Pm37 as a susceptible Sr22 allele confers resistance to wheat powdery mildew and leaf rust

Domain: chemistry
Published: 2026-02-17
Editorial outcome: minor_revision

Abstract
Wheat relatives harbour abundant variations that can be used for genetic improvement against diseases through complex domestication processes. Here, we report the isolation of the powdery mildew resistance gene Pm37 in common wheat through a strategy integrating map-based cloning, PacBio SMRT long-read genome sequencing, mutagenesis, barley stripe mosaic virus-induced gene silencing and stable transformation. Pm37 encodes a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) receptor. The cell death-inducing activity of PM37 in Nicotiana benthamiana is mainly mediated by its CC domain. De novo genome sequencing and haplotype analysis reveal that Pm37 may be derived from Triticum monococcum, and transferred into common wheat through T. timopheevii. Interestingly, Pm37 is a susceptible allele of the stem rust resistance locus Sr22, and also confers moderate resistance to leaf rust. Our findings identify a gene characterized by allelic variation-driven functional divergence in response to different wheat diseases.

Introduction
Common wheat (Triticum aestivum L., 2n=6×=42, AABBDD) is a major cereal crop that is consumed by approximately 30% of the global population. Powdery mildew, caused by the obligate biotrophic pathogen Blumeria graminis f. sp. tritici (Bgt), poses a significant threat to wheat production, causing yield losses often ranging from 10% to 15% globally1. Dynamic coevolution between pathogens and their hosts frequently renders resistance genes (R genes) ineffective because of the emergence of virulent isolates2. Consequently, dissecting the genetic and molecular mechanisms underlying diversified resistance is essential for disease management.

To date, more than 140 officially named powdery mildew resistance (Pm) genes/alleles (Pm1-Pm71) have been identified in wheat and its relatives3,4. Among these genes, the majority encode typical nucleotide-binding domain leucine-rich repeat (NLR) proteins, which play a central role in plant immunity by recognizing pathogen-derived effectors and triggering robust defense responses5. These NLR proteins, characterized by a modular architecture comprising an N-terminal signaling domain, a nucleotide-binding (NB) domain, and a leucine-rich repeat (LRR) domain, exhibit remarkable adaptability and specificity. Recent analyses of wheat genomes have revealed the extensive diversity of NLRs, suggesting the existence of up to 7780 unique NLR-encoding genes across the wheat gene pool, although fewer than 10% of these genes have been characterized to date5. In addition to NLRs, a growing number of resistance genes with unique domain architectures have also been identified. These include Pm13, encoding a kinase fusion protein6,7; Pm4, encoding a chimeric protein with serine-threonine kinase and C2 domains8; Pm24, Pm36, Pm57, and WTK4, encoding tandem kinase proteins9–12; Pm38/Yr18/Lr34/Sr57, encoding ATP-binding cassette transporter13; and Pm46/Yr46/Lr67/Sr55, encoding a hexose transporter14. Currently, NLR genes remain the primary focus of resistance breeding because of their dominant role in conferring race-specific immunity through direct or indirect effector recognition and their high potential for enhancing disease resistance in wheat breeding programs.

The cloning of wheat genes has long been hindered by the complex genome and evolutionary history of this allohexaploid species. Challenges such as large fragment deletion, insertion and duplication on chromosomes between different Triticum species complicate the gene identification efforts in wheat. Although the rapid development of high-throughput sequencing technologies has accelerated the cloning of wheat genes, abundant structural variations (SVs) among different genotypes are still major obstacles to gene identification in complex genomes3. To address these challenges, sequencing of the donor genome/transcriptome and de novo assembly have proven effective in mitigating issues arising from genomic variation, thereby expediting the identification of target genes. For instance, sequencing trait-associated mutations (STAM) enabled the cloning of YrNAM, encoding a noncanonical resistance protein with a NAM domain and a ZnF-BED domain15; transcriptome sequencing analysis of the loss-of-function mutants of the donor enabled the cloning of Pm13, encoding a mixed lineage kinase domain-like (MLKL) protein from Aegilops longissima in a wheat background. PacBio SMRT long-read genome sequencing allowed the cloning of Pm36, which was generated from a ~900 kb insertion and absent in available wheat reference genomes; and similar uses of this strategy also facilitated the cloning of Pm6916 and Yr2717, demonstrating the power of advanced sequencing techniques in overcoming the inherent challenges of wheat gene cloning.

T. timopheevii (Zhuk.) Zhuk. subsp. timopheevii and its wild form T. timopheevii subsp. armeniacum (Jakubz.) van Slageran (AtAtGG, 2n=28) are the close species of common wheat and possesses abundant disease resistance genes, such as the stem rust (Sr) resistance genes Sr36 and Sr3718; the leaf rust (Lr) resistance genes Lr1819 and Lr5020; and the Pm genes Pm621, Pm2722, and Pm3723. Among them, Pm37 was originally reported to be derived from T. timopheevii and was subsequently transferred into the common wheat background to develop the hexaploid germplasm line NC99BGTAG1123. NC99BGTAG11 is resistant to powdery mildew, making Pm37 a valuable genetic resource for wheat improvement23. Pm37 was preliminarily mapped to a 1.0 cM genetic interval on the chromosome arm 7AL23.

In this work, we clone Pm37 from NC99BGTAG11 by employing an integrated strategy combining map-based cloning and De novo genome sequencing. Pm37 resistance is mediated by a typical CC-NBS-LRR receptor. Pm37 plays a critical role in triggering cell death, mainly mediated by its CC domain. Pm37 may be originally evolved from T. monococcum, and transferred into common wheat through the bridge T. timopheevii. Pm37 is identified as a susceptible allele of the stem rust resistance locus Sr22, and also confers moderate resistance to leaf rust. Collectively, our results uncover a resistance locus whose function diverges through allelic variation, enabling distinct responses to different wheat pathogens.

Results
Map-based cloning of Pm37
For the fine mapping of Pm37, NC99BGTAG11 was crossed with the susceptible wheat parent Wuhuatou to generate a segregating population. Phenotyping of the segregating population across multiple generations using the Bgt isolate E09 revealed a clear monogenic segregation ratio, indicating the suitability of this population for fine mapping (Supplementary Data 1). Two flanking markers, Pm37-G2 and Pm37-163, were first confirmed and used for screening the derivative population comprising 2558 F families of NC99BGTAG11 × Wuhuatou (Supplementary Data 2). We obtained 28 recombinants, which can be used for fine mapping (Supplementary Data 3). Using bulked segregated RNA sequencing (BSR-Seq), more polymorphic markers linked to Pm37 were developed to narrow the fine mapping interval (Supplementary Data 2). Finally, Pm37 was delimited to a 0.0045 cM genetic interval flanked by the markers Pm37-151 and KASP-SNP1 on chromosome arm 7AL, corresponding to a 147.6 kb physical interval referred to the Chinese Spring (CS) reference genome (RefSeq 2.1) (Fig. 1a–c, Supplementary Data 3). Three high-confidence genes were annotated in this interval, including TraesCS7A03G1212600 (encoding a putative B3 domain-containing protein), TraesCS7A03G1212700 (encoding a CC-NBS-LRR, abbreviated as NLR-1), and TraesCS7A03G1212800 (encoding a DYW family of nucleic acid deaminases) (Fig. 1d). We then performed a time-course qRT-PCR analysis of these three genes following inoculation with the Bgt isolate E09. All three genes were expressed in the leaves of both NC99BGTAG11 and Wuhuatou seedlings (Supplementary Fig. 1). Among the three candidates, only TraesCS7A03G1212700 (NLR-1) exhibited higher expression levels in the resistant parent NC99BGTAG11 than in the susceptible parent Wuhuatou across multiple post-inoculation time points, whereas the expression of TraesCS7A03G1212600 and TraesCS7A03G1212800 did not significantly differ between these two genotypes. Homologous sequences of these three genes were then isolated from the cDNA of NC99BGTAG11 and Wuhuatou. No sequence variations were detected in TraesCS7A03G1212600 and TraesCS7A03G1212800 between NC99BGTAG11 and Wuhuatou, suggesting that they were not the Pm37 gene. However, we failed to obtain the full-length sequence of NLR-1 in NC99BGTAG11 after many attempts using a homology-based cloning strategy, indicating possibly complex structural or sequence variations in this interval.

De novo genome assembly of NC99BGTAG11 and candidate gene isolation
To address the above issue, we sequenced NC99BGTAG11 using the PacBio SMRT long-read sequencing approach. PacBio CCS (HiFi) reads corresponding to approximately 16-fold coverage of the CS reference genome were generated. After sequence assembly, 3820 contigs were obtained, with a contig N50 of 25.7 Mb and N90 of 5.3 Mb. The longest contig length was 202.2 Mb, and the total length of the genome assembly was 14.6 Gb, covering the whole genome of the CS reference genome. Those assembly contigs were further scaffolded to chromosome level using RagTag with the CS reference genome, achieving a 98.5% scaffolding rate (Supplementary Data 4). A 17.9 Mb contig spanning the entire Pm37 physical mapping interval was captured by aligning the flanking markers Pm37-151 and KASP-SNP1 around the Pm37 locus (Fig. 1e). Comparison of the target interval among the Triticeae genomes of T. monococcum TA299 and common wheat CS indicated that no new gene was present (Fig. 1d).

Combining the information from PacBio SMRT long-read sequencing, we obtained the full-length sequence of NLR-1 in NC99BGTAG11. Sequence alignment revealed 287 SNPs and 88 InDels between NC99BGTAG11 and Wuhuatou (Supplementary Data 5). A structural variation was identified in the second intron of NLR-1: compared to the susceptible allele in IWGSC RefSeq v2.1, an approximately 3000 bp fragment insertion was present in NC99BGTAG11, which was then validated by PCR amplification and Sanger sequencing (Supplementary Fig. 2). Therefore, NLR-1 was considered as the pivotal candidate gene for Pm37. The full-length sequence of NLR-1 is 9012 bp from the start codon to the stop codon and contains a coding sequence (CDS) of 2808 bp with four exons (Supplementary Fig. 2a).

Validation of NLR-1 by loss-of-function mutants
To clarify whether NLR-1 is required for the resistance to powdery mildew, we screened 894 M2 lines derived from ethyl methanesulfonate (EMS) mutagenesis of NC99BGTAG11 using the Bgt isolate E09, and obtained five independent susceptible homozygous mutant lines (Fig. 2a). All five independent susceptible mutants had SNPs in NLR-1 that resulted in amino acid substitutions or premature stop codons (Fig. 2b). Among them, Mut-1 was a nonsense mutation that produced premature stop codons at the 616th amino acid, resulting in the loss of the entire LRR domain; a SNP was detected in the CC domain of Mut-3, causing p.E46K; Mut-2 and Mut-4 harbored missense mutations with c.C1120T and c.G626A, leading to p.P374S and p.G209D in the NB-ARC domain, respectively; and Mut-5 had a missense point mutation producing p.G494D and a synonymous point mutation of c.G2070A. Structural analysis demonstrated that variations of NLR-1 in these five mutant lines all resulted in three-dimensional (3D) structural changes in the encoded protein (Fig. 2c).

Silencing of NLR-1 eliminates the powdery mildew resistance of NC99BGTAG11
To further validate whether NLR-1 mediates resistance to powdery mildew, we used barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) to specifically knock down the expression of NLR-1 in NC99BGTAG11. qRT-PCR analysis revealed that the expression level of NLR-1 was reduced by 77%–92% in the silenced plants treated with BSMV:NLR-1, revealing the high efficiency of the BSMV-VIGS system (Fig. 2d). After inoculation with the Bgt isolate E09, obvious fungal sporulation occurred on the leaves of the silenced plants, whereas no spores were observed on the leaves of the plants treated with the empty vector BSMV:00 (Fig. 2e, f). These results indicated that the silencing of NLR-1 compromised the powdery mildew resistance of NC99BGTAG11.

Stable transformation of NLR-1 results in a high level of powdery mildew resistance
To further validate whether NLR-1 is sufficient to confer powdery mildew resistance, we transformed a susceptible wheat cultivar Fielder with the CDS of NLR-1 driven by the maize ubiquitin (Ubi) promoter using Agrobacterium-mediated transformation (Fig. 3a). A total of 10 independent positive T0 transgenic plants were identified by PCR and qRT-PCR analysis (Supplementary Fig. 3) and subsequently self-pollinated to produce the T1 generation. Similar to NC99BGTAG11, these positive T1 progenies were immune to the Bgt isolate E09 and an additional 30 tested Bgt isolates (Fig. 3b and Supplementary Data 6). These results demonstrate that NLR-1 is sufficient to confer powdery mildew resistance and serves as the functional allele underlying Pm37.

Expression profile of Pm37
From 2014–2024, NC99BGTAG11 was tested against a total of 108 Bgt isolates collected from different years and regions in China. NC99BGTAG11 displayed immune activity to all the Bgt isolates with infection type (IT) 0 at the seedling stage (Supplementary Data 7). Moreover, NC99BGTAG11 was also immune to 30 mixed Bgt isolates at the adult plant stage under the field conditions in different regions of China over the past decade.

We also traced the pathological index after inoculation with the Bgt isolate E09. No visible spores were detected on the leaves of NC99BGTAG11 at any time, whereas Wuhuatou was highly susceptible, with abundant visible spores and secondary hyphae in the time course of 0–72 h post-inoculation (hpi) (Fig. 4a), in parallel with the increased concentration of H2O2 in NC99BGTAG11 (Fig. 4b). These results suggest the involvement of Pm37 in the regulation of cell death and pathogen defense responses. To further characterize the expression profile of Pm37, we performed qRT-PCR analysis across different tissues. The results revealed that the expression of Pm37 was greatest in the flag leaves, which can further protect the leaves from Bgt destruction for grain-filling (Fig. 4c). Moreover, the expression of Pm37 in NC99BGTAG11 was upregulated by Bgt infection, peaked at 8 hpi, and then maintained a higher expression level than that in Wuhuatou (Fig. 4d), which was relatively low and stable throughout the whole experiment.

We subsequently evaluated the impact of Pm37 on the expression of seven main pathogenesis-related (PR) genes (PR1, PR2, PR3, PR4, PR5, PR9, and PR10) involved in plant defense in NC99BGTAG11 and Wuhuatou at different times after inoculation with the Bgt isolate E09. Significant up-regulation of these PR genes was observed in NC99BGTAG11 upon Bgt invasion, peaking at later time-points than Pm37, except for PR4, whose expression peaked at 6 hpi (Fig. 4e). The coordinated expression patterns of Pm37 and these PR genes demonstrated the active role of Pm37 in plant immunity against fungal infection and suggested that Pm37 can trigger the pathway of the powdery mildew defense response soon after Bgt invasion.

PM37 mediates cell death-inducing activity analysis
PM37 and its individual functional domains (CC, NBS, LRR) and domain combinations (CC-NBS, NBS-LRR) were transiently overexpressed in Nicotiana benthamiana via Agrobacterium-mediated infiltration. The results demonstrated that overexpression of full-length PM37 induced obvious cell death in N. benthamiana leaves. Both the CC domain alone and the CC-NBS domain combination of PM37 were also capable of triggering cell death in N. benthamiana. In contrast, overexpression of the single NBS domain, single LRR domain, or NBS-LRR domain combination failed to induce any observable cell death in N. benthamiana leaves. These results indicate that the cell death-inducing activity of PM37 in N. benthamiana is mainly mediated by its CC domain (Supplementary Fig. 4).

Functional divergence of Pm37
Phylogenetic analysis revealed that Pm37 was an allele of Sr22 locus, which was derived from T. monococcum (Fig. 5a). PM37 and SR22b shared 94.22% of amino acid sequence identity, but distinct functional divergence occurred (Supplementary Data 8). Specifically, Pm37 confers resistance to powdery mildew but susceptibility to stem rust (Fig. 5b), while Sr22 locus was resistant to stem rust but susceptible to powdery mildew (Fig. 5c). Sequence alignment also revealed that PM37 shared the diagnostic amino acids for the stem rust-susceptible alleles (V384L, S608F/Y and G658D), which was consistent with its stem rust-susceptible phenotype (Supplementary Data 8). Further multiple disease assessment revealed that NC99BGTAG11 and NLR-1 transgenic lines were susceptible to all the 14 tested Puccinia striiformis f. sp. tritici (Pst) races, which cause stripe rust (Supplementary Data 9). However, when tested with seven Puccinia triticina (Pt) races that cause leaf rust, NC99BGTAG11 and NLR-1 transgenic lines conferred specifically moderate resistance to the Pt race THDS, while the transgenic receptor Fielder was susceptible (Fig. 5d). In conclusion, NLR-1 produces rare allelic variation-driven functional divergence in response to different wheat pathogens.

