-ZmUbi::Pm68-1 and pLGY02-ZmUbi::Pm68-2. These constructs were separately transformed into wheat cv. Fielder using the Agrobacterium tumefaciens-mediated transformation method. We obtained five independent transgenic T₀ events carrying Pm68-1 and Pm68-2 each. RT-PCR and qRT-PCR analyses verified the expression of the Pm68-1 and Pm68-2 in transgenic T₀ plants (Supplementary Fig. 5; Supplementary Fig. 6). However, all these transgenic T₀ and T₁ lines were susceptible to BgtYZ01 at both seedling and adult-plant stages (Supplementary Fig. 7). We then pyramided Pm68-1 and Pm68-2 transgenes by crossing the T₁ plants carrying Pm68-1 or Pm68-2. The two independent F₁ plants and their selfed progenies carrying both Pm68-1 and Pm68-2 showed resistance to BgtYZ01 at both the seedling and adult plant stages (Fig. 1e; Supplementary Fig. 8). Trypan blue staining revealed that Bgt-induced cell death first observed in transgenic Pm68-1/Pm68-2 Line 1 and TRI 1796 plants at 12 h post-inoculation (hpi), became more pronounced at 24 hpi and 48 hpi, whereas no cell death was not detected in transgenic lines expressing single NLR genes (Supplementary Fig. 9). Transgenic Pm68-1/Pm68-2 Line 1 and TRI 1796 exhibited resistance to eight Bgt isolates collected from eight different regions across five provinces in China (Supplementary Fig. 10). It is concluded that Pm68 resistance is controlled by the NLR pair Pm68-1 and Pm68-2.

Pm68-2 or its N-terminal CC-like (CC_l) domain positively regulate Pm68-1 in triggering cell death

Based on the prediction of NCBI Conserved Domain Search, Pm68-1 encodes a typical NLR with an N-terminal CC domain, a central NBS domain and a C-terminal LRR domain, while Pm68-2 encodes a NLR without CC domain. We predicted the three-dimensional structure of the two NLRs using AlphaFold 3. Pm68-2 were predicted to have a four-helix bundle that is similar to a typical CC domain, hereby designated CC-like (CC_l) domain (Fig. 2a, b).

Fig. 2: Pm68-2 or its N-terminal CC-like (CC_l) domain positively regulate Pm68-1 -mediated cell death in *Nicotiana benthamiana.*

Functional characterization of NLR pairs is frequently performed using transient expression in N. benthamiana^41,42. Therefore, we performed a series of A. tumefaciens-mediated transient overexpression assays of Pm68-1 and Pm68-2 in N. benthamiana leaves (Fig. 2c). We observed that the individual expression of Pm68-1 or Pm68-2 did not induce cell death in N. benthamiana leaves at 72 h post-infiltration. However, co-expression of the two full-length proteins triggered cell death response. Meanwhile, the mutant Pm68-1_D505V, which contains an MHD motif substitution known to cause autoactivation⁴³, also led to significant cell death, whereas the mutant Pm68-2_D545V did not exhibit this phenotype (Fig. 2d; Supplementary Fig. 11). Further analysis involved the introduction of the K210R mutation, known to impair ATP binding⁴⁴, into Pm68-1_D505V and Pm68-1. The mutant Pm68-1_K210R/D505V was found to lost the ability to induce cell death compared with the autoactivation mutant Pm68-1_D505V, suggesting that the K210R mutation in Pm68-1 resulted in a loss of function. As expected, the expression of Pm68-1_K210R alone or the co-expression of Pm68-1_K210R and Pm68-2 did not trigger cell death. In contrast, co-expression of Pm68-1 with the mutant Pm68-2_K255R, where K255R corresponds to the K210R in Pm68-1, still resulted in observable cell death (Fig. 2d; Supplementary Fig. 11). Co-immunoprecipitation (Co-IP) assays revealed that Pm68-1 physically interacts with Pm68-2 and each of its three individual domains. (Supplementary Fig. 12). These findings indicate that Pm68-1 is a pivotal player in the initiation of cell death and that its activity can be regulated by Pm68-2.

To identify the domain of Pm68-2 responsible for regulating the cell death-inducing activity of Pm68-1, we co-expressed Pm68-1 or Pm68-2_CC_l with the different truncated fragments of Pm68-2 or Pm68-1 separately in N. benthamiana leaves. Individual expression of truncated fragments of Pm68-1 or Pm68-2, and CC domain of Pm21 were used as controls. The results revealed that only the combination of Pm68-1 and Pm68-2_CC_l induced cell death (Fig. 2d; Supplementary Fig. 11). This suggests that the CC_l domain of Pm68-2 is necessary for modulating the activity of Pm68-1.

