Intracellular Reactive Oxygen Species-Aided Localized Cell Death Contributing to Immune Responses Against Wheat Powdery Mildew Pathogen.

Reactive oxygen species (ROS)- and hypersensitive response (HR)-mediated cell death have long been known to play critical roles in plant immunity to pathogens. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive wheat pathogen. Here, we report a quantitative analysis of the proportion of infected cells with local apoplastic ROS (apoROS) versus intracellular ROS (intraROS) accumulation in various wheat accessions that carry different disease resistance genes (R genes) at a series of time points postinfection. The proportion of apoROS accumulation was 70 to 80% of the infected wheat cells detected in both compatible and incompatible host-pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses was detected in 11 to 15% of the infected wheat cells, mainly in wheat lines that carried nucleotide-binding leucine-rich repeat R genes (e.g., Pm3F, Pm41, TdPm60, MIIW72, and Pm69). The lines that carry unconventional R genes, Pm24 (Wheat Tandem Kinase 3) and pm42 (a recessive R gene), showed fewer intraROS responses, whereas 11% of Pm24 line-infected epidermis cells still showed HR cell death, suggesting that different resistance pathways are activated there. Here, we also demonstrated that ROS could not act as a strong systemic signal for inducing high resistance to Bgt in wheat, although it induced the expression of pathogenesis-related genes. These results provide new insights into the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew.

Genome-Wide Association Study in Bread Wheat Identifies Genomic Regions Associated with Grain Yield and Quality under Contrasting Water Availability.

The objectives of this study were to identify genetic loci in the bread wheat genome that would influence yield stability and quality under water stress, and to identify accessions that can be recommended for cultivation in dry and hot regions. We performed a genome-wide association study (GWAS) using a panel of 232 wheat accessions spanning diverse ecogeographic regions. Plants were evaluated in the Israeli Northern Negev, under two environments: water-limited (D; 250 mm) and well-watered (W; 450 mm) conditions; they were genotyped with ~71,500 SNPs derived from exome capture sequencing. Of the 14 phenotypic traits evaluated, 12 had significantly lower values under D compared to W conditions, while the values for two traits were higher under D. High heritability (H2 = 0.5-0.9) was observed for grain yield, spike weight, number of grains per spike, peduncle length, and plant height. Days to heading and grain yield could be partitioned based on accession origins. GWAS identified 154 marker-trait associations (MTAs) for yield and quality-related traits, 82 under D and 72 under W, and identified potential candidate genes. We identified 24 accessions showing high and/or stable yields under D conditions that can be recommended for cultivation in regions under the threat of global climate change.

Variation in phosphorus and sulfur content shapes the genetic architecture and phenotypic associations within the wheat grain ionome.

Dissection of the genetic basis of wheat ionome is crucial for understanding the physiological and biochemical processes underlying mineral accumulation in seeds, as well as for efficient crop breeding. Most of the elements essential for plants are metals stored in seeds as chelate complexes with phytic acid or sulfur-containing compounds. We assume that the involvement of phosphorus and sulfur in metal chelation is the reason for strong phenotypic correlations within ionome. Adjustment of element concentrations for the effect of variation in phosphorus and sulfur seed content resulted in drastic change of phenotypic correlations between the elements. The genetic architecture of wheat grain ionome was characterized by quantitative trait loci (QTL) analysis using a cross between durum and wild emmer wheat. QTL analysis of the adjusted traits and two-trait analysis of the initial traits paired with either P or S considerably improved QTL detection power and accuracy, resulting in the identification of 105 QTLs and 617 QTL effects for 11 elements. Candidate gene search revealed some potential functional associations between QTLs and corresponding genes within their intervals. Thus, we have shown that accounting for variation in P and S is crucial for understanding of the physiological and genetic regulation of mineral composition of wheat grain ionome and can be implemented for other plants.

Tandem Protein Kinases Emerge as New Regulators of Plant Immunity.