Allelic variations of Pm37 in diverse germplasms
To evaluate the allelic variations of Pm37 in diverse germplasms, the homologous sequence of NLR-1 was amplified from 144 wheat accessions and wheat relative accessions, including 36 einkorn wheat, 42 emmer wheat, seven T. timopheevii accessions and 59 hexaploid wheat accessions. Target bands were produced in only five einkorn wheat, three emmer wheat, one T. timopheevii and 15 hexaploid wheat (Supplementary Data 10). Sequence alignment revealed complex sequence variations in this gene among these accessions, including 104 SNPs and nine InDels. These variations are distributed across all the functional domains (CC, NB-ARC, and LRR) of PM37 protein, with effects such as premature stop codons, amino acid substitutions in key functional regions, and frame shifts. Furthermore, these 24 accessions are classified into five haplotypes according to their major sequence variations (Supplementary Data 10). Consistent sequence variations were identified between NC99BGTAG11 and susceptible types, including a 6 bp deletion at the 270-275 position, G284A, G1104A and C2392G. Presumably, these variations are pivotal for the resistance of Pm37, and could be used as diagnostic sequences for the powdery mildew-resistant alleles. Using this information, we further observed that Sr22b possesses susceptibility alleles at these loci, which is consistent with its susceptibility to powdery mildew. Moreover, identical genomic sequences and CDS of NLR-1 as HapI were detected in one T. monococcum accession AA359 (CWI18758), and one T. timopheevii (Zhuk.) Zhuk. subsp. timopheevii accession AAGG477 (CWI17281) (Supplementary Data 10). Powdery mildew phenotype assessment also indicated that AA359 and AAGG477 were both immune to all the 31 tested Bgt isolates as same as Pm37 donor NC99BGTAG11 and the transgenic lines (Supplementary Data 11).

Evolutionary tracing of Pm37
To explore the possible donor of Pm37, the chromosome 7A sequence of NC99BGTAG11 was aligned to the genomes of T. monococcum (accession TA299) and T. timopheevii (accession PI 94760). Five genomic segments (in total 14.2 Mb) of NC99BGTAG11 on the terminal of chromosome arm 7AL were identified exhibiting high similarity to the corresponding region of TA299. Furthermore, we found that Pm37 was derived from a 2.2 Mb introgressed segment from the chromosome 7A of T. monococcum (Fig. 1e). In contrast, no alignment (≥150 kb) was identified between NC99BGTAG11 and T. timopheevii PI 94760 on chromosome 7A (Fig. 6a–c). Although no NLR-1 sequence was identified in T. timopheevii accession PI 94760, NLR-1 was isolated from another T. timopheevii accession AAGG477 (Supplementary Data 10). Taken together, we speculated that Pm37 in the hexaploid line NC99BGTAG11 may be originally derived from T. monococcum and then transferred into bread wheat through T. timopheevii. Additionally, homologs or alleles alignment of Pm37 in different A subgenomes revealed Pm37 clusters with Tm.TA299.r1.7AG0168310 from T. monococcum TA299 (Fig. 6d), further confirming the T. monococcum origin.

Development of diagnostic marker and elite introgression lines for Pm37
To facilitate the detection of Pm37 on the basis of its unique variations, a diagnostic InDel marker, Pm37-FM, was developed based on the unique sequence polymorphism in the second intron. Amplification with this PCR marker generated an amplicon of 473 bp only when the gene Pm37 was present (Supplementary Fig. 5). Using this diagnostic marker, we developed a series of advanced breeding lines carrying Pm37 in different genetic backgrounds, such as the cultivars Shixin 828, Kenong 199, Yannong 999 and Yannong 199. Field investigation of these advanced breeding lines revealed no obvious negative effects on key agronomic or yield traits, such as spike length, spike numbers per plant, spikelet numbers per spike, kernel numbers per spike or thousand-kernel weight (Supplementary Data 12).

Methods
Plant materials
The wheat line NC99BGTAG11 (Reg. no. GP-729, PI 615588), which is available in the United States Department of Agriculture Agricultural Research Service, is an F7-derived line with the pedigree “Saluda”*3/PI 427315, of which Saluda is a soft red winter wheat, and PI 427315 is a T. timopheevii accession34. It carries a single dominant gene, Pm37, that confers resistance to powdery mildew and has been mapped to a 1.0 cM genetic interval on chromosome arm 7AL23. The F1, F2, and F2:3 segregating populations were developed from a cross between NC99BGTAG11 and the Chinese landrace Wuhuatou, which is susceptible to all Bgt isolates tested in our laboratory. The spring wheat cultivar Fielder, which is susceptible to powdery mildew, was used as the transgenic receptor. The wheat accession Sr22TB (donor of Sr22a) and three transgenic families T3-Tm514-2, T3-Tm515-6, and T3-Tm517-1 homozygous for the Sr22b transgene, provided by Prof. Shisheng Chen from the Peking University Institute of Advanced Agricultural Sciences35, were used as a positive control for stem rust resistance evaluation and for analyzing the functional divergence between Pm37 and Sr22, respectively. Wheat samples for haplotype analysis, including five einkorn wheat, three emmer wheat, one T. timopheevii accession and 15 hexaploid wheat accessions, were provided by Prof. Hongxing Xu, Henan University, Kaifeng, China (Supplementary Data 10). The common wheat cultivar Tainong 18 was used as the susceptible control and spreader of Bgt isolates in the phenotype assessment against powdery mildew.

Phenotyping of powdery mildew response
A total of 108 Bgt isolates from different wheat-producing regions in China were used to evaluate the wheat accessions for resistance against powdery mildew (Supplementary Data 7). Among these Bgt isolates, the Bgt isolate E09 was used for phenotyping of NC99BGTAG11, Wuhuatou and their genetic segregating populations, EMS-induced mutants of NC99BGTAG11, and Pm37 transgenic and BSMV-VIGS plants. To map Pm37, more than 20 seedlings of each F2:3 family were used for phenotype evaluation using Bgt isolate E09. At least 20 individuals of the T1 transgenic lines were inoculated with Bgt isolate E09, and another 30 Bgt isolates (Supplementary Data 6). For the evaluations, wheat seedlings at the two-leaf stage were inoculated with Bgt isolates by dusting fungal conidiospores from Tainong 18 leaves36,37. Disease severity was evaluated 14 days after inoculation on a scale from IT 0 to IT 4, where ITs 0, 0;, 1, and 2 indicated a resistant phenotype, and ITs 3 and 4 corresponded to a susceptible reaction38.

Fine mapping of Pm37
To fine map Pm37, equal amounts of RNA from 30 homozygous resistant and 30 homozygous susceptible F2:3 plants of the cross NC99BGTAG11 × Wuhuatou, were mixed to construct the resistant and susceptible bulks, respectively. cDNA libraries of NC99BGTAG11, Wuhuatou, and the two bulks were constructed using TruSeq Stranded mRNA LT Sample Prep Kits (Illumina, San Diego, CA, USA). High-quality cDNA libraries were subsequently sequenced using an Illumina HiSeq 4000 at Beijing Biomarker Technologies Co., Ltd. (Beijing, China). A minimum of 30 Gb of clean data for each bulk and 10 Gb for each parent was set as the thresholds for data production. Afterwards, the sequence alignment, SNP calling, calculation of the ΔSNP index for each SNP, conformation of the candidate intervals and development of polymorphic markers and genotyping detection were performed39. The linkage relationship between polymorphic markers and Pm37 was established using Mapmaker 3.0 software, with the Kosambi map function and the logarithm of odd (LOD) score threshold was set at 3.040. Putative genes were identified through preexisting annotations retrieved through JBrowse (http://202.194.139.32/jbrowse.html) and Ensembl Plants (http://plants.ensembl.org).

PacBio HiFi sequencing and genome assembly of the Pm37 donor
Fresh leaf tissues of NC99BGTAG11 were sampled at the four-leaf stage for DNA extraction and purity assessment following an established procedure11. Eligible DNA with an appropriate size range (15–20 kb) was used for the construction of PacBio HiFi sequencing libraries and subsequent sequencing on the Pacific Biosciences Revio system using HiFi sequencing protocols at Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Libraries were sequenced on three PacBio SMART cells, and more than 90 Gb of data was produced in each cell. A primary assembly was generated from those HiFi sequencing reads using hifiasm (v0.20.0)41. Using RagTag (v2.1.0)42 and the T. aestivum (CS) reference (RefSeq 2.1)43, the assembly contigs were scaffolded to the chromosome level.

Genome-wide alignment analysis with T. monococcum and T. timopheevii
The RagTag-scaffold of chromosome 7A of NC99BGTAG11 was aligned to the reference genomes of T. monococcum TA29944, and T. timopheevii PI 9476045 using MUMmer (v4.0.0rc1)46. The resulting alignments were stringently filtered (≥10 kb, ≥90% identity) to ensure high accuracy of introgression identification. To define contiguous introgressed segments, a more stringent 150 kb length filter was applied, and adjacent retained alignments were merged into blocks with a maximum gap of 200 kb.

Extraction of RNA and qRT-PCR
To investigate the expression patterns of the candidate genes of Pm37 and the pathogenesis-related genes PR1, PR2, PR3, PR4, PR5, PR9 and PR1047, total RNA was extracted from two-leaf-stage leaves of NC99BGTAG11 and Wuhuatou inoculated with the Bgt isolate E09 at 0, 0.5, 2, 4, 6, 8, 12, 16, 24, 36, 48, and 72 hpi using a FastPure Universal Plant Total RNA Isolation Kit (TransGen, Beijing, China) following the instructions. Furthermore, to determine the expression pattern of the Pm37 gene in different tissues, total RNA was extracted from flag leaves, second leaves from the top, third leaves from the top, fourth leaves from the top, fifth leaves from the top, spikes, stems, and sheaths. Approximately 2 μg of RNA was used for reverse transcription to produce cDNA with a HiScript® III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). The qRT-PCR assays were performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a Bio-Rad CFX Connect real-time PCR system (Bio-Rad, USA). The relative expression level per gene was calculated as the fold change using the comparative CT method48. Three technical replications of each sample were performed. The gene TaActin was used as the internal control for standardization. Statistically significant differences: two-way ANOVA with Tukey’s test, ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Primers used in this study are listed in Supplementary Data 2.

EMS mutagenesis and mutant validation
To produce loss-of-function mutants, 4672 seeds of NC99BGTAG11 were first soaked in distilled water for 18 hours at room temperature. Afterwards, they were immersed in freshly prepared 0.8% EMS on a shaker for 14 hours. Prior to sowing, the treated seeds were rinsed with running water for 12 h and dried in a fume hood49. These treated seeds were then sown in the field to produce M2 plants, and a total of 894 M2 plants were harvested. To screen for loss-of-function mutants, the response of each M3 family to the Bgt isolate E09 was tested. The susceptible plants were transplanted into the field to harvest the M4 generation, which was used for phenotypic verification using the same Bgt isolate. To verify the candidate genes, the homologous genomic sequences of candidate gene(s) in these mutants were amplified and then compared for nucleotide variations with those of NC99BGTAG11 by multiple sequence alignment using DNAMAN 8 software (Lynnon Biosoft, San Ramon, CA, USA). Furthermore, the amino acid sequences of Pm37 in NC99BGTAG11 and its mutants were used as inputs to predict the 3D structure of Pm37, and AlphaFold v3.0 was used to model the 3D structure of PM3750. A 3D structure diagram was generated using PyMOL (v.2.6.0) to visualize the molecular structure.

BSMV-VIGS
To silence the candidate gene of Pm37, a 245-bp specific sequence within its CC domain was identified through sequence alignment and selected as the target for silencing (Supplementary Data 13). This fragment was then amplified and inserted in reverse orientation into the barley stripe mosaic virus RNA to generate the recombinant vector BSMV:Pm37. Infected NC99BGTAG11 harboring the empty vector BSMV:00 was used as the negative control47. When the plants grew to the two-leaf stage, the second leaves of the plants were inoculated with the barley stripe mosaic virus9. At 7–12 days post inoculation (dpi), the plants were inoculated with the Bgt isolate E09. Approximately 14 days later, the powdery mildew reactions were evaluated with the 0-4 standard as described above. Spore development was observed with Coomassie blue staining. Relative expression levels of Pm37 in the lines NC99BGTAG11 treated with BSMV:00 (control) and BSMV:Pm37 were examined by qRT-PCR. The expression values are presented as the means ± SDs of three biological replicates and three technical replicates for each leaf. Statistically significant differences: two-way ANOVA with Tukey’s test, ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Stable transformation of the candidate gene
The CDS of the candidate gene flanked by a BamHI restriction site was amplified via PCR from the ZTOPO-Blunt/TA-Pm37_CDS construct and cloned into the BamHI site of the pWMB110 vector downstream of the maize (Zea mays L.) ubiquitin (Ubi) promoter with the nopaline synthase (Nor) terminator via the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) to generate the overexpression vector ProUbi:Pm37. The colonies were subsequently sequenced to confirm the accuracy of the cDNA insertions. The ProUbi:Pm37 plasmid was subsequently transformed into the Agrobacterium tumefaciens strain EHA105, which was subsequently transformed into the susceptible wheat cultivar Fielder51. The transgenic plants were confirmed by PCR amplification and gene sequencing. T0 plants and more than 10 plants of each T1 transgenic family were first tested for their responses to the Bgt isolate E09, and then the resistant T1 plants were further assessed using an additional 30 Bgt isolates, with NC99BGTAG11 and Fielder used as controls. The expression patterns of the transgenic plants were also measured using qRT-PCR. Leaves of each T0 seedling plant at the two-leaf stage were collected. TaActin was used as an endogenous control. All the primers used for vector construction, confirmation of positive transgenic plants, and gene expression are listed in Supplementary Data 2.

Cell death assay in N. benthamiana
Pm37 and its domains were constructed into the pS1300-NOS vector for overexpression in N. benthamiana, respectively. Agrobacterium-mediated transient expression was conducted following the method described by Liu et al. with slight modifications52. In brief, Agrobacterium tumefaciens GV3101 strains carrying the specified vector constructs were cultured at 28 °C in Luria-Bertani medium (containing 0.5% yeast extract, 0.25% NaCl, 1% tryptone) with Kanamycin sulfate and rifampicin. The cultures were then centrifuged, and the pellets were resuspended in infiltration buffer (composed of 10 mM MgCl2, and 10 mM MES [pH 5.7]), supplemented with 200 μM acetosyringone. The suspensions were adjusted to an OD600 of 0.8 using the same infiltration buffer and incubated at room temperature for 2–3 h prior to infiltration. Leaves of approximately 4-week-old N. benthamiana plants were infiltrated, with each strain infiltrated into three leaves from different plants. After infiltration, plants were kept under controlled conditions in growth chambers and photographed at 48 hpi53.

Sequence and haplotype analysis
Wheat and its relatives54, including five einkorn wheat accessions, three emmer wheat accessions, one T. timopheevii accession, and 15 hexaploid wheat accessions, were used for haplotype analysis. They were first evaluated by 31 different Bgt isolates to analyze the resistance spectrum as mentioned above (Supplementary Data 11). PacBio HiFi sequencing and genome assembly of the Pm37 donor NC99BGTAG11 provided a high-quality genomic reference. This assembly allowed to accurately identify the CDS of Pm37, enabling the design of highly specific primers targeting conserved regions within the CDS. The PCR primer pairs used for amplifying the entire Pm37 gene are listed in Supplementary Data 2. Total RNA was extracted from each accession, and cDNA was synthesized as the template for amplification. The amplification was performed in a 50 μl reaction mixture containing 25 μl of 2× TransStart FastPfu PCR SuperMix (+dye) (TransGen, Beijing, China), 12 ng of each primer, and 250 ng of genomic DNA. DNA amplification was performed at 95 °C for 3 min, followed by 35 cycles at 95 °C for 15 s, 55–58 °C for 15 s depending on the annealing temperatures of the primer pairs, and 72 °C for 30 s/kb, with a final extension at 72 °C for 5 min. All the PCR products were Sanger sequenced at Sangon Biotech (Shanghai, China) Co., Ltd. The sequences were assembled and aligned using SeqMan Pro in the LaserGene 11 package.

Phylogenetic analysis
The protein sequences encoded by cloned wheat NLR resistance genes, including 30 Pm genes, 12 Sr genes, seven Lr genes, and two Yr genes, were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/). These genes were selected based on their confirmed NLR domain architecture to ensure sequence homology. For multiple sequence alignment, we utilized MEGA11 and specifically chose the Align by MUSCLE (Codons) option. After the alignment, poorly aligned regions were manually trimmed to ensure the reliability of subsequent analyses. The phylogenetic cladogram of PM37 with these NLR resistance proteins was constructed using the neighbor-joining method in MEGA11, with the JTT substitution model and 1000 bootstrap replicates to assess branch reliability (node values represent bootstrap support values, indicating the reliability of the topological relationships)55. The cladogram was visualized using iTOL (https://itol.embl.de/)56.

