Genome-assisted identification of wheat leaf rust resistance gene Lr30 (synonym Lr.ace-4A)

Genome-assisted identification of wheat leaf rust resistance gene Lr30 (synonym Lr.ace-4A) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genome-assisted identification of wheat leaf rust resistance gene Lr30 (synonym Lr.ace-4A) Shisheng Chen, Jinwei Yang, Hongna Li, Mengyu Li, Rui Song, Tao Shen, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6289485/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Leaf rust is a devastating fungal disease of wheat. Planting resistant wheat cultivars is the most effective strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr30 / Lr.ace-4A encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem NB-ARC domains. This gene proves both necessary and sufficient to confer resistance to Puccinia triticina , as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr30 provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr30 haplotypes, with transgenic plants carrying either variant exhibiting susceptibility. Cloning of Lr30 will accelerate its deployment in wheat breeding programs. Biological sciences/Genetics/Plant breeding Biological sciences/Biotechnology/Plant biotechnology/Agricultural genetics Biological sciences/Plant sciences/Plant stress responses/Biotic Biological sciences/Genetics/Agricultural genetics wheat leaf rust chromosome-scale assembly resistance gene CC-NBS-LRR Lr30 (synonym Lr.ace-4A) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Wheat is a major staple food crop, contributing about one-fifth of the total calories and proteins consumed by humankind. An effective strategy to increase wheat production is to mitigate losses caused by fungal pathogens. Puccinia triticina Eriksson ( Pt ), the causal agent of wheat leaf rust, represents one of the most formidable threats to global wheat production. This disease affects most wheat-growing areas and can significantly reduce yields in susceptible wheat varieties under favorable weather conditions 1 . Due to the impacts of global warming and the evolving virulence of the pathogens, leaf rust has significantly expanded its geographical range and poses a grave threat to global wheat production 2 . Among the strategies to control this devastating disease, breeding for leaf rust resistance is considered the most feasible and sustainable approach. To date, more than 80 leaf rust resistance ( Lr ) genes have been assigned official designations in wheat and its wild relatives 3 , 4 . Nevertheless, owing to the vast size and complexity of the wheat genome, only twelve Lr genes have been successfully cloned, using either traditional map-based cloning methods ( Lr1 , Lr10 , Lr21 , Lr34 , Lr42 , Lr67 ) 5 – 8 or advanced gene-cloning techniques, including MutRenSeq ( Lr13 ) 9 , 10 , TACCA ( Lr22a ) 11 , MutChromSeq ( Lr14a and Lr85 ) 4 , 12 , MutIsoSeq ( Lr9 ) 13 , and MutRNASeq ( Lr47 ) 5 . Cloning additional Lr genes is highly desirable, as it would enable the development of more diverse Pt resistance gene combinations in transgenic cassettes or gene pyramids, promoting more durable resistance. Wheat resistance to leaf rust can be classified into two primary categories: race-specific resistance and slow rusting resistance 3 . Race-specific resistance is based on the gene-for-gene hypothesis. Most race-specific Lr genes encode coiled-coil nucleotide-binding leucine-rich repeat (NLR) proteins 4 – 6 , with the exceptions of Lr14a 12 and Lr9 13 , which encode proteins featuring twelve ankyrin repeats and an N-terminal tandem kinase domain followed by vWA/Vwaint domains, respectively. The slow rusting resistance genes, Lr34 and Lr67 , encode a putative ATP-binding cassette transporter 7 and a hexose transporter 8 , respectively. Recent advances in protein crystallization have demonstrated that the wheat stem rust resistance protein Sr35, upon recognizing the avirulence protein AvrSr35 from the pathogen, forms a homo-pentameric resistosome, resulting in hypersensitive responses (HR) or necrosis on wheat leaves 14 . Triticum turgidum ssp. durum , a key member of the primary gene pool of wheat, is cultivated across approximately 18 million hectares worldwide 15 . In 2019, a draft assembly of the T. durum genome (accession Svevo, BBAA) was released 16 . However, the assembly remains in low contiguity (contig N50 = 0.06 Mb), with a total of 309,814 gaps 16 . A more contiguous genome assembly is essential for enhancing gene identification in durum wheat. Several cataloged Lr genes, such as Lr3a , Lr14a , Lr27 + Lr31 , Lr61 , Lr72 , Lr79 , and LrCamayo 17 , 18 , have been detected in durum wheat cultivars. However, among the Lr genes present in durum wheat, only Lr14a has been successfully cloned so far 12 , likely reflecting the challenges posed by the large and complex wheat genomes. Seedlings of the Portuguese landrace of durum wheat, PI 192051, exhibited robust resistance [infection type (IT) = 0;] to four Pt pathotypes originating from the USA, Tunisia, Morocco, and Ethiopia 18 . Genetic analysis using recombinant inbred lines (RILs) derived from the cross Rusty (susceptible) × PI 192051 (resistant) and the Illumina iSelect 9K wheat SNP array, revealed a dominant Lr gene, designated as Lr.ace-4A , located in the centromeric region of chromosome arm 4AS 18 . Conversely, the Lr30 gene, which confers intermediate resistance (ITs = 1 to 2), was identified as a recessive resistance gene in the hexaploid wheat cultivar Terenzio 19 . This gene was initially mapped to chromosome arm 4BL 19 , but its locus was later corrected to chromosome 4AL 20 . Lr30 has not been reported in durum wheat and seems to be quite rare in bread wheat germplasm 18 , 21 . Recently, three Canadian spring wheat cultivars (AAC Prevail, AAC Concord, and Lillian) were postulated to carry Lr30 22 . While Lr30 continues to exhibit resistance against approximately half of the Pt pathotypes collected in China 23 , 24 , progress in its genetic interpretation has significantly lagged. Here, we generate a high-quality genome assembly of the durum wheat landrace PI 192051 (contig N50 = 42.53 Mb) and successfully clone the leaf rust resistance gene Lr.ace-4A . This gene encodes a non-canonical NLR receptor with tandem NB-ARC domains. Further investigations demonstrate that Lr.ace-4A in durum wheat corresponds to the Lr30 gene previously identified in common wheat. Transformation of a 9.3-kb genomic sequence containing Lr.ace-4A into the susceptible T. aestivum cultivar Fielder and the T. durum ethyl methanesulfonate (EMS) mutant line m1 from PI 192051 demonstrate that this gene is less effective in a hexaploid background compared to in a tetraploid background. Two amino acid polymorphisms distinguish the resistant and susceptible Lr30 haplotypes. Transformation with the 9.3-kb genomic sequences carrying either of these variations results in susceptibility. Additionally, we develop a diagnostic molecular marker for Lr30 , which will facilitate its deployment in wheat breeding programs. Results Durum wheat landrace PI 192051 exhibited robust resistance to multiple Pt pathotypes. Seedlings of the durum wheat landrace PI 192051 exhibited robust resistance to eight Pt pathotypes (Supplementary Table 1) collected in China. In contrast, another durum wheat genotype, Rusty, displayed high susceptibility to all Pt pathotypes tested (Fig. 1 a). When challenged with Pt pathotype PHQS, Rusty seedlings showed visible rust spores at six days post-inoculation (dpi), whereas PI 192051 exhibited a typical HR in the leaves (Fig. 1 b). Microscopic analysis with WGA-FITC staining revealed that although rust fungi successfully formed haustoria in PI 192051, the expansion of secondary rust hyphae was restricted (Fig. 1 c). In contrast, in the susceptible line Rusty, the rust hyphae expanded extensively, forming a diffuse network of fungal growth at the infection sites. At all four time points, the average infection areas in PI 192051 were significantly smaller ( P < 0.001) than in Rusty (Fig. 1 d). Among the 286 F 2 plants derived from the cross PI 192051 × Rusty and evaluated with Pt pathotype PHQS, we identified 76 resistant plants and 210 susceptible ones (Supplementary Fig. 1a). This distribution corresponded to the expected 1:3 segregation ratio for a single recessive gene ( χ 2 = 0.38, P = 0.54). However, a subset of 315 F 2 plants from the cross between PI 192051 and m1 (a susceptible EMS-induced mutant line derived from the PI 192051 mutant population) was also evaluated with PHQS. Among these, 240 plants were resistant and 75 were susceptible (Supplementary Fig. 1b), which fits well the 3:1 segregation ratio expected for a single dominant gene ( χ 2 = 0.24, P = 0.63). Genetic mapping of Lr.ace-4A . Lr.ace-4A was previously mapped to chromosome 4A of durum wheat PI 192051 18 , located within a 4.0 cM region flanked by markers IWA232 and IWA1793 (145.24–562.83 Mb; Svevo RefSeq v1.0; Supplementary Fig. 2a). In this study, we first performed RNA-seq analysis to identify single nucleotide polymorphisms (SNPs) between the tetraploid parental lines PI 192501 and Rusty. Using the 286 F 2:3 families evaluated with Pt pathotype PHQS and six newly developed PCR markers (Supplementary Data 1) on chromosome 4A, Lr.ace-4A was refined to a 2.45 cM genetic interval flanked by markers IWA232 and pku2574 (Supplementary Fig. 2b). In this population, recombination suppression was observed in a region spanning approximately 145.24 to 562.83 Mb of chromosome 4A. Such suppression could arise from alien introgressions or inverted chromosomal segments or the centromeric region. Cytogenetic analyses revealed no evidence of alien introgression in PI 192051 or chromosomal inversions between the parental lines PI 192051 and Rusty (Supplementary Fig. 3), indicating that the gene is likely located within the centromeric region. To further eliminate the possibility of chromosomal inversions or alien introgression within the Lr.ace-4A mapping region, we used the 315 F 2 plants derived from the cross PI 192051 × m1 to map Lr.ace-4A . RNA sequencing of both PI 192051 and m1 allowed us to identify EMS-induced SNPs (Supplementary Table 2) and develop seven new PCR markers on chromosome 4A (Supplementary Data 1). In this population, we mapped Lr.ace-4A between markers pku8123 and pku4169 (101.33–536.89 Mb; Supplementary Fig. 2d). According to the mapping results from both populations, the markers from pku1280 (162.15 Mb; Svevo RefSeq v1.0) to pku0332 (519.32 Mb) were completely linked to Lr.ace-4A (Supplementary Fig. 2b-d). These results indicate that recombination suppression likely extends across a substantial portion of chromosome 4A, covering at least the region from 162.15 to 519.32 Mb. This recombination-suppressed region encompasses the centromere region (Supplementary Table 3). De novo genome assembly of PI 192051 and genome-assisted identification of Lr.ace-4A . To clone Lr.ace-4A , we constructed a chromosome-scale reference genome for the resistant parent PI 192051. Using 451.11 Gb of PacBio High-Fidelity (HiFi) reads and 451.46 Gb of high-throughput chromosome conformation capture sequencing (Hi-C) reads (Supplementary Table 4), we generated a high-quality genome assembly with a total size of 10.51 Gb. This assembly features a scaffold N50 of 749.44 Mb, includes all 14 chromosomes, and captures 26 telomeres (Fig. 2 a; Supplementary Table 5). The contiguity of this PI 192051 genome assembly, with a contig N50 of 42.53 Mb, significantly exceeds that of T. dicoccoides accession Zavitan (contig N50, 0.06 Mb) and T. durum genotype Svevo (contig N50, 0.06 Mb), establishing it as one of the highest-quality durum wheat genomes to date (Fig. 2 b; Supplementary Tables 5 and 6). The assembly quality was further validated by a high LTR Assembly Index (LAI) score of 19.71, a Quality Assessment (QA) score of 64.54, and a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score of 98.4% (Supplementary Table 5), collectively indicating a highly continuous and complete genome. A total of 65,860 protein-coding genes were predicted in PI 192051 using a combination of homoeologous protein sequences, Iso-seq, and RNA-seq data derived from four tissues (leaf, root, stem, and spike) across multiple developmental stages (Supplementary Table 4). Synteny analysis based on high-confidence annotated genes demonstrated strong collinearity between the PI 192051 genome and the A/B subgenomes of Zavitan ( T. dicoccoides ), Svevo ( T. durum ), and Chinese Spring (CS; T. aestivum ) (Fig. 2 c). To investigate genetic variation within the recombination-suppressed region, we obtained whole-genome resequencing data of a tetraploid wheat panel, comprising six accessions each of T. dicoccon , T. durum , and T. dicoccoides (GenBank accession number PRJEB61424). Using the identified SNPs, we analyzed variant density on chromosome 4A and observed a significant reduction in genetic variation spanning approximately 150 Mb to 530 Mb (Supplementary Fig. 4). This region coincides with the identified recombination-suppressed interval (Supplementary Fig. 2). Reduced variation is expected in a centromeric region lacking recombination, as the entire region is selected as a single block. EMS mutagenesis was carried out on PI 192051, resulting in the generation of 1,853 independent M 2 mutant families. Screening of these M 2 mutant families with Pt pathotype PHQS identified seven independent families segregating susceptible plants (Fig. 3 a), with susceptibility further validated using progeny testing. Genotyping with six 4A-genome specific markers confirmed that these mutant lines retained the PI 192051 allele. To identify the candidate gene for Lr.ace-4A , we generated RNA-seq reads from Pt -inoculated leaves of seven independent susceptible M 3 mutants. The resulting clean reads were mapped to the annotated genes within the Lr.ace-4A candidate region on chromosome 4A of the PI 192051 genome. This analysis led to the identification of an NLR gene, PI192051.r1.4AG0210600 , which displayed EMS-induced (G/C-to-A/T) point mutations in all seven susceptible mutants (Fig. 3 b, c). Using three primer pairs pku4AF1R1 , pku4AF2R2 , and pku4AF3R3 (Supplementary Data 1) developed from this candidate gene, we performed PCR amplification of the regions harboring the mutations and confirmed the presence of nucleotide transitions in these susceptible mutants (Supplementary Fig. 5). All these EMS-induced mutations resulted in nonsynonymous amino acid substitutions (Fig. 3 c). PI192051.r1.4AG0210600 is located at 513.5 Mb in the Svevo reference genome and co-segregated with the phenotype in both mapping populations. This gene comprises three exons and two introns, encoding a non-canonical NLR protein of 1,174 amino acids (GenBank accession number PV159345), characterized by tandem NB-ARC domains (Fig. 3 d). Using 5 ′ and 3 ′ rapid amplification of cDNA ends (RACE; Supplementary Fig. 6), we determined that the 5′-untranslated region (UTR) of Lr.ace-4A is 87 bp and the 3′-UTR spans 329 bp. These findings identify PI192051.r1.4AG0210600 as the candidate functional gene responsible for Lr.ace-4A -mediated resistance to Pt . Functional validation of Lr.ace-4A using CRISPR/Cas9-mediated gene editing and transgenic complementation. To knockout PI192051.r1.4AG0210600 in PI 192051, a gene editing system was initially established in tetraploid wheat. This system was refined by incorporating Cas9-Trex2 and GRF4-GIF1 fusion proteins 25 , 26 to enhance both editing and regeneration efficiency. A guide RNA (gRNA) targeting the second exon of PI192051.r1.4AG0210600 was designed (Fig. 4 a). To assess potential off-target effects, we conducted BLASTN searches using the designed gRNA and PAM sequence (5'-GCCAATGAGACTATTAACCGTGG-3') as queries against both the Svevo and PI 192051 genomes. No off-target sites were detected in either genome. We successfully generated 39 independent transgenic T 0 plants. Genotyping of these T 0 plants revealed that 32 plants (82.1% of the total) contained mutations at the target site (Supplementary Fig. 7). Among these, two homozygous edited T 0 plants, T 0 KO-1 (an "A" insertion) and T 0 KO-2 (an "A" deletion), were selected for further analysis (Fig. 4 a). All T 1 progeny from these two knockout T 0 plants were susceptible to the Pt pathotype PHQS, while the WT PI 192051 retained its resistance (Fig. 4 b). These results suggest that PI192051.r1.4AG0210600 is required for Lr.ace-4A -mediated resistance to leaf rust in durum wheat. To determine whether PI192051.r1.4AG0210600 is sufficient to confer resistance to leaf rust, a 9,264 bp genomic DNA fragment (GenBank accession number PV159345) derived from PI 192051, encompassing the complete transcribed region and native regulatory sequences (Fig. 4 c), was introduced into the susceptible EMS mutant line m1 via Agrobacterium tumefaciens -mediated transformation. A total of 25 independent transgenic T 0 plants were generated, of which nine were randomly selected for further analysis. The presence and expression of the transgene in these selected T 0 plants were confirmed through PCR and qRT-PCR analyses (Supplementary Fig. 8a). All transgenic T 0 and T 1 plants carrying the transgene exhibited robust resistance to Pt pathotype PHQS, whereas the untransformed m1 control displayed susceptibility (Fig. 4 d and Supplementary Fig. 8b). Taken together, the genetic mapping, EMS and CRISPR/Cas9-induced mutations, and transgenic complementation results demonstrated that PI192051.r1.4AG0210600 is Lr.ace-4A . Lr.ace-4A is synonymous with Lr30 , exhibiting diminished resistance in a hexaploid genetic background. Lr30 is the only Lr gene currently cataloged on chromosome arm 4AL. According to the Lr30 map on the GrainGenes website ( https://wheat.pw.usda.gov/GG3/ ), this gene is flanked by IWA4359 (514.10 Mb; Svevo RefSeq v1.0) and