Plant-pathogen interactions result in disease development in a susceptible host. Plants actively resist pathogens via a complex immune system comprising both surface-localized receptors that sense the extracellular space as well as intracellular receptors recognizing pathogen effectors. To date, the majority of cloned resistance genes encode intracellular nucleotide-binding leucine-rich repeat receptor proteins. Recent discoveries have revealed tandem kinase proteins (TKPs) as another important family of intracellular proteins involved in plant immune responses. Five TKP genes-barley Rpg1 and wheat WTK1 (Yr15), WTK2 (Sr60), WTK3 (Pm24), and WTK4-protect against devastating fungal diseases. Moreover, a large diversity and numerous putative TKPs exist across the plant kingdom. This review explores our current knowledge of TKPs and serves as a basis for future studies that aim to develop and exploit a deeper understanding of innate plant immunity receptor proteins.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

TdPm60 identified in wild emmer wheat is an ortholog of Pm60 and constitutes a strong candidate for PmG16 powdery mildew resistance.

KEY MESSAGE: We identified TdPm60 alleles from wild emmer wheat (WEW), an ortholog of Pm60 from T. urartu, which constitutes a strong candidate for PmG16 mildew resistance. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta improved the resistance to several local Bgt isolates. Wild emmer wheat (WEW), the tetraploid progenitor of durum and bread wheat, is a valuable genetic resource for resistance to powdery mildew fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). PmG16 gene, derived from WEW, confers high resistance to most tested Bgt isolates. We mapped PmG16 to a 1.4-cM interval between the flanking markers uhw386 and uhw390 on Chromosome 7AL. Based on gene annotation of WEW reference genome Zavitan_V1, 34 predicted genes were identified within the ~ 3.48-Mb target region. Six genes were annotated as associated with disease resistance, of which TRIDC7AG077150.1 was found to be highly similar to Pm60, previously cloned from Triticum urartu, and resides in the same syntenic region. The functional molecular marker (FMM) for Pm60 (M-Pm60-S1) co-segregated with PmG16, suggesting the Pm60 ortholog from WEW (designated here as TdPm60) as a strong candidate for PmG16. Sequence alignment identified only eight SNPs that differentiate between TdPm60 and TuPm60. Furthermore, TdPm60 was found to be present also in the WEW donor lines of the powdery mildew resistance genes MlIW172 and MlIW72, mapped to the same region of Chromosome 7AL as PmG16, suggesting that TdPm60 constitutes a candidate also for these genes. Furthermore, screening of additional 230 WEW accessions with Pm60 specific markers revealed 58 resistant accessions from the Southern Levant that harbored TdPm60, while none of the susceptible accessions showed the presence of this gene. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta conferred resistance against several local Bgt isolates.

Genomic Architecture of Phenotypic Plasticity in Response to Water Stress in Tetraploid Wheat.

Phenotypic plasticity is one of the main mechanisms of adaptation to abiotic stresses via changes in critical developmental stages. Altering flowering phenology is a key evolutionary strategy of plant adaptation to abiotic stresses, to achieve the maximum possible reproduction. The current study is the first to apply the linear regression residuals as drought plasticity scores while considering the variation in flowering phenology and traits under non-stress conditions. We characterized the genomic architecture of 17 complex traits and their drought plasticity scores for quantitative trait loci (QTL) mapping, using a mapping population derived from a cross between durum wheat (Triticum turgidum ssp. durum) and wild emmer wheat (T. turgidum ssp. dicoccoides). We identified 79 QTLs affected observed traits and their plasticity scores, of which 33 reflected plasticity in response to water stress and exhibited epistatic interactions and/or pleiotropy between the observed and plasticity traits. Vrn-B3 (TaTF1) residing within an interval of a major drought-escape QTL was proposed as a candidate gene. The favorable alleles for most of the plasticity QTLs were contributed by wild emmer wheat, demonstrating its high potential for wheat improvement. Our study presents a new approach for the quantification of plant adaptation to various stresses and provides new insights into the genetic basis of wheat complex traits under water-deficit stress.