Phenotype divergence assessment for powdery mildew, stem rust, stripe rust and leaf rust
To further determine the relationship between Pm37 and Sr22, the response of the transgenic lines carrying Pm37 and transgenic lines carrying Sr22b to stem rust and powdery mildew was tested using the Chinese Puccinia graminis f. sp. tritici (Pgt) race 34C3RKGQM and the Bgt isolate E09, respectively. Moreover, to explore whether Pm37 is resistant to other diseases, the transgenic lines carrying Pm37 were also inoculated with seven Pt races, including THKS, PHJT, DGJT, PCTT, THTT, THDS and THTS, which cause leaf rust and 14 Pst races, which cause stripe rust (Supplementary Data 9). At least 10 individuals of each transgenic line were inoculated using NC99BGTAG11 and Fielder as controls. Inoculation and phenotype assessment of powdery mildew were performed as described above. Inoculation and phenotype assessment of stem rust were carried out following the procedure previously reported by Luo et al. with minor modifications35: Inoculation was conducted in the greenhouse when the first leaf was fully expanded, after which it was incubated at 25 °C/8 h darkness 22 °C for 16 h. And, 12-14 dpi later, the ITs were recorded on the 0–4 scale57. Inoculation and phenotype assessment of leaf rust were performed in Hebei Agricultural University, Baoding, China, according to previously described methods as Liu et al.58. Inoculation was conducted in the greenhouse when the first leaf was fully expanded. Inoculated seedlings were then incubated at 18–22 °C and 100% relative humidity (RH) for about 24 h. They were then transferred to a growth chamber maintained under 12 h darkness/12 h light at 18–22 °C and 70% RH. Inoculation and phenotype assessment of stripe rust were carried out at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, following the procedure previously reported by Li et al.59. Inoculation was conducted under controlled conditions of 20 °C, 80% humidity and 14 h daily light when the plants were at the fully expanded one-leaf stage, and fresh urediniospores suspended in an aqueous solution containing 0.05% v/v Tween 20 were used for this process. After inoculation, the seedlings were placed in a dew chamber at 10 °C for 24 h and then transferred into a controlled greenhouse with a diurnal cycle of 14 h light at 18 °C and 10 h darkness at 12 °C. ITs were determined on a 0–4 scale57. All the phenotypic assessments were performed at least three independent replications.