IWA2585 18 (536.89 Mb). The location of Lr30 closely coincides with that of Lr.ace-4A (513.5 Mb; Svevo RefSeq v1.0). Sequencing of Lr.ace-4A in the Lr30 monogenic line RL6049 confirmed the presence of a gene 100% identical to Lr.ace-4A . However, as documented in previous studies 23 , 24 , 27 , Lr30 confers only intermediate levels of resistance (ITs = 1 to 2) when present alone. Indeed, we observed that RL6049 exhibited intermediate resistance to Pt pathotypes FHJL, FHJR, THDB, and PHJS (Supplementary Fig. 9). We hypothesized that Lr.ace-4A is less effective in a hexaploid background compared to a tetraploid background. To test this hypothesis, the same 9,264 bp genomic fragment carrying Lr.ace-4A from PI 192051 was transformed into the hexaploid wheat cultivar Fielder via A. tumefaciens -mediated transformation (Fig. 5 a). We successfully generated 37 independent transgenic T 0 plants, with nine randomly selected for detailed characterization. The presence and expression of the transgene in these selected T 0 plants were verified through PCR and qRT-PCR assays (Supplementary Fig. 10). In this hexaploid background, most transgenic T 0 plants (e.g., T 0 C652-3 and T 0 C652-10) exhibited intermediate resistance to Pt pathotype PHQS, resembling the responses observed in RL6049. In contrast, the untransformed Fielder control was completely susceptible (Fig. 5 b). Evaluation of the transgenic T 1 families, each comprising approximately 20 plants per event revealed that the resistant plants consistently carried the transgene (Fig. 5 b and Supplementary Fig. 11). In the transgenic families T 1 C652-12 and T 1 C652-27, all T 1 plants showed resistance and were confirmed to possess the transgene (Fig. 5 b and Supplementary Fig. 11). Furthermore, the resistant line PI 192051 was crossed and backcrossed two times with the Chinese common wheat variety Yangmai21 (YM21; Supplementary Fig. 12a), which is susceptible to multiple Pt pathotypes 5 . The BC 2 F 3 plants homozygous for the natural Lr.ace-4A gene exhibited intermediate resistance against Pt pathotype PHQS, similar to the responses observed in RL6049 (Supplementary Fig. 12b). In contrast, the YM21 control displayed susceptible infection types (ITs = 4) to the same race. These findings confirm that Lr.ace-4A is synonymous with Lr30 , though its effectiveness is reduced in a hexaploid background. In the Fielder background, several transgenic events (e.g., T 1 C652-12 and T 1 C652-27) with higher levels of transgene expression (Supplementary Fig. 10) displayed enhanced resistance, comparable to the responses observed in the tetraploid wheat PI 192051 (Fig. 5 b and Supplementary Fig. 11). This result suggests that the resistance levels in transgenic plants may be correlated with the number of Lr30 insertions or transgene expression levels. To investigate the impact of Lr30 overexpression on resistance, we generated 50 independent T 0 transgenic plants overexpressing Lr30 driven by the maize ubiquitin ( UBI ) promoter (Fig. 5 c). From these, nine T 0 plants were randomly selected for detailed analysis. Transcript levels of Lr30 were significantly higher in all examined transgenic T 0 plants compared to the Fielder control ( P < 0.001; Supplementary Fig. 13). Upon inoculation with Pt pathotype PHQS, both T 0 and T 1 transgenic plants overexpressing Lr30 exhibited robust resistance, whereas the Fielder control displayed complete susceptibility (Fig. 5 d and Supplementary Fig. 14). To determine whether the transgene exhibits a resistance profile similar to that of the natural Lr30 gene, two transgenic lines (T 1 C652-12 and T 1 C652-27; Fig. 5 b), displaying higher levels of transgene expression, were challenged with five Pt pathotypes known to be highly virulent to Fielder 5 . T 1 plants harboring the transgene from these two transgenic lines exhibited strong resistance to all five tested Pt pathotypes (Fig. 6 a). These results suggest that the transgene replicates the resistance profile of the natural Lr30 (synonym Lr.ace-4A ) gene. Haplotype analysis and diagnostic marker of Lr30 . A BLASTp similarity search against the published genomes of hexaploid, tetraploid, and diploid wheat, as well as the National Center for Biotechnology Information (NCBI) database, revealed that homologous proteins on chromosome arm 4AL shared over 99.4% similarity with Lr30 (Supplementary Fig. 15). To further investigate the sequence variation in Lr30 , we sequenced its coding regions from 59 accessions of Triticum turgidum subsp. dicoccon , which had been inoculated with Pt pathotype PHQS. This analysis revealed that none of these T. dicoccon accessions carried the Lr30 gene. From these T. dicoccon accessions and the published wheat genomes, we identified seven susceptible haplotypes (Hap2 – Hap8) based on their susceptible reactions and/or shared amino acid sequences (Supplementary Fig. 15 and Supplementary Table 7). Lr30 (Hap1) differed from the other seven haplotypes by three (Hap2), four (Hap3 and Hap6), two (Hap4), five (Hap5), and seven (Hap7 and Hap8) amino acid changes, respectively. Two cDNA polymorphisms, G1597A and C1984T (corresponding amino acid changes E533K and R662C), distinguish Lr30 from all other susceptible haplotypes (Supplementary Fig. 15). Additionally, the 1597G and 1984C alleles in Lr30 were identified in a single genotype, PI 619381, from approximately 1,000 wheat accessions with available exome sequencing data 28 . When challenged with Pt pathotype PHQS, PI 619381 displayed a resistance response similar to that conferred by RL6049 (Supplementary Fig. 16). These results indicate that these two unique polymorphisms are likely critical for the functionality of Lr30 and its ability to recognize pathogen effectors. To further investigate the functional significance of each variation, we constructed single-mutation versions of Lr30 at these two sites using the vector containing the 9,264 bp genomic fragment from PI 192051. We then generated transgenic wheat plants expressing the 9,264 bp genomic fragment with either the 533K (Lr30 533K ) or 662C (Lr30 662C ) amino acid substitution. Positive transgenic T 0 and T 1 plants expressing Lr30 533K and Lr30 662C exhibited complete susceptibility to Pt pathotype PHQS (Fig. 6 b, c and Supplementary Fig. 17). In contrast, transgenic wheat plants expressing the original 9,264 bp genomic fragment exhibited resistance (Fig. 5 a, b). These results underscore the indispensable role of both natural variations for the resistance function of Lr30 . A dominant marker, Lr30MAS-47FR (Supplementary Data 1), was developed based on these two unique cDNA polymorphisms in PI 192051 (Supplementary Fig. 15). PCR amplification using this marker at an annealing temperature of 54˚C yields a 916-bp fragment when Lr30 is present (Supplementary Fig. 18). This marker was used to assess a collection of 309 wheat accessions, including 158 T. aestivum , 82 T. turgidum , and 69 T. monococcum . PCR amplicons of the expected size were detected only in three genotypes (PI 192051, RL6049, and PI 619381), with no amplification observed in any other wheat genotypes tested (Supplementary Table 8). Characterization of Lr30 encoding an NLR immune receptor protein. The transcript levels of Lr30 in PI 192051 were quantified relative to the TaActin reference gene using qRT-PCR. Our analysis revealed a significant upregulation of Lr30 expression in plants infected with the Pt pathotype PHQS from 1 to 4 dpi (Fig. 7 a), suggesting that Lr30 is induced by the presence of Pt . Additionally, no significant difference in Lr30 transcript levels was detected between the hexaploid line RL6049 and the tetraploid PI 192051 (Supplementary Fig. 19). To investigate the relationships between Lr30 and other known NLR proteins, a phylogenetic analysis was conducted using Lr30 and 186 known NLR proteins from the Gramineae family. The analysis revealed that Lr30 was most closely related to Pib from rice (Supplementary Fig. 20). However, sequence alignment revealed a sequence similarity of less than 38.7% between Lr30 and Pib. To determine the subcellular localization of Lr30, a green fluorescent protein (GFP) tag was fused to Lr30 to visualize its signals within plant cells. Both cytoplasmic and nuclear fluorescence were detected in Nicotiana benthamiana leaves expressing the GFP-Lr30_CDS (coding region) and Lr30_CDS-GFP fusion proteins (Fig. 7 b). Similarly, a dual cytoplasmic and nuclear localization of GFP-Lr30_CDS was observed in wheat protoplasts (Fig. 7 c). The expression of the GFP-fused proteins was validated via Western blot analysis using an α-GFP antibody (Supplementary Fig. 21). To evaluate whether the full-length Lr30 or its individual domains can trigger cell death in N. benthamiana , an A. tumefaciens -mediated transient expression analysis was conducted in N. benthamiana leaves. No cell death or noticeable yellowing was observed in the leaf regions expressing Lr30 or its individual protein domains (Fig. 7 d). In contrast, pronounced cell death was observed in leaf regions expressing BAX, which served as a positive control (Fig. 7 d). Additionally, we used AlphaFold2 and AlphaFold-Multimer to predict the potential resistosome structures of Lr30. Both the coiled-coil (CC) and NB-NB-LRR domains of Lr30 demonstrated the ability to form pentamers (Supplementary Fig. 22). The critical amino acid substitution R662C is located within the binding interface of the pentamer, while the other key amino acid substitution, E533K, is positioned on the exterior surface of the predicted resistosome (Fig. 7 e). Discussion Durum wheat is a vital cereal grain primarily cultivated for pasta production and serves as an important reservoir of genetic diversity for traits such as disease resistance 29 – 32 and nutritional quality 33 . Despite its agricultural significance, the genomic resources for durum wheat remain limited, with only a few draft genome assemblies currently available 16 . The development of additional high-quality reference genomes for durum wheat is crucial for isolating valuable genes and advancing functional and evolutionary genomic studies. To facilitate the practical exploitation of durum wheat, we developed a high-quality reference genome for the Portugal durum wheat landrace PI 192051 (Fig. 2 ). Using this genome and EMS-induced susceptible mutants, we successfully cloned the leaf rust resistance gene Lr30 (synonym Lr.ace-4A ), which is located within a recombination-sparse region on chromosome 4A in durum wheat. Previous studies suggested that the leaf rust resistance genes Lr.ace-4A and Lr30 were distinct, based on differences in their genetic origins, chromosomal locations, infection types, and inheritance patterns (dominant/recessive) 18,19 . However, in this study, we provide evidence that Lr.ace-4A and Lr30 are, in fact, the same gene. This conclusion is supported by multiple lines of experimental evidence. First, Lr.ace-4A and Lr30 are located at the same locus and share an identical genomic sequence. The discrepancies in the previous gene mapping study 18 likely arise from a large recombination-suppressed region on chromosome 4A (Supplementary Fig. 2) in the cross between PI 192051 and Rusty, which may have obscured the true genetic relationship. Second, functional validation through transgenic complementation experiments demonstrated that Lr.ace-4A confers infection types similar to Lr30 when expressed in the susceptible hexaploid wheat cultivar Fielder. The introgression of Lr.ace-4A into the common wheat variety YM21 resulted in a resistance phenotype comparable to that of Lr30 in the hexaploid line RL6049 (Supplementary Fig. 12). Third, while Lr30 in the hexaploid wheat cultivar Terenzio was reported as a recessive resistance gene 19 , Lr.ace-4A in the tetraploid PI 192051 was initially identified as a dominant resistant gene 18 . In our study, segregation analysis of seedling resistance in the F 2 population from the PI 192051 × Rusty cross revealed that Lr.ace-4A was recessive against the Pt pathotype PHQS. Conversely, Lr.ace-4A exhibited dominant inheritance in the PI 192051 × m1 mapping population. These contrasting inheritance patterns can be explained by dominance reversals, a phenomenon previously observed in several wheat rust resistance genes, such as Sr8155B1 , Yr6 , and Sr6 34–37 . It is hypothesized that factors such as genetic backgrounds, pathogen isolates, and environmental conditions could contribute to this reversal 34 , 36 – 38 . Furthermore, allelic interactions and dominant-negative effects may also influence the observed dominance reversal 39 , 40 . Yeast two-hybrid assays revealed no direct interactions between the Lr30 protein and its susceptible haplotypes (Supplementary Fig. 23). Further studies are needed to clarify the mechanisms underlying this dominance reversal in Lr30. The results presented herein demonstrate that Lr30 is less effective when present in a hexaploid background than in a tetraploid background. A similar trend has been observed for several other wheat resistance genes, including Sr21 41,42 , Sr13 29 , Pm8 43 , and YrAS2388 44 , which also confer lower levels of resistance when transferred from lower-ploidy relatives into hexaploid wheat. This reduction in resistance has been attributed to the presence of inhibitors or modifiers in the hexaploid wheat genetic backgrounds that suppress the function of the transferred resistance genes. In certain cases, such as Pm8 and YrAS2388 , the resistance reduction of the transferred genes was found to vary across different hexaploid backgrounds 43 , 44 . In the case of wheat powdery mildew, the Pm8 resistance gene from rye was suppressed in hexaploid wheat by a susceptible allele of its wheat ortholog, Pm3 45 . Identification of the chromosomal region responsible for the reduced effectiveness of Lr30 in hexaploid wheat would be of significant interest, as this knowledge may be useful to enhance the gene's performance in hexaploid backgrounds. Although Lr30 and Lr.ace-4A were first identified in 1981 19 and 2019 18 , respectively, these genes have not been widely utilized in the breeding of bread and durum wheat. Our haplotype analysis and diagnostic marker evaluation (Supplementary Table 8) reveal that Lr30 is present in only one (1.2%) T. turgidum accession and two (1.3%) T. aestivum accessions, while it is absent in all other diploid, tetraploid, and hexaploid wheat genotypes. This finding indicates that the incorporation of Lr30 can benefit a wide range of commercial durum wheat varieties, particularly due to its robust resistance against multiple Pt races. However, the utility of Lr30 is constrained by its limited effectiveness against several Pt pathotypes and its partial resistance in hexaploid wheat. To address these limitations, Lr30 would need to be deployed in combination with other Lr genes or integrated into transgenic cassettes containing multiple resistance genes, particularly once transgenic approaches gain broader public acceptance. Given the enhanced resistance in transgenic hexaploid plants with higher gene copy numbers or transgene expression (Fig. 5 ), it might be valuable to incorporate multiple copies of Lr30 into such transgenic cassettes. The cloning of Lr30 and its successful introgression from the original durum wheat PI 192051 into the Chinese commercial bread wheat cultivar YM21 (Supplementary Fig. 12) represent significant steps toward its future application in global wheat breeding programs. Moreover, our analysis identified only two critical cDNA polymorphisms (G1597A and C1984T; Supplementary Fig. 15) that distinguish resistant and susceptible haplotypes of Lr30 . This suggests that the foundational structure of this gene is broadly present in global wheat germplasms. With the rapid advancement of base editing technologies, Lr30 could become a promising target for precise base editing, enabling the direct enhancement of wheat resistance to leaf rust. The Lr30 gene encodes a non-canonical NLR protein with tandem NB-ARC domains (CC-NB-NB-LRR; Fig. 3 d). Despite the presence of an additional NB domain in this protein, a typical pentameric resistosome was successfully predicted using AlphaFold2 and AlphaFold-Multimer (Fig. 7 e). This structural prediction suggests that Lr30 retains the ability to form a resistosome 46 . The NB domain of NLR proteins is known to play a central role in binding nucleoside triphosphates (NTPs), facilitating the exchange between NTPs and nucleoside diphosphates (NDPs), and regulating the oligomerization and activation switch of NLR proteins 47 . The presence of an additional NB domain in Lr30 might enable more nuanced and sensitive regulation of its activity, potentially enhancing its responsiveness to pathogen signals. Remarkably, the two critical natural variations, amino acids 553E and 662R, are located within or adjacent to the secondary NB domain. Point mutations at these two amino acid residues completely abolished the resistance function of the Lr30 protein. These findings suggest that the secondary NB domain, together with the associated critical residues, plays a crucial role in the structural integrity and functional regulation of Lr30. In summary, we demonstrated the feasibility of generating chromosome-scale wheat assemblies in combination with EMS-induced susceptible mutants to facilitate the cloning of resistance genes located within recombination-sparse regions of the wheat genome. We confirmed that Lr.ace-4A in tetraploid wheat and Lr30 in hexaploid wheat represent the same gene, although Lr30 exhibits reduced effectiveness in a hexaploid background. The cloning of Lr30 , the introgression line generated, and the diagnostic marker developed in this study provide invaluable resources for diversifying the deployment of Lr genes and accelerating the integration of Lr30 into wheat breeding programs. Methods Plant materials