Wheat tandem kinases provide insights on disease-resistance gene flow and host-parasite co-evolution.

Stripe (yellow) rust, caused by the fungus Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat spread globally. Wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) is known as a source for novel Pst resistance genes (R-gene), but our knowledge on wheat-Pst co-evolution in natural populations is limited. Yr15 is a WEW (accession G25) gene, which confers a broad-spectrum resistance to Pst, and encodes a tandem kinase-pseudokinase protein designated as WTK1. Exon-intron comparisons of multiple WTK1 homoeologous and paralogous copies scattered in allopolyploid wheat genomes enabled us to develop functional molecular markers (FMMs), which were used for population genetic study. The functional allele (Wtk1) was absent in a worldwide collection of 513 wheat cultivars, except for 32 introgression lines with Yr15 from G25, as well as in 84% of the 382 tested WEW accessions collected across the Fertile Crescent. Yr15 was found to be distributed along a narrow axis from Mt Carmel to the Anti-Lebanon Mountains ridge, mostly at elevations above c. 500 m, where the climatic conditions are favorable for disease development, therefore providing insights on gene flow and host-parasite co-evolution in WEW natural habitats. Moreover, the worldwide absence of Wtk1 in cultivated wheat and in WEW natural populations from southeast Turkey, where wheat is believed to have been domesticated, proposes that Yr15 was rather left behind, than lost during domestication. Our results highlight the importance of conservation of WEW populations in their natural habitats for discovery of novel R-genes and studies of host-parasite co-evolution.

A CNL protein in wild emmer wheat confers powdery mildew resistance.

Powdery mildew, a fungal disease caused by Blumeria graminis f. sp. tritici (Bgt), has a serious impact on wheat production. Loss of resistance in cultivars prompts a continuing search for new sources of resistance. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, WEW), the progenitor of both modern tetraploid and hexaploid wheats, harbors many powdery mildew resistance genes. We report here the positional cloning and functional characterization of Pm41, a powdery mildew resistance gene derived from WEW, which encodes a coiled-coil, nucleotide-binding site and leucine-rich repeat protein (CNL). Mutagenesis and stable genetic transformation confirmed the function of Pm41 against Bgt infection in wheat. We demonstrated that Pm41 was present at a very low frequency (1.81%) only in southern WEW populations. It was absent in other WEW populations, domesticated emmer, durum, and common wheat, suggesting that the ancestral Pm41 was restricted to its place of origin and was not incorporated into domesticated wheat. Our findings emphasize the importance of conservation and exploitation of the primary WEW gene pool, as a valuable resource for discovery of resistance genes for improvement of modern wheat cultivars.

Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1.

The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303, and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and show here that these three resistance loci co-segregate in fine-mapping populations and share an identical full-length genomic sequence of functional Wtk1. Independent ethyl methanesulfonate (EMS)-mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303, and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303-, and YrH52-mediated resistances to yellow rust are encoded by a single locus, Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks.

Grain protein content and thousand kernel weight QTLs identified in a durum × wild emmer wheat mapping population tested in five environments.

KEY MESSAGE: Genetic dissection of GPC and TKW in tetraploid durum × WEW RIL population, based on high-density SNP genetic map, revealed 12 GPC QTLs and 11 TKW QTLs, with favorable alleles for 11 and 5 QTLs, respectively, derived from WEW. Wild emmer wheat (Triticum turgidum ssp. dicoccoides, WEW) was shown to exhibit high grain protein content (GPC) and therefore possess a great potential for improvement of cultivated wheat nutritional value. Genetic dissection of thousand kernel weight (TKW) and grain protein content (GPC) was performed using a high-density genetic map constructed based on a recombinant inbred line (RIL) population derived from a cross between T. durum var. Svevo and WEW acc. Y12-3. Genotyping of 208 F6 RILs with a 15 K wheat single nucleotide polymorphism (SNP) array yielded 4166 polymorphic SNP markers, of which 1510 were designated