Detection of H2O2 reactions and plant cell death
To detect the accumulation of H2O2, the first leaves were collected from NC99BGTAG11 and Wuhuatou at 0, 0.5, 2, 4, 6, 8, 12, 16, 24, 36, 48, and 72 hpi following invasion of the Bgt isolate E09 and incubated in a 3,3′-diaminobenzidine (DAB) solution (1 mg/mL, pH 5.8) in the dark for 12 h and then bleached in Carnoy’s fixative (absolute alcohol: glacial acetic acid, 3:1, v/v)60. Before the accumulation of H2O2 was assessed, the bleached leaves were incubated in a 0.6% (w/v) Coomassie blue solution for 30 s–120 s and then washed with water. Finally, the samples were observed under an Axioscope 5 microscope (ZEISS, Germany).
To further monitor the invasion process, NC99BGTAG11 and Wuhuatou were inoculated with the Bgt isolate E09, and 2 cm of seedling leaves were collected at 0, 0.5, 2, 4, 6, 8, 12, 16, 24, 36, 48, and 72 hpi, fixed in 5 mL of Carnoy’s fixative at 37 °C for 24 h, and stained in 5 mL of 0.6% (w/v) Coomassie blue liquid for 5 min. Excess dye on the leaf samples was rinsed with distilled water. Finally, the samples were observed under an Axioscope 5 microscope (ZEISS, Germany).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript the authors report a comprehensive analysis of Pm37, a gene for resistance to wheat powdery mildew.\nThey identified a Pm37 candidate through map-based cloning. The candidate gene was confirmed to be Pm37 through\nmutagenesis, barley stripe mosaic virus-induced gene silencing, and stable transformation. A major contribution of this study\nis the finding that Pm37 is an allele of the stem rust resistance gene Sr22, and also confers resistance to leaf rust. This is an\nexample that diversification of a resistance gene has produced alleles effective against various pathogens. The manuscript\nis well-organized, and the results are convincing. Therefore, I suggest that it merits for publication after minor revisions\ndescribed below.\n(1) Line 86: Timopheevii (Zhuk.) Zhuk.\n=>T. timopheevi (Zhuk.) Zhuk. subsp. timopheevi (?)\n(2) Lines 86-89: “….were the ancestral species of common wheat, possessing abundant disease resistance genes, such as\nstem rust (Sr) resistance genes Sr36 and Sr37, stripe rust (Yr) resistance genes Yr10, and Pm genes Pm6, Pm27 and\nPm37.”\n=>Why has there been no report of resistance genes against leaf rust, one of the major wheat diseases? I am wondering\nwhy researchers have not noticed the effect of Pm37 against leaf rust ?\n(3) Line 104: Supplementary Table 3 can be modified to help readers understand the position of Pm37 (determined by\nphenotypes) quickly. Please refer to the attachment as an example.\nIn Supplementary Table 3 and Fig. 1c, the same designation should be used for the types of recombinants. In the second\ncolumn of the revised version of Supplementary Table 3 (attached), I noted corresponding types (R1-R6) which I speculated.\nPlease check them.\n(4) Lines 128-129: “The longest contig length of 202.2 Mb, and the total length of the genome assembly of 14.6 Gb, which\ncovered the whole genome of CS reference genome.”\n=>Please add a verb to this sentence.\n(5) Lines 164-166: “T1 progenies from all these transgenic positive line were immune to the Bgt isolate E09 and additional\n30Bgt isolates as same as NC99BGTAG11.”\n=>Did each T1 line contain no susceptible seedlings?\n(6) Lines 202-204: “Notably, further disease assessment revealed that NC99BGTAG11 and NLR-1 transgenic lines also\nconfers resistance to Puccinia triticina (Pt) race that causes leaf rust, while the transgenic receptor Fielder is susceptible.”\n=>How (or why) did you pick up this pathogen (Pt) as a possible target of Pm37? Did you also test some other pathogens?\nHow many Pt races did you test? Only one (THTT)? If you tested several races, did you find race specificity?\nYou may sure that this gene is different from Lr genes that have been identified to date. Please explain the basis for this\nthought.\n(7) Lines 210-211: “Only CC domain of PM37 induced cell death,…. (Supplementary Fig. 7). However, overexpression of\ndifferent domains of SR22 in N. benthamiana leaves did not induce cell death whereas the full-length SR22 did cause cell\ndeath (Supplementary Fig. 7).”\n=>The description of the results for SR22 (shown in Supplementary Fig. 7c) is reasonable. However, it seems difficult to\ninterpret the results for PM37 (shown in Supplementary Fig. 7a) as “Only CC domain of PM37 induced cell death”. For\nexample, the lesion in Supplementary Fig. 7a-2 is very small and similar to those in Supplementary Fig. 7c-2 and 4.\n(8) Supplementary Table 7, 8, 9: Please check the table numbers in the files for Supplementary Table 7, 8, and 9. (They are\ndescribed as Table 8, 9, and 10, respectively.)\n(9) L248: Even through\n=>Even though?\n(10) L317: Rgm8 =>Rmg8\n(11) L384: Wuhautou at 0, …….. 72 hpi inoculating with Bgt isolate E09\n=>Wuhautou inoculated with Bgt isolate E09 at 0, …….. 72 hpi\n(12) L677 (Fig. 6): It may be better to divide Fig. 6 into three parts, i.e. Fig. 6a (stem rust), Fig. 6b (powdery mildew), and Fig.\n6c (leaf rust).", "reviewer_2": "The manuscript reports isolation of the Pm37 resistance gene from NC99BGTAG11. Authors made good use of standard\ngenomic tools for fine-mapping to isolate and verify the gene sequence underlying resistance gene, including long read\nsequencing of the resistant line NC99BGTAG11. They validate isolation of the Pm37 resistance gene through identification\nof EMS mutants, VIGS of Pm37 and stable transgenics. They demonstrate that Pm37 confers resistance to a large collection\nof powdery mildew isolates from different parts of China. They also determined that NC99BGTAG11 and Fielder transgenics\nhaving Pm37 are resistant to leaf rust isolates from China. They explore homology of Pm37 derived in this case from\ntetraploid T. timopheevii subsp. armeniacum (AtAtGG) with the Sr22 gene that was transferred to common wheat from an A-\ngenome diploid. They identify two accessions of T. monococcum having an identical Pm37 coding sequence, including\nTA299 for which a genome sequence is available. They also identified an accession of T. timopheevii (subsp.\narmeniacum?) having Pm37.\nMethodology and presentation of cloning results are appropriate. The work is robust and significant in identifying an allele of\nSr22 that confers resistance to powdery mildew and as well as a leaf rust.\nThe most original and significant aspect of the paper is the determination that Pm37 is an allele of stem rust resistance gene\nSr22 present in both a diploid and tetraploid wheat relative, and that this allele confers resistance to powdery mildew and\nleaf rust (but not to stem rust). This result is broadly interesting to wheat researchers as well as those working in other crop\nsystems. However, there are areas where statements about global utility of Pm37 conferred resistance may be overstated\n(see information below).\nOverall, the presentation of the manuscript suffers from instances of poor English grammar and language usage. It could be\nimproved by professional copy editing.\nAccess to NC99BGTAG11 long read sequence and assembly are necessary to reproduce some results. They do not appear\nto be shared.\nFor all figures having error bars (Figs. 2, 4, S1, and S5), the error bars are not defined in the figure legends.\nThe presentation of work in Figure S7 is somewhat out of the scope of my expertise. However, the conclusion that\noverexpression of the CC domain of Pm37 induced cell death does not seem supported by Figure S7a.\nWere Pm37 lines evaluated for resistance to yellow rust? If so, it may be interesting to report a reaction to this rust pathogen\nas well.\nFigure S2 - The figure legend should include the source of einkron and timopheevii assemblies (TA299 and PI 94760,\nrespectively) to which NC99BGTAG11 reads were mapped. It is interesting that there were more regions (beyond the\nterminal portion of 7AL) where higher read depths were observed between NC99BGTAG11 with wild einkorn TA299 than\nwith PI 94760. While this may be because PI 94760 is not the donor of Pm37, the overall pattern makes one wonder about\nthe pedigree of NC99BGTAG11. I would expect more regions of similarity with T. timopheevii in a BC2-derived line. And\nfewer with T. monococcum.\nAdditionally, Fig 1e indicates that the Pm37 region in NC99BGTAG11 is more like TA299 than PI94760. Lines 247 -252\nnote the Pm37 was derived from T. monococcum via the bridge species T. timopheevii. The authors did find evidence of\nPm37 in the accession of AAGG477 of T. timopheevii (is this an accession of subspecies armeniacum? or subsp\ntimpopheevii?).\nSeed of PI 427315, the T. timopheevii subsp armeniacum parent of the NC99BGTAG11, is available from the USDA\nNational Small Grains collection. In order to verify that the Pm37 was indeed derived from T. timopheevii, the authors could\ninclude accession PI 427315 in their analysis of allelic variation.\nIn Figs. 5 and S8, branch lengths appear to be set as equal. Therefore, it would be best to label these as cladograms in the\nfigure legends. It would be interesting to see these presented as phylograms with bar length indicating sequence similarity.\nRather than comparing with several cloned R genes belonging to other clades in Figure 5, it may be more interesting to\ncompare Pm37 with the Sr22 alleles/homologs present in A-genome diploids and reported in the Asyraf et al. 2020. At the\nleast, this paper should be included in the discussion.\nExtensive Genetic Variation at the Sr22 Wheat Stem Rust Resistance Gene Locus in the Grasses Revealed Through\nEvolutionary Genomics and Functional Analyses. Molecular Plant-Microbe Interactions 2020 33:11, 1286-1298\nIt should be mentioned in the manuscript that the sample of powdery mildew and leaf rust isolates evaluated were not global.\nKloppe et al. 2022 (doi: 10.3389/fpls.2022.954958) reported Pm37 virulence in 9 and 10% of Bgt isolates evaluated from\nEqypt and Turkey, respectively, as well as 1 and 2% of isolates from the United States and Russia/Kazakhstan. They\nconclude that the higher levels of virulence observed in Turkey to Pm37 (and other newly introgressed Pm genes) may be\nrelated to the presence of donor species in the region as the gene has not been used commercially. It would be useful to\nmention this work in the manuscript.\nThe NC99BGTAG11 germplasm was grown in the field over multiple years in the US state of North Carolina. In at least three\nyears where we experienced natural leaf rust epidemics, high levels of leaf rust were observed on recurrent parent Saluda\nand NC99BGTAG11. These data are not reported in literature, so would not be known to the authors. But this indicates that\nthe Pm37 mediated leaf rust resistance reported in the manuscript is not expected to be globally effective, even in the\nabsence of commercial use of the gene.\nThere are a number of places in the manuscript where the writing could be improved. Also, please see comments below.\nLine 86 – T. timopheevii (genome designations AtAtGG) and its wild species armeniacum are not direct ancestors of T.\naestivum (AABBDD); they are related species. The ‘sister’ species of T. timopheevii is T. turgidum (AABB) and it is the\nancestor of hexaploid wheat.\nIt may be useful to clarify the relationship of T. timopheevii and T. monococcum to each other and with T. aestivum, perhaps\nas a diagram in the supplemental material.\nLine 89 - Yr10 is not a T. timopheevii derived gene – from T. spelta and T. vavilovi (both are AABBDD genome sub-species)\nLine 92 – “ ultra-broad-spectrum resistance for more than a decade”. This statement is misleading as Pm37 has not been\ndeployed in commercial varieties.\nLine 117 - In figure S1 ,only TraesCS7A12127000 (NLR-1) has significantly higher expression in the resistant line - this is\nnot mentioned in the text.\nLine 170-”...108 BGT isolates collected from different years and regions…” should specify that all isolates were collected\nfrom China\nLine174-175 - As noted above, the statement about “ultra-broad spectrum powdery mildew resistance at the whole growth\nperiod” should be qualified as Pm37 was not deployed in the region over this period.\nLines 268- 272 - This sentence suggests that the authors consider T. timopheevii to be in the secondary gene pool of wheat,\nbut would not be a wheat relative where introgression would be associated with linkage drag?\nIt is interesting that recombination rates were quite good in the Pm37 region. This differs somewhat from the Sr22\ntransfer where suppressed recombination was reported and initial introgression lines were not used due to poor agronomic\nperformance (linkage drag).\nLines 275-277: I am not sure what the authors mean here. I don’t think that this work provides a new “framework” for using\nresistance genes from wild relatives or isolation of resistance genes.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "Review on “Jin et al., Pm37, a susceptible allele of Sr22 from Triticum monocuccm, confers resistance to powdery mildew\nand leaf rust in common wheat\"\nThe article from Jin and colleagues describes the cloning of Pm37 in a resistant wheat cultivar (abbreviated NC99 here in\nmy report) that derives from a cross between a winter wheat and a T. timopheevii accession carrying this gene. The paper\ndescribes the fine mapping steps leading to a very small genetic interval and all the supporting evidence that the candidate\nNLR present in the interval is actually the gene controlling the resistance phenotype i.e., production of EMS mutants,\ntransformation in Fielder, overexpression of the protein with recombined domains. In addition, the paper also describes\nsequence analyses performed in order to understand the diversity/evolution of the gene and the locus.\nThe results about fine mapping and functional validation are interesting, well performed, scientifically sound, and\nconvincing. In contrast, the sequence analyses and evolutionary interpretations are questionable. Since this later point is my\narea of expertise, I will focus my comments/remarks on these aspects which, I think, require significant improvements to be\nunderstood, first, and convincing. I have main concerns about the methods used for sequence analyses and phylogeny.\nPlease see my remarks below.\n## POINT-BY-POINT COMMENTS\nI guess there is a generalized mistake with writing the \"monococcum\" species name here. At several places, the authors\nwrote \"monocuccm\" or even \"monocuccum\". It requires corrections all along the text and especially in the Title.\n# ABSTRACT\n- \"Pm37 is susceptible to stem rust, and Sr22 is susceptible to powdery mildew\".\nA gene (or a protein) is not susceptible nor resistant to a disease. A plant is. The writing needs some serious edits.\n- \"Pm37 and Sr22 produced functional divergence that only overexpression of the CC domain of PM37 and the full length\nSR22 could trigger cell-death activity.\"\nI read the sentence several times but cannot make any sense of it.\n# INTRODUCTION\n- Line 91: A reference is needed when presenting NC99.\n# RESULTS\n+ Map-based cloning of Pm37\n- \"comparison of the target interval [..] indicated that no new gene was present (Fig. 1d).\"\nThis is clearly not supported by what is shown at Fig 1d. I found this panel d requires significant improvement so that the\nreader is able to understand the Figure by itself. As far as I get it, there are several nonsyntenic genes in the genomes\ncompared here, no? I see some genes related with \"links\" and some without link, so I conclude they are non-conserved. In\naddition, it is not obvious to me that the genomic segments compared here are the syntenic/orthologous ones. Since the\nsegments are not flanked by orthologous/collinear genes in all genomes compared, it raises questions. For instance, gene\n03G1214100 (CS) could be just next to 168350 (monococcum) but not represented in the segment represented here. Such\ncomparative genomics picture is not clear if the segments are not bordered by exact collinear genes. To me, it is not possible\nto conclude about gene presence/absence based on the work performed here.\n+ De novo genome assembly of NC99BGTAG11 and candidate gene isolation\n- \"Sequence alignment revealed a mass of SNPs and InDels between NC99BGTAG11 and Wuhuatou (Supplementary\nTable 4).\"\nFirst, the supplemental figures and tables are not numbered, so it is not easy to follow.\nSecond, Sup Table 4 is cited to support the fact that the two alleles differ a lot. This is a table with 1 line per SNP… A basic\nsequence alignment would probably have been much more informative. And in the text, I suggest to just give the numbers of\nvariations/length or percent identity, which would be much more informative than \"a mass\".\n- Supplemental Fig 3 is misleading with the scale not respected. It must be corrected. It appears that there is a 6 kb intron,\nwhich is somehow unusual in these Triticeae genomes. In CS, this gene is different because this intron is about 2.8kb. Why\nnot having shown these variations? Is there a TE inserted which would explain this difference? What about monococcum\nand timopheevii? This variation could be highly valuable to help with reconstructing haplotypes and trace the origin of the\ngene.\nSince this gene is almost 9 kb in NC99 versus 6 kb in CS, it is not surprising that all attempts to get a full-length amplicon\nsystematically failed. This could be added as a probable explanation.\n+ Validation of NLR-1 by loss-of-function mutants\n- \"we […] obtained five independent homozygous mutant lines\".\nGuess the authors mean five independent \"susceptible\" homozygous mutant lines.\n- Results are convincing. Five susceptible mutants, all with 1 deleterious mutation, perfectly clear.\n+ Silencing of NLR-1 eliminated powdery mildew resistance of NC99BGTAG11\nResults are convincing. The description of the work performed is, nevertheless, quite expeditive. NLRs are highly repeated\nin terms of copy number and silencing 1 single copy is probably not that trivial. I found we lack some details about this\nparticular NLR, how many copies share similarity etc. what about homeologous copies? And was it obvious to design\nprobes that specifically target this copy? I suggest to give some more details about that. Although, the previous part with fine\nmapping + 5 independent EMS mutants is already quite convincing that this candidate is the causal gene.\n+ Stable transformation of NLR-1 produced high level of powdery mildew resistance\nAgain, expeditive. It would have been interesting, at least, to describe the status of this NLR-1 in the Fielder genome since\nan assembled sequence is available.\n+ Functional divergence of Pm37\n- \"PM37 and SR22 shared 94.22% amino acid sequences identity, but occurred distinctly functional divergence\n(Supplementary Fig. 6).\"\n94% aa identity is very low, even lower than what is usually observed between A-B-D homeologs, for instance i.e., genes\nthat have diverged millions of years ago. Such high level of divergence between genes that are supposed to derive from a\nAA common ancestor is questionable. One should not simply conclude that Pm37 is an allele of Sr22. This must be\ndiscussed in regards to \"allelic\" variations of orthologous genes of AA genomes. This is very unusual and would require\nadditional analyses before proposing conclusions on the evolution of this gene.\n+ Allelic variations of Pm37 in diversified germplasms\nThe amplification of this gene failed (as explained earlier) in NC99, so why is this limit not considered when trying to amplify\nit in 144 accessions? This is a major limit obviously which prevents from concluding on the presence/absence. The Methods\nrelated to that experiment are not detailed enough and it is not possible to evaluate. Maybe the amplification was limited to a\npart of the gene only?\n- Line 227: \"Sequence alignment revealed complex sequence variations in this gene among these accessions\".\nWhy complex?\n- Line 230: \"sequence variations […] including c. GGAACAC276-282- - - - C - -\"\nWritten as it is here, it is not easy to understand the variation. To me, this is doubtful variation with a single C flanked by 2\nindels… This typically due to a problem with the alignment: the C must have been aligned with the last C of GGAACAC. The\nindel corresponds to GGAACA only. This is typical from a multiple alignment between sequences that are too variable. The\nalignment must be corrected before haplotype classification.\n- Line 238: \"Additionally, sequence variation analysis of NLR-1 in different wheat […] revealed complex sequence variations\n[…]\"\nAgain, same conclusion as the sentence before. \"Complex\" is not a result. What are the results here?\n+ Evolutionary tracing of Pm37\nMethods and results are quite questionable. Several remarks:\n- First, I suggest a make a Figure showing how the authors identified the introgressed segment.