and mapping populations. The leaf rust resistance gene Lr.ace-4A was sourced from the durum wheat accession PI 192051, a landrace originating from Lisboa, Portugal 18 . To map this gene, two F 2 mapping populations were developed: one comprising 286 F 2 plants from a cross between PI 192051 and the susceptible genotype Rusty 18 , 29 , and another with 315 F 2 plants from a cross between PI 192051 and m1, a susceptible EMS-induced mutant line derived from the PI 192051 mutant population. The Lr30 monogenic line RL6049 (GSTR 430; Thatcher*6/Terenzio) and PI 619381 were obtained from the U.S. Department of Agriculture National Small Grains Collection. A diverse wheat panel, including 158 T. aestivum , 69 T. monococcum , and 82 T. turgidum accessions (Supplementary Table 8), was used to evaluate the effectiveness of the diagnostic marker for marker-assisted selection. Additionally, SNPs from exome capture data of ~ 1,000 wheat genotypes 28 were analyzed to study natural variations in Lr30 . Leaf rust assays. Eight Pt pathotypes (THSP, FHJR, HCJR, PHQS, PHJS, PHST, THDB, and FHJL; Supplementary Table 1) were inoculated onto PI 192051 and Rusty. Seedlings at the three-leaf stage were treated with Pt urediniospores mixed with talcum powder (1:25 ratio), using the shaking-off method 5 . Inoculated plants were placed in a dark dew chamber at 22°C for 24 hours and then transferred to a growth chamber maintained at 22–24°C with a 16-hour photoperiod. Disease symptoms were scored around 12 days post-inoculation (dpi) using a 0–4 scale 48 . For pathogen growth quantification, leaf segments from PI 192051 and Rusty inoculated with PHQS were sampled at 2, 4, 6, and 8 dpi, cleared with KOH (37°C, 12 hours), stained with WGA-FITC (Cat. No. L4895-10MG, Sigma-Aldrich, USA), and examined under a Zeiss Discovery V20 fluorescence dissecting microscope (Zeiss, Jena, Germany). Cytogenetic assays. Fluorescence in situ hybridization (FISH) karyotyping experiments were conducted using four oligonucleotide probes: pTa535 5 , pSc119.2 5 , (CTT) 10 49 , and pTa713 5 . These probes, labeled with FAM or TAMRA, were synthesized by TsingKe Biological Technology Co., Ltd. (Chengdu, Sichuan, China). EMS mutagenesis and mutant screening. Approximately 10,000 seeds of PI 192051 were evenly distributed into three flasks and treated with 250 mL solutions of 0.6%, 0.5%, and 0.4% EMS (Cat. No. M0880-25G, Sigma-Aldrich, USA). All surviving M 1 plants were grown in the greenhouse, resulting in the development of 1,853 independent M 2 families. Approximately 20 M 2 seeds from each family were challenged with the Pt pathotype PHQS in growth chambers. M 3 seeds derived from susceptible M 2 plants were re-evaluated to confirm phenotypic consistency across generations. To rule out the possibility of seed contamination, all identified mutants were genotyped using 4A-genome specific PCR markers linked to Lr.ace-4A . DNA extraction, library construction, and sequencing. PI 192051 plants were grown in a controlled growth chamber, and fresh leaves from 10-day-old seedlings were harvested for high molecular weight (HMW) DNA extraction. The DNA was sheared into ~ 20 kb fragments using g-TUBE, and libraries were constructed using the PacBio SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA). Libraries were sequenced on the PacBio Revio sequencing platform at Biomarker Technologies Corporation (Qingdao, China). Hi-C libraries were prepared following a modified standard protocol 50 and sequenced on the Illumina HiSeq X platform at the same facility. RNA-seq and Iso-seq. RNA was extracted from the leaves, roots, stems, and spikes of PI 192051 at three growth stages (seedling, booting, and heading). Total RNA extraction, RNA-seq library construction, and sequencing were carried out by Novogene Bioinformatics Technology Co., Ltd. (Tianjin, China), yielding approximately 11 Gb of raw data per sample (Supplementary Table 4). Full-length transcriptome sequencing (PacBio Iso-seq) was also conducted at Novogene (Tianjin, China). Genome assembly and validation. A preliminary genome assembly was generated from 451.11 Gb of PacBio HiFi reads using hifiasm v0.19.5-r587 51 . To achieve a chromosome-scale assembly, 451.46 Gb of Hi-C data were used to resolve contigs into chromosomal scaffolds based on spatial proximity information. The raw Hi-C reads were filtered using fastp v0.23.2 52 and aligned to the contig assembly using BWA mem v0.7.17-r1188 53 . Contigs were anchored into chromosomes using YaHS v1.2a.1 54 , and the assembly was manually refined using Juicebox v1.11 ( https://github.com/aidenlab/Juicebox ). Assembly quality was assessed by analyzing LTR retrotransposons with LTR_FINDER_parallel v1.2 55 (-in harvest) and LTR_retriever 56 , evaluating consensus accuracy with Merqury v1.3 57 (21-mer frequency analysis), and assessing completeness with BUSCO (poales_odb10 dataset) 58 . Gene model prediction. Gene annotation of PI 192051 integrated homology-based protein data, Pacio Iso-seq, and RNA-seq datasets. Gene annotations from T. durum , T. dicoccoides , and the hexaploid wheat variety CS were transferred to the PI 192051 genome using Liftoff 59 . RNA-seq data from roots, stems, leaves, and spikes were processed using fastp 52 , mapped to the PI 192051 genome using HISAT2 v2.2.1, and assembled using Stringtie v2.2.1 60 . Iso-seq data were processed using Lima and IsoSeq3 and mapped on the assembled genome using pbmm2 v1.10.0 ( https://github.com/PacificBiosciences/pbmm2 ). Redundant transcripts were collapsed using cDNA_Cupcake ( https://github.com/Magdoll/cDNA_Cupcake ). RNA-seq and Iso-seq results were merged using the Stringtie–merge function, and open reading frames were identified with TransDecoder v5.5.0 ( https://github.com/TransDecoder/TransDecoder ). Output GFF files were consolidated into a single file using the Perl script “agat_sp_merge_annotation.pl” from the AGAT toolkit ( https://github.com/NBISweden/AGAT ). Redundant transcripts for each gene were removed using CD-HIT v.4.8.1. Annotation completeness was evaluated using BUSCO v1.7.131 58 , and functional annotation was performed using eggNOG-mapper v2.1.1232 61 . Comparative synteny and SNP density assays. Collinearity relationships between the A and B subgenomes of PI 192051 and their diploid, tetraploid, and hexaploid counterparts was analyzed using JCVI 62 . Synteny blocks were identified through an all-against-all BLAST search, with homologous hits concatenated using a 20-gene distance cutoff. To investigate variant density on chromosome 4A, SNPs from whole-genome resequencing of a tetraploid wheat panel (including six accessions each of T. dicoccon , T. durum , and T. dicoccoides ) were used. Sequencing reads (NCBI BioProject No. PRJEB61424) were trimmed using fastp 52 , mapped to the PI 192051 genome using BWA-mem 53 , and deduplicated with GATK 63 . Variants were called using Freebayes 64 and filtered (read depth > 5, quality > 10) with BCFtools. Variant density was calculated as the number of SNPs and indels per 1 Mb region. Candidate gene identification. Total RNA was extracted from leaves of seven susceptible M 3 mutant families inoculated with Pt pathotype PHQS. RNA-Seq was conducted by Novogene Bioinformatics Technology Co., Ltd. (Tianjin, China). To identify the Lr.ace-4A candidate gene, RNA-seq reads from the seven mutants were individually aligned to the PI 192051 genome using STAR 65 . The resulting BAM files were sorted and indexed using SAMtools 66 . Variants were called using Freebayes 64 , followed by filtering (quality score > 10) using BCFtools. CRISPR/Cas9-based gene editing. The CRISPR/Cas9 gene-editing system was used to validate the Lr.ace-4A candidate gene ( PI192051.r1.4AG0210600 ) in the resistant parent PI 192051. The Cas9-Trex2 25 and GRF4-GIF1 26 fusion proteins were used to enhance editing and regeneration efficiency, respectively. gRNAs were designed using the CRISPR-Cereal website ( http://crispr.hzau.edu.cn/CRISPR-Cereal/index.php ). Sequence alignment of homeologous and paralogous of PI192051.r1.4AG0210600 was performed using MUSCLE in MEGA v7.0, followed by specificity validation via BLASTN searches against the Svevo and PI 192051 genomes. The gRNA was synthesized, and then the TaU3p:target-optimized gRNA scaffold was generated through two rounds of PCR using pOPGR-TS1 as a template 67 . PCR amplifications were carried out using the primer pairs Cas9-4AF1R1 , Cas9-4AF2R2 , and Cas9-4AF1R2 (Supplementary Data 1). The resulting fragment containing the gRNA was cloned into the modified pCas9T vector at the Stu I site using the In-Fusion® HD Cloning Kit (Clontech, CA, USA). The final construct was transformed into PI 192051 via A. tumefaciens -mediated transformation. Mutations in transgenic plants were identified using the 4A-genome specific primer pairs pku4AF2R2 (Supplementary Data 1). Wheat transformation. A 9,264-bp genomic fragment, including the complete coding region and introns (4,652 bp), along with 2,949 bp upstream of the start codon and 1,663 bp downstream of the stop codon, was amplified from PI 192051 by PCR using PrimeStar Max DNA Polymerase (TaKaRa, Kyoto, Japan). Overlapping PCR products, generated with the primer pairs p1300‑Lr4AF1R1 and p1300‑Lr4AF2R2 (Supplementary Data 1), were inserted into the linearized binary vector pCAMBIA1300 using the In-Fusion® HD Cloning Kit (Clontech, CA, USA). This construct was transformed into the EMS-induced mutant line m1 and the hexaploid wheat cultivar Fielder via A. tumefaciens -mediated transformation. Four primer pairs pku23F1R1 , pku23F2R2 , pku24F1R1 , and pku24F2R2 (Supplementary Data 1) were used to generate two single-mutation versions of the constructs, each containing one of the critical variations (G1597A or C1984T; amino acid changes E533K or R662C). Primer pairs pku65FR , pku66FR , pku4AF2R2 , and pku4AF3R3 (Supplementary Data 1) were used to confirm the presence of transgenes. Transcript levels in the transgenic plants were quantified using primer pairs pku51FR (Supplementary Data 1). Additionally, the complete coding sequence (3,525 bp) of Lr.ace-4A was amplified from the cDNA of PI 192051 using primer pairs OE-4AFR (Supplementary Data 1). This fragment was cloned into the pCAMBIA1300-OE vector under the maize UBI promoter for overexpression in Fielder. Transgene presence and transcript levels were verified using primer pairs pku16FR , pku20FR , and pku51FR (Supplementary Data 1). Transferring of T. durum segment carrying Lr.ace-4A into hexaploid wheat. PI 192051 was crossed with the bread wheat cultivar YM21. The resulting F 1 plants were backcrossed twice with YM21 to produce BC 2 F 1 . These BC 2 F 1 plants were self-pollinated for two generations to produce BC 2 F 3 . The presence of Lr.ace-4A was validated using the PCR marker Lr30MAS-47FR (Supplementary Data 1). Selected BC 2 F 3 plants were inoculated with the Pt pathotype PHQS. Haplotyping and phylogenetic analysis. Homeologs or orthologs of Lr30 were sourced from publicly accessible Triticeae genomes ( http://plants.ensembl.org/ ) and the NCBI database. Moreover, 59 T. dicoccon accessions were inoculated with the Pt pathotype PHQS, and the coding regions of Lr30 were amplified using 4A-genome specific primer pairs pku4AF1R1 , pku4AF2R2 , and pku4AF3R3 (Supplementary Data 1), followed by Sanger sequencing. Cloned R protein sequences from Gramineae species were obtained from the NCBI database ( http://www.ncbi.nlm.nih.gov/ ). A neighbor-joining phylogenetic tree was constructed using MEGA v7 and visualized with iTOL v7 ( https://itol.embl.de/ ). qRT-PCR analysis. At the three-leaf stage, PI 192051 seedlings were either mock- or Pt -inoculated in two independent growth chambers under identical conditions (24°C day/22°C night, 16 h light/8 h dark). Leaf samples were collected at four time points: 0 h (immediately before inoculation), 1, 2, and 4 dpi. Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, MA, USA) and purified using the Direct-zol RNA MiniPrepPlus Kit (ZymoResearch, CA, USA). qRT-PCR was performed on an ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems, CA, USA) using the primer pairs pku22FR (Supplementary Data 1). The endogenous control TaActin 29 was used to normalize the RNA expression levels using the 2 −ΔCT method 29 , 42 . Significance was estimated using Student’s t -test. In planta expression of GFP-fused Lr30 protein and western blotting analysis. The coding region of the Lr30 protein (amino acids 1-1174) and its CC domain (amino acids 1-188) were cloned into vectors pBIN, pJIM19-GFP, and pJIT163-Ubi-GFP 5 , 68 . These recombinant constructs, along with the empty vector (EV) control, were transiently expressed in N. benthamiana leaves via A. tumefaciens infiltration (OD = 0.6). Wheat protoplasts from Fielder were isolated and transformed using the polyethylene glycol (PEG)-mediated method 69 . Fluorescence imaging was performed using a confocal microscope (A1 HD25 Nikon, Tokyo, Japan). Proteins from A. tumefaciens -transformed tobacco leaves were extracted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using an anti-GFP primary antibody (1:2500; Abcam, Cambridge, UK) and a Goat anti-Rabbit IgG-HRP secondary antibody (1:10000; Abmart, Shanghai, China). Cell death induction assay. The coding region of the mammalian cell death inducer BAX 70 was cloned into pJIM19GFP as a positive control. A. tumefaciens cultures (OD = 1.0) carrying the constructs CDS-GFP, GFP-CDS, CC-GFP, GFP-CC, NB-GFP, GFP-NB, LRR-GFP, GFP-LRR, BAX, and GFP were infiltrated into N. benthamiana leaves. Necrosis induced by BAX was observed at 48 h post inoculation (hpi). Yeast two-hybrid assays. The Lr30 coding sequence and its susceptible haplotypes were cloned into pGBKT7 and pGADT7, respectively. The constructs were co-transformed into Y2H Gold yeast, cultured on SD/-Trp/-Leu medium, and plated on selective medium SD/-Trp/-Leu/-His + 40 µg/mL X-α-gal and SD/-Trp/-Leu/-His/-Ade + 40 µg/mL X-α-gal. Prediction of protein structure. The structural models of Lr30 were generated using AlphaFold2 and AlphaFold-Multimer, specifically configured to predict multimeric assemblies. The computational modeling was conducted on a high-performance computing platform. The resulting models were subsequently visualized and refined using ChimeraX software. Declarations Data availability Data supporting the findings of this work are available within the paper and its supplementary information files. All raw sequencing data, genome assembly, and gene annotation for this project are archived at the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, under BioProject accession number PRJCA036461. The sequence of the Lr30 gene was deposited in NCBI GenBank under accession number PV159345. Source data are provided with this paper. Acknowledgements Work at SC laboratory was supported by the National Key Research and Development Program of China (2022YFD1201300), the Key R&D Program of Shandong Province (2024LZGC034 and 2023LZGC022), and the Shandong Provincial Natural Science Foundation (SYS202206), the National Natural Science Foundation of China (32472159), and the Taishan Scholars Program. Work at XW laboratory was supported by National Key Research and Development Program of China (2023YFD1201002), Provincial Natural Science Foundation of Hebei (C2022204010). State Key Laboratory of North China Crop Improvement and Regulation (NCCIR2024ZZ-5), and S&T Program of Hebei (23567601H). Author contributions S.C. designed the research; J.Y., H.L., M.L., and R.S. conducted the experiments; T.S. and S.R. contributed to EMS mutants; B.S., H.L., and D.X. performed the genome assembly; G.W., L.H., and Y.L. contributed to vector construction; M.H. conducted cytogenetic assays; A.J. and C.L. contributed to AlphaFold prediction; C.L., X.W.D., and J.D. provided scientific support; S.C., J.Y., H.L., and R.S. analyzed the data. S.C., J.Y., and X.W. wrote the initial manuscript; S.C. generated the final manuscript; S.C., X.W., and B.S. supervised the project. All authors reviewed and revised the manuscript. Competing interests S.C., J.Y., L.H., R.S., and S.L. are inventors on a Chinese provisional patent application (China patent filing No.202510124660.9) relating to the use of the Lr30 gene in wheat breeding programs. 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S.C., J.Y., L.H., R.S., and S.L. are inventors on a Chinese provisional patent application (China patent filing No.202510124660.9) relating to the use of the Lr30 gene in wheat breeding programs. The remaining authors declare no competing interests. Supplementary Files SupplementaryData1.xlsx Dataset 1 Lr30reportingsummary1new11.pdf Report Summary SupplementaryFiguresandTables32025.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6289485","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":434486812,"identity":"773dfb14-0cdb-4a34-82d0-009c0c9fa7f4","order_by":0,"name":"Shisheng 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Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Aolin","middleName":"","lastName":"Jia","suffix":""},{"id":434486822,"identity":"a5a31bc1-981a-4e09-8917-e21f2d45ff09","order_by":10,"name":"Shams ur Rehman","email":"","orcid":"","institution":"State Key Laboratory of Wheat Improvement, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Shandong 261325, China","correspondingAuthor":false,"prefix":"","firstName":"Shams","middleName":"ur","lastName":"Rehman","suffix":""},{"id":434486823,"identity":"0a1b7e98-96b6-4e5e-a625-b73454d9a018","order_by":11,"name":"Lei Hua","email":"","orcid":"https://orcid.org/0000-0002-6141-7649","institution":"State Key Laboratory of Wheat Improvement, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in 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Song","email":"","orcid":"https://orcid.org/0000-0003-1478-9228","institution":"Peking University Institute of Advanced Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Baoxing","middleName":"","lastName":"Song","suffix":""},{"id":434486830,"identity":"93fda6b0-2652-4b71-b191-aa253115840a","order_by":18,"name":"Xiaodong Wang","email":"","orcid":"https://orcid.org/0000-0002-4304-5482","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-03-23 17:00:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6289485/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6289485/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-64428-5","type":"published","date":"2025-10-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79330818,"identity":"038f0783-7da5-4a7c-89ad-cd0bef9b9478","added_by":"auto","created_at":"2025-03-27 06:36:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2319733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDurum wheat landrace PI 192051 exhibits high resistance to multiple \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epathotypes.