The Pm68 locus can be traced back to a specific wild emmer subpopulation

Comparative k-mer analysis revealed that TRI 1796 carries a ~ 1.6 Mb haplotype block that is highly diverged from the durum reference cultivar Svevo³⁹ and more similar to the reference assembly of wild emmer line Zavitan⁴⁰ (Supplementary Fig. 13). Identity-by-state (IBSpy) analyses⁴⁵ confirmed that the Pm68-containing haplotype block originated from the genetically distinct and geographically restricted judaicum wild emmer subpopulation⁴⁶, to which Zavitan belongs⁴⁷ (Supplementary Fig. 14; Supplementary Data 5).

Then we use the dominant marker Xdw08.8, corresponding to Pm68-2, to screen 120 durum wheat accessions and 118 wild emmer accessions (including Zavitan) (Supplementary Data 3). A total of 32 accessions, including 6 durum wheat and 26 wild emmer accessions, were positive for marker Xdw08.8. We amplified and Sanger sequenced the two NLR genes from these 32 accessions, which revealed nine Pm68 haplotypes (Haplotypes A to I), consisting of six Pm68-1 alleles (Supplementary Data 6) and six Pm68-2 alleles (Supplementary Data 7). We co-infiltrated different Pm68 allelic NLR pairs representing Haplotypes A to I into N. benthamiana, all of which induced cell death (Supplementary Fig. 15). All six durum wheat accessions (TRI 1796, TRI 1876, TRI 10092, PI 70716, DR64, and DR343) have the identical NLR pair (Pm68-1/Pm68-2), designated Haplotype A. Haplotype F (Pm68-1^IW21/Pm68-2 ^IW21), found in two wild emmer accessions (IW21 and IW29), is identical to the powdery mildew resistance gene MlIW39 reported in the companion study⁴⁸. Compared with Haplotype A, Haplotype F/MlIW39 only has one nucleotide variation G2324A in Pm68-1 and one nucleotide variation T1604C in Pm68-2 leading to amino acid substitutions R775H and M535T, respectively. The nonfunctional Haplotype C (Pm68-1/Pm68-2^Zavitan) exists in four BgtYZ01-susceptible wild emmer accessions including Zavitan, and it differs for one nucleotide (G1111T of Pm68-2) from Haplotype A, leading to a premature stop codon of Pm68-2 (Fig. 1d). Another six haplotypes (Haplotypes B, D, E, G, H and I) were detected only in wild emmer accessions that showed immunity to BgtYZ01 (Supplementary Data 8; Supplementary Fig. 16), suggesting additional gene resources for the wheat powdery mildew resistance breeding.

Pm68 shows potential to be used for powdery mildew-resistance breeding in wheat

To expand the utilization of Pm68, we crossed Pm68 into Chinese elite cultivar YM158, and generated two independent BC₃F₅ introgression lines. The two lines showed resistance to Bgt isolate BgtYZ01 at both the seedling and adult-plant stages (Fig. 3a). We evaluated their major agronomic traits and compared with the recurrent parent YM158. The traits including plant height, spike number per plant, grain number per spike, and 1000-grain weight of the introgression lines were significantly higher than those of the recurrent parent YM158 (P < 0.05), whereas spike length and spikelet number per spike exhibited no obvious difference between the introgression lines and YM158 (Fig. 3b–d). We also investigated the agronomic performance for the T₄ homozygous transgenic line carrying both Pm68-1 and Pm68-2. All the tested agronomic traits showed no significant difference between the Pm68 transgenic lines and the untransformed Fielder (Supplementary Fig. 17). In addition, the overexpression of Pm68-1/Pm68-2 in transgenic wheat lines did not cause visible hypersensitive response (HR) or spontaneous necrosis in leaves (Supplementary Fig. 18). These results suggest that Pm68 introgression is not associated with deleterious linkage drag and Pm68 has no obvious negative effects on the major agronomic traits, which highlights the potential value of Pm68 in powdery mildew resistance breeding of wheat.

**Fig. 3: *Pm68* introgression shows no deleterious linkage drag.**

Discussion

Pm68 was mapped to the terminal part of 2BS in Greek durum wheat accession TRI 1796³⁸. In this study, we adopted an integrated approach, involving genetic fine mapping using a biparental population, association analysis of durum wheat natural population, and PacBio sequencing and assembling of TRI 1796 genome, to isolate Pm68. Stable transgenic complementation assays revealed that Pm68 resistance is mediated by a pair of NLR genes, Pm68-1/Pm68-2. Biparental population based fine-mapping and natural population-based association mapping delimited Pm68 to 266-kb or 297-kb intervals correspond to different reference genomes. Low-coverage (~10 x) PacBio genome sequencing of resistance donor accession provided reference for candidate gene mining, and RNA-seq reads mapping helped to reduce the number of candidate genes. As sequencing costs are continuously decreasing, the integrated approach could accelerate gene cloning in plant species with large genome, such as wheat.