\n- Then, why having used read mapping method while there is an assembled genome sequence available? chr7A of NC99\ncould have been scaffolded (with RagTag or whatever) and compared to chr7A of timopheevii and\nmonococcum.\n- Supplemental Fig 2 is somehow useless and does not provide any evidence of the introgression.\n- More importantly, I could not understand the results… In summary it is written: NC99 read mapping revealed similarity over\n78Mb (monococcum) and 69Mb (timopheevii), so Pm37 derives from a 32Mb introgressed segment??? How did the author\nreach this value? In addition, the Figure 1e does not show any 32Mb or 78Mb segment… and it even shows no similarity\nwith timopheevii at all… Completely confusing. I suggest to redo the analyses and describe the results in a way that readers\ncan understand.\n- Line 250: \"Taking all these information together, we speculated that Pm37 […] was originally derived from T. monocuccm\nand transferred into bread wheat through the bridge species T. timopheevii\"\nI do not see the results supporting this conclusion. There are some T. monococcum with and some without Pm37, so it could\nhave been inherited from a cross with timopheevii, no?\n# DISCUSSION\n- \"Compared to the susceptible allele in IWGSC RefSeqv2.1, an approximately 4000 bp fragment insertion was present on\nthe second intron of NLR-1 in NC99BGTAG11.\"\nThis is a result not mentioned in the Results section. This is exactly what I have commented above.\n# METHODS\n- \"Putative genes were annotated with the JBrowse pipeline (http://202.194.139.32/jbrowse.html)\"\nJBrowse is a viewer, not a pipeline for annotating genes. Suggest to clarify.\n- \"All the sequencing reads were assembled following the established procedure (11)\"\nReference 11 is not about HiFi sequence assembly. For bioinformatic analyses, I suggest to describe\ntools/methods/thresholds used instead of citing papers that have performed the same type of analyses.\n- Line 280: \"introduced segments\"\nGuess the authors mean \"introgressed\" no?\n- \"a 204-bp fragment of the gene was amplified and inserted\"\nFor reproducibility purposes, I think it is required to provide the sequence used here for silencing.\n- \"Sequence and haplotype analysis\"\nIn this paragraph, I do not read any information about sequence analysis or haplotype reconstruction. In contrast, the details\nof PCR reactions are given. Methods used for sequence analysis are expected for the results to be reproducible.\n- \"Phylogenetic analysis\"\nThe methods are not provided again. A tree was built based on a multiple alignment I guess, so methodological details are\nneeded here. The authors just mention that the tree was built using NJ method. However, all branches of the tree (Figure 5)\nhave the same length… it is likely a UPGMA tree, no? And this is also the case for the tree provided as a Supplemental\nFigure. If UPGMA have been used, I suggest to rebuild the tree with NJ. More importantly, bootstrap values are needed to be\nable to conclude on the phylogeny observed.\nTo me, there is a problem because I suspect that the multiple sequence alignment was computed based on sequences that\nsomehow do not share any similarity i.e., the 30 Pm, 16 Sr, 10 Lr, and 7 Yr proteins downloaded from NCBI are probably too\ndivergent to be aligned properly. Or, they were selected on purpose because all these proteins are homologous? In that\ncase, it must be indicated. But even homologs may contain domains that completely differ. Usually, what is aligned is just\ndomains that really align (or alignment is cleaned from misaligned positions) so that a tree could be calculated on an\nalignment that is correct to calculate genetic distances. So, it looks like what has been done is not scientifically sound. For\ninstance, STB6 is not a NBS-LRR so I doubt this could be aligned properly with the others. Since evolution of this gene is a\nmain message of the paper, this part requires significant improvement to be convincing.\n# FIGURES\n- Fig1: Panel e does not provide any added value to the written message. Suggest to delete or combine with panel d but I am\nafraid the two scales are too different.\n- Fig5: I doubt a multiple sequence alignment could be computed with such divergent sequences. If yes, this multiple\nalignment used for building the tree should be provided.\n- There is a confusion between gene IDs between Figures. Gene IDs in trees do not correspond to gene IDs in Fig1 (different\nversions). This should be fixed."}, "author_response": "ResponsetoReviewer#1\nIn this manuscript the authors report a comprehensive analysis of Pm37, a gene for resistance to wheat powdery\nmildew. They identified a Pm37 candidate through map-based cloning. The candidate gene was confirmed to be\nPm37 throughmutagenesis, barleystripemosaic virus-inducedgenesilencing, andstabletransformation.Amajor\ncontribution of this study is the finding that Pm37 is an allele of the stem rust resistance gene Sr22, and also\nconfers resistance to leaf rust. This is an example that diversification of a resistance gene has produced alleles\neffectiveagainstvariouspathogens.Themanuscriptiswell-organized,andtheresultsareconvincing.Therefore,I\nsuggestthatitmeritsforpublicationafterminorrevisionsdescribedbelow.\nResponse:Thanksforthereviewer’spositivecommentsonourwork.\n(1)Line86:Timopheevii(Zhuk.)Zhuk.\n=>T.timopheevi(Zhuk.)Zhuk.subsp.timopheevi\nResponse: Thank you foryour carefulsuggestion.Wehave revised the taxonomic name to T. timopheevi(Zhuk.)\nZhuk.subsp.timopheeviinthemanuscriptasrecommended.\n(2) Lines 86-89: “….were the ancestral species of common wheat, possessing abundant disease resistance genes,\nsuch as stem rust (Sr) resistance genes Sr36 and Sr37, stripe rust (Yr) resistance genes Yr10, and Pm genes Pm6,\nPm27andPm37.”\n=>Why has there been no report of resistance genes against leaf rust, one of the major wheat diseases? I am\nwonderingwhyresearchershavenotnoticedtheeffectofPm37againstleafrust?\nResponse: We greatly appreciate you raising this important issue, which allows us to improve the description of\nresistancegenesinT.timopheevii.WehaveaddedthedescriptionaboutLrgenes,suchasLr18(Yamamori,1994)\nand Lr50 (Brown-Guedira et al. 2003) which have been identified on chromosome 6G and 2G in T. timopheevi\n(Zhuk.) Zhuksubsp.timopheevi,respectively.RegardingtheeffectofPm37againstleafrust,ourinitialfocuswas\non powdery mildew resistance, and the leaf rust resistance was incidentally detected during extensive screening\nfor multiple diseases. We tested major diseases including stem rust, stripe rust, and leaf rust, and only observed\nspecificresponsestoleafrust.ThedonorandtransgeniclinesofPm37weretestedagainstsevendifferentvirulent\nPt races. The results showed that Pm37 had narrow-spectrum resistance to leaf rust and only provided moderate\nresistance to the Pt race THDS. As the Reviewer 2 pointed out, the level of leaf rust resistance holds limited\npractical significance in agricultural production. For this reason, the researchers could easily neglect its leaf rust\nresistance in production. So, the part of leaf rust resistance occupies only a minor portion of our study. The\nprimary significance of our work focus on cloning of the powdery mildew resistance gene Pm37 and its huge\nfunctional differentiation from Sr22. It is rare that allelic variation of NLR produces functional divergence to\ndifferent pathogens in wheat, also it is the first report about the functional divergence between powdery mildew\nresistance and stem rust resistance caused by allelic variation of NLR, highlighting a novel mechanism of\ndiversification within NLRs. Our findings lay a solid foundation for the molecular mechanism underlying\nfunctional divergence to different diseases, which opens a window for engineering novel NLR protein with more\nkinds of multiple diseases (even super resistance) resistance using site-saturation mutagenesis or precise genome\nediting.\n(3) Line 104: Supplementary Table 3 can be modified to help readers understand the position of Pm37\n(determinedbyphenotypes)quickly.Pleaserefertotheattachmentasanexample.\nIn Supplementary Table 3 and Fig. 1c, the same designation should be used for the types of recombinants. In the\nsecond column of the revised version of Supplementary Table 3 (attached), I noted corresponding types (R1-R6)\nwhichIspeculated.Pleasecheckthem.\nResponse: Sincerely thank you for your suggestion. The example you provided is extremely helpful. We have\nreorganizedSupplementaryTable3,unifyingthenomenclatureofrecombinanttypes tofullycorrespondwithFig.\n1c, making it easier for readers to quickly locate Pm37 through phenotypes. The revised table has significantly\nimprovedinlogicalclarity.\n(4) Lines 128-129: “The longest contig length of 202.2 Mb, and the total length of the genome assembly of 14.6\nGb,whichcoveredthewholegenomeofCSreferencegenome.”\n=>Pleaseaddaverbtothissentence.\nResponse: Thank you for pointing out this grammatical oversight. Wehave revised this sentence to “The longest\ncontig length was 202.2 Mb and the total length of the genome assembly was 14.6 Gb, covering the whole\ngenomeofCSreferencegenome.”\n(5) Lines 164-166:“T progenies from all these transgenic positive lines were immune to the Bgt isolate E09 and\n1\nadditional30BgtisolatesassameasNC99BGTAG11.”\n=>DideachT linecontainnosusceptibleseedlings?\ntheT families derived from selfing of 10T positive plants, 3 families showedsegregation (verified by PCR and\n1 0\nqRT-PCR). In the powdery mildew resistance assessment, we only selected positive seedlings for phenotypic\nevaluation, so the \"positive T progenies\" in the text all showed immunity, consistent with NC99BGTAG11. We\n1\nhaveclarifiedthisinformationintherevisedmanuscripttoensuretheaccuracyofthedescription.\n(6) Lines 202-204: “Notably, further disease assessment revealed that NC99BGTAG11 and NLR-1 transgenic\nlines also confers resistance to Puccinia triticina (Pt) race that causes leaf rust, while the transgenic receptor\nFielderissusceptible.”\n=>How (or why) did you pick up this pathogen (Pt) as a possible target of Pm37? Did you also test some other\npathogens?\nResponse: Thank you for pointing out this issue. Given that Pm37 is an allele of the stem rust resistance gene\nSr22, this study further evaluated the resistance profile of Pm37 against stem rust, leaf rust, and stripe rust. Our\nresults demonstrated that among the three diseases, Pm37 exhibited resistance exclusively to leaf rust, while\nshowingsusceptibilitytobothstemrustandstriperust.\n(7) HowmanyPtracesdidyoutest?Onlyone(THTT)?Ifyoutestedseveralraces,didyoufindracespecificity?\nYou may sure that this gene is different from Lr genes that have been identified to date. Please explain the basis\nforthisthought.\nResponse: Thank you for your valuable comments. In our originally submitted manuscript, we found Pm37 was\nresistant to the race THTT. However, the identification condition of this race is very complex due to winter\nholiday, and the overall low temperature conditions make the process difficult to replicate, thereby limiting its\npracticalvalueinagriculturalproduction.ToclarifytheleafrustresistanceofPm37,wespentagreatdealoftime\nto assess and repeat the resistance to different Pt races with different virulence under normal temperature, which\ncould truly reflect its breeding value in agricultural production. Seven prevalent Pt races were used to test Pm37,\nincluding THKS, PHJT, DGJT, PCTT, THTT, THDS and THTS. Pm37 was only moderately resistant to Pt race\nTHDS, reflecting race specificity and limited value in breeding against leaf rust.Additionally, the only reported\nleaf rust resistance gene on 7AL, Lr20, originates from hexaploid wheat and exhibits strong resistance, whereas\nPm37is derivedfromTriticum monococcumwithrelatively weakresistance,specificallyeffective onlyagainstPt\nraceTHDS. Meanwhile, Lr20 was located atthe Pm1 locus (Neuet al. 2002), whichis alsodifferentfrom Pm37.\nThesedifferencesinorigin,resistancelevelandlocusconfirmthatPm37isdistinctfromidentifiedLrgenes.\n(8) Lines 210-211: “Only CC domain of PM37 induced cell death (Supplementary Fig. 7). However,\noverexpression of different domains of SR22 in N. benthamiana leaves did not induce cell death whereas the\nfull-lengthSR22didcausecelldeath(SupplementaryFig.7).”\n=>The description of the results for SR22 (shown in Supplementary Fig. 7c) is reasonable. However, it seems\ndifficult to interpret the results for PM37 (shown in Supplementary Fig. 7a) as “Only CC domain of PM37\ninduced cell death”. For example, the lesion in Supplementary Fig. 7a-2 is very small and similar to those in\nSupplementaryFig.7c-2and4.\nResponse:Wesincerely appreciate your carefulattention to the results of cell death-inducing activity andcritical\ncomments on Supplementary Figure S7 (renamed Supplementary Fig. 5 in the revised version). To address this\nconcern and further reduce the potential impact of tags on the intrinsic function of PM37 and its domains, we\nredesigned the experiment: we removed the tag sequence from all constructs, including full-length PM37, its\nCC/NBS/LRR domains, and CC-NBS/NBS-LRR domain combinations, while retaining the MAS promoter for\ntransient overexpression in Nicotiana benthamiana leaves. Results from three independent biological repetitions\nconsistentlyshowedthatfull-lengthPM37inducedobviousandstablecelldeathlesions;theCCdomainofPM37\nalone triggered clear cell death, with lesion size and necrosis severity significantly increased compared to the\ninitialtaggedexperiment;andtheCC-NBS domaincombinationofPM37alsoinducedcelldeath;thesingleNBS\ndomain,singleLRRdomain,andNBS-LRRdomaincombinationofPM37stilldidnotinduceanyobservablecell\ndeath.Theserevisedresults,nowpresentedinSupplementaryFig.5,clearlyconfirmthattheCCdomainofPM37\nacts as the key module mediating cell death induction. This observation aligns with the functional characteristics\nof several previously reported NLR proteins, such as PM12 and PM21, where the CC domain also serves as the\ncoremodulefortriggeringcelldeathinN.benthamiana.\n(8) SupplementaryTable 7,8, 9:Please checkthetable numbers in the files for SupplementaryTable 7, 8,and9.\n(TheyaredescribedasTable8,9,and10,respectively.)\nResponse: We apologize for the oversight of inconsistent numbering. We have comprehensively checked and\ncorrected the numbering of supplementary tables to ensure that all the Supplementary Tables fully correspond\nwiththefiles,avoidingconfusionforreaders.\n(9)L248:Eventhrough\n=>Eventhough?\nResponse: Thank you for pointing out this typo. We have corrected \"Even through\" to \"Even though\" and\nrecheckedthefulltextforsimilarspellingissuestoensureaccuracy.\n(9) L317:Rgm8=>Rmg8\nResponse: Thank you for finding this spelling error. We have corrected \"Rgm8\" to the correct \"Rmg8\" and\ncheckedthespellingofallgenenamestoavoidsimilarmistakes.\n(11)L384:Wuhautouat0,……..72hpiinoculatingwithBgtisolateE09\n=>WuhautouinoculatedwithBgtisolateE09at0,……..72hpi\nResponse:Thanksforyourrevision.WehavereviseditaccordinglyintheMS.\n(12) L677 (Fig. 6): It may be better to divide Fig. 6 into three parts, i.e. Fig. 6a (stem rust), Fig. 6b (powdery\nmildew),andFig.6c(leafrust).\nResponse: Thank youfor your valuable suggestion.Wehave splitFig. 6 into three subfigures (Fig. 6a:stem rust;\nFig. 6b:powderymildew;Fig. 6c:leafrust), makingthepresentationofresults foreachdiseaseclearerandeasier\nforreaderstounderstand.\nResponse to Reviewer #2The manuscript reports isolation of the Pm37 resistance gene from NC99BGTAG11.\nAuthors made good use of standard genomic tools for fine-mapping to isolate and verify the gene sequence\nunderlying resistance gene, including long read sequencing of the resistant line NC99BGTAG11. They validate\nisolation of the Pm37 resistance gene through identification of EMS mutants, VIGS of Pm37 and stable\ntransgenics.TheydemonstratethatPm37confersresistancetoalarge collectionofpowderymildew isolatesfrom\ndifferent parts of China. They also determined that NC99BGTAG11 and Fielder transgenics having Pm37 are\nresistant to leaf rust isolates from China. They explore homology of Pm37 derived in this case from tetraploid T.\ntimopheevii subsp. armeniacum (AtAtGG) with the Sr22 gene that was transferred to common wheat from an\nA-genome diploid. They identify two accessions of T. monococcum having an identical Pm37 coding sequence,\nincluding TA299 for which a genome sequence is available. They also identified an accession of T. timopheevii\n(subsp.armeniacum?)havingPm37.\nMethodology andpresentationofcloningresults areappropriate.Theworkisrobustandsignificantinidentifying\nanalleleofSr22thatconfersresistancetopowderymildewandaswellasaleafrust.\nResponse:Thanksforthereviewer’spositivecommentsonourwork.\n1. The most original and significant aspect of the paper is the determination that Pm37 is an allele of stem rust\nresistance gene Sr22 presentin both a diploid and tetraploid wheat relative, and that this allele confers resistance\nto powdery mildew and leaf rust (but not to stem rust). This result is broadly interesting to wheat researchers as\nwell as those working in other crop systems. However, there are areas where statements about global utility of\nPm37conferredresistancemaybeoverstated(seeinformationbelow).\nResponse:Thanks foryoursuggestion.WehaverevisedthemaccordinglyintheMS.See thedetailedrepliesand\nrevisionsbelow.\n2. Overall,thepresentationofthemanuscriptsuffersfrominstancesofpoorEnglishgrammarandlanguageusage.\nItcouldbeimprovedbyprofessionalcopyediting.\nResponse:ThanksforyourvaluablecommentregardingtheEnglishlanguage.Themanuscripthasbeencarefully\nrevised by a native English-speaking colleague to improve the clarity, grammar, and overall fluency. We also\nthoroughlycheckedthewholetexttoensureallterminologyandphrasingalignwithacademicstandards.\n3. AccesstoNC99BGTAG11longreadsequenceandassembly arenecessarytoreproducesomeresults.Theydo\nnotappeartobeshared.\nResponse: Thank you for highlighting the importance of data accessibility for reproducibility. The contig\nsequence harboring Pm37 has been deposited in the National Center for Biotechnology Information (NCBI)\ndatabase under accession number SRR34967779. These data are publicly available, and the accession number is\nspecifiedintherevisedmanuscripttofacilitatereproductionofourresults.\n4. Forallfigureshavingerrorbars(Figs.2,4,S1,andS5),theerrorbarsarenotdefinedinthefigurelegends.\nResponse:Thankyoufornotingthisoversight.Wehaveaddedexplicitdescriptionsoftheerrorbars,inthefigure\nlegendsintherevisedmanuscript,ensuringreaderscanclearlyinterpretthedatavariability.\n5. Thepresentation ofwork in Figure S7 is somewhat outof the scopeof my expertise. However,the conclusion\nthatoverexpressionoftheCCdomainofPm37inducedcelldeathdoesnotseemsupportedbyFigureS7a.\nResponse:Wesincerely appreciate your carefulattention to the results of cell death-inducing activity andcritical\ncomments on Supplementary Figure S7 (renamed Supplementary Fig. 5 in the revised version). To address this\nconcern and further reduce the potential impact of tags on the intrinsic function of PM37 and its domains, we\nredesigned the experiment: we removed the tag sequence from all constructs, including full-length PM37, its\nCC/NBS/LRR domains, and CC-NBS/NBS-LRR domain combinations, while retaining the MAS promoter for\ntransient overexpression in Nicotiana benthamiana leaves. Results from three independent biological repetitions\nconsistentlyshowedthatfull-lengthPM37inducedobviousandstablecelldeathlesions;theCCdomainofPM37\nalone triggered clear cell death, with lesion size and necrosis severity significantly increased compared to the\ninitialtaggedexperiment;andtheCC-NBS domaincombinationofPM37alsoinducedcelldeath;thesingleNBS\ndomain,singleLRRdomain,andNBS-LRRdomaincombinationofPM37stilldidnotinduceanyobservablecell\ndeath.Theserevisedresults,nowpresentedinSupplementaryFig.5,clearlyconfirmthattheCCdomainofPM37\nacts as the key module mediating cell death induction. This observation aligns with the functional characteristics\nof several previously reported NLR proteins, such as PM12 and PM21, where the CC domain also serves as the\ncoremodulefortriggeringcelldeathinN.benthamiana\n6. WerePm37lines evaluated for resistance to yellow rust? If so, itmay beinteresting to reportareaction to this\nrustpathogenaswell.\nResponse:Thanksforthisinsightfulsuggestion.Todothis,wecollaboratedwiththeInstituteofPlantProtection,\nChinese Academy of Agricultural Sciences, to evaluate the resistance of Pm37 against 14 virulent races of\nPuccinia striiformis f. sp. tritici (Pst). Results showed high susceptibility (infection types 3-4) to all the tested\nraces.ThesephenotypicdatahavebeenaddedtoSupplementaryTable7intherevisedmanuscript.