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Seedlings of PI 192051 exhibited robust resistance to eight \u003cem\u003ePt\u003c/em\u003e pathotypes, while the durum line Rusty displayed high susceptibility. R, resistant; S, susceptible. \u003cstrong\u003eb\u003c/strong\u003e Infection types in PI 192051 and Rusty in response to \u003cem\u003ePt\u003c/em\u003e pathotypePHQS at 2, 4, 6, and 8 dpi. \u003cstrong\u003ec\u003c/strong\u003e Fluorescent staining of fungal structures. Leaves were collected at 2, 4, 6, and 8 dpi, cleared with KOH, and stained with WGA-FITC. Scale bars represent 100 μm. \u003cstrong\u003ed\u003c/strong\u003e Average infection areas observed microscopically were significantly smaller in PI 192051 (blue) compared to Rusty (pink) across all four time points. Data were obtained from 15 independent fungal infection sites (n = 15). Black dots represent single data points.Asterisks denote the level of significance by two-sided unpaired \u003cem\u003et\u003c/em\u003e-test. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Error bars represent standard errors of the means. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/54255b9d8a78f1594b422aeb.png"},{"id":79332075,"identity":"0f64201c-8ef1-4daa-8bc9-5f9a88552fe7","added_by":"auto","created_at":"2025-03-27 06:52:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2030016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA high-quality genome assembly of the durum wheat landrace PI 192051. a \u003c/strong\u003eCircular diagram showing the assembly features of PI 192051. The tracks, arranged from outermost to innermost, include: (i) chromosomes, (ii) GC content, (iii) gene density, and (iv) syntenic connections between A and B subgenomes. \u003cstrong\u003eb \u003c/strong\u003eComparison of genome assembly quality (contig N50) between PI 192051and other published tetraploid (turquoise blue) and hexaploid (pink) wheat genomes. The horizontal axis represents the year, and the vertical axis represents the contig N50 size. Each circle represents one genome, with the diameter of the circle corresponding to the genome size. PI 192051 is highlighted with a red arrow. \u003cstrong\u003ec \u003c/strong\u003eSyntenic blocks among \u003cem\u003eT. urartu\u003c/em\u003e (G1812 v2.0), \u003cem\u003eT. monococcum\u003c/em\u003e (PI 306540), \u003cem\u003eT. durum\u003c/em\u003e (PI 192051 and Svevo v1.0), \u003cem\u003eT. dicoccoides \u003c/em\u003e(Zavitan v1.0),\u003cem\u003e Ae. searsii \u003c/em\u003e(TE01), \u003cem\u003eAe. speltoides \u003c/em\u003e(TS01), and the A/B subgenomes of the bread wheat variety Chinese Spring (CS, RefSeq v2.1). Each line represents a syntenic block of 15 or more gene pairs with ≥ 80% identity.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/307440a50095b04e3374a8a1.png"},{"id":79330822,"identity":"f21fab8b-a7e2-4981-8ce1-2bd05b41d174","added_by":"auto","created_at":"2025-03-27 06:36:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1597939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-assisted identification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLr.ace-4A\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Susceptible EMS mutants used to isolate\u003cem\u003e \u003c/em\u003ethe\u003cem\u003eLr.ace-4A\u003c/em\u003e gene. Infection types for PI 192051, seven independent susceptible EMS mutants (m13, m12, m22, m1, m23, m2, and m10), and Rusty inoculated with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. R, resistant; S, susceptible. \u003cstrong\u003eb\u003c/strong\u003e Integrative Genomics Viewer (IGV) snapshots showing RNA-seq readsfrom susceptible EMS mutantsmapped to thePI 192051 genome. Blue arrows indicate the positions of EMS-induced point mutations (G/C to A/T) identified in the sevensusceptible mutants. WT, wild-type PI 192051. \u003cstrong\u003ec \u003c/strong\u003eGene\u003cstrong\u003e \u003c/strong\u003estructure of \u003cem\u003eLr.ace-4A\u003c/em\u003e. The positions of the EMS-induced mutations are indicated byblue arrows. Gray boxes represent untranslated regions, blue boxes indicate coding exons, and gray lines denote introns. The start and stop codons are indicated by black arrows. \u003cstrong\u003ed \u003c/strong\u003eProtein structure of Lr.ace-4A. The coiled-coil (CC), nucleotide-binding site (NB), and leucine-rich repeat (LRR) domains are highlighted in yellow, green, and pink, respectively, according to the Pfam protein families database.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/56bd65bd4d97accced0c0a75.png"},{"id":79330832,"identity":"8a642513-a57f-461e-8724-d8d12e3e5429","added_by":"auto","created_at":"2025-03-27 06:36:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2548540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional validation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLr.ace-4A\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by CRISPR/Cas9-mediated gene editing and transgenic complementation.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Sequencing chromatogram showing the induced polymorphisms between the WT and selected CRISPR/Cas9-induced editing mutants (T\u003csub\u003e0\u003c/sub\u003eKO-1 and T\u003csub\u003e0\u003c/sub\u003eKO-2). The red box indicates the gRNA targeting \u003cem\u003eLr.ace-4A\u003c/em\u003e. The mutated regions are underlined, and the PAM sequence is highlighted in red. +1/-1 bp indicates an insertion or deletion of one base pair. \u003cstrong\u003eb\u003c/strong\u003e Infection types of homozygous edited transgenic T\u003csub\u003e1\u003c/sub\u003e plants, PI 192051, and Rusty in response to \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. R, resistant; S, susceptible. \u003cstrong\u003ec\u003c/strong\u003e A 9,264-bp genomic DNA fragment carrying \u003cem\u003eLr.ace-4A\u003c/em\u003e was used for genetic transformation. This fragment includes the complete coding region and introns (4652 bp), 2949 bp upstream of the start codon, and 1663 bp downstream of the stop codon. \u003cstrong\u003ed\u003c/strong\u003e Infection types of transgenic T\u003csub\u003e1\u003c/sub\u003e plants, PI 192051, and Rusty in response to \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. -, absence of \u003cem\u003eLr.ace-4A\u003c/em\u003e; +, presence of \u003cem\u003eLr.ace-4A\u003c/em\u003e. R, resistant; S, susceptible.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/b198262eb34b7ee8ade62f14.png"},{"id":79330831,"identity":"62125a79-578b-410d-998f-52a306bc8058","added_by":"auto","created_at":"2025-03-27 06:36:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5022205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransgenic complementation in the hexaploid wheat background. a\u003c/strong\u003e A 9,264-bp genomic DNA fragment containing \u003cem\u003eLr.ace-4A\u003c/em\u003e was introduced into the susceptible wheat cultivar Fielder. This fragment encompasses the complete coding region and introns (4652 bp), 2949 bp upstream of the start codon, and 1663 bp downstream of the stop codon. \u003cstrong\u003eb\u003c/strong\u003e Infection types of transgenic T\u003csub\u003e0\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e plants in the Fielder background when challenged with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. The transgenic family T\u003csub\u003e1\u003c/sub\u003eC652-27 exhibited significantly higher transgene expression levels (Supplementary Fig. 10), whereas family T\u003csub\u003e1\u003c/sub\u003eC652-3 showed segregation. RL6049 was used as the positive control, and Fielder served as the negative control. \u003cstrong\u003ec\u003c/strong\u003e The 3525-bp coding sequence (CDS) of \u003cem\u003eLr.ace-4A\u003c/em\u003e, driven by the \u003cem\u003eUBI\u003c/em\u003e promoter, was transformed into Fielder. The black arrow indicates the coding region. LB and RB\u0026nbsp;denote the left and right borders of the T-DNA, respectively. \u003cstrong\u003ed \u003c/strong\u003eInfection types of transgenic T\u003csub\u003e0\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e plants overexpressing \u003cem\u003eLr.ace-4A\u003c/em\u003e in the Fielder background in response to \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. The presence (+) or absence (−) of \u003cem\u003eLr.ace-4A\u003c/em\u003e is indicated. R, resistant; S, susceptible.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/bdba5624b0130912678eebc9.png"},{"id":79330825,"identity":"a47c91ba-5c90-45ea-9f72-6389418b9d8a","added_by":"auto","created_at":"2025-03-27 06:36:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3951100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResistance profile of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLr30\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgene and transgenic wheat lines expressing the 9,264 bp genomic fragment with either the Lr30\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e533K\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e or Lr30\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e662C\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e amino acid substitution. a\u003c/strong\u003e Phenotypic evaluation of two transgenic T\u003csub\u003e1\u003c/sub\u003e families (T₁C652-27 and T₁C652-12) with five different \u003cem\u003ePt\u003c/em\u003e pathotypes (FHJL, HCJR, FHJR, PHST, PHJS). R, resistant; S, susceptible. \u003cstrong\u003eb \u003c/strong\u003eTransgenic wheat lines expressing the 9,264 bp genomic fragment with the Lr30\u003csup\u003e533K\u003c/sup\u003e amino acid substitution. The critical variation 1597A (amino acid 533K) is highlighted with a red arrow. Transgenic T\u003csub\u003e0\u003c/sub\u003e plants and the Fielder control were susceptible when challenged with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, whereas PI 192051 exhibited strong resistance. \u003cstrong\u003ec\u003c/strong\u003e Transgenic wheat lines expressing the 9,264 bp genomic fragment with the Lr30\u003csup\u003e662C\u003c/sup\u003e amino acid substitution. The critical variation 1984T (amino acid 662C) is indicated by a red arrow. Transgenic T\u003csub\u003e0\u003c/sub\u003e plants and the Fielder control were susceptible, while PI 192051 displayed high resistance. R, resistant; S, susceptible.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/c920c5c899bcfbc030f8c430.png"},{"id":79330839,"identity":"6af003b9-7c4c-4108-81ac-94b10675665d","added_by":"auto","created_at":"2025-03-27 06:36:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5239581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional characterization of Lr30. a\u003c/strong\u003e Transcript levels of \u003cem\u003eLr30\u003c/em\u003e in mock-inoculated and \u003cem\u003ePt\u003c/em\u003e-inoculated PI 192051 plants. Transcript levels were quantified in threebiological replicates (n = 3) and expressed as fold-\u003cem\u003eActin\u003c/em\u003e. Black open dots represent individual data points. Statistical significance was determined using a two-sided unpaired \u003cem\u003et\u003c/em\u003e-test. Error bars indicate standard errors of the mean (SEM). ns = not significant; \u003cem\u003e*\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eb\u003c/strong\u003e \u0026nbsp;Subcellular localization of Lr30 in \u003cem\u003eN. benthamiana \u003c/em\u003eleaves using GFP-tagged constructs. This experiment was repeated three times with consistent results. BF, bright field; GFP, green fluorescent protein. Scale bars represent 50 μm. \u003cstrong\u003ec\u003c/strong\u003e Subcellular localization of the GFP-fused Lr30 protein in wheat protoplasts. This experiment was repeated three times with consistent results. Scale bars represent 10 μm. \u003cstrong\u003ed\u003c/strong\u003e \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transient expression analysis of Lr30 and its domains in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves.CC, coiled-coil; NB, nucleotide binding; LRR, leucine-rich repeat; CDS, coding sequence of Lr30; BAX, a mammalian cell death induceras a positive control. \u003cstrong\u003ee \u003c/strong\u003ePredicted resistosome structures of Lr30 generated using AlphaFold2 and AlphaFold-Multimer. Red arrows indicate the critical amino acid substitutions E533 and R662. Source data are provided in the Source Data file.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/beb068c0ec7d57c4e7b48021.png"},{"id":94169280,"identity":"f47b1fb7-5a1c-4d6f-aff0-84fc11985644","added_by":"auto","created_at":"2025-10-23 07:06:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33760237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/972f347b-43ab-474b-a515-fc6943040a7c.pdf"},{"id":79331641,"identity":"7fede064-be58-4175-a6c6-f4f08ccaa6c8","added_by":"auto","created_at":"2025-03-27 06:44:03","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15304,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"SupplementaryData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/873d6aaf44c1ab56d0c9a1d0.xlsx"},{"id":79330824,"identity":"8fecf0f9-ce9c-47a6-9ff7-d8f7e98a683b","added_by":"auto","created_at":"2025-03-27 06:36:03","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":255450,"visible":true,"origin":"","legend":"Report Summary","description":"","filename":"Lr30reportingsummary1new11.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/2a85ef0f2db97d576e695db4.pdf"},{"id":79331643,"identity":"6f14a527-e468-417e-aa93-99e8482afc7a","added_by":"auto","created_at":"2025-03-27 06:44:04","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3694924,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresandTables32025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6289485/v1/5d5e1900f07842bf24d60900.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nS.C., J.Y., L.H., R.S., and S.L. are inventors on a Chinese provisional patent application (China patent filing No.202510124660.9) relating to the use of the Lr30 gene in wheat breeding programs. The remaining authors declare no competing interests.","formattedTitle":"Genome-assisted identification of wheat leaf rust resistance gene Lr30 (synonym Lr.ace-4A)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWheat is a major staple food crop, contributing about one-fifth of the total calories and proteins consumed by humankind. An effective strategy to increase wheat production is to mitigate losses caused by fungal pathogens. \u003cem\u003ePuccinia triticina\u003c/em\u003e Eriksson (\u003cem\u003ePt\u003c/em\u003e), the causal agent of wheat leaf rust, represents one of the most formidable threats to global wheat production. This disease affects most wheat-growing areas and can significantly reduce yields in susceptible wheat varieties under favorable weather conditions\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Due to the impacts of global warming and the evolving virulence of the pathogens, leaf rust has significantly expanded its geographical range and poses a grave threat to global wheat production\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the strategies to control this devastating disease, breeding for leaf rust resistance is considered the most feasible and sustainable approach. To date, more than 80 leaf rust resistance (\u003cem\u003eLr\u003c/em\u003e) genes have been assigned official designations in wheat and its wild relatives\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Nevertheless, owing to the vast size and complexity of the wheat genome, only twelve \u003cem\u003eLr\u003c/em\u003e genes have been successfully cloned, using either traditional map-based cloning methods (\u003cem\u003eLr1\u003c/em\u003e, \u003cem\u003eLr10\u003c/em\u003e, \u003cem\u003eLr21\u003c/em\u003e, \u003cem\u003eLr34\u003c/em\u003e, \u003cem\u003eLr42\u003c/em\u003e, \u003cem\u003eLr67\u003c/em\u003e)\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e or advanced gene-cloning techniques, including MutRenSeq (\u003cem\u003eLr13\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, TACCA (\u003cem\u003eLr22a\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, MutChromSeq (\u003cem\u003eLr14a\u003c/em\u003e and \u003cem\u003eLr85\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, MutIsoSeq (\u003cem\u003eLr9\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and MutRNASeq (\u003cem\u003eLr47\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Cloning additional \u003cem\u003eLr\u003c/em\u003e genes is highly desirable, as it would enable the development of more diverse \u003cem\u003ePt\u003c/em\u003e resistance gene combinations in transgenic cassettes or gene pyramids, promoting more durable resistance.