In plants, NLR proteins belong to the major class of immune receptors that recognize pathogen effectors and activate effector-triggered immunity (ETI)⁴⁹. Plant NLRs likely evolved from singleton NLRs to NLR pairs and then to NLR networks⁵⁰. Singleton NLRs can both recognize effectors directly or indirectly and activate cell death and defence responses, such as the wheat stem rust resistance protein Sr35⁵¹ and Arabidopsis Pseudomonas syringae resistance protein ZAR1^52,53. Some NLRs function in genetically linked pairs, which consist of a sensor NLR and a helper/executor NLR that are specialized to recognize the pathogen and initiate immune signaling, respectively. For examples, the rice (Oryza sativa L.) NLR pairs RGA4/RGA5⁴¹ and Pikp1/Pikp2⁵⁴ confer resistance to Magnaporthe oryzae. Transient overexpression of the helper/executor NLR RGA4 could induce cell death in N. benthamiana leaves, which can be inhibited by the co-expressed sensor NLR RGA5, through forming a hetero-complex. Co-expression of RGA4, RGA5, and the corresponding effector AVR-Pia also triggered cell death⁵⁵. In the Pikp-1/Pikp-2 pair, neither co-expression of them or expression of them alone trigger cell death, while co-expression of Pikp-1, Pikp-2, and the corresponding effector AVR-PikD induced cell death, suggesting that the Pikp-1/Pikp-2 complex needs to be activated by AVR-PikD⁴⁴. Unlike previous reported NLR pairs, transient co-expression of Pm68-1 and Pm68-2 triggered cell death in N. benthamiana leaves, while expression of either single NLR did not induce cell death, and co-expression of Pm68-1 and N-terminal CC_l domain of Pm68-2 also induced cell death, suggesting a different activation mechanism of this NLR pair.

Our results also showed that an autoactivation variant of Pm68-1, rather than Pm68-2, induced cell death in N. benthamiana leaves, indicating only Pm68-1 is capable of inducing cell death upon the activated state. In addition, co-expression of the loss-of-function mutant Pm68-1_K210R with Pm68-2 did not induce cell. This observation indicates that the Pm68-1 functions as a helper/executor NLR in Pm68-1/Pm68-2-mediated cell death. Pm68-2 could be a sensor NLR that directly or indirectly recognizes effectors from avirulent Bgt pathogens. Haplotype analysis reveals greater diversification in Pm68-2 alleles than in Pm68-1 alleles, consistent with sensor NLR divergence versus helper/executor NLR conservation (Supplementary Data 6 and 7). Pm68-2 is an atypical NLR that its CC_l domain shows no sequence homology, but structural resemblance to typical CC domains of NLRs. Expression of Pm68- 2 or its CC_l domain activated Pm68-1 and triggered cell death in N. benthamiana leaves. Therefore, the CC_l domain of Pm68-2 is essential for modulating the activity of Pm68-1. We suppose that the CC_l domain serve as a regulatory interface, facilitating the interactions between Pm68-1 and Pm68-2 and controlling the activation of cell death response. Unlike the heterologous expression system, transgenic plants carrying both maize ubiquitin promoter-driven Pm68-1 and Pm68-2 exhibited no autoimmunity or spontaneous necrosis while gaining resistance to powdery mildew (Supplementary Fig. 18). Trypan blue staining showed sporadic cell death at Bgt infection sites in TRI 1796 and transgenic Pm68-1/Pm68-2 Line 1, whereas no cell death occurred in transgenic lines expressing single NLR genes (Supplementary Fig. 9). The above results indicate that the Pm68-1/Pm68-2 complex is constitutively active in heterologous systems but maintains an inactive and primed state in homologous systems. This suggests that a regulatory component absent from heterologous systems might be required for suppression of auto-activation. Our findings provide insights into the molecular mechanism underlying the regulation of activity by this unique pair of NLRs⁴⁸.

Distant hybridization has played a significant role in the improvement and breeding of wheat, which has greatly enriched wheat genetic diversity. For example, more than 40% of the disease resistance genes used in common wheat breeding are introgressed from outside the common wheat gene pool⁵. However, recombination suppression resulted linkage drag has become a major obstacle that restricts the wide application of this strategy. Wild emmer, which is considered a progenitor of cultivated tetraploid and hexaploid wheat, and durum wheat shared the same sets of A and B genome with common wheat⁵⁶. Therefore, relatively mild or no recombination suppression allows gene introgression without linkage drag. Considering the effectiveness of Pm68 against all the 22 Bgt isolates tested³⁸, we transferred Pm68 from durum wheat TRI 1796 into the elite common wheat cultivar YM158 through interspecific cross and multiple backcrosses and self-crosses. When challenged by Bgt isolate BgtYZ01, the introgression lines carrying Pm68 showed effective resistance (scored IT 1) at the seedling stage and immunity at the adult plant stage (Fig. 3a). The introgression lines did not show any obviously adverse effects on the major agronomic traits, except for a slight increase of plant height, which may be inherited from TRI 1796 ( ~ 152 cm). Conversely, the introgression lines exhibited superior agronomic traits, which was probably an outcome of larger biomass. We also did not observe significant differences between transgenic plants with the overexpressed Pm68-1/Pm68-2 pair and the untransformed control Fielder. The above results highlight the disease-resistance breeding value of Pm68 in both durum wheat and common wheat.