\n7. Figure S2-The figure legend shouldincludethesourceofeinkron andtimopheeviiassemblies (TA299andPI\n94760, respectively) to which NC99BGTAG11 reads were mapped. It is interesting that there were more regions\n(beyond the terminal portion of 7AL) where higher read depths were observed between NC99BGTAG11 with\nwild einkorn TA299 than with PI 94760. While this may be because PI 94760 is not the donor of Pm37, the\noverall pattern makes one wonder about the pedigree of NC99BGTAG11. I would expect more regions of\nsimilarity with T. timopheevii in a BC2-derived line, and fewer with T. monococcum. Additionally, Fig 1e\nindicates that the Pm37 region in NC99BGTAG11 is more like TA299 than PI94760. Lines 247 -252 note the\nPm37 was derived from T. monococcum via the bridge species T. timopheevii. The authors did find evidence of\nPm37 in the accession ofAAGG477 of T. timopheevii (is this an accession of subspecies armeniacum? or subsp\ntimpopheevii?).\nResponse: Thanks for your in-depth insights and professional comments. We have added the description of the\nsourceofeinkronandtimopheeviiassemblies(TA299andPI94760,respectively)towhichNC99BGTAG11reads\nwere mapped in Figure S2 (Figure 6a&6b in the revised version) legend.Additionally, we fully agree with your\ninsightful comments and suggestions. Multiple genomic regions (extending beyond the terminal portion of 7AL)\nshowed higher read depth similarity between NC99BGTAG11 and wild einkorn TA299 compared to PI 94760.\nThe reference genome of T. timopheevii PI 94760 is indeed not the donor of Pm37, the true pedigree of\nNC99BGTAG11 is T. timopheevii PI 427315 and soft red wheat Saluda. In our study, De novo genome assembly\nof NC99BGTAG11 was used to isolate the target gene of Pm37. Then, the evolutionary origin of Pm37 was\nfurther analyzed byleveraging theresequencingdatacombined with haplotypeanalysis. In future,we willfurther\nanalyze genomic introgressions in BC -derived line.AccessionAAGG477 is T. timopheevi (Zhuk.) Zhuk. subsp.\n2\ntimopheevi.withitsCIMMYTaccessionnumberCWI17281andUSDAPlantIntroductionnumberPI306557.\n8. Seed of PI 427315, the T.timopheevii subsp. armeniacum parentof the NC99BGTAG11, is available from the\nUSDANationalSmallGrains collection.InordertoverifythatthePm37wasindeedderived fromT.timopheevii,\ntheauthorscouldincludeaccessionPI427315intheiranalysisofallelicvariation.\nResponse: Thanksfor yoursuggestion.Wehavetried ourbestto obtaintheseedof PI 427315,the T.timopheevii\nsubsp. armeniacum parent of the NC99BGTAG11. However, as a key agricultural germplasm resource, plant\nseeds fall under China's Catalog of Prohibited and Restricted Import Plant Species. Their import is restricted to\nauthorized research institutions with special permits. Currently, our organization does not hold the required\nAgricultural GMO Safety Certificate for this purpose. Meanwhile, seed exports from USDA to other countries\nalsoisalsosubjecttostrictcontrol.Itisnotpossibletocompleterelatedprocedureswithinashortperiodoftime.\n9. In Figs. 5 and S8, branch lengths appear to be set as equal. Therefore, it would be best to label these as\ncladograms in the figure legends. It would be interesting to see these presented as phylograms with bar length\nindicating sequence similarity. Rather than comparing with several cloned R genes belonging to other clades in\nFigure 5, it may be more interesting to compare Pm37 with the Sr22 alleles/homologs present in A-genome\ndiploidsandreportedintheAsyrafetal.2020.Attheleast,thispapershouldbeincludedinthediscussion.\nExtensive Genetic Variation at the Sr22 Wheat Stem Rust Resistance Gene Locus in the Grasses Revealed\nThrough Evolutionary Genomics and Functional Analyses. Molecular Plant-Microbe Interactions 2020 33:11,\n1286-1298\nResponse: Thanks for your valuable suggestion regarding the phylogenetic presentations in Figs. 5 and S8 (Fig.\n5a&6cintherevisedversion).Wehaverevisedbothfigurestoaddressyourcomments:bothFigs.5andS8(Fig.\n5a & 6c in the revised version) are now presented as phylograms with branch lengths explicitly indicating\nevolutionary distances (average number of amino acid substitutions per site), scale bars have been added to\nquantify these distances, and bootstrap support values (from 1000 replicates) have been included at each node to\nreflectthereliabilityofbranchingpatterns,enhancingtherobustnessoftheevolutionaryrelationshipsdepicted.In\nline with your recommendation, we have focused additional comparative analysis on Pm37 and Sr22\nalleles/homologs from A-genome diploids, as reported in Asyraf et al. (2020), which reveals that Pm37 clusters\ncloselywithSr22homologsfromTriticummonococcumbutcontainsdistinctsubstitutionsintheLRRdomainthat\ncorrelate with its functional divergence from Sr22 (specifically, resistance to powdery mildew rather than stem\nrust); we have incorporated this discussion in the revised manuscript and cited Asyraf et al. (2020) to\ncontextualize the evolutionary dynamics of the Sr22/Pm37 locus. The revised legend for Fig. S8 (Fig. 6c in the\nrevised version) clearly describes the phylogenetic methodology, including the JTT substitution model and\nbootstrapvalidation,ensuringtransparencyandreproducibility.\n10. It should be mentioned in the manuscript that the sample of powdery mildew and leaf rust isolates evaluated\nwerenotglobal.Kloppeetal.2022(doi:10.3389/fpls.2022.954958)reportedPm37virulencein9and10%ofBgt\nisolatesevaluatedfromEgyptandTurkey,respectively,aswellas1and2%ofisolatesfromtheUnitedStatesand\nRussia/Kazakhstan. They conclude that the higher levels of virulence observed in Turkey to Pm37 (and other\nnewly introgressed Pm genes) may be related to the presence of donor species in the region as the gene has not\nbeenusedcommercially.Itwouldbeusefultomentionthisworkinthemanuscript.\nResponse: Thanks for this valuable information. In this study, the Bgt isolates and Pt races were collected from\ndifferent wheat producing regions in China, and were used to evaluate the resistance of wheat entries against\npowderymildewandleafrust,respectively.ThedonorparentandtransferringprogeniesofPm37wereresistantto\nChina.As stated in the references you provided, some studies showed that Pm37 was susceptible to 9% and 10%\nof Bgt isolates evaluated from Egypt andTurkey, respectively, as well as 1% and 2% of isolates from the United\nStatesandRussia/Kazakhstan.Itmayberelatedtothevariationoflocalvirulentisolatesindifferentcountriesand\nregions.Toprovideamoreobjectivedescriptionoftheeffectivenessagainstpowderymildew,wehaverevisedthe\ncorresponding section in the revised manuscript: confined the resistance to 108 isolates in China and have also\nadded the resistance frequency information in other countries in Discussion part.This information could help the\nreadersandbreedersobjectivelyevaluateitsbreedingvalueagainstpowderymildew.\n11. TheNC99BGTAG11germplasmwasgrowninthefieldovermultipleyearsintheUSstateofNorthCarolina.\nIn atleastthree years where weexperienced naturalleaf rustepidemics, high levels ofleaf rustwere observed on\nrecurrent parent Saluda and NC99BGTAG11.These data are not reported in literature, so would notbe known to\nthe authors. But this indicates that the Pm37 mediated leaf rust resistance reported in the manuscript is not\nexpectedtobegloballyeffective,evenintheabsenceofcommercialuseofthegene.\nResponse: Thanks for your valuable information about the Saluda and NC99BGTAG11 against leaf rust in US\nstate of North Carolina. To objectively clarify the leaf rust resistance of Pm37 under agricultural production\ncondition, we spent a great deal of time to assess and repeat the resistance to different Pt races with different\nvirulence under normal production temperature, which could truly reflect its breeding value in production. Seven\nprevalent Pt races were used to test NC99BGTAG11, Pm37 transgenesis lines and transgenesis receptor Fielder,\nincludingracesTHKS, PHJT,DGJT,PCTT,THTT,THDS andTHTS. Pm37 was onlymoderately resistantto the\nPt race THDS, reflecting race specificity and limited value in breeding against leaf rust. This scenario bears a\ncertain resemblance to the situation observed in the United States. So, we fully agree that this level of resistance\nholds limited practical significance in agricultural production as you pointed out.As for the leaf rust resistance,\nwe suspect that it may be functional redundancy caused by NLR variation, which easily overlooked by the high\nlevel of powdery mildew resistance. Theoretically, NLR variations produced functional differentiation from stem\nrust to powdery mildew and may also to leaf rust. So, leaf rust resistance was found by accident, and only\noccupies a minor portion of our study. The primary significance of our work focus on cloning of the powdery\nmildew resistance gene Pm37 and its functional differentiation from Sr22. It is rare that allelic variation of NLR\nproducesfunctionaldivergencetodifferentpathogens,especiallybetweenmildewandrustinwheat,whichisalso\nthe first report about the functional divergence between powdery mildew resistance and stem rust resistance\ncaused by allelic variation of NLR, highlighting a novel mechanism of diversification within NLRs and\nbroadening our understanding on NLR. So, our findings lay a solid foundation for the molecular mechanism\nunderlying functionaldivergence to differentdiseases, which opens a window for engineering novel NLR protein\nwith multiple diseases (even super resistance) resistance using site-saturation mutagenesis or precise genome\nediting.\n12. There are a number of places in the manuscript where the writing could be improved. Also, please see\ncommentsbelow.\nLine86–T.timopheevii(genomedesignationsAtAtGG)anditswild speciesarmeniacumarenotdirectancestors\nof T.aestivum (AABBDD); they are related species.The ‘sister’species of T.timopheeviiis T.turgidum (AABB)\nand it is the ancestor of hexaploid wheat. It may be useful to clarify the relationship of T. timopheevii and T.\nmonococcumtoeachotherandwithT.aestivum,perhapsasadiagraminthesupplementalmaterial.\nResponse: Thanks very much for your valuable suggestion. We fully agree that clarifying the relationships\nbetweenT.timopheevii,T.monococcum,andT.aestivumiscrucialforenhancingtheclarityofthemanuscript.As\nrecommended, we have supplemented a diagram (Supplementary Fig. 8) in the revised manuscript to visually\nillustratetheserelationships,makingtheevolutionarycontextmoreintuitiveandaccessible.\n13. Line 89 - Yr10 is not a T. timopheevii derived gene – from T. spelta and T. vavilovi (both are AABBDD\ngenomesub-species)\nResponse: Thanks for your critical correctionregarding theorigin of Yr10. Uponrevisiting the relevantliterature\nand genomic evidence, we confirm that Yr10 is not derived from T. timopheevii but is associated with T. spelta\nandT.vavilovi.Intherevisedmanuscript,wehaverevisedthisinformation.\n14. Line 92 – “ ultra-broad-spectrum resistance for more than a decade”. This statement is misleading as Pm37\nhasnotbeendeployedincommercialvarieties.\nResponse: Thanks for pointing out this misleading statement. In the revised version, we have revised this\nstatement as follows: NC99BGTAG11 exhibited disease resistance against all 108 Bgt isolates of powdery\nmildewcollectedinChina,indicatingthatPm37isavaluablegeneticresourceforwheatimprovement.\n15. Line 117 - In figure S1, only TraesCS7A12127000 (NLR-1) has significantly higher expression in the\nresistantline-thisisnotmentionedinthetext.\nResponse: Thanks for your comment on Figure S1.Wehave revised it in the manuscriptto explicitly include the\nresultregardingtheexpressionofTraesCS7A03G1212700(NLR-1).Specifically,weaddedthefollowingcontent:\nNotably, among the three candidates, only TraesCS7A03G1212700 (NLR-1) exhibited higher expression\nlevels in the resistant parent NC99BGTAG11 than in the susceptible parent Wuhuatou across multiple\npost-inoculation time points, whereasthe expressionof TraesCS7A03G1212600 and TraesCS7A03G1212800\ndid not significantly differ between these two genotypes. This ensures alignment between the text and Figure\nS1.\n16. Line 170-”...108 BGT isolates collected from different years and regions…” should specify that all isolates\nwerecollectedfromChina.\nResponse:Weappreciateyourattentiontoprecision.Thetexthasbeenrevisedtospecifythatthe108Bgtisolates\nwerecollectedfromvariousregionsinChina,ensuringclarityaboutthescopeofourresistanceevaluations.\n17. Line174-175 -As noted above, the statement about “ultra-broad spectrum powdery mildew resistance at the\nwholegrowthperiod”shouldbequalifiedasPm37wasnotdeployedintheregionoverthisperiod.\nResponse: Thanks for your valuable suggestion regarding the statement about \"ultra-broad spectrum powdery\nmildewresistanceatthewholegrowthperiod\".Wefullyagreethatthisdescriptionrequiresqualification,asPm37\nwas not deployed in the region during the evaluation period. To address this, we have removed the original\nstatementin the revised manuscriptto preventover generalization. Instead, wenow focusonpresentingobjective\nexperimentalresults, specifying thatNC99BGTAG11andits transgeniclines exhibitedresistanceto alltested Bgt\nisolates at both seedling and adult plant stages in our evaluations. This revision ensures our description aligns\nstrictlywiththedata,enhancingtheaccuracyandrigorofourfindings.\n18. Lines 268- 272 - This sentence suggests that the authors consider T. timopheevii to be in the secondary gene\npoolof wheat, butwould notbe awheatrelative where introgression wouldbeassociated with linkage drag? It is\ninteresting that recombination rates were quite good in the Pm37 region. This differs somewhat from the Sr22\ntransfer where suppressed recombination was reported and initial introgression lines were not used due to poor\nagronomicperformance(linkagedrag).\nResponse: Thanks for your insightful comment. This is indeed an interesting question. As for the suppressed\nrecombination between chromosomes 7Aof T. monococcum and common wheat, we found that 7Achromosome\nof T. monococcum was not completely suppressed recombination, and the levels of recombination was also\ndifferent among different generations of populations and different cross combinations based on the reported\ninformation (Olson et al. Development of wheat lines having a small introgressed segment carrying stem rust\nresistance gene Sr22. Crop Science. 2010, 50:1823-1830). In NC99BGTAG11, introgression segment of\nchromosome7AofT.monococcumisverysmall, andthischromosomalregionmighthavenormalrecombination\nwith common wheat. The small introgression segment itself also indicated that recombination could occur\nbetween chromosomes 7Aof T. monococcum andT. aestivum. In addition, the genetic backgroundofthe softred\nwinterwheatSaludamaybeeasilyrecombinedwithT.monococcumthanothergeneticbackground.Infact,inour\nprocess of fine mapping, we also developed other segregation populations with different susceptible parents, and\nsuppressedrecombinationwasalsonotobservedinthesesegregationpopulations.\nAs forthe linkage drag, the introgression segmentof T. monococcumis small andmayhave nolinkage drag\nin this introgression segment, while the Sr22 introgression lines often have large introgression segment, even the\nwholelongarmofchromosome7AofT.monococcum,whichcarrieslinkagedrag.\n19. Lines 275-277: I am not sure what the authors mean here. I don’t think that this work provides a new\n“framework”forusingresistancegenesfromwildrelativesorisolationofresistancegenes.\nResponse:Thanksforyourvaluablecomment.WeagreewithyouropinionandhaverevisedLines275-277inthe\nmanuscript to avoid overstating the contribution. The original text to providing a \"new framework\" has been\nadjusted to clarify thatour studydemonstrates the utility of integrating map-basedcloning, long-read sequencing,\nandfunctionalvalidation for isolating resistance genes from secondary gene pools,andhighlights the potentialof\nsuch genes in breeding. Specifically,the revised text now states: \"Our findings not only highlight the utility of\nPm37in wheat breeding programs, but alsounderscoreits advantagesas aresistanceresource, making it a\nvaluable asset forenhancing disease resistance in wheat improvement efforts.\"This revision more accurately\nreflectspracticalinsightsratherthanclaiminganewframework.\nResponsetoReviewer#3Ico-reviewedthismanuscriptwithoneofthereviewerswhoprovidedthelistedreports.\nThisispartoftheNatureCommunicationsinitiativetofacilitatetraininginpeerreviewandtoprovideappropriate\nrecognitionforEarlyCareerResearcherswhoco-reviewmanuscripts.\nResponse: We sincerely appreciate your insightful comments and constructive suggestions, which have\nsignificantly improved the quality of our manuscript. We have revised all of them item by item based on your\ncommentsandsuggestions.\nResponse to Reviewer #4Review on “Jin et al., Pm37, a susceptible allele of Sr22 from Triticum monococcum,\nconfersresistancetopowderymildewandleafrustincommonwheat\"\nThearticlefromJinandcolleaguesdescribesthecloningofPm37inaresistantwheatcultivar(abbreviatedNC99\nhere in my report) that derives from a cross between a winter wheat and a T. timopheevii accession carrying this\ngene. The paper describes the fine mapping steps leading to a very small genetic interval and all the supporting\nevidence that the candidate NLR present in the interval is actually the gene controlling the resistance phenotype\ni.e., production of EMS mutants, transformation in Fielder, overexpression of the protein with recombined\ndomains. In addition, the paper also describes sequence analyses performed in order to understand the\ndiversity/evolutionofthegeneandthelocus.\nTheresults aboutfinemappingandfunctionalvalidationare interesting, wellperformed,scientifically sound,and\nconvincing. In contrast, the sequence analyses and evolutionary interpretations are questionable. Since this later\npoint is my area of expertise, I will focus my comments/remarks on these aspects which, I think, require\nsignificant improvements to be understood, first, and convincing. I have main concerns about the methods used\nforsequenceanalysesandphylogeny.Pleaseseemyremarksbelow.\nResponse:Thanksforthereviewer’spositivecommentsonourwork.\n##POINT-BY-POINTCOMMENTS\n1. Iguessthereisageneralizedmistakewithwritingthe\"monococcum\"speciesnamehere.Atseveralplaces,the\nauthorswrote\"monocuccm\"oreven\"monocuccum\".Itrequirescorrectionsallalongthetextandespeciallyinthe\nTitle.\nResponse: Thanks for pointing out spelling error of \"monococcum\" (previously miswritten as \"monocuccm\" or\n\"monocuccum\"). We have carefully corrected this mistake throughout the entire manuscript, and conducted a\nthoroughchecktoensurenofurtherinstancesremain.\n#ABSTRACT\n2. -\"Pm37issusceptibletostemrust,andSr22issusceptibletopowderymildew\".\nAgene(oraprotein)isnotsusceptiblenorresistanttoadisease.Aplantis.Thewritingneedssomeseriousedits.\nResponse: Thanks for pointing out this issue. We have edited the sentence as follows:Yet the carrier of Pm37 is\nsusceptibletostemrust,andthecarrierofSr22issusceptibletopowderymildew.\n3. -\"Pm37andSr22producedfunctionaldivergencethatonlyoverexpressionoftheCCdomainofPM37andthe\nfulllengthSR22couldtriggercell-deathactivity.\"\nIreadthesentenceseveraltimesbutcannotmakeanysenseofit.\nResponse: Thank you for pointing this issue.Wehave revised it as follows: The cell death-inducing activity of\nPM37inNicotianabenthamianaismainlymediatedbyitsCCdomain.\n#INTRODUCTION\n4. -Line91:AreferenceisneededwhenpresentingNC99.\nResponse: Thanks for your suggestion. We have added the reference (23: Perugini et al. 2008) when presenting\nNC99.\n#RESULTS\n5. +Map-basedcloningofPm37\n-\"comparisonofthetargetinterval[..]indicatedthatnonewgenewaspresent(Fig.1d).\"\nThis is clearlynotsupportedbywhatis shownatFig 1d.Ifoundthis paneldrequiressignificantimprovementso\nthat the reader is able to understand the Figure by itself.As far as I get it, there are several nonsyntenic genes in\nthe genomes compared here, no?I see some genes related with \"links\" and some without link, so I conclude they\nare non-conserved. In addition, it is not obvious to me that the genomic segments compared here are the\nsyntenic/orthologous ones. Since the segments are not flanked by orthologous/collinear genes in all genomes\ncompared, it raises questions. For instance, gene 03G1214100 (CS) could be just next to 168350 (monococcum)\nbut not represented in the segment represented here. Such comparative genomics picture is not clear if the\nsegments are not bordered by exact collinear genes. To me, it is not possible to conclude about gene\npresence/absencebasedontheworkperformedhere.\nResponse: Thanks for pointing out this problem. This phenomenon is caused by the removing of low confidence\ngene set of reference genomes of common wheat Chinese Spring (CS) and monococcum TA299. We filtered out\nlow-confidence genes from the T. aestivum (CS) and T. monococcum (TA299) gene annotations prior to the\nanalysis. Their absence from the initial comparison broke the collinearity at the flanks of the syntenic block. We\nhave added back those two low confidence genes, and now the orthologous/collinear relationship is much clear.