\u003c/p\u003e \u003cp\u003eWheat resistance to leaf rust can be classified into two primary categories: race-specific resistance and slow rusting resistance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Race-specific resistance is based on the gene-for-gene hypothesis. Most race-specific \u003cem\u003eLr\u003c/em\u003e genes encode coiled-coil nucleotide-binding leucine-rich repeat (NLR) proteins\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, with the exceptions of \u003cem\u003eLr14a\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eLr9\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e, which encode proteins featuring twelve ankyrin repeats and an N-terminal tandem kinase domain followed by vWA/Vwaint domains, respectively. The slow rusting resistance genes, \u003cem\u003eLr34\u003c/em\u003e and \u003cem\u003eLr67\u003c/em\u003e, encode a putative ATP-binding cassette transporter\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and a hexose transporter\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, respectively. Recent advances in protein crystallization have demonstrated that the wheat stem rust resistance protein Sr35, upon recognizing the avirulence protein AvrSr35 from the pathogen, forms a homo-pentameric resistosome, resulting in hypersensitive responses (HR) or necrosis on wheat leaves\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTriticum turgidum\u003c/em\u003e ssp. \u003cem\u003edurum\u003c/em\u003e, a key member of the primary gene pool of wheat, is cultivated across approximately 18\u0026nbsp;million hectares worldwide\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In 2019, a draft assembly of the \u003cem\u003eT. durum\u003c/em\u003e genome (accession Svevo, BBAA) was released\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, the assembly remains in low contiguity (contig N50\u0026thinsp;=\u0026thinsp;0.06 Mb), with a total of 309,814 gaps\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A more contiguous genome assembly is essential for enhancing gene identification in durum wheat. Several cataloged \u003cem\u003eLr\u003c/em\u003e genes, such as \u003cem\u003eLr3a\u003c/em\u003e, \u003cem\u003eLr14a\u003c/em\u003e, \u003cem\u003eLr27\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eLr31\u003c/em\u003e, \u003cem\u003eLr61\u003c/em\u003e, \u003cem\u003eLr72\u003c/em\u003e, \u003cem\u003eLr79\u003c/em\u003e, and \u003cem\u003eLrCamayo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, have been detected in durum wheat cultivars. However, among the \u003cem\u003eLr\u003c/em\u003e genes present in durum wheat, only \u003cem\u003eLr14a\u003c/em\u003e has been successfully cloned so far\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, likely reflecting the challenges posed by the large and complex wheat genomes.\u003c/p\u003e \u003cp\u003eSeedlings of the Portuguese landrace of durum wheat, PI 192051, exhibited robust resistance [infection type (IT)\u0026thinsp;=\u0026thinsp;0;] to four \u003cem\u003ePt\u003c/em\u003e pathotypes originating from the USA, Tunisia, Morocco, and Ethiopia\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Genetic analysis using recombinant inbred lines (RILs) derived from the cross Rusty (susceptible) \u0026times; PI 192051 (resistant) and the Illumina iSelect 9K wheat SNP array, revealed a dominant \u003cem\u003eLr\u003c/em\u003e gene, designated as \u003cem\u003eLr.ace-4A\u003c/em\u003e, located in the centromeric region of chromosome arm 4AS\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Conversely, the \u003cem\u003eLr30\u003c/em\u003e gene, which confers intermediate resistance (ITs\u0026thinsp;=\u0026thinsp;1 to 2), was identified as a recessive resistance gene in the hexaploid wheat cultivar Terenzio\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This gene was initially mapped to chromosome arm 4BL\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, but its locus was later corrected to chromosome 4AL\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eLr30\u003c/em\u003e has not been reported in durum wheat and seems to be quite rare in bread wheat germplasm\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Recently, three Canadian spring wheat cultivars (AAC Prevail, AAC Concord, and Lillian) were postulated to carry \u003cem\u003eLr30\u003c/em\u003e\u003csup\u003e22\u003c/sup\u003e. While \u003cem\u003eLr30\u003c/em\u003e continues to exhibit resistance against approximately half of the \u003cem\u003ePt\u003c/em\u003e pathotypes collected in China\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, progress in its genetic interpretation has significantly lagged.\u003c/p\u003e \u003cp\u003eHere, we generate a high-quality genome assembly of the durum wheat landrace PI 192051 (contig N50\u0026thinsp;=\u0026thinsp;42.53 Mb) and successfully clone the leaf rust resistance gene \u003cem\u003eLr.ace-4A\u003c/em\u003e. This gene encodes a non-canonical NLR receptor with tandem NB-ARC domains. Further investigations demonstrate that \u003cem\u003eLr.ace-4A\u003c/em\u003e in durum wheat corresponds to the \u003cem\u003eLr30\u003c/em\u003e gene previously identified in common wheat. Transformation of a 9.3-kb genomic sequence containing \u003cem\u003eLr.ace-4A\u003c/em\u003e into the susceptible \u003cem\u003eT. aestivum\u003c/em\u003e cultivar Fielder and the \u003cem\u003eT. durum\u003c/em\u003e ethyl methanesulfonate (EMS) mutant line m1 from PI 192051 demonstrate that this gene is less effective in a hexaploid background compared to in a tetraploid background. Two amino acid polymorphisms distinguish the resistant and susceptible \u003cem\u003eLr30\u003c/em\u003e haplotypes. Transformation with the 9.3-kb genomic sequences carrying either of these variations results in susceptibility. Additionally, we develop a diagnostic molecular marker for \u003cem\u003eLr30\u003c/em\u003e, which will facilitate its deployment in wheat breeding programs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDurum wheat landrace PI 192051 exhibited robust resistance to multiple\u003c/b\u003e \u003cb\u003ePt\u003c/b\u003e \u003cb\u003epathotypes.\u003c/b\u003e Seedlings of the durum wheat landrace PI 192051 exhibited robust resistance to eight \u003cem\u003ePt\u003c/em\u003e pathotypes (Supplementary Table\u0026nbsp;1) collected in China. In contrast, another durum wheat genotype, Rusty, displayed high susceptibility to all \u003cem\u003ePt\u003c/em\u003e pathotypes tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). When challenged with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, Rusty seedlings showed visible rust spores at six days post-inoculation (dpi), whereas PI 192051 exhibited a typical HR in the leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Microscopic analysis with WGA-FITC staining revealed that although rust fungi successfully formed haustoria in PI 192051, the expansion of secondary rust hyphae was restricted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In contrast, in the susceptible line Rusty, the rust hyphae expanded extensively, forming a diffuse network of fungal growth at the infection sites. At all four time points, the average infection areas in PI 192051 were significantly smaller (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than in Rusty (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the 286 F\u003csub\u003e2\u003c/sub\u003e plants derived from the cross PI 192051 \u0026times; Rusty and evaluated with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, we identified 76 resistant plants and 210 susceptible ones (Supplementary Fig.\u0026nbsp;1a). This distribution corresponded to the expected 1:3 segregation ratio for a single recessive gene (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.38, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.54). However, a subset of 315 F\u003csub\u003e2\u003c/sub\u003e plants from the cross between PI 192051 and m1 (a susceptible EMS-induced mutant line derived from the PI 192051 mutant population) was also evaluated with PHQS. Among these, 240 plants were resistant and 75 were susceptible (Supplementary Fig.\u0026nbsp;1b), which fits well the 3:1 segregation ratio expected for a single dominant gene (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.63).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic mapping of\u003c/b\u003e \u003cb\u003eLr.ace-4A\u003c/b\u003e. \u003cem\u003eLr.ace-4A\u003c/em\u003e was previously mapped to chromosome 4A of durum wheat PI 192051\u003csup\u003e18\u003c/sup\u003e, located within a 4.0 cM region flanked by markers \u003cem\u003eIWA232\u003c/em\u003e and \u003cem\u003eIWA1793\u003c/em\u003e (145.24\u0026ndash;562.83 Mb; Svevo RefSeq v1.0; Supplementary Fig.\u0026nbsp;2a). In this study, we first performed RNA-seq analysis to identify single nucleotide polymorphisms (SNPs) between the tetraploid parental lines PI 192501 and Rusty. Using the 286 F\u003csub\u003e2:3\u003c/sub\u003e families evaluated with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS and six newly developed PCR markers (Supplementary Data 1) on chromosome 4A, \u003cem\u003eLr.ace-4A\u003c/em\u003e was refined to a 2.45 cM genetic interval flanked by markers \u003cem\u003eIWA232\u003c/em\u003e and \u003cem\u003epku2574\u003c/em\u003e (Supplementary Fig.\u0026nbsp;2b). In this population, recombination suppression was observed in a region spanning approximately 145.24 to 562.83 Mb of chromosome 4A. Such suppression could arise from alien introgressions or inverted chromosomal segments or the centromeric region. Cytogenetic analyses revealed no evidence of alien introgression in PI 192051 or chromosomal inversions between the parental lines PI 192051 and Rusty (Supplementary Fig.\u0026nbsp;3), indicating that the gene is likely located within the centromeric region.\u003c/p\u003e \u003cp\u003eTo further eliminate the possibility of chromosomal inversions or alien introgression within the \u003cem\u003eLr.ace-4A\u003c/em\u003e mapping region, we used the 315 F\u003csub\u003e2\u003c/sub\u003e plants derived from the cross PI 192051 \u0026times; m1 to map \u003cem\u003eLr.ace-4A\u003c/em\u003e. RNA sequencing of both PI 192051 and m1 allowed us to identify EMS-induced SNPs (Supplementary Table\u0026nbsp;2) and develop seven new PCR markers on chromosome 4A (Supplementary Data 1). In this population, we mapped \u003cem\u003eLr.ace-4A\u003c/em\u003e between markers \u003cem\u003epku8123\u003c/em\u003e and \u003cem\u003epku4169\u003c/em\u003e (101.33\u0026ndash;536.89 Mb; Supplementary Fig.\u0026nbsp;2d). According to the mapping results from both populations, the markers from \u003cem\u003epku1280\u003c/em\u003e (162.15 Mb; Svevo RefSeq v1.0) to \u003cem\u003epku0332\u003c/em\u003e (519.32 Mb) were completely linked to \u003cem\u003eLr.ace-4A\u003c/em\u003e (Supplementary Fig.\u0026nbsp;2b-d). These results indicate that recombination suppression likely extends across a substantial portion of chromosome 4A, covering at least the region from 162.15 to 519.32 Mb. This recombination-suppressed region encompasses the centromere region (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDe novo\u003c/b\u003e \u003cb\u003egenome assembly of PI 192051 and genome-assisted identification of\u003c/b\u003e \u003cb\u003eLr.ace-4A\u003c/b\u003e. To clone \u003cem\u003eLr.ace-4A\u003c/em\u003e, we constructed a chromosome-scale reference genome for the resistant parent PI 192051. Using 451.11 Gb of PacBio High-Fidelity (HiFi) reads and 451.46 Gb of high-throughput chromosome conformation capture sequencing (Hi-C) reads (Supplementary Table\u0026nbsp;4), we generated a high-quality genome assembly with a total size of 10.51 Gb. This assembly features a scaffold N50 of 749.44 Mb, includes all 14 chromosomes, and captures 26 telomeres (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Supplementary Table\u0026nbsp;5). The contiguity of this PI 192051 genome assembly, with a contig N50 of 42.53 Mb, significantly exceeds that of \u003cem\u003eT. dicoccoides\u003c/em\u003e accession Zavitan (contig N50, 0.06 Mb) and \u003cem\u003eT. durum\u003c/em\u003e genotype Svevo (contig N50, 0.06 Mb), establishing it as one of the highest-quality durum wheat genomes to date (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb; Supplementary Tables\u0026nbsp;5 and 6). The assembly quality was further validated by a high LTR Assembly Index (LAI) score of 19.71, a Quality Assessment (QA) score of 64.54, and a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score of 98.4% (Supplementary Table\u0026nbsp;5), collectively indicating a highly continuous and complete genome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 65,860 protein-coding genes were predicted in PI 192051 using a combination of homoeologous protein sequences, Iso-seq, and RNA-seq data derived from four tissues (leaf, root, stem, and spike) across multiple developmental stages (Supplementary Table\u0026nbsp;4). Synteny analysis based on high-confidence annotated genes demonstrated strong collinearity between the PI 192051 genome and the A/B subgenomes of Zavitan (\u003cem\u003eT. dicoccoides\u003c/em\u003e), Svevo (\u003cem\u003eT. durum\u003c/em\u003e), and Chinese Spring (CS; \u003cem\u003eT. aestivum\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eTo investigate genetic variation within the recombination-suppressed region, we obtained whole-genome resequencing data of a tetraploid wheat panel, comprising six accessions each of \u003cem\u003eT. dicoccon\u003c/em\u003e, \u003cem\u003eT. durum\u003c/em\u003e, and \u003cem\u003eT. dicoccoides\u003c/em\u003e (GenBank accession number PRJEB61424). Using the identified SNPs, we analyzed variant density on chromosome 4A and observed a significant reduction in genetic variation spanning approximately 150 Mb to 530 Mb (Supplementary Fig.\u0026nbsp;4). This region coincides with the identified recombination-suppressed interval (Supplementary Fig.\u0026nbsp;2). Reduced variation is expected in a centromeric region lacking recombination, as the entire region is selected as a single block.\u003c/p\u003e \u003cp\u003eEMS mutagenesis was carried out on PI 192051, resulting in the generation of 1,853 independent M\u003csub\u003e2\u003c/sub\u003e mutant families. Screening of these M\u003csub\u003e2\u003c/sub\u003e mutant families with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS identified seven independent families segregating susceptible plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), with susceptibility further validated using progeny testing. Genotyping with six 4A-genome specific markers confirmed that these mutant lines retained the PI 192051 allele.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the candidate gene for \u003cem\u003eLr.ace-4A\u003c/em\u003e, we generated RNA-seq reads from \u003cem\u003ePt\u003c/em\u003e-inoculated leaves of seven independent susceptible M\u003csub\u003e3\u003c/sub\u003e mutants. The resulting clean reads were mapped to the annotated genes within the \u003cem\u003eLr.ace-4A\u003c/em\u003e candidate region on chromosome 4A of the PI 192051 genome. This analysis led to the identification of an NLR gene, \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e, which displayed EMS-induced (G/C-to-A/T) point mutations in all seven susceptible mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). Using three primer pairs \u003cem\u003epku4AF1R1\u003c/em\u003e, \u003cem\u003epku4AF2R2\u003c/em\u003e, and \u003cem\u003epku4AF3R3\u003c/em\u003e (Supplementary Data 1) developed from this candidate gene, we performed PCR amplification of the regions harboring the mutations and confirmed the presence of nucleotide transitions in these susceptible mutants (Supplementary Fig.\u0026nbsp;5). All these EMS-induced mutations resulted in nonsynonymous amino acid substitutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e is located at 513.5 Mb in the Svevo reference genome and co-segregated with the phenotype in both mapping populations. This gene comprises three exons and two introns, encoding a non-canonical NLR protein of 1,174 amino acids (GenBank accession number PV159345), characterized by tandem NB-ARC domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Using \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e5\u003c/span\u003e\u0026prime; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand 3\u003c/span\u003e\u0026prime; rapid amplification of cDNA ends (RACE; Supplementary Fig.\u0026nbsp;6), we determined that the 5\u0026prime;-untranslated region (UTR) of \u003cem\u003eLr.ace-4A\u003c/em\u003e is 87 bp and the 3\u0026prime;-UTR spans 329 bp. These findings identify \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e as the candidate functional gene responsible for \u003cem\u003eLr.ace-4A\u003c/em\u003e-mediated resistance to \u003cem\u003ePt\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFunctional validation of\u003c/b\u003e \u003cb\u003eLr.ace-4A\u003c/b\u003e \u003cb\u003eusing CRISPR/Cas9-mediated gene editing and transgenic complementation.\u003c/b\u003e To knockout \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e in PI 192051, a gene editing system was initially established in tetraploid wheat. This system was refined by incorporating Cas9-Trex2 and GRF4-GIF1 fusion proteins\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e to enhance both editing and regeneration efficiency. A guide RNA (gRNA) targeting the second exon of \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e was designed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). To assess potential off-target effects, we conducted BLASTN searches using the designed gRNA and PAM sequence (5'-GCCAATGAGACTATTAACCGTGG-3') as queries against both the Svevo and PI 192051 genomes. No off-target sites were detected in either genome. We successfully generated 39 independent transgenic T\u003csub\u003e0\u003c/sub\u003e plants. Genotyping of these T\u003csub\u003e0\u003c/sub\u003e plants revealed that 32 plants (82.1% of the total) contained mutations at the target site (Supplementary Fig.