Methods

Plant materials and growth conditions

A panel of 120 durum wheat accessions was ordered from the Genebank Information System of the IPK Gatersleben (GBIS-IPK), the U.S. National Plant Germplasm System (NPGS), and the Chinese Crop Germplasm Resources Information System (CCGRIS) (Supplementary Data 3). A total of 118 wild emmer accessions were ordered from the NPGS or kindly provided by Dr. Z. Y. Liu of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China and Dr. H. Li of Henan University, Kaifeng, China (Supplementary Data 3). The resistant durum wheat accession TRI 1796, carrying Pm68³⁸, was crossed with the susceptible accession PI 584832, and the derived 1382 F₂ individuals were used for genetic mapping of Pm68. The susceptible wheat cultivar Yangmai 23 (YM23) was used for reproducing Bgt conidiospores to prepare inoculum. The susceptible wheat cultivar Fielder was used as the recipient for transformation. The susceptible wheat cultivar Yangmai 158 (YM158) was used as the recurrent parent when transferring Pm68 from durum wheat TRI 1796 into common wheat. N. benthamiana was used for transient expression assays. All plants were grown in a greenhouse with LED lighting under long-day conditions (16 h light/8 h dark) at 24 °C.

Evaluation of powdery mildew resistance

A total of 1382 F₂ plants and 20 plants of each F₃ family that exhibited recombination events at the one-leaf stage were inoculated with the Bgt isolate BgtYZ01, which was collected in Yangzhou, China⁵⁷, by dusting fungal conidiospores from YM23 leaves. Responses to powdery mildew were evaluated 8–10 days post-inoculation (dpi) according to the infection types (IT) scored on a scale from 0 to 4, where ITs 0, 0, 1, and 2 were classified as resistant, and ITs 3 and 4 as susceptible⁵⁸. Powdery mildew IT values of ten plants of each durum wheat accession and the Xdw08.9-positive wild emmer were also tested.

The resistance spectrum of transgenic Pm68-1/Pm68-2 Line 1 and durum wheat TRI 1796 were was examined in seedling tests with three replicates using BgtYZ01 and the other seven Bgt isolates collected from different regions of China by Dr. Yinghui Li (Triticeae Research Institut, Sichuan Agricultural University, Chengdu, China). Common wheat Fielder and durum wheat PI 584832 were used as the controls. Leaves were cut into segments ~3 cm in length and placed in Petri dishes containing 8.0 g/L agar and 50 mg/L benzimidazole. Leaves on Petri dishes were inoculated with the above eight Bgt isolates separately and maintained ex vivo at 22 °C on 16 h light/8 h dark cycle⁵⁹. Pictures of powdery mildew responses were taken at 8 days post inoculation.

Development of and application of molecular markers for genotyping

Ten co-dominant markers (Xdw03 − Xdw10, Xdw12 and Xdw15) were reported in our previous work³⁸ and one co-dominant markers (Xdw08.9) were developed in this study according to the surrounding sequences of the InDel regions between durum wheat cultivars Svevo³⁹ (https://www.interomics.eu/durum-wheat-genome) and Kronos (https://opendata.earlham.ac.uk/opendata/data/Triticum_turgidum/EI/v1.1). In addition, five PI 584832-dominant markers (Xdw08.1 − Xdw08.5) and three TRI 1796-dominant markers (Xdw08.6 − Xdw08.8) were designed based on the differential genes between the reference genomes of durum wheat cultivar Svevo and wild emmer accession Zavitan⁴⁰. The primers of all polymorphic markers can be found in Supplementary Data 1.

Genomic DNA used for genotyping was extracted from seedling leaves using the CTAB method. PCR amplification was carried out in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) using an initial denaturation at 94 °C for 3 min, 35 cycles of 10 s at 94 °C, 30 s at 60 °C, 1 min at 72 °C, and a final extension for 5 min at 72 °C. Twenty-five microlitres of reaction mixture contained 1× PCR buffer, 0.2 mM of each dNTP, 2 μM of each primer, 50 ng genomic DNA, and 1 Unit of Taq DNA polymerase (TaKaRa, Shiga, Japan, Catalog No. R001A). PCR products were separated in 8% non-denaturing polyacrylamide gels, followed by silver staining, or separated in 1.2% agarose gel stained with GelRed.

Fine genetic mapping of Pm68

A total of 1382 F₂ individuals derived from the cross between TRI 1796 and PI 584832 were used to map Pm68. The flanking markers Xdw03 and Xdw15 were initially used for screening recombination events in the above mapping population. Then, 41 individuals containing recombination events were phenotyped at the one-leaf stage with Bgt isolate BgtYZ01 and genotyped by a set of markers described above (Supplementary Data 1 and 2). Finally, 41 F₃ families were generated from the F₂ individuals containing recombination events, and 20 plants of each F₃ family at the one-leaf stage were phenotyped again using BgtYZ01.