\nThe two low confidence genes are: Tm.TA299.r1.7AG0168360 (TA299), which is orthologous to\nTraesCS7A03G1214100 (CS); TraesCS7A03G1214000LC (CS), which is orthologous to\nTm.TA299.r1.7AG0168350(TA299).\nRegarding theexclusion of low-confidencegenes inFigure 1d:this choicewasmadeto prioritize clarity and\navoid visual clutter, as including all low-confidence genes would have obscured the core comparison of the\ncandidate gene interval. Werecognize, however,that this exclusion led to ambiguity in syntenic boundaries (e.g.,\nthe missing link between TraesCS7A03G1214100 (CS) and Tm.TA299.r1.7AG0168360 (TA299)). In the current\ngraph,ifoneofthetwocollineargenes(fromthetwogenomes)isahigh-confidencegene,weretainthatgene.\n6. +DenovogenomeassemblyofNC99BGTAG11andcandidategeneisolation\n- \"Sequence alignment revealed a mass of SNPs and InDels between NC99BGTAG11 and Wuhuatou\n(SupplementaryTable4).\"\nFirst,thesupplementalfiguresandtablesarenotnumbered,soitisnoteasytofollow.\nSecond, Sup Table 4 is cited to support the fact that the two alleles differ a lot. This is a table with 1 line per\nSNP…Abasic sequence alignment would probably have been much more informative.And in the text, I suggest\nto just give the numbers of variations/length or percent identity, which would be much more informative than \"a\nmass\".-SupplementalFig3ismisleadingwiththescalenotrespected.Itmustbecorrected.\nResponse: Thanks for your valuable comments on the presentation of sequence variations and supplementary\nmaterials. We have carefully addressed each issue as follows: firstly, all supplementary figures and tables have\nbeenuniformly numbered in the revised manuscriptto ensureeasy cross-referencing; secondly,to better illustrate\nthe sequence differences between NC99BGTAG11 and Wuhuatou, we have replaced the original Supplementary\nTable 4 with a sequence alignment figure (now Supplementary Fig. 2), which clearly shows 287 SNPs and 88\nIndelsbetweenthetwogenotypes.\nRegarding Supplementary Fig. 3 the scale issue is indeed misleading. Due to the exceptionally long intron\n(5,996 bp in Pm37), maintaining a uniform scale for the entire gene would distort the visualization of exonic\nregions and compromise the overall clarity. To resolve this issue, we have modified the figure to represent the\nintron with a dashed line, explicitly labeled with its actual length. This approach retains the accuracy of length\ninformationwhileensuringtheexonicregionsareclearlyvisibleandthefigureremainsvisuallycoherent.\n7. Itappearsthatthereisa6kbintron,whichissomehowunusualintheseTriticeaegenomes.InCS,thisgeneis\ndifferentbecausethisintronisabout2.8kb.Whynothavingshownthesevariations?IsthereaTEinsertedwhich\nwouldexplain this difference?Whataboutmonococcumandtimopheevii?This variation couldbehighly valuable\ntohelpwithreconstructinghaplotypesandtracetheoriginofthegene.\nResponse: Thanks very much for your professionalinsights.Wehave addedthe variations of NLR-1 betweenCS\nand NC99BGTAG11 in Figure S3. According to your suggestion, we employed the RepeatMasker (v4.0.5)\nsoftwaretoannotateintronsequencestoinvestigatethepotentialpresenceoftransposableelement(TE)insertions\nwithintheintronicregions.However,ouranalysisrevealednoevidenceofTEinsertionsintheintron.Inaddition,\nthrough homologous sequence alignment of monococcum and timopheevii, we isolated the identical genomic\nsequenceofPm37inT.monococcumTA299.InadditiontoanSNPdifferencebetweensecondintron,thegenomic\nsequence of T.monococcumTA10622 and T.monococcum PI306540 are completely identical, and have SNPs and\nInDels with Pm37 in both the CDS region and the intron region. Reference genome T. timopheevii PI94760\nshowed great differences with Pm37 in CDS and second intron, and second intron showed great variation in\nT.monococcumandT.timopheeviiPI94760.\n8. Since this gene is almost 9 kb in NC99 versus 6 kb in CS, it is not surprising that all attempts to get a\nfull-lengthampliconsystematicallyfailed.Thiscouldbeaddedasaprobableexplanation.\nResponse: Thanks for this insightful suggestion. We fully agree that the huge length difference between the two\nalleles is a convincing reason for the initial failure in amplifying the full-length sequence.As recommended, we\nhaveaddedthisexplanationintheDiscussionsectionasfollows:\"Thisfailureislikelyattributabletoamarked\nlengthpolymorphism:theNLR-1locusinNC99BGTAG11spans9,012bp,incontrasttothe5,918bplength\nof its counterpart in the Chinese Spring reference genome.\" This insight helps us clarify the technical\nchallengeencounteredduringtheexperiment.\n9. +ValidationofNLR-1byloss-of-functionmutants\n-\"we[…]obtainedfiveindependenthomozygousmutantlines\".\nGuesstheauthorsmeanfiveindependent\"susceptible\"homozygousmutantlines.\n-Resultsareconvincing.Fivesusceptiblemutants,allwith1deleteriousmutation,perfectlyclear.\nResponse: Thank you very much for your careful review and valuable suggestion. As you suggested, we have\nrevised the corresponding sentence in the manuscript to \"we […] obtained five independent susceptible\nhomozygousmutantlines(Fig.2a)\"toexplicitlyindicatethephenotypiccharacteristicofthesemutantlines.\n10. +SilencingofNLR-1eliminatedpowderymildewresistanceofNC99BGTAG11\nResults areconvincing.Thedescriptionoftheworkperformed is,nevertheless,quiteexpeditive.NLRs arehighly\nrepeated in terms of copy number and silencing 1 single copy is probably not that trivial. I found we lack some\ndetails about this particular NLR, how many copies share similarity etc. what about homeologous copies? And\nwas it obvious to design probes that specifically target this copy? I suggest to give some more details about that.\nAlthough, thepreviouspartwithfinemapping+5independentEMSmutantsis alreadyquiteconvincingthatthis\ncandidateisthecausalgene.\nResponse: Thanks foryour approvalandvaluable comments onthis part.Wefully agree with the suggestion that\nadditionaldetailsontheNLRgenesilencingexperimentneedtobesupplemented.Giventhecharacteristicsofthe\nNLR gene family, such as high copy numbers and abundant homologous sequences, to ensure the specificity of\nsilencing, we firstly conducted a comprehensive homology alignment analysis between NLR-1 and other\nhomologoussequencesintheNC99BGTAG11genome,includingcopiesonhomeologouschromosomes.Through\nthe alignment, a 245 bp sequence with high specificity in the CC domain of this gene was identified. So, we\nselected this 245 bp specific sequence as the target sequence for gene silencing to design specific probes. This\ndesign ensured that the silencing experiment could specifically target the NLR-1 copy without significantly\naffecting other homologous or paralogous sequences. We have incorporated the aforementioned details into the\nrevisedmanuscripttomoreclearlyelucidatethespecificdesignrationaleoftheNLR-1silencingexperiment.\n11. +StabletransformationofNLR-1producedhighlevelofpowderymildewresistance\nAgain, expeditive. It would have been interesting, at least, to describe the status of this NLR-1 in the Fielder\ngenomesinceanassembledsequenceisavailable.\nResponse: Thanks for your approval and valuable comments. We appreciate your insight regarding the need to\nelaborate on the status of NLR-1 (Pm37) in the Fielder genome. Firstly, as a well-known powdery\nmildew-susceptible cultivar,Fielder hasa publicly available genome assembly,which we have analyzed in detail.\nOur study found that Fielder carries the susceptible allele of Pm37 in its genome, and specifically, it belongs to\nhaplotype 5 of this susceptible Pm37 allele. This result is also consistent with its susceptible phenotype. This\ngeneticbackgroundmakesitanidealrecipientforourtransformationexperiment,asitallowsclearobservationof\nresistanceconferredbytheintroducedPm37.Secondly,Fielderiswidelyrecognizedasamodelcultivarforwheat\ntransformation due to its high genetic transformation efficiency, clear genetic background, and stable agronomic\ntraits. These characteristics ensure the reliability and reproducibility oftransgenic experiments, which is why it is\nfrequentlychosenasarecipientinwheatgeneticstudies.Inourtransgenicexperiments,theinsertionofPm37into\nthe Fielder genome was random. However, we performed qRT-PCR analysis on the positive transgenic lines,\nwhich confirmed high expression levels of Pm37 in all the 10 independent T lines (Supplementary Fig. 4).\nconsistent with the high expression of Pm37. We have incorporated these details into the revised manuscript to\nprovideamorecomprehensivedescriptionofthetransgenicvalidation.\n12. +FunctionaldivergenceofPm37\n- \"PM37 and SR22 shared 94.22% amino acid sequences identity, but occurred distinctly functional divergence\n(SupplementaryFig.6).\"\n94%aaidentityisverylow,evenlowerthanwhatisusuallyobservedbetweenA-B-Dhomeologs,forinstancei.e.,\ngenes thathave diverged millions ofyears ago. Suchhigh levelof divergence betweengenes thatare supposedto\nderive from a AA common ancestor is questionable. One should not simply conclude that Pm37 is an allele of\nSr22. This must be discussed in regards to \"allelic\" variations of orthologous genes ofAAgenomes. This is very\nunusualandwouldrequireadditionalanalysesbeforeproposingconclusionsontheevolutionofthisgene.\nResponse: We appreciate your insightful discussion regarding the evolutionary relationship between Pm37 and\nSr22.Addressing the concern aboutwhether 94.22% amino acid sequence identity supports their classification as\nalleles, we note that a recent study published on 22 July 2025 (Wu et al. Genomics-driven discovery of superior\nalleles and genes for yellow rust resistance in wheat. Nature Genetics, 57:2017-2027) provides similar situation:\nwheat resistance genes Yr6 and Pm5e, which share 94.37% protein sequence identity, were explicitly designated\nas alleles. This finding aligns closely with our observations: the sequence identity between PM37 and SR22\n(94.22%) iscomparable tothatofYR6andPM5e(94.37%). Sofar,significantfunctionaldivergencebetweenthe\nresistances to mildew and rust caused by the allelic variation of a single NLR is rare and is of great significance.\nBothPm37/Sr22andYr6/Pm5e,maybeproducedbyanoutcomeoflong-termevolutionforplantresistancegenes\nadapting to diverse pathogen pressures. Critically, the core criteria for allelism (sequence conservation and\ncollineargenomiclocalization)aresatisfiedinbothcases.\nFurthermore, basedonthe genomic collinearity analysis andprevious reports (Luo etal, 2022, Identification\nandcharacterizationofSr22b, anewallele ofthewheatstem rustresistancegene Sr22effective againstthe Ug99\nrace group.), the NLR-1 (Pm37), is the direct homolog gene of Sr22, that is, the allele. In light of this precedent,\nwe maintain that classifying Pm37 and Sr22 as alleles is justified. We will incorporate this citation in the revised\nmanuscript and expand the discussion on molecular mechanisms underlying allelic functional divergence to\nstrengthenthisconclusion.\n13. +AllelicvariationsofPm37indiversifiedgermplasms\nThe amplification of this gene failed (as explained earlier) in NC99, so why is this limit not considered when\ntrying to amplify it in 144 accessions? This is a major limit obviously which prevents from concluding on the\npresence/absence. The Methods related to that experiment are not detailed enough and it is not possible to\nevaluate.Maybetheamplificationwaslimitedtoapartofthegeneonly?\nResponse: Thanks for your valuable comment.We appreciate your attention to the amplification of Pm37 alleles\nfrom diverse germplasms. We performed de novo genome assembly, which provided a high-quality genomic\nreference. This assembly allowed us to accurately identify the coding sequence (CDS) of Pm37, enabling the\ndesignofhighlyspecificprimerstargetingconservedregionswithintheCDS.Importantly,allamplificationswere\nconducted using cDNA, which further ensured specificity by focusing on expressed regions and avoiding\nnon-coding or repetitive sequences that might interfere with amplification. We have revised the Methods section\nto explicitly describe the use of CDS-derived primers and cDNA as the template, providing detailed primer\nsequencestoenhancetransparencyandreproducibility.\n14. -Line227:\"Sequencealignmentrevealedcomplexsequencevariationsinthisgeneamongtheseaccessions\".\nWhycomplex?\nResponse: Thanks for your comment.As for the description of \"complex sequence variations\" in Line 227:This\nis based on the detailed sequence analysis of the 24 accessions that successfully amplified Pm37 alleles.\nSpecifically,we classified the amplificationresults ofthese 24accessionsinto five haplotypes,and identified 104\nSNPs and 9 InDels across these accessions. These variations are distributed across all functional domains (CC,\nNB-ARC, and LRR) of the gene, with notable effects on the encoded protein, leading to premature stop codons,\naltering key amino acids in functional domains, and frame shifts. Moreover, according to the characteristics of\nthesevariations,weclassifiedtheamplificationresultsofthe24accessionsintofivehaplotypes.Amongthem,the\nfirst haplotype is completely identical to the Pm37 reference sequence; the fifth haplotype is a susceptible\nhaplotype, which shows the largest difference from Pm37, with 72 SNPs and 9 InDels. These multi-dimensional\nvariationscollectivelyreflectthecomplexityofsequencevariationsinthisgeneamongtheaccessions.\n15. -Line230:\"sequencevariations[…]includingc.GGAACAC276-282----C--\"\nWritten as it is here, it is not easy to understand the variation. To me, this is doubtful variation with a single C\nflanked by 2 indels… This typically due to a problem with the alignment: the C must have been aligned with the\nlast C of GGAACAC. The inDel corresponds to GGAACA only. This is typical from a multiple alignment\nbetweensequencesthataretoovariable.Thealignmentmustbecorrectedbeforehaplotypeclassification.\nResponse: Thanks for your meticulous comment on the clarity of the sequence variation description. After\ncarefully considering your comment, we conducted in-depth checks on the sequence data using SeqMAN,\nSnapGene, NCBI tools, and MEGA11, and confirmed the root cause of the ambiguity: the target region contains\ntwo consecutive tandem repeats of the GGAACA. This tandem repeat structure led to misalignment during the\ninitial multiple sequence alignment process, resulting in the isolated C beingincorrectly positioned relative to the\nInDels.As you astutely pointed out, the C should indeed be aligned with the last C of the GGAACAC segment,\nand the InDel corresponds only to the GGAACA motif-our verification using these tools fully supports this\nconclusion.Basedonthiscorrectedalignment,wehaverevisedthesequencevariationdescriptioninthemaintext\ntoeliminateambiguityandupdatedtherelevantsupplementarytabletoreflecttheaccuratehaplotypeinformation.\nWe greatly appreciate your critical feedback, which has not only help us resolve a key technical ambiguity but\nalsoenhancestherigorofourresults.Yourexpertiseisinvaluabletotheimprovementofthismanuscript.\n16. - Line 238: \"Additionally, sequence variation analysis of NLR-1 in different wheat […] revealed complex\nsequencevariations[…]\"\nAgain,sameconclusionasthesentencebefore.\"Complex\"isnotaresult.Whataretheresultshere?\nResponse: Thanks for your comment regarding the sequence variation analysis of NLR-1. To further enrich the\nevidence for Pm37’s evolutionary origin, we have revised the relevant content and supplemented key analysis\nresults: We added evolutionary analysis of Pm37 and its homologous sequences in different A subgenomes—\nspecifically by performing homologs or alleles alignment of Pm37 across theseAsubgenomes. The results, now\npresentedinFigure6coftherevisedmanuscript,showthatPm37clusterscloselywithTm.TA299.r1.7AG0168310,\na sequence derived from T. monococcum ssp. aegilopoides accession TA299. This clustering result confirms that\nPm37 may be originated from T. monococcum, which not only complements the sequence variation analysis of\nNLR-1butalsostronglysupportstheearlierspeculationthatPm37incommonwheatisoriginallyderivedfromT.\nmonococcum.\n17. +EvolutionarytracingofPm37\nMethodsandresultsarequitequestionable.Severalremarks:\n-First,IsuggestamakeaFigureshowinghowtheauthorsidentifiedtheintrogressedsegment.\n-Then,whyhavingusedreadmappingmethodwhilethereisanassembled genome sequenceavailable?chr7Aof\nNC99 could have been scaffolded (with RagTag or whatever) and compared to chr7A of timopheevii and\nmonococcum.\n-SupplementalFig2issomehowuselessanddoesnotprovideanyevidenceoftheintrogression.\n- More importantly, I could not understand the results… In summary it is written: NC99 read mapping revealed\nsimilarity over 78Mb (monococcum) and 69Mb (timopheevii), so Pm37 derives from a 32Mb introgressed\nsegment??? How did the author reach this value? In addition, the Figure 1e does not show any 32Mb or 78Mb\nsegment… and it even shows no similarity with timopheevii at all… Completely confusing. I suggest to redo the\nanalysesanddescribetheresultsinawaythatreaderscanunderstand.\nResponse: Thanks very much for these valuable and insightful comments and suggestions. We have thoroughly\nrevised our analysis following your recommendations to improve the clarity.As you suggested, we used RagTag\nto scaffold our NC99BGTAG11 assembly contigs to the chromosome level using the T. aestivum (CS) reference,\nachieving a 98.5% scaffolding rate. Then, we performed a comparative analysis against the Chr7A from T.\nmonococcum and T. timopheevii. This assembly-based approach provided a much clearer interpretation than our\ninitialread-mappingmethod.\nWe have removed the original Supplemental Fig. 2 and replaced it with an updated analysis version (Fig.\n6a&b in revised version). By applying a more stringent alignment length filter (>150 kb), we reduced noise and\nunambiguously detected an introgressed segment from T. monococcum at the end of Chr7A, which harbors the\nPm37locus.\nFurthermore, we have updated Fig. 1e to zoom in on a 2.2 Mb segment—identified in Supplemental Fig.\n2—thatharborsthePm37locus.Thisrevisedpanelprovidesaclearerviewofthenear-completesequenceidentity\nbetween NC99BGTAG11 and T. monococcum across this region. In comparison, this segment shows limited\nsimilarity to T. timopheevii even under relaxed alignment parameters (≥10 kb). These results provide strong\nevidencethatthePm37segmentwasoriginallyintrogressedfromT.monococcum.\nDuetothepotentialgeneticdiversitybetweenthereferencegenome(TA299)andtheactualdonor,aswellas\nourstringentdetectioncriteria,wecannotruleoutfalsenegativesinagenome-widescanofintrogressedsegments.\nTherefore, to ensure the accuracy and readability of the manuscript, we have focused our revised analysis\nspecificallyonthePm37introgressionsegmentratherthandescribinggenome-wideintrogressionpatterns.\n18. - Line 250:\"Takingalltheseinformation together,we speculatedthatPm37 […]was originally derived from\nT.monocuccmandtransferredintobreadwheatthroughthebridgespeciesT.timopheevii\"\nI donotsee theresults supportingthis conclusion.There aresomeT.monococcum with andsome withoutPm37,\nsoitcouldhavebeeninheritedfromacrosswithtimopheevii,no?\nResponse: Thanks for your comment. Regarding why we make this assumption, we firstly know the pedigree of\nNC99BGTAG11, and speculated the resistance was derived from T. timopheevii, and we also amplified identical\nhaplotype with Pm37 in a T. timopheevii accession. Then, we confirmed that Pm37 was located in a 2.2 Mb\nintrogression segment of T. monococcum through PacBio SMRT long-read genome sequencing. So, combined\nwith these information, we speculated that Pm37 may be originally derived from T. monococcum and transferred\nintobreadwheatthroughthebridgespeciesT.timopheevii.\nThere are some T.monococcumwith andsome withoutPm37, itis true. Pm37 shouldbederived from the T.\nmonococcum with Pm37, and the result of PacBio SMRT long-read genome sequencing has confirm this\ninformation. Combined with the pedigree “Saluda”*3/PI 427315, the common wheat parent Saluda of\nNC99BGTAG11 was susceptible, so Pm37 was derived from the T. timopheevii parent PI 427315. Based on the\nevolutionary history of common wheat, we speculated that Pm37 may be originally derived from T.monococcum\nandtransferredintobreadwheatthroughthebridgespeciesT.timopheevii.\n#DISCUSSION\n19. - \"Compared to the susceptible allele in IWGSC RefSeqv2.1, an approximately 4000 bp fragment insertion\nwaspresentonthesecondintronofNLR-1inNC99BGTAG11.\"\nThisisaresultnotmentionedintheResultssection.ThisisexactlywhatIhavecommentedabove.