\u0026nbsp;7). Among these, two homozygous edited T\u003csub\u003e0\u003c/sub\u003e plants, T\u003csub\u003e0\u003c/sub\u003eKO-1 (an \"A\" insertion) and T\u003csub\u003e0\u003c/sub\u003eKO-2 (an \"A\" deletion), were selected for further analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). All T\u003csub\u003e1\u003c/sub\u003e progeny from these two knockout T\u003csub\u003e0\u003c/sub\u003e plants were susceptible to the \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, while the WT PI 192051 retained its resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These results suggest that \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e is required for \u003cem\u003eLr.ace-4A\u003c/em\u003e-mediated resistance to leaf rust in durum wheat.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e is sufficient to confer resistance to leaf rust, a 9,264 bp genomic DNA fragment (GenBank accession number PV159345) derived from PI 192051, encompassing the complete transcribed region and native regulatory sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), was introduced into the susceptible EMS mutant line m1 via \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation. A total of 25 independent transgenic T\u003csub\u003e0\u003c/sub\u003e plants were generated, of which nine were randomly selected for further analysis. The presence and expression of the transgene in these selected T\u003csub\u003e0\u003c/sub\u003e plants were confirmed through PCR and qRT-PCR analyses (Supplementary Fig.\u0026nbsp;8a). All transgenic T\u003csub\u003e0\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e plants carrying the transgene exhibited robust resistance to \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, whereas the untransformed m1 control displayed susceptibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;8b). Taken together, the genetic mapping, EMS and CRISPR/Cas9-induced mutations, and transgenic complementation results demonstrated that \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e is \u003cem\u003eLr.ace-4A\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLr.ace-4A\u003c/b\u003e \u003cb\u003eis synonymous with\u003c/b\u003e \u003cb\u003eLr30\u003c/b\u003e, \u003cb\u003eexhibiting diminished resistance in a hexaploid genetic background.\u003c/b\u003e \u003cem\u003eLr30\u003c/em\u003e is the only \u003cem\u003eLr\u003c/em\u003e gene currently cataloged on chromosome arm 4AL. According to the \u003cem\u003eLr30\u003c/em\u003e map on the GrainGenes website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wheat.pw.usda.gov/GG3/\u003c/span\u003e\u003cspan address=\"https://wheat.pw.usda.gov/GG3/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), this gene is flanked by \u003cem\u003eIWA4359\u003c/em\u003e (514.10 Mb; Svevo RefSeq v1.0) and \u003cem\u003eIWA2585\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003e (536.89 Mb). The location of \u003cem\u003eLr30\u003c/em\u003e closely coincides with that of \u003cem\u003eLr.ace-4A\u003c/em\u003e (513.5 Mb; Svevo RefSeq v1.0). Sequencing of \u003cem\u003eLr.ace-4A\u003c/em\u003e in the \u003cem\u003eLr30\u003c/em\u003e monogenic line RL6049 confirmed the presence of a gene 100% identical to \u003cem\u003eLr.ace-4A\u003c/em\u003e. However, as documented in previous studies\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eLr30\u003c/em\u003e confers only intermediate levels of resistance (ITs\u0026thinsp;=\u0026thinsp;1 to 2) when present alone. Indeed, we observed that RL6049 exhibited intermediate resistance to \u003cem\u003ePt\u003c/em\u003e pathotypes FHJL, FHJR, THDB, and PHJS (Supplementary Fig.\u0026nbsp;9). We hypothesized that \u003cem\u003eLr.ace-4A\u003c/em\u003e is less effective in a hexaploid background compared to a tetraploid background.\u003c/p\u003e \u003cp\u003eTo test this hypothesis, the same 9,264 bp genomic fragment carrying \u003cem\u003eLr.ace-4A\u003c/em\u003e from PI 192051 was transformed into the hexaploid wheat cultivar Fielder via \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). We successfully generated 37 independent transgenic T\u003csub\u003e0\u003c/sub\u003e plants, with nine randomly selected for detailed characterization. The presence and expression of the transgene in these selected T\u003csub\u003e0\u003c/sub\u003e plants were verified through PCR and qRT-PCR assays (Supplementary Fig.\u0026nbsp;10). In this hexaploid background, most transgenic T\u003csub\u003e0\u003c/sub\u003e plants (e.g., T\u003csub\u003e0\u003c/sub\u003eC652-3 and T\u003csub\u003e0\u003c/sub\u003eC652-10) exhibited intermediate resistance to \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, resembling the responses observed in RL6049. In contrast, the untransformed Fielder control was completely susceptible (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Evaluation of the transgenic T\u003csub\u003e1\u003c/sub\u003e families, each comprising approximately 20 plants per event revealed that the resistant plants consistently carried the transgene (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;11). In the transgenic families T\u003csub\u003e1\u003c/sub\u003eC652-12 and T\u003csub\u003e1\u003c/sub\u003eC652-27, all T\u003csub\u003e1\u003c/sub\u003e plants showed resistance and were confirmed to possess the transgene (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;11). Furthermore, the resistant line PI 192051 was crossed and backcrossed two times with the Chinese common wheat variety Yangmai21 (YM21; Supplementary Fig.\u0026nbsp;12a), which is susceptible to multiple \u003cem\u003ePt\u003c/em\u003e pathotypes\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e plants homozygous for the natural \u003cem\u003eLr.ace-4A\u003c/em\u003e gene exhibited intermediate resistance against \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, similar to the responses observed in RL6049 (Supplementary Fig.\u0026nbsp;12b). In contrast, the YM21 control displayed susceptible infection types (ITs\u0026thinsp;=\u0026thinsp;4) to the same race. These findings confirm that \u003cem\u003eLr.ace-4A\u003c/em\u003e is synonymous with \u003cem\u003eLr30\u003c/em\u003e, though its effectiveness is reduced in a hexaploid background.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the Fielder background, several transgenic events (e.g., T\u003csub\u003e1\u003c/sub\u003eC652-12 and T\u003csub\u003e1\u003c/sub\u003eC652-27) with higher levels of transgene expression (Supplementary Fig.\u0026nbsp;10) displayed enhanced resistance, comparable to the responses observed in the tetraploid wheat PI 192051 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;11). This result suggests that the resistance levels in transgenic plants may be correlated with the number of \u003cem\u003eLr30\u003c/em\u003e insertions or transgene expression levels. To investigate the impact of \u003cem\u003eLr30\u003c/em\u003e overexpression on resistance, we generated 50 independent T\u003csub\u003e0\u003c/sub\u003e transgenic plants overexpressing \u003cem\u003eLr30\u003c/em\u003e driven by the maize \u003cem\u003eubiquitin\u003c/em\u003e (\u003cem\u003eUBI\u003c/em\u003e) promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). From these, nine T\u003csub\u003e0\u003c/sub\u003e plants were randomly selected for detailed analysis. Transcript levels of \u003cem\u003eLr30\u003c/em\u003e were significantly higher in all examined transgenic T\u003csub\u003e0\u003c/sub\u003e plants compared to the Fielder control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Supplementary Fig.\u0026nbsp;13). Upon inoculation with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, both T\u003csub\u003e0\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e transgenic plants overexpressing \u003cem\u003eLr30\u003c/em\u003e exhibited robust resistance, whereas the Fielder control displayed complete susceptibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;14).\u003c/p\u003e \u003cp\u003eTo determine whether the transgene exhibits a resistance profile similar to that of the natural \u003cem\u003eLr30\u003c/em\u003e gene, two transgenic lines (T\u003csub\u003e1\u003c/sub\u003eC652-12 and T\u003csub\u003e1\u003c/sub\u003eC652-27; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), displaying higher levels of transgene expression, were challenged with five \u003cem\u003ePt\u003c/em\u003e pathotypes known to be highly virulent to Fielder\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. T\u003csub\u003e1\u003c/sub\u003e plants harboring the transgene from these two transgenic lines exhibited strong resistance to all five tested \u003cem\u003ePt\u003c/em\u003e pathotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). These results suggest that the transgene replicates the resistance profile of the natural \u003cem\u003eLr30\u003c/em\u003e (synonym \u003cem\u003eLr.ace-4A\u003c/em\u003e) gene.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHaplotype analysis and diagnostic marker of\u003c/b\u003e \u003cb\u003eLr30\u003c/b\u003e. A BLASTp similarity search against the published genomes of hexaploid, tetraploid, and diploid wheat, as well as the National Center for Biotechnology Information (NCBI) database, revealed that homologous proteins on chromosome arm 4AL shared over 99.4% similarity with Lr30 (Supplementary Fig.\u0026nbsp;15). To further investigate the sequence variation in \u003cem\u003eLr30\u003c/em\u003e, we sequenced its coding regions from 59 accessions of \u003cem\u003eTriticum turgidum\u003c/em\u003e subsp. \u003cem\u003edicoccon\u003c/em\u003e, which had been inoculated with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. This analysis revealed that none of these \u003cem\u003eT. dicoccon\u003c/em\u003e accessions carried the \u003cem\u003eLr30\u003c/em\u003e gene. From these \u003cem\u003eT. dicoccon\u003c/em\u003e accessions and the published wheat genomes, we identified seven susceptible haplotypes (Hap2 \u0026ndash; Hap8) based on their susceptible reactions and/or shared amino acid sequences (Supplementary Fig.\u0026nbsp;15 and Supplementary Table\u0026nbsp;7). Lr30 (Hap1) differed from the other seven haplotypes by three (Hap2), four (Hap3 and Hap6), two (Hap4), five (Hap5), and seven (Hap7 and Hap8) amino acid changes, respectively. Two cDNA polymorphisms, G1597A and C1984T (corresponding amino acid changes E533K and R662C), distinguish \u003cem\u003eLr30\u003c/em\u003e from all other susceptible haplotypes (Supplementary Fig.\u0026nbsp;15). Additionally, the 1597G and 1984C alleles in \u003cem\u003eLr30\u003c/em\u003e were identified in a single genotype, PI 619381, from approximately 1,000 wheat accessions with available exome sequencing data\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. When challenged with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, PI 619381 displayed a resistance response similar to that conferred by RL6049 (Supplementary Fig.\u0026nbsp;16). These results indicate that these two unique polymorphisms are likely critical for the functionality of \u003cem\u003eLr30\u003c/em\u003e and its ability to recognize pathogen effectors.\u003c/p\u003e \u003cp\u003eTo further investigate the functional significance of each variation, we constructed single-mutation versions of \u003cem\u003eLr30\u003c/em\u003e at these two sites using the vector containing the 9,264 bp genomic fragment from PI 192051. We then generated transgenic wheat plants expressing the 9,264 bp genomic fragment with either the 533K (Lr30\u003csup\u003e533K\u003c/sup\u003e) or 662C (Lr30\u003csup\u003e662C\u003c/sup\u003e) amino acid substitution. Positive transgenic T\u003csub\u003e0\u003c/sub\u003e and T\u003csub\u003e1\u003c/sub\u003e plants expressing Lr30\u003csup\u003e533K\u003c/sup\u003e and Lr30\u003csup\u003e662C\u003c/sup\u003e exhibited complete susceptibility to \u003cem\u003ePt\u003c/em\u003e pathotype PHQS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c and Supplementary Fig.\u0026nbsp;17). In contrast, transgenic wheat plants expressing the original 9,264 bp genomic fragment exhibited resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). These results underscore the indispensable role of both natural variations for the resistance function of \u003cem\u003eLr30\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA dominant marker, \u003cem\u003eLr30MAS-47FR\u003c/em\u003e (Supplementary Data 1), was developed based on these two unique cDNA polymorphisms in PI 192051 (Supplementary Fig.\u0026nbsp;15). PCR amplification using this marker at an annealing temperature of 54˚C yields a 916-bp fragment when \u003cem\u003eLr30\u003c/em\u003e is present (Supplementary Fig.\u0026nbsp;18). This marker was used to assess a collection of 309 wheat accessions, including 158 \u003cem\u003eT. aestivum\u003c/em\u003e, 82 \u003cem\u003eT. turgidum\u003c/em\u003e, and 69 \u003cem\u003eT. monococcum\u003c/em\u003e. PCR amplicons of the expected size were detected only in three genotypes (PI 192051, RL6049, and PI 619381), with no amplification observed in any other wheat genotypes tested (Supplementary Table\u0026nbsp;8).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eLr30\u003c/b\u003e \u003cb\u003eencoding an NLR immune receptor protein.\u003c/b\u003e The transcript levels of \u003cem\u003eLr30\u003c/em\u003e in PI 192051 were quantified relative to the \u003cem\u003eTaActin\u003c/em\u003e reference gene using qRT-PCR. Our analysis revealed a significant upregulation of \u003cem\u003eLr30\u003c/em\u003e expression in plants infected with the \u003cem\u003ePt\u003c/em\u003e pathotype PHQS from 1 to 4 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea), suggesting that \u003cem\u003eLr30\u003c/em\u003e is induced by the presence of \u003cem\u003ePt\u003c/em\u003e. Additionally, no significant difference in \u003cem\u003eLr30\u003c/em\u003e transcript levels was detected between the hexaploid line RL6049 and the tetraploid PI 192051 (Supplementary Fig.\u0026nbsp;19).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the relationships between Lr30 and other known NLR proteins, a phylogenetic analysis was conducted using Lr30 and 186 known NLR proteins from the Gramineae family. The analysis revealed that Lr30 was most closely related to Pib from rice (Supplementary Fig.\u0026nbsp;20). However, sequence alignment revealed a sequence similarity of less than 38.7% between Lr30 and Pib.\u003c/p\u003e \u003cp\u003eTo determine the subcellular localization of Lr30, a green fluorescent protein (GFP) tag was fused to Lr30 to visualize its signals within plant cells. Both cytoplasmic and nuclear fluorescence were detected in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves expressing the GFP-Lr30_CDS (coding region) and Lr30_CDS-GFP fusion proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Similarly, a dual cytoplasmic and nuclear localization of GFP-Lr30_CDS was observed in wheat protoplasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The expression of the GFP-fused proteins was validated via Western blot analysis using an α-GFP antibody (Supplementary Fig.\u0026nbsp;21).\u003c/p\u003e \u003cp\u003eTo evaluate whether the full-length Lr30 or its individual domains can trigger cell death in \u003cem\u003eN. benthamiana\u003c/em\u003e, an \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transient expression analysis was conducted in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. No cell death or noticeable yellowing was observed in the leaf regions expressing Lr30 or its individual protein domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). In contrast, pronounced cell death was observed in leaf regions expressing BAX, which served as a positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eAdditionally, we used AlphaFold2 and AlphaFold-Multimer to predict the potential resistosome structures of Lr30. Both the coiled-coil (CC) and NB-NB-LRR domains of Lr30 demonstrated the ability to form pentamers (Supplementary Fig.\u0026nbsp;22). The critical amino acid substitution R662C is located within the binding interface of the pentamer, while the other key amino acid substitution, E533K, is positioned on the exterior surface of the predicted resistosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDurum wheat is a vital cereal grain primarily cultivated for pasta production and serves as an important reservoir of genetic diversity for traits such as disease resistance\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and nutritional quality\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Despite its agricultural significance, the genomic resources for durum wheat remain limited, with only a few draft genome assemblies currently available\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The development of additional high-quality reference genomes for durum wheat is crucial for isolating valuable genes and advancing functional and evolutionary genomic studies. To facilitate the practical exploitation of durum wheat, we developed a high-quality reference genome for the Portugal durum wheat landrace PI 192051 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Using this genome and EMS-induced susceptible mutants, we successfully cloned the leaf rust resistance gene \u003cem\u003eLr30\u003c/em\u003e (synonym \u003cem\u003eLr.ace-4A\u003c/em\u003e), which is located within a recombination-sparse region on chromosome 4A in durum wheat.