Marker analysis of different durum wheat accessions

To further narrow the Pm68 locus, 85 susceptible and 6 resistant durum wheat accessions were genotyped with 15 markers, including seven co-dominant markers, five PI 584832-dominant markers (Xdw08.1 − Xdw08.5) and three TRI 1796-dominant markers (Xdw08.6 − Xdw08.8) (Supplementary Data 1 and 4).

PacBio HiFi sequencing and assembling of TRI 1796 genome

PacBio HiFi sequencing was conducted by BGI (Shenzhen, China). High molecular weight genomic DNA was extracted from TRI 1796 seedling leaves using the CTAB method and then sheared to 15−20 kb using Diagenode Megaruptor system (Diagenode, Seraing, Belgium). PacBio HiFi sequencing library was constructed using SMRTbell Express Template Prep Kit 2.0 and DNA fragment size was selected with the BluePippin System (Sage Science, Beverly, MA, USA). High-throughput sequencing was conducted on PacBio Sequel IIe system (Pacific Biosciences, Menlo Park, CA, USA). The PacBio HiFi reads were assembled using hifiasm (v.0.19.8) with default parameters and then evaluated using seqkit (v.2.6.1)^60,61.

Transcriptome sequencing of TRI 1796 leaves and read-mapping analysis

The seedling of TRI 1716 was inoculated with Bgt isolate BgtYZ01 and the leaf sample was collected at 24 h post inoculation. Total RNA was extracted using Illumina TruSeq RNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA), and then used for RNA sequencing on the Illumina NovaSeq 6000 platform. The low quality reads and adapter contaminations were removed with Trimmomatic v0.40⁶². Subsequently, the high quality reads were aligned to the TRI 1716 genome assembly using STAR (2.7.11b) with default parameters. The BAM file corresponding to the 305-kb Pm68 interval flanked by markers Xdw08 and Xdw09 in contig ptg004078l was extracted using Geneious Prime (v.2020.2.4). Then, the average read coverage depth per 100 bp for the 305-kb interval was calculated using samtools (v.1.19.2) and then visualized by R package ggplot2 (v.3.5.0).

K-mer analysis

The 51-mers datasets were generated from the durum wheat Svevo and wild emmer wheat Zavitan genomes using kmc (3.2.4), respectively, and then mapped to the TIR 1796 assembly using bwa (0.7.17), with only perfect alignments outputted (-T 51). Non-overlapping bins of length 100 bp were created using bedtools (2.31.0), and the k-mer mapping coverage within each bin was calculated using samtools (1.19.2). For visualization, the R package ggplot2 (v.3.5.0) was employed to plot the mapping coverage distribution of k-mers from Svevo and Zavitan, and the distribution of the mapping coverage differences of k-mers from Svevo and Zavitan.

Identity-by-state (IBSpy) analyses

The 68 publicly available wild emmer whole genome sequences^63,64,65,66 and the reference genome Zavitan⁴⁰ (Supplementary Data 5) were downloaded and the whole genome sequencing (WGS) raw data were trimmed with Trimmomatic (v. 0.39) with the following settings: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:25 MINLEN:75. From the trimmed WGS sequences and the Zavitan reference genome, all the canonical 31-mers were counted using kmc (v 3.1.2). We compared the 69 wild emmer accession to the contig ptg004078l containing the Pm68 from the TRI 1796 assembly using IBSpy (v0.4.6) in 50 kb windows. We also run IBSpy with the Zavitan genome assembly as reference with the same settings to identify the judaicum accessions present in the panel.

Protein domain and three-dimensional model prediction

Domains of Pm68-1 and Pm68-2 were predicted using NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Three-dimensional models of Pm68-1 and Pm68-2 were predicted by AlphaFold 3⁶⁷.

RT-PCR and quantitative real-time RT-PCR (qRT-PCR) analysis

The seedlings of durum wheat cultivar TRI 1796 were inoculated with Bgt isolate BgtYZ01 as described above. Leaf samples were collected at 0, 12 and 24 h post-inoculation (hpi) for extracting total RNA with the RNAiso Plus Kit (TaKaRa, Shiga, Japan, Catalog No. 9109). Two micrograms of total RNA were then used to synthesize the first-strand cDNA with the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan, Catalog No. 6210 A). The candidate genes Pm68-1 and Pm68-2 were PCR amplified from cDNAs (0 hpi) using the high fidelity PrimeSTAR Max Premix (TaKaRa, Shiga, Japan, Catalog No. R045A) with primer pairs P1/P2 and P3/P4, followed by Sanger sequencing with primers P5 to P8 and P9 to P13, respectively. qRT-PCR was performed using TB Green Premix Ex Taq™ II (TaKaRa, Shiga, Japan, Catalog No. RR820A) in Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, USA) with primer pairs P14/P15 and P16/P17 for Pm68-1 and Pm68-2, respectively. The gene actin was used as the internal control. The 2^-∆∆Ct method⁶⁸ was used to evaluate relative transcription levels of transgenes.