\nResponse: Thanks for drawing attention to this oversight. As you recommended, we have incorporated this\nstructural variation result into the Results section with detailed context: \"Notably, a structural variation was\nidentified in the second intron of NLR-1: compared to the susceptible allele in IWGSC RefSeqv2.1, an\napproximately 3,000 bp fragment insertion was present in NC99BGTAG11, which was validated by PCR\namplification and Sangersequencing.\"This addition connects the insertion event to the technical challenges in\ncloningandreinforcesNLR-1asthecandidategene,comprehensivelyaddressingyourcomment.\n#METHODS\n20. -\"PutativegeneswereannotatedwiththeJBrowsepipeline(http://202.194.139.32/jbrowse.html)\"\nJBrowseisaviewer,notapipelineforannotatinggenes.Suggesttoclarify.\nResponse: Thanks for your carefulcomment. Weapologize for the misleading description in our manuscript.We\ndid not perform gene annotation for this gene; instead, we viewed its existing annotation information using\nJBrowse.Intherevisedmanuscript,wehavecorrectedtherelevantdescriptiontoaccuratelyreflectthis.\n21. -\"Allthesequencingreadswereassembledfollowingtheestablishedprocedure(11)\"\nReference 11 is not about HiFi sequence assembly. For bioinformatic analyses, I suggest to describe\ntools/methods/thresholdsusedinstead ofcitingpapersthathaveperformedthesametypeofanalyses.\nResponse: Thanks for pointing out the mis-citation. Following your suggestion, we have revised the sentence to\nhighlightthetoolhifiasmanditscorrespondingcitation.\n22. -Line280:\"introducedsegments\"\nGuesstheauthorsmean\"introgressed\"no?\nResponse: Thanks for comment. We have revised \"introduced segments\" to \"introgressed segments\" throughout\nthemanuscripttoensureaccuracy.\n23. -\"a204-bpfragmentofthegenewasamplifiedandinserted\"\nForreproducibilitypurposes,Ithinkitisrequiredtoprovidethesequenceusedhereforsilencing.\nResponse:Thanksforyourvaluablesuggestionregarding reproducibility.Wewouldliketoclarifythatthelength\nofthesilencingfragmentwasincorrectlystatedas204bp,theaccuratelengthis245bp,andweapologizeforthis\nerror, which has been corrected in the revised manuscript. The primers used to amplify this 245 bp fragment are\nalreadyprovidedinSupplementaryTable 2.Tofurther enhancereproducibility,weaddedthespecificsequenceof\nthis 245 bp silencing fragment in the supplementary materials of the revised manuscript. This ensures that all\nnecessaryinformationrequiredtoreplicatetheexperimentisclearlyaccessible.\n24. -\"Sequenceandhaplotypeanalysis\"\nIn this paragraph, I do not read any information about sequence analysis or haplotype reconstruction. In contrast,\nthe details of PCR reactions are given. Methods used for sequence analysis are expected for the results to be\nreproducible.\nResponse: Thanks for your valuable comment. We fully agree that detailed methods for sequence analysis are\nessential for the reproducibility of our results. In \"Sequence and haplotype analysis\" section of the revised\nmanuscript, we have supplemented the methods for sequence analysis: all the PCR products were Sanger\nsequenced at Sangon Biotech (Shanghai, China) Co., Ltd. The sequences were assembled and aligned using\nSeqManProintheLaserGene11package.\n25. -\"Phylogeneticanalysis\"\nThe methods are not provided again.A tree was built based on a multiple alignment I guess, so methodological\ndetails are needed here. The authors just mention that the tree was built using NJ method. However, all branches\nof the tree (Figure 5) have the same length… it is likely a UPGMAtree, no?And this is also the case for the tree\nprovided as a Supplemental Figure. If UPGMA have been used, I suggest to rebuild the tree with NJ. More\nimportantly,bootstrapvaluesareneededtobeabletoconcludeonthephylogenyobserved.\nTo me, there is a problem because I suspect that the multiple sequence alignment was computed based on\nsequences that somehow do not share any similarity i.e., the 30 Pm, 16 Sr, 10 Lr, and 7Yr proteins downloaded\nfrom NCBI areprobablytoodivergenttobealigned properly.Or,theywere selectedonpurposebecauseallthese\nproteins are homologous? In that case, it must be indicated. But even homologs may contain domains that\ncompletely differ. Usually, what is aligned is just domains that really align (or alignment is cleaned from\nmisaligned positions) so that a tree could be calculated on an alignment that is correct to calculate genetic\ndistances. So, it looks like what has been done is not scientifically sound. For instance, STB6 is not a NBS-LRR\nso I doubt this could be aligned properly with the others. Since evolution of this gene is a main message of the\npaper,thispartrequiressignificantimprovementtobeconvincing.\nResponse: Thanks for your critical and constructive comments on the phylogenetic analysis, which have\nsignificantly improved this section. We have thoroughly revised the methodology, sequence selection, and tree\nvisualizationto addressyour concerns,asdetailedbelow:Firstly,regardingsequenceselectionandhomology: we\nhave refined the dataset to include only confirmed NLR resistance proteins, explicitly excluding non-NLR\nproteins such as STB6. The final dataset comprises 30 Pm, 12 Sr, 7 Lr, and 2 Yr genes, all with verified NLR\ndomain architectures (NB-ARC and LRR domains) to ensure sequence homology.This selection strategy ensures\nthe aligned sequences share conserved functional domains, addressing the concern about excessive divergence.\nSecondly, regarding multiple sequence alignment: we have clarified that alignment was performed using MEGA\n11withtheAlignbyMUSCLE(Codons)option,whichis moresuitableforprotein-codingsequencesandensures\naccurate codon-based alignment. To enhance accuracy, poorly aligned regions (especially in non-conserved\ndomains) were manually trimmed, ensuring the remaining alignment reflects true genetic distances—critical for\nreliable phylogenetic inference. Thirdly, regarding tree construction methods: we confirm that the tree is built\nusing the neighbor-joining (NJ) method in MEGA 11, with the JTT substitution model and 1000 bootstrap\nreplicates. Now, the revised Figure 5 includes bootstrap values at all nodes to indicate branch reliability, and\nbranch lengths are proportional to genetic distances (no longer uniform), correcting the earlier misrepresentation.\nThisdirectlyaddressestheconcernaboutpotentialUPGMAusage.Finally,therevisedFigure5alegendexplicitly\nstates the NJ method, and the meaning of bootstrap values, ensuring consistency between the text and\nvisualization.\n#FIGURES\n26. - Fig1: Panel e does not provide any added value to the written message. Suggest to delete or combine with\npaneldbutIamafraidthetwoscalesaretoodifferent.\nResponse:ThanksforyourvaluablecommentonFigure1e.WeagreethattheoriginalFigure1elackedsufficient\naddedvalueandwehaverevisedittopresentacomparisonofthegenomesequencesofchromosome7AamongT.\nmonococcumTA299, T.aestivumNC99BGTAG11,andT.timopheeviiPI94760.Therevised panelclearlyshows\nthe 2.2 Mb Pm37-harboring fragment introgressed from T. monococcum TA299 into NC99BGTAG11, and no\n≥150 kb alignment between NC99BGTAG11 and T. timopheevii PI 94760 on chromosome 7A — directly\nsupporting Pm37’s origin from T. monococcum and T. timopheevii as the introgression bridge.We have carefully\nconsidered your concern aboutpotential scale inconsistencies between Figure 1d and 1e: since Figure 1d focuses\non the local 147.6-kb candidate gene annotation interval (small-scale genetic context) while Figure 1e depicts\nlarge-scale (megabase-level) genomic segment comparisons across species to trace evolutionary origins, we\nretained Figure 1e as an independent panel to ensure the clarity of each dataset. The updated Figure 1e now\nprovides direct visual evidence for the evolutionary trajectory of Pm37, strongly complementing the written\nresults on Pm37’s origin and introgression pathway without redundancy. We believe this revision can effectively\naddressyourconcernandenhancetheinformativevalueofFigure1.\n27. - Fig5: I doubt a multiple sequence alignment could be computed with such divergent sequences. If yes, this\nmultiplealignmentusedforbuildingthetreeshouldbeprovided.\nResponse: Thanks for your valuable comment regarding the validity of the multiple sequence alignment for\ndivergent sequences. To address your concern, we have included the complete multiple sequence alignment used\nforconstructingFigure5aasSupplementaryData1.This alignmentwasgeneratedusingthe\"Align byMUSCLE\n(Codons)\" option in MEGA11, which is specifically optimized for protein-coding sequences and helps to handle\nmoderate sequence divergence by preserving codon reading frames. The alignment file (Supplementary Data 1)\nincludes all 43 sequences (30 Pm, 12 Sr, 7 Lr, and 2 Yr proteins). We believe this transparency confirms that the\nsequences share sufficient homology in functional domains to support meaningful phylogenetic inference,\nreinforcingthereliabilityoftheevolutionaryrelationshipspresentedinFigure5a.\n28. -ThereisaconfusionbetweengeneIDsbetweenFigures.GeneIDsintreesdonotcorrespondtogeneIDsin\nFig1(differentversions).Thisshouldbefixed.\nResponse: Thanks for your comment on the inconsistency of gene IDs across figures. We have revised the gene\nIDs in relevant figures (including Fig. 1 and Fig. 6c) to unify them using the Chinese Spring RefSeq v2.1\nannotationsystem,ensuringconsistentgeneIDsacrossallfiguresinthemanuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript seems to be revised adequately and is acceptable for publication in nature communications.", "reviewer_3": "Thank you to the authors for addressing our previous comments. The manuscript appears much improved. Following these changes, I have a few additional requests for revision listed below.\n1. The contig assembly was deposited in NCBI Sequence Read Archives (SRA), which is intended for raw sequencing data, not assembled contigs. For assembled contig data, it is more appropriate to submit the sequence to GenBank. This should be corrected. Since the scaffold-level assembly is reported, it seems appropriate to submit the full assembly and raw sequencing data to GenBank and SRA, respectively.\n2. Figure 6 – In Figure 6a, widely different scales in the x-axis and y-axis prevent comparison and interpretation of these plots. This is not adequately explained in the text. The new Figure 6b removes some concerns about the previous Figure S2 by excluding alignment information outside of Pm57. It would have been preferable to include a T. timophevii line with the introgression such as PI 427315, though it is understandable that this may not be possible within a reasonable timeframe.\nFigure 6c uses a mixture of accession names, cultivar names, common gene names, and annotation gene names. The authors should improve the labels and description of this figure to make it clear to the reader what these sequences are and how they were selected. Description would also be better worded as: “Alignment of Pm37/Sr22 homologs and alleles…”\nMethod of branch support value estimation should be added to the figure legend.\n3. Number of bootstrap replicates should also be indicated in the Figure 5a legend.\n4. Line 221, it would be preferable to write, “Pm37 confers resistance to powdery mildew but susceptibility to stem rust…”\n5. Methods (line 527) state that phylogenetic tree branch lengths reflect genetic distances. However, it appears that all branch lengths are equal in Figures 5a and 6c with no scale bars. Please either update the methods to reflect the cladogram generation or replace the cladograms with phylograms with appropriate scale bars.\n6. Figure S8, T. turgidum is misspelled.", "reviewer_4": "The authors provided precise answers to all my previous concerns and revised the manuscript accordingly. The comparative genomics analyses appear much clearer and the gene tree is now based only on NLRs that align correctly. The impact of the large intron size variation is also now mentioned and discussed. Same for the weird indel caused by misaligned sequences.\nI find the paper significantly increased in quality and I acknowledge the authors for the work performed to revised the manuscript, rather than resubmitting the original version to another journal."}, "author_response": "ResponsetoReviewers\nReviewer#1:\nThe manuscript seems to be revised adequately and is acceptable for publication in\nnature communications.\nResponse: We sincerely appreciate your positive approval on our work and valuable\nsuggestions toimprovethe qualityof ourmanuscript.\nReviewer#3:\nThank you to the authors for addressing our previous comments. The manuscript\nappears much improved. Following thesechanges, Ihave afew additional requests for\nrevision listed below.\nResponse: We sincerely appreciate your approval and valuable comments on this\nwork. We have carefully studied all your comments and suggestions and thoroughly\nrevised all of them item by item, which were listed as follows and also revised in the\nmanuscript accordingly.\n1. The contig assembly was deposited in NCBI Sequence Read Archives (SRA),\nwhich is intended for raw sequencing data, not assembled contigs. For assembled\ncontig data, it is more appropriate to submit the sequence to GenBank. This should be\ncorrected. Since the scaffold-level assembly is reported, it seems appropriate to\nsubmitthefull assembly and raw sequencing datato GenBank and SRA,respectively.\nResponse: Thank you for your valuable comment. We apologize for the inappropriate\nsubmissionof thecontig assembly to theNCBI Sequence ReadArchive (SRA), which\nstemmed from a misunderstanding of SRA's data deposition guidelines. Following\nyour suggestion, we have deposited the data as follows: Raw HiFi reads in the NCBI\nSequence Read Archive (SRA) and the scaffold assembly in GenBank, under\nBioProject accession PRJNA1365683. The updated data availability information has\nbeen revised in the manuscript to reflect these corrections, and we appreciate your\nattention tothis detail, which helps upholdtheaccuracy and compliance ofpublicdata\nrepositories.\n2. Figure 6 – In Figure 6a, widely different scales in the x-axis and y-axis prevent\ncomparison and interpretation of these plots. This is not adequately explained in the\ntext. The new Figure 6b removes some concerns about the previous Figure S2 by\nexcluding alignment information outside of Pm37. It would have been preferable to\ninclude a T. timophevii line with the introgression such as PI 427315, though it is\nunderstandable that this may not be possible within a reasonable timeframe. Figure 6c\nuses a mixture of accession names, cultivar names, common gene names, and\nannotation gene names. The authors should improve the labels and description of this\nfigure to make it clear to the reader what these sequences are and how they were\nselected. Description would also be better worded as: “Alignment of Pm37/Sr22\nhomologs and alleles…”\nMethod of branch support value estimation should be added\ntothe figure legend.\nResponse: Thank you for your insightful comments on Figure 6. We highly\nappreciateyour attention to thecomprehensiveness of theplots.\nRegarding the original Figure 6a, we have revised the x-axis and y-axis scales to\nensure consistency (the y-axis now uses a unified log -transformed count scale, and\n10\nthe x-axis range aligns with the updated Figure 6b). This modification, along with a\ndetailed explanation of the axis settings, has been added to the figure legend and\ncorresponding text to address the prior lack of clarity. We have restructured the\nvisualization: the original Figure 6a has been split into the updated Figure 6a and 6b,\nwhich show alignments to T. monococcum TA299 and T. timopheevii PI 94760,\nrespectively.The updated legend is as follows: Distribution of MUMmer alignment\nlengths from chromosome 7A of NC99BGTAG11 against homologous\nchromosomes in the T. monococcum TA299 (a) and T. timopheevii PI 94760 (b)\nreference genomes, with log -transformed bin counts on the Y-axis. A 150-kb\n10\nthreshold (dashed line) defines introgressed blocks in (a), while no alignments\nsurpassthis length in(b).Segments longerthan1.5Mb arehighlighted inred.\nRegarding your suggestion for previous Figure 6b, we wholeheartedly agree this\naddition would greatly enrich the analysis. We deeply appreciate you pointing out this\nvaluable improvement. As you pointed out, we are confronted with practical\nconstraints. Due to the dual restrictions of domestic import policies and international\nexport regulations, the accession PI 427315 may not be obtained within a reasonable\ntimeframe.We sincerely thank you forunderstanding thepractical barriers we faced.\nRegarding your suggestions for previous Figure 6c (now Figure 6d), we have\ncarefully addressed the issues you raised regarding label consistency, description\nclarity, and methodological details. The revisions are as follows: First, to resolve the\nmixed use of accession names, cultivar names, and gene names in the figure, we have\nunified the labels. We replaced gene names (e.g., “PM37”) and annotated gene names\n(e.g., previously used locus IDs like “TRAESCS7A03G1212700”) with specific\ngermplasm identifiers tied to their donor materials. This ensures readers can directly\nassociate each sequence with its genetic source. Second, we have optimized the figure\ndescription to align with your input. The original description has been revised to\n“Alignment of sequences from Pm37 donors, Sr22b and its allele-carrying donor\ngermplasms, andA-genome-containing germplasms.\" On one hand, it includes the\ncore Pm37/Sr22b allele donor materials. On the other hand, it emphasizes that the\nadditional materials were selected specifically because they carry the A-genome,\ncovering representative Triticeae species with an A-genome background. Finally, we\nhave added the method for estimating branch support values to the figure legend. We\nspecified: “Branch support values were calculated using the neighbor-joining\nmethod with 1,000 bootstrap replicates.” This provides complete methodological\ncontextfor theevolutionary relationships shown inthefigure.\n3. Numberof bootstrap replicates shouldalso beindicated inthe Figure 5alegend.\nResponse: Thank you for your valuable comment. We have revised the legend of\nFigure 5a to include the number of bootstrap replicates. The updated legend now\nspecifies that the phylogenetic tree was constructed with 1,000 bootstrap replicates to\nassess branch reliability.\n4. Line 221, it would be preferable to write, “Pm37 confers resistance to powdery\nmildewbutsusceptibility to stemrust…”\nResponse: Thank you for your valuable suggestion. We have revised Line 221 as\nrecommended, and the sentence now reads: “Pm37 confers resistance to powdery\nmildewbutsusceptibility to stem rust...”.\n5. Methods (line 527) state that phylogenetic tree branch lengths reflect genetic\ndistances. However, it appears that all branch lengths are equal in Figures 5a and 6c\nwith no scale bars. Please either update the methods to reflect the cladogram\ngeneration orreplace thecladograms with phylograms with appropriate scale bars.\nResponse: Thank you for your meticulous review and valuable comment. We\nappreciate your attention to the consistency between the methods description and the\nphylogenetic tree presentation. We apologize for the inconsistency in the initial\nmanuscript. Figures 5a and 6c are indeed cladograms (where branch lengths are\nstandardized and do not reflect actual genetic distances) rather than phylograms. The\nerror arose from an imprecise description in the methods section. To address this, we\nhave revised the relevant content in the methods to accurately reflect the tree\ngeneration approach. The updated description reads: “The phylogenetic cladogram\nof PM37 with these NLR resistance proteins was constructed using the\nneighbour-joining method in MEGA 11, with the JTT substitution model and\n1,000 bootstrap replicates to assess branch reliability (node values represent\nbootstrap support values, indicating the reliability of the topological\nrelationships)”.\n6. Figure S8, T.turgidumis misspelled.\nResponse: Thank you for your careful review and valuable comment. We have\nchecked Figure S8 andcorrected themisspelling ofT.turgidumas suggested.\nReviewer#4:\nThe authors provided precise answers to all my previous concerns and revised the\nmanuscript accordingly. The comparative genomics analyses appear much clearer and\nthe gene tree is now based only on NLRs that align correctly. The impact of the large\nintron size variation is also now mentioned and discussed. Same for the weird indel\ncaused bymisaligned sequences.\nI find the paper significantly increased in quality and I acknowledge the authors for\nthe work performed to revised the manuscript, rather than resubmitting the original\nversion to another journal.\nResponse:We sincerely appreciate your positive approval and constructive comments\nand suggestions,which greatly improvethequality ofourmanuscript."}]

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