\u003c/p\u003e \u003cp\u003ePrevious studies suggested that the leaf rust resistance genes \u003cem\u003eLr.ace-4A\u003c/em\u003e and \u003cem\u003eLr30\u003c/em\u003e were distinct, based on differences in their genetic origins, chromosomal locations, infection types, and inheritance patterns (dominant/recessive)\u003csup\u003e18,19\u003c/sup\u003e. However, in this study, we provide evidence that \u003cem\u003eLr.ace-4A\u003c/em\u003e and \u003cem\u003eLr30\u003c/em\u003e are, in fact, the same gene. This conclusion is supported by multiple lines of experimental evidence. First, \u003cem\u003eLr.ace-4A\u003c/em\u003e and \u003cem\u003eLr30\u003c/em\u003e are located at the same locus and share an identical genomic sequence. The discrepancies in the previous gene mapping study\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e likely arise from a large recombination-suppressed region on chromosome 4A (Supplementary Fig.\u0026nbsp;2) in the cross between PI 192051 and Rusty, which may have obscured the true genetic relationship. Second, functional validation through transgenic complementation experiments demonstrated that \u003cem\u003eLr.ace-4A\u003c/em\u003e confers infection types similar to \u003cem\u003eLr30\u003c/em\u003e when expressed in the susceptible hexaploid wheat cultivar Fielder. The introgression of \u003cem\u003eLr.ace-4A\u003c/em\u003e into the common wheat variety YM21 resulted in a resistance phenotype comparable to that of \u003cem\u003eLr30\u003c/em\u003e in the hexaploid line RL6049 (Supplementary Fig.\u0026nbsp;12). Third, while \u003cem\u003eLr30\u003c/em\u003e in the hexaploid wheat cultivar Terenzio was reported as a recessive resistance gene\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eLr.ace-4A\u003c/em\u003e in the tetraploid PI 192051 was initially identified as a dominant resistant gene\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In our study, segregation analysis of seedling resistance in the F\u003csub\u003e2\u003c/sub\u003e population from the PI 192051 \u0026times; Rusty cross revealed that \u003cem\u003eLr.ace-4A\u003c/em\u003e was recessive against the \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. Conversely, \u003cem\u003eLr.ace-4A\u003c/em\u003e exhibited dominant inheritance in the PI 192051 \u0026times; m1 mapping population. These contrasting inheritance patterns can be explained by dominance reversals, a phenomenon previously observed in several wheat rust resistance genes, such as \u003cem\u003eSr8155B1\u003c/em\u003e, \u003cem\u003eYr6\u003c/em\u003e, and \u003cem\u003eSr6\u003c/em\u003e\u003csup\u003e34\u0026ndash;37\u003c/sup\u003e. It is hypothesized that factors such as genetic backgrounds, pathogen isolates, and environmental conditions could contribute to this reversal\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Furthermore, allelic interactions and dominant-negative effects may also influence the observed dominance reversal\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Yeast two-hybrid assays revealed no direct interactions between the Lr30 protein and its susceptible haplotypes (Supplementary Fig.\u0026nbsp;23). Further studies are needed to clarify the mechanisms underlying this dominance reversal in Lr30.\u003c/p\u003e \u003cp\u003eThe results presented herein demonstrate that \u003cem\u003eLr30\u003c/em\u003e is less effective when present in a hexaploid background than in a tetraploid background. A similar trend has been observed for several other wheat resistance genes, including \u003cem\u003eSr21\u003c/em\u003e\u003csup\u003e41,42\u003c/sup\u003e, \u003cem\u003eSr13\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e, \u003cem\u003ePm8\u003c/em\u003e\u003csup\u003e43\u003c/sup\u003e, and \u003cem\u003eYrAS2388\u003c/em\u003e\u003csup\u003e44\u003c/sup\u003e, which also confer lower levels of resistance when transferred from lower-ploidy relatives into hexaploid wheat. This reduction in resistance has been attributed to the presence of inhibitors or modifiers in the hexaploid wheat genetic backgrounds that suppress the function of the transferred resistance genes. In certain cases, such as \u003cem\u003ePm8\u003c/em\u003e and \u003cem\u003eYrAS2388\u003c/em\u003e, the resistance reduction of the transferred genes was found to vary across different hexaploid backgrounds\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In the case of wheat powdery mildew, the \u003cem\u003ePm8\u003c/em\u003e resistance gene from rye was suppressed in hexaploid wheat by a susceptible allele of its wheat ortholog, \u003cem\u003ePm3\u003c/em\u003e\u003csup\u003e45\u003c/sup\u003e. Identification of the chromosomal region responsible for the reduced effectiveness of \u003cem\u003eLr30\u003c/em\u003e in hexaploid wheat would be of significant interest, as this knowledge may be useful to enhance the gene's performance in hexaploid backgrounds.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eLr30\u003c/em\u003e and \u003cem\u003eLr.ace-4A\u003c/em\u003e were first identified in 1981\u003csup\u003e19\u003c/sup\u003e and 2019\u003csup\u003e18\u003c/sup\u003e, respectively, these genes have not been widely utilized in the breeding of bread and durum wheat. Our haplotype analysis and diagnostic marker evaluation (Supplementary Table\u0026nbsp;8) reveal that \u003cem\u003eLr30\u003c/em\u003e is present in only one (1.2%) \u003cem\u003eT. turgidum\u003c/em\u003e accession and two (1.3%) \u003cem\u003eT. aestivum\u003c/em\u003e accessions, while it is absent in all other diploid, tetraploid, and hexaploid wheat genotypes. This finding indicates that the incorporation of \u003cem\u003eLr30\u003c/em\u003e can benefit a wide range of commercial durum wheat varieties, particularly due to its robust resistance against multiple \u003cem\u003ePt\u003c/em\u003e races.\u003c/p\u003e \u003cp\u003eHowever, the utility of \u003cem\u003eLr30\u003c/em\u003e is constrained by its limited effectiveness against several \u003cem\u003ePt\u003c/em\u003e pathotypes and its partial resistance in hexaploid wheat. To address these limitations, \u003cem\u003eLr30\u003c/em\u003e would need to be deployed in combination with other \u003cem\u003eLr\u003c/em\u003e genes or integrated into transgenic cassettes containing multiple resistance genes, particularly once transgenic approaches gain broader public acceptance. Given the enhanced resistance in transgenic hexaploid plants with higher gene copy numbers or transgene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), it might be valuable to incorporate multiple copies of \u003cem\u003eLr30\u003c/em\u003e into such transgenic cassettes. The cloning of \u003cem\u003eLr30\u003c/em\u003e and its successful introgression from the original durum wheat PI 192051 into the Chinese commercial bread wheat cultivar YM21 (Supplementary Fig.\u0026nbsp;12) represent significant steps toward its future application in global wheat breeding programs. Moreover, our analysis identified only two critical cDNA polymorphisms (G1597A and C1984T; Supplementary Fig.\u0026nbsp;15) that distinguish resistant and susceptible haplotypes of \u003cem\u003eLr30\u003c/em\u003e. This suggests that the foundational structure of this gene is broadly present in global wheat germplasms. With the rapid advancement of base editing technologies, \u003cem\u003eLr30\u003c/em\u003e could become a promising target for precise base editing, enabling the direct enhancement of wheat resistance to leaf rust.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLr30\u003c/em\u003e gene encodes a non-canonical NLR protein with tandem NB-ARC domains (CC-NB-NB-LRR; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Despite the presence of an additional NB domain in this protein, a typical pentameric resistosome was successfully predicted using AlphaFold2 and AlphaFold-Multimer (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). This structural prediction suggests that Lr30 retains the ability to form a resistosome\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The NB domain of NLR proteins is known to play a central role in binding nucleoside triphosphates (NTPs), facilitating the exchange between NTPs and nucleoside diphosphates (NDPs), and regulating the oligomerization and activation switch of NLR proteins\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The presence of an additional NB domain in Lr30 might enable more nuanced and sensitive regulation of its activity, potentially enhancing its responsiveness to pathogen signals. Remarkably, the two critical natural variations, amino acids 553E and 662R, are located within or adjacent to the secondary NB domain. Point mutations at these two amino acid residues completely abolished the resistance function of the Lr30 protein. These findings suggest that the secondary NB domain, together with the associated critical residues, plays a crucial role in the structural integrity and functional regulation of Lr30.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrated the feasibility of generating chromosome-scale wheat assemblies in combination with EMS-induced susceptible mutants to facilitate the cloning of resistance genes located within recombination-sparse regions of the wheat genome. We confirmed that \u003cem\u003eLr.ace-4A\u003c/em\u003e in tetraploid wheat and \u003cem\u003eLr30\u003c/em\u003e in hexaploid wheat represent the same gene, although \u003cem\u003eLr30\u003c/em\u003e exhibits reduced effectiveness in a hexaploid background. The cloning of \u003cem\u003eLr30\u003c/em\u003e, the introgression line generated, and the diagnostic marker developed in this study provide invaluable resources for diversifying the deployment of \u003cem\u003eLr\u003c/em\u003e genes and accelerating the integration of \u003cem\u003eLr30\u003c/em\u003e into wheat breeding programs.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePlant materials and mapping populations.\u003c/b\u003e The leaf rust resistance gene \u003cem\u003eLr.ace-4A\u003c/em\u003e was sourced from the durum wheat accession PI 192051, a landrace originating from Lisboa, Portugal\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. To map this gene, two F\u003csub\u003e2\u003c/sub\u003e mapping populations were developed: one comprising 286 F\u003csub\u003e2\u003c/sub\u003e plants from a cross between PI 192051 and the susceptible genotype Rusty\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and another with 315 F\u003csub\u003e2\u003c/sub\u003e plants from a cross between PI 192051 and m1, a susceptible EMS-induced mutant line derived from the PI 192051 mutant population. The \u003cem\u003eLr30\u003c/em\u003e monogenic line RL6049 (GSTR 430; Thatcher*6/Terenzio) and PI 619381 were obtained from the U.S. Department of Agriculture National Small Grains Collection. A diverse wheat panel, including 158 \u003cem\u003eT. aestivum\u003c/em\u003e, 69 \u003cem\u003eT. monococcum\u003c/em\u003e, and 82 \u003cem\u003eT. turgidum\u003c/em\u003e accessions (Supplementary Table\u0026nbsp;8), was used to evaluate the effectiveness of the diagnostic marker for marker-assisted selection. Additionally, SNPs from exome capture data of ~\u0026thinsp;1,000 wheat genotypes\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e were analyzed to study natural variations in \u003cem\u003eLr30\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLeaf rust assays.\u003c/b\u003e Eight \u003cem\u003ePt\u003c/em\u003e pathotypes (THSP, FHJR, HCJR, PHQS, PHJS, PHST, THDB, and FHJL; Supplementary Table\u0026nbsp;1) were inoculated onto PI 192051 and Rusty. Seedlings at the three-leaf stage were treated with \u003cem\u003ePt\u003c/em\u003e urediniospores mixed with talcum powder (1:25 ratio), using the shaking-off method\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Inoculated plants were placed in a dark dew chamber at 22\u0026deg;C for 24 hours and then transferred to a growth chamber maintained at 22\u0026ndash;24\u0026deg;C with a 16-hour photoperiod. Disease symptoms were scored around 12 days post-inoculation (dpi) using a 0\u0026ndash;4 scale\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For pathogen growth quantification, leaf segments from PI 192051 and Rusty inoculated with PHQS were sampled at 2, 4, 6, and 8 dpi, cleared with KOH (37\u0026deg;C, 12 hours), stained with WGA-FITC (Cat. No. L4895-10MG, Sigma-Aldrich, USA), and examined under a Zeiss Discovery V20 fluorescence dissecting microscope (Zeiss, Jena, Germany).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytogenetic assays.\u003c/b\u003e Fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) karyotyping experiments were conducted using four oligonucleotide probes: pTa535\u003csup\u003e5\u003c/sup\u003e, pSc119.2\u003csup\u003e5\u003c/sup\u003e, (CTT)\u003csub\u003e10\u003c/sub\u003e\u003csup\u003e49\u003c/sup\u003e, and pTa713\u003csup\u003e5\u003c/sup\u003e. These probes, labeled with FAM or TAMRA, were synthesized by TsingKe Biological Technology Co., Ltd. (Chengdu, Sichuan, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEMS mutagenesis and mutant screening.\u003c/b\u003e Approximately 10,000 seeds of PI 192051 were evenly distributed into three flasks and treated with 250 mL solutions of 0.6%, 0.5%, and 0.4% EMS (Cat. No. M0880-25G, Sigma-Aldrich, USA). All surviving M\u003csub\u003e1\u003c/sub\u003e plants were grown in the greenhouse, resulting in the development of 1,853 independent M\u003csub\u003e2\u003c/sub\u003e families. Approximately 20 M\u003csub\u003e2\u003c/sub\u003e seeds from each family were challenged with the \u003cem\u003ePt\u003c/em\u003e pathotype PHQS in growth chambers. M\u003csub\u003e3\u003c/sub\u003e seeds derived from susceptible M\u003csub\u003e2\u003c/sub\u003e plants were re-evaluated to confirm phenotypic consistency across generations. To rule out the possibility of seed contamination, all identified mutants were genotyped using 4A-genome specific PCR markers linked to \u003cem\u003eLr.ace-4A\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA extraction, library construction, and sequencing.\u003c/b\u003e PI 192051 plants were grown in a controlled growth chamber, and fresh leaves from 10-day-old seedlings were harvested for high molecular weight (HMW) DNA extraction. The DNA was sheared into ~\u0026thinsp;20 kb fragments using g-TUBE, and libraries were constructed using the PacBio SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA). Libraries were sequenced on the PacBio Revio sequencing platform at Biomarker Technologies Corporation (Qingdao, China). Hi-C libraries were prepared following a modified standard protocol\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and sequenced on the Illumina HiSeq X platform at the same facility.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq and Iso-seq.\u003c/b\u003e\u0026nbsp;RNA was extracted from the leaves, roots, stems, and spikes of PI 192051 at three growth stages (seedling, booting, and heading). Total RNA extraction, RNA-seq library construction, and sequencing were carried out by Novogene Bioinformatics Technology Co., Ltd. (Tianjin, China), yielding approximately 11 Gb of raw data per sample (Supplementary Table\u0026nbsp;4). Full-length transcriptome sequencing (PacBio Iso-seq) was also conducted at Novogene (Tianjin, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome assembly and validation.\u003c/b\u003e A preliminary genome assembly was generated from 451.11 Gb of PacBio HiFi reads using hifiasm v0.19.5-r587\u003csup\u003e51\u003c/sup\u003e. To achieve a chromosome-scale assembly, 451.46 Gb of Hi-C data were used to resolve contigs into chromosomal scaffolds based on spatial proximity information. The raw Hi-C reads were filtered using fastp v0.23.2\u003csup\u003e52\u003c/sup\u003e and aligned to the contig assembly using BWA mem v0.7.17-r1188\u003csup\u003e53\u003c/sup\u003e. Contigs were anchored into chromosomes using YaHS v1.2a.1\u003csup\u003e54\u003c/sup\u003e, and the assembly was manually refined using Juicebox v1.11 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/aidenlab/Juicebox\u003c/span\u003e\u003cspan address=\"https://github.com/aidenlab/Juicebox\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Assembly quality was assessed by analyzing LTR retrotransposons with LTR_FINDER_parallel v1.2\u003csup\u003e55\u003c/sup\u003e (-in harvest) and LTR_retriever\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, evaluating consensus accuracy with Merqury v1.3\u003csup\u003e57\u003c/sup\u003e (21-mer frequency analysis), and assessing completeness with BUSCO (poales_odb10 dataset)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene model prediction.\u003c/b\u003e Gene annotation of PI 192051 integrated homology-based protein data, Pacio Iso-seq, and RNA-seq datasets. Gene annotations from \u003cem\u003eT. durum\u003c/em\u003e, \u003cem\u003eT. dicoccoides\u003c/em\u003e, and the hexaploid wheat variety CS were transferred to the PI 192051 genome using Liftoff\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. RNA-seq data from roots, stems, leaves, and spikes were processed using fastp\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, mapped to the PI 192051 genome using HISAT2 v2.2.1, and assembled using Stringtie v2.2.1\u003csup\u003e60\u003c/sup\u003e. Iso-seq data were processed using Lima and IsoSeq3 and mapped on the assembled genome using pbmm2 v1.10.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/PacificBiosciences/pbmm2\u003c/span\u003e\u003cspan address=\"https://github.com/PacificBiosciences/pbmm2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Redundant transcripts were collapsed using cDNA_Cupcake (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Magdoll/cDNA_Cupcake\u003c/span\u003e\u003cspan address=\"https://github.com/Magdoll/cDNA_Cupcake\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). RNA-seq and Iso-seq results were merged using the Stringtie\u0026ndash;merge function, and open reading frames were identified with TransDecoder v5.5.