Plasmid construction and wheat transformation

The coding sequences of Pm68-1 and Pm68-2 were amplified from genomic DNA of TRI 1796 using the high fidelity PrimeSTAR Max Premix (TaKaRa, Shiga, Japan, Catalog No. R045A) and primer pairs P1/P2 and P3/P4, respectively (Supplementary Data 9). The PCR products of the two genes were cut with SmaI and SpeI and ligated into the downstream of maize ubiquitin promoter in the binary vector pLGY02¹³ to generate pLGY02-ZmUbi::Pm68-1 and pLGY02-ZmUbi::Pm68-2, respectively. After confirming by Sanger sequencing using primers P5 − P8 for Pm68-1 and primers P9 − P13 for Pm68-2 (Supplementary Data 9), the two constructs were separately transformed into the susceptible wheat cultivar Fielder by the Agrobacterium tumefaciens-mediated transformation method⁶⁹.

Transgenic assays

The T₀ plants of Pm68-1 and Pm68-2 were detected for the presence and absence of the transgene by PCR using primer pairs P8/P20 and P13/P20, respectively. Total RNA was extracted from T₀ leaves for the synthesis of the first-strand cDNA with the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan, Catalog No. 6210 A). qRT-PCR was performed with primer pairs P14/P15 and P16/P17 for Pm68-1 and Pm68-2, respectively (Supplementary Data 9). Wheat gene actin was used as the internal control. The 2^-∆∆Ct method⁶⁸ was used to evaluate relative transcription levels of transgenes. Ten transgene-positive plants in each T₁ families of Pm68-1 and Pm68-2 at the seedling and the adult-plant stages were successively inoculated with Bgt isolate BgtYZ01 to test their responses to powdery mildew. The Pm68-1-positive T₁ lines 1 and 2 were crossed with the Pm68-2-positive T₁ lines 1 and 2, respectively. The generated F₁ to F₄ plants were detected with primer pairs P8/P20 for Pm68-1 and P13/P20 for Pm68-2, respectively. Finally, the F₄ families of the two crosses pyramiding the pair of Pm68-1 and Pm68-2 were obtained. Ten plants of each F₄ family derived the two crosses were inoculated with Bgt isolate BgtYZ01 at the seedling and the adult-plant stages to assess their powdery mildew responses.

Histochemical detection of cell death in transgenic wheat and durum wheat leaves

Transgenic lines with single Pm68-1, single Pm68-2 and pair Pm68-1/Pm68-2 and durum wheat TRI 1796 were inoculated with Bgt isolate BgtYZ01 at the one-leaf stage. At 0, 12 24 and 48 hpi, leaves were cut and immersed in a trypan blue staining solution (2.5 mg/ml trypan blue, 25% lactic acid, and 23% saturated phenol) for 30 min in boiling wate, destained with Chloral hydrate solution (2.5 g/ml) for 3 d and stained with 0.6% Comassie brilliant blue R-250 solution for 10 s. Images were taken using a Leica DM2500 microscope.

Cell death assays on N. benthamiana

The CDS of Pm68-1 and Pm68-2, along with their respective domain-encoding sequences, were amplified from TRI 1796 using high fidelity PrimeSTAR GXL DNA polymerase (TaKaRa, Shiga, Japan, Catalog No. R050A). The CDSs without stop codons were cloned into the pS1300-Flag-Nos vector (C-terminally fused with Flag) with an expression cassette driven by the constitutive mannopine synthase (MAS) promoter utilizing the ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China; Catalog No. C115-01). Individual domain-encoding fragments were cloned into either the pS1300-Flag-Nos or pS1300-GFP-Nos vectors to obtain C-terminally Flag- or GFP-tagged fusions. Fragments containing the P-loop mutations (K210R for Pm68-1 and K255R for Pm68-2) and/or the autoactive mutations (D505V for Pm68-1, D545V for Pm68-2) were generated using the overlap extension PCR method⁷⁰, resulting in specific amino acid substitutions at the target positions. These mutated PCR products were subsequently cloned into the pS1300-Flag-Nos vector. All constructs were confirmed by Sanger sequencing using primers listed in Supplementary Data 9. In cell death assays, the construct containing the encoding sequence of the CC domain of Pm21 was used as the positive control⁷¹.

The above constructs were transformed into Agrobacterium strain GV3101, separately. Transformed Agrobacterium was cultured overnight at 28 °C in Luria-Bertani broth supplemented with 50 mg/mL rifampicin and 50 mg/mL kanamycin. The bacteria were harvested by centrifugation and suspended in infiltration medium (2% sucrose, 0.5% MS basal salts, 10 mM MES, pH 5.6, and 200 μM acetosyringone) to an OD₆₀₀ of 1. The suspension was incubated at room temperature for 1–3 h prior to infiltration into N. benthamiana leaves. For co-infiltration experiments, Agrobacterium cultures were mixed in a 1:1 ratio with either another construct or infiltration medium. The infiltrated N. benthamiana plants were maintained at room temperature and photographed at 48 hpi. The infiltrated leaves were stained using a trypan blue solution (10 mL lactic acid, 10 ml glycerol, 10 g phenol, 10 mg trypan blue, and 50 mL ethanol, dissolved in 30 mL distilled water) in boiling water for 5 min. Stained leaves were immersed in chloral hydrate solution (2.5 g/mL) until the background was no longer visible. Agrobacterium strain GV3101 harboring the empty vector was used as a negative control.