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/TransDecoder/TransDecoder\u003c/span\u003e\u003cspan address=\"https://github.com/TransDecoder/TransDecoder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Output GFF files were consolidated into a single file using the Perl script \u0026ldquo;agat_sp_merge_annotation.pl\u0026rdquo; from the AGAT toolkit (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/NBISweden/AGAT\u003c/span\u003e\u003cspan address=\"https://github.com/NBISweden/AGAT\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Redundant transcripts for each gene were removed using CD-HIT v.4.8.1. Annotation completeness was evaluated using BUSCO v1.7.131\u003csup\u003e58\u003c/sup\u003e, and functional annotation was performed using eggNOG-mapper v2.1.1232\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative synteny and SNP density assays.\u003c/b\u003e Collinearity relationships between the A and B subgenomes of PI 192051 and their diploid, tetraploid, and hexaploid counterparts was analyzed using JCVI\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Synteny blocks were identified through an all-against-all BLAST search, with homologous hits concatenated using a 20-gene distance cutoff. To investigate variant density on chromosome 4A, SNPs from whole-genome resequencing of a tetraploid wheat panel (including six accessions each of \u003cem\u003eT. dicoccon\u003c/em\u003e, \u003cem\u003eT. durum\u003c/em\u003e, and \u003cem\u003eT. dicoccoides\u003c/em\u003e) were used. Sequencing reads (NCBI BioProject No. PRJEB61424) were trimmed using fastp\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, mapped to the PI 192051 genome using BWA-mem\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, and deduplicated with GATK\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Variants were called using Freebayes\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e and filtered (read depth\u0026thinsp;\u0026gt;\u0026thinsp;5, quality\u0026thinsp;\u0026gt;\u0026thinsp;10) with BCFtools. Variant density was calculated as the number of SNPs and indels per 1 Mb region.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCandidate gene identification.\u003c/b\u003e Total RNA was extracted from leaves of seven susceptible M\u003csub\u003e3\u003c/sub\u003e mutant families inoculated with \u003cem\u003ePt\u003c/em\u003e pathotype PHQS. RNA-Seq was conducted by Novogene Bioinformatics Technology Co., Ltd. (Tianjin, China). To identify the \u003cem\u003eLr.ace-4A\u003c/em\u003e candidate gene, RNA-seq reads from the seven mutants were individually aligned to the PI 192051 genome using STAR\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The resulting BAM files were sorted and indexed using SAMtools\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Variants were called using Freebayes\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, followed by filtering (quality score\u0026thinsp;\u0026gt;\u0026thinsp;10) using BCFtools.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRISPR/Cas9-based gene editing.\u003c/b\u003e The CRISPR/Cas9 gene-editing system was used to validate the \u003cem\u003eLr.ace-4A\u003c/em\u003e candidate gene (\u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e) in the resistant parent PI 192051. The Cas9-Trex2\u003csup\u003e25\u003c/sup\u003e and GRF4-GIF1\u003csup\u003e26\u003c/sup\u003e fusion proteins were used to enhance editing and regeneration efficiency, respectively. gRNAs were designed using the CRISPR-Cereal website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/CRISPR-Cereal/index.php\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/CRISPR-Cereal/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Sequence alignment of homeologous and paralogous of \u003cem\u003ePI192051.r1.4AG0210600\u003c/em\u003e was performed using MUSCLE in MEGA v7.0, followed by specificity validation via BLASTN searches against the Svevo and PI 192051 genomes. The gRNA was synthesized, and then the TaU3p:target-optimized gRNA scaffold was generated through two rounds of PCR using pOPGR-TS1 as a template\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. PCR amplifications were carried out using the primer pairs \u003cem\u003eCas9-4AF1R1\u003c/em\u003e, \u003cem\u003eCas9-4AF2R2\u003c/em\u003e, and \u003cem\u003eCas9-4AF1R2\u003c/em\u003e (Supplementary Data 1). The resulting fragment containing the gRNA was cloned into the modified pCas9T vector at the \u003cem\u003eStu\u003c/em\u003eI site using the In-Fusion\u0026reg; HD Cloning Kit (Clontech, CA, USA). The final construct was transformed into PI 192051 via \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transformation. Mutations in transgenic plants were identified using the 4A-genome specific primer pairs \u003cem\u003epku4AF2R2\u003c/em\u003e (Supplementary Data 1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eWheat transformation.\u003c/b\u003e A 9,264-bp genomic fragment, including the complete coding region and introns (4,652 bp), along with 2,949 bp upstream of the start codon and 1,663 bp downstream of the stop codon, was amplified from PI 192051 by PCR using PrimeStar Max DNA Polymerase (TaKaRa, Kyoto, Japan). Overlapping PCR products, generated with the primer pairs \u003cem\u003ep1300‑Lr4AF1R1\u003c/em\u003e and \u003cem\u003ep1300‑Lr4AF2R2\u003c/em\u003e (Supplementary Data 1), were inserted into the linearized binary vector pCAMBIA1300 using the In-Fusion\u0026reg; HD Cloning Kit (Clontech, CA, USA). This construct was transformed into the EMS-induced mutant line m1 and the hexaploid wheat cultivar Fielder via \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transformation. Four primer pairs \u003cem\u003epku23F1R1\u003c/em\u003e, \u003cem\u003epku23F2R2\u003c/em\u003e, \u003cem\u003epku24F1R1\u003c/em\u003e, and \u003cem\u003epku24F2R2\u003c/em\u003e (Supplementary Data 1) were used to generate two single-mutation versions of the constructs, each containing one of the critical variations (G1597A or C1984T; amino acid changes E533K or R662C). Primer pairs \u003cem\u003epku65FR\u003c/em\u003e, \u003cem\u003epku66FR\u003c/em\u003e, \u003cem\u003epku4AF2R2\u003c/em\u003e, and \u003cem\u003epku4AF3R3\u003c/em\u003e (Supplementary Data 1) were used to confirm the presence of transgenes. Transcript levels in the transgenic plants were quantified using primer pairs \u003cem\u003epku51FR\u003c/em\u003e (Supplementary Data 1). Additionally, the complete coding sequence (3,525 bp) of \u003cem\u003eLr.ace-4A\u003c/em\u003e was amplified from the cDNA of PI 192051 using primer pairs \u003cem\u003eOE-4AFR\u003c/em\u003e (Supplementary Data 1). This fragment was cloned into the pCAMBIA1300-OE vector under the maize \u003cem\u003eUBI\u003c/em\u003e promoter for overexpression in Fielder. Transgene presence and transcript levels were verified using primer pairs \u003cem\u003epku16FR\u003c/em\u003e, \u003cem\u003epku20FR\u003c/em\u003e, and \u003cem\u003epku51FR\u003c/em\u003e (Supplementary Data 1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransferring of\u003c/b\u003e \u003cb\u003eT. durum\u003c/b\u003e \u003cb\u003esegment carrying\u003c/b\u003e \u003cb\u003eLr.ace-4A\u003c/b\u003e \u003cb\u003einto hexaploid wheat.\u003c/b\u003e PI 192051 was crossed with the bread wheat cultivar YM21. The resulting F\u003csub\u003e1\u003c/sub\u003e plants were backcrossed twice with YM21 to produce BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e. These BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e plants were self-pollinated for two generations to produce BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e. The presence of \u003cem\u003eLr.ace-4A\u003c/em\u003e was validated using the PCR marker \u003cem\u003eLr30MAS-47FR\u003c/em\u003e (Supplementary Data 1). Selected BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e plants were inoculated with the \u003cem\u003ePt\u003c/em\u003e pathotype PHQS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHaplotyping and phylogenetic analysis.\u003c/b\u003e Homeologs or orthologs of \u003cem\u003eLr30\u003c/em\u003e were sourced from publicly accessible Triticeae genomes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plants.ensembl.org/\u003c/span\u003e\u003cspan address=\"http://plants.ensembl.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the NCBI database. Moreover, 59 \u003cem\u003eT. dicoccon\u003c/em\u003e accessions were inoculated with the \u003cem\u003ePt\u003c/em\u003e pathotype PHQS, and the coding regions of \u003cem\u003eLr30\u003c/em\u003e were amplified using 4A-genome specific primer pairs \u003cem\u003epku4AF1R1\u003c/em\u003e, \u003cem\u003epku4AF2R2\u003c/em\u003e, and \u003cem\u003epku4AF3R3\u003c/em\u003e (Supplementary Data 1), followed by Sanger sequencing. Cloned R protein sequences from Gramineae species were obtained from the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A neighbor-joining phylogenetic tree was constructed using MEGA v7 and visualized with iTOL v7 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR analysis.\u003c/b\u003e At the three-leaf stage, PI 192051 seedlings were either mock- or \u003cem\u003ePt\u003c/em\u003e-inoculated in two independent growth chambers under identical conditions (24\u0026deg;C day/22\u0026deg;C night, 16 h light/8 h dark). Leaf samples were collected at four time points: 0 h (immediately before inoculation), 1, 2, and 4 dpi. Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, MA, USA) and purified using the Direct-zol RNA MiniPrepPlus Kit (ZymoResearch, CA, USA). qRT-PCR was performed on an ABI QuantStudio 5 Real-Time PCR System (Applied Biosystems, CA, USA) using the primer pairs \u003cem\u003epku22FR\u003c/em\u003e (Supplementary Data 1). The endogenous control \u003cem\u003eTaActin\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e was used to normalize the RNA expression levels using the 2\u003csup\u003e\u0026minus;ΔCT\u003c/sup\u003e method\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Significance was estimated using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn planta\u003c/b\u003e \u003cb\u003eexpression of GFP-fused Lr30 protein and western blotting analysis.\u003c/b\u003e The coding region of the Lr30 protein (amino acids 1-1174) and its CC domain (amino acids 1-188) were cloned into vectors pBIN, pJIM19-GFP, and pJIT163-Ubi-GFP\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. These recombinant constructs, along with the empty vector (EV) control, were transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves via \u003cem\u003eA. tumefaciens\u003c/em\u003e infiltration (OD\u0026thinsp;=\u0026thinsp;0.6). Wheat protoplasts from Fielder were isolated and transformed using the polyethylene glycol (PEG)-mediated method\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Fluorescence imaging was performed using a confocal microscope (A1 HD25 Nikon, Tokyo, Japan). Proteins from \u003cem\u003eA. tumefaciens\u003c/em\u003e-transformed tobacco leaves were extracted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. Immunoblotting was performed using an anti-GFP primary antibody (1:2500; Abcam, Cambridge, UK) and a Goat anti-Rabbit IgG-HRP secondary antibody (1:10000; Abmart, Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell death induction assay.\u003c/b\u003e The coding region of the mammalian cell death inducer BAX\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e was cloned into pJIM19GFP as a positive control. \u003cem\u003eA. tumefaciens\u003c/em\u003e cultures (OD\u0026thinsp;=\u0026thinsp;1.0) carrying the constructs CDS-GFP, GFP-CDS, CC-GFP, GFP-CC, NB-GFP, GFP-NB, LRR-GFP, GFP-LRR, BAX, and GFP were infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Necrosis induced by BAX was observed at 48 h post inoculation (hpi).\u003c/p\u003e \u003cp\u003e \u003cb\u003eYeast two-hybrid assays.\u003c/b\u003e The Lr30 coding sequence and its susceptible haplotypes were cloned into pGBKT7 and pGADT7, respectively. The constructs were co-transformed into Y2H Gold yeast, cultured on SD/-Trp/-Leu medium, and plated on selective medium SD/-Trp/-Leu/-His\u0026thinsp;+\u0026thinsp;40 \u0026micro;g/mL X-α-gal and SD/-Trp/-Leu/-His/-Ade\u0026thinsp;+\u0026thinsp;40 \u0026micro;g/mL X-α-gal.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrediction of protein structure.\u003c/b\u003e The structural models of Lr30 were generated using AlphaFold2 and AlphaFold-Multimer, specifically configured to predict multimeric assemblies. The computational modeling was conducted on a high-performance computing platform. The resulting models were subsequently visualized and refined using ChimeraX software.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this work are available within the paper and its supplementary information files. All raw sequencing data, genome assembly, and gene annotation for this project are archived at the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, under BioProject accession number PRJCA036461. The sequence of the \u003cem\u003eLr30\u003c/em\u003e gene was deposited in NCBI GenBank under accession number PV159345. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWork at SC laboratory was supported by the National Key Research and Development Program of China (2022YFD1201300), the Key R\u0026amp;D Program of Shandong Province (2024LZGC034 and 2023LZGC022), and the Shandong Provincial Natural Science Foundation (SYS202206), the National Natural Science Foundation of China (32472159), and the Taishan Scholars Program. Work at XW laboratory was supported by National Key Research and Development Program of China (2023YFD1201002), Provincial Natural Science Foundation of Hebei (C2022204010). State Key Laboratory of North China Crop Improvement and Regulation (NCCIR2024ZZ-5), and S\u0026amp;T Program of Hebei (23567601H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.C. designed the research; J.Y., H.L., M.L., and R.S. conducted the experiments; T.S. and S.R. contributed to EMS mutants; B.S., H.L., and D.X. performed the genome assembly; G.W., L.H., and Y.L. contributed to vector construction; M.H. conducted cytogenetic assays; A.J. and C.L. contributed\u0026nbsp;to AlphaFold prediction;\u0026nbsp;C.L., X.W.D., and J.D. provided scientific support; S.C., J.Y., H.L., and R.S. analyzed the data. S.C., J.Y., and X.W. wrote the initial manuscript; S.C. generated the final manuscript; S.C., X.W., and B.S. supervised the project. All authors reviewed and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.C., J.Y., L.H., R.S., and S.L. are inventors on a Chinese provisional patent application (China patent filing No.202510124660.9) relating to the use of the \u003cem\u003eLr30\u003c/em\u003e gene in wheat breeding programs. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuerta-Espino, J.\u003cem\u003e et al.\u003c/em\u003e Global status of wheat leaf rust caused by Puccinia triticina. \u003cem\u003eEuphytica\u003c/em\u003e \u003cstrong\u003e179\u003c/strong\u003e, 143-160 (2011).\u003c/li\u003e\n\u003cli\u003eHelfer, S. 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Biol.\u003c/em\u003e \u003cstrong\u003e2464\u003c/strong\u003e, 131-141 (2022).\u003c/li\u003e\n\u003cli\u003eYoshinaga, K.\u003cem\u003e et al.\u003c/em\u003e Mammalian Bax initiates plant cell death through organelle destruction. \u003cem\u003ePlant Cell Rep.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 408-417 (2005).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"wheat, leaf rust, chromosome-scale assembly, resistance gene, CC-NBS-LRR, Lr30 (synonym Lr.ace-4A)","lastPublishedDoi":"10.21203/rs.3.rs-6289485/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6289485/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLeaf rust is a devastating fungal disease of wheat. Planting resistant wheat cultivars is the most effective strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene \u003cem\u003eLr.ace-4A\u003c/em\u003e within a recombination-sparse region of PI 192051 and demonstrate that \u003cem\u003eLr.ace-4A\u003c/em\u003e is identical to the previously designated \u003cem\u003eLr30\u003c/em\u003e gene in hexaploid wheat. \u003cem\u003eLr30\u003c/em\u003e/\u003cem\u003eLr.ace-4A\u003c/em\u003e encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem NB-ARC domains. This gene proves both necessary and sufficient to confer resistance to \u003cem\u003ePuccinia triticina\u003c/em\u003e, as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. \u003cem\u003eLr30\u003c/em\u003e provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible \u003cem\u003eLr30\u003c/em\u003e haplotypes, with transgenic plants carrying either variant exhibiting susceptibility. Cloning of \u003cem\u003eLr30\u003c/em\u003e will accelerate its deployment in wheat breeding programs.\u003c/p\u003e","manuscriptTitle":"Genome-assisted identification of wheat leaf rust resistance gene Lr30 (synonym Lr.ace-4A)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 06:35:59","doi":"10.21203/rs.3.rs-6289485/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b44eb78c-224b-4675-9c01-ccb76f079553","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46266548,"name":"Biological sciences/Genetics/Plant breeding"},{"id":46266549,"name":"Biological sciences/Biotechnology/Plant biotechnology/Agricultural genetics"},{"id":46266550,"name":"Biological sciences/Plant sciences/Plant stress responses/Biotic"},{"id":46266551,"name":"Biological sciences/Genetics/Agricultural genetics"}],"tags":[],"updatedAt":"2025-10-23T07:05:42+00:00","versionOfRecord":{"articleIdentity":"rs-6289485","link":"https://doi.org/10.1038/s41467-025-64428-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-10-22 04:00:00","publishedOnDateReadable":"October 22nd, 2025"},"versionCreatedAt":"2025-03-27 06:35:59","video":"","vorDoi":"10.1038/s41467-025-64428-5","vorDoiUrl":"https://doi.org/10.1038/s41467-025-64428-5","workflowStages":[]},"version":"v1","identity":"rs-6289485","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6289485","identity":"rs-6289485","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}