Protein extraction and Western blotting analysis

Leaf tissues for protein extraction were sampled at 24 h post-infiltration. A total of 0.1 g of frozen leaf tissues were ground to fine powder for each sample. Total proteins were extracted in 0.5 mL extraction buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.25% deoxycholate, 10 mM dithiothreitol (DTT), and 1× cocktail (protease inhibitor, Roche, Basel, Switzerland, Catalog No. 4693116001)]. The lysates were centrifuged at 18,000 × g for 20 min at 4 °C. The supernatants were collected and incubated in 1× Laemmli buffer at 95 °C for 5 min.

The heated protein samples were then separated by 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Boston, MA, USA; Catalog No. IPVH08100). Immunoblots were probed with either anti-Flag (Proteintech, Wuhan, China; Catalog No. 66008-4-lg), anti-GFP (Proteintech, Wuhan, China; Catalog No. 66002-2-lg) or anti-HA (Proteintech, Wuhan, China; No. 51064-2-AP) antibodies diluted to 1:5000. The secondary antibody, HRP-conjugated Affinipure Goat Anti-Mouse IgG (Proteintech, Wuhan, China; Catalog No. SA00001-1), was applied at a dilution of 1:5000 to detect the primary antibody. Blots were visualized using SuperSignal maximum sensitivity substrate (Proteintech, Wuhan, China, Catalog No. PK10002) and a ChemiDog imaging system (Tanon, Shanghai, China). Ponceau staining was employed to ensure equal loading across protein samples.

Co-immunoprecipitation (Co-IP) assays

The CDS of Pm68-1 was cloned into pS1300-HA-Nos (C-terminal HA fusion) vector via homologous recombination described above. After Sanger sequencing verification, the resulting construct was transformed into Agrobacterium strain GV3101. Other constructs used for Co-IP assays have been described in the method section of cell death assays. Equal volumes (1:1, v/v) of Agrobacterium cultures harboring the Pm68-1-HA and the Pm68-2-Flag, Pm68-1-Flag and Pm68-2-CC_l-GFP, Pm68-1-Flag and Pm68-2-NBS-GFP, Pm68-1-Flag and Pm68-2-LRR-GFP, Pm68-1-Flag and GFP constructs were mixed and co-infiltrated into N. benthamiana leaves, respectively. To avoid cell death induced by overexpression of Pm68-1 and Pm68-2, Agrobacteria-infiltrated N. benthamiana tissue was collected at 24 h post infiltration. Total protein was extracted from 0.2 g of infiltrated leaf tissue using 1 mL of extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.25% sodium deoxycholate, 10 mM dithiothreitol (DTT), and 1× protease inhibitor cocktail (Roche). Following centrifugation at 20,000 × g for 15 min at 4 °C, the supernatant was incubated with 10 μL of pre-washed anti-Flag magnetic beads (Yeasen, Catalog No. 20765ES40) under constant rotation overnight at 4 °C. Beads were collected using a magnetic stand (DynaMag™-2, Thermo Fisher Scientific) and washed three times with TBS buffer. Bound proteins were eluted with 50 μL of 1× Laemmli buffer by boiling at 95 °C for 5 min. Proteins from crude extracts (input) and immunoprecipitated proteins were analyzed by immunoblotting as described above.

Sanger sequencing of Pm68-1 and Pm68-2 isolated from durum wheat and wild emmer

The genomic DNAs of the collection of 118 wild emmer accessions were first genotyped with the dominant marker Xdw08.8, corresponding to Pm68-2. The genomic DNAs of 20 positive wild emmer and 6 positive durum wheat accessions found above were used as templates for PCR cloning of Pm68-1 and Pm68-2. PCR amplifications were performed using the high fidelity PrimeSTAR Max DNA polymerase (TaKaRa, Shiga, Japan, Catalog No. R045A) with primer pairs P1/P2 and P3/P4 followed by Sanger sequencing using primers P5–P8 and P9–P13 for Pm68-1 and Pm68-2, respectively.

Development of wheat introgression lines carrying Pm68

To transfer Pm68 from durum wheat into wheat, crosses were carried out using the elite wheat cultivar YM158 and TRI 1796 as the female and male parents, respectively. The progenies were then backcrossed with YM158 three times, followed five generations of selfing in greenhouse conditions. In each generation, the co-segregating marker Xdw08.9 was used to screen the individuals carrying Pm68. Finally, BC₃F₅ lines with Pm68 introgression were obtained and tested with BgtYZ01 at the seedling and the adult-plant stages as described above.

Agronomic traits evaluation of Pm68 introgression lines and Pm68 transgenic lines

In growing season 2023–2024, two Pm68 introgression lines and recurrent parent YM158, two Pm68 transgenic lines and non-transgenic Fielder were planted in experimental fields at Songjiang, Shanghai and Jiangsu University (Zhenjiang, China), respectively, for agronomic trait evaluation. Twenty seeds of each wheat line were sown in a row designed as 1.5 m in length with 20 cm inter-row space. Each line was sown in three replicate rows. The major agronomic traits, including plant height (cm), spike length (cm), spike numbers per plant, spikelet numbers per spike, grain numbers per spike, and 1000-grain weight were determined using five plants of each row. The significance of differences among means of the above agronomic traits was analyzed using two-tailed Student’s t-test.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The PacBio HiFi reads and Illumina RNA-Seq data of TRI 1796 carrying Pm68 were deposited in the Sequence Read Archive (SRA) database under accession SRR31057414 and SRR31534046, respectively. The sequences of Pm68-1 and Pm68-2 alleles isolated from durum wheat and wild emmer wheat accessions have been deposited in GenBank under the accession numbers PQ655406–PQ655417 [https://www.ncbi.nlm.nih.gov/nuccore/PQ655417]. Source data are provided with this paper.

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Acknowledgements

This study was supported by grants from National Natural Science Foundation of China (32171990 to H.H.), Natural Science Foundation of Jiangsu Province (BK20231321 to H.H.; BK20240839 to J.W.), Key Technology R&D Program of Henan Province of China (241111110900, 231111112900 and 225200810024 to A.G.), State Key Laboratory of Plant Cell and Chromosome Engineering (PCCE-KF-2022-07 to S.Z.), State Key Laboratory of Crop Biology in Shandong Agricultural University (2021KF01 to H.H.), Start-up funding of Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences (to Y.W.), Project of the Faculty of Agricultural Engineering of Jiangsu University (NZXB20200102 to C.W.) and Key and General Projects of Jiangsu Province (BE2022338 to C.W.). The authors thank the Genebank Information System of the IPK Gatersleben (GBIS-IPK), the U.S. National Plant Germplasm System (NPGS), the Chinese Crop Germplasm Resources Information System (CCGRIS), Dr. Zhiyong Liu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) and Dr. Hao Li (Henan University, Kaifeng, China) for providing durum wheat and wild emmer accessions, Dr. Genying Li (Crop Research Institution, Shandong Academy of Agricultural Sciences, Jinan, China) for providing the binary vector pLGY02, Dr. Yinghui Li (Triticeae Research Institut, Sichuan Agricultural University, Chengdu, China) for assistance of powdery mildew resistance spectrum analysis, Yi Ouyang (Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China) for protein structure modeling and Dr. Li Wan (Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China) for advice on the discussion of NLR immune receptors.

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Authors and Affiliations

School of Life Sciences, Jiangsu University, Zhenjiang, China
Huagang He, Qiulian Tang, Qianyuan Zhang, Jiale Wang & Jin Wang
School of Environment and Safety Engineering, Jiangsu University, Zhenjiang, China
Shanying Zhu & Chundu Wu
School of Life Sciences, Henan University, Kaifeng, China
Sijia Lv, Hongxing Xu & Anli Gao
College of Agronomy, Shandong Agricultural University, Taian, China
Yinguang Bao
State Key Laboratory of Plant Trait Design, CAS Centre for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
Jiabao Liang & Yajun Wang
Plant Science Program, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
Emile Cavalet-Giorsa & Simon G. Krattinger
Institute of Biotechnology, Xianghu Laboratory, Hangzhou, China
Hongjie Li

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Huagang He
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Qiulian Tang
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Qianyuan Zhang
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Shanying Zhu
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Hongxing Xu
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Hongjie Li
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Chundu Wu
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Anli Gao
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Contributions

H.H., A.G., and Y.W. conceived and designed the research. H.H., Q.T., Q.Z., S.Z., S.L., Y.B., J.L.W., J.W., and H.X. performed experiments. H.H., J.L., Y.W., E.C.-G., S.G.K., C.W., and A.G. analyzed the data. H.H., H.L., A.G., and Y.W. wrote the manuscript.

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Correspondence to Huagang He, Anli Gao or Yajun Wang.

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Nature Communications thanks Adachi Hiroaki, Javier Sánchez-Martín and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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He, H., Tang, Q., Zhang, Q. et al. An NLR pair in the Pm68 locus confers powdery mildew resistance in durum and common wheat. Nat Commun 16, 9039 (2025). https://doi.org/10.1038/s41467-025-64048-z

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Received: 14 December 2024
Accepted: 06 September 2025
Published: 10 October 2025
DOI: https://doi.org/10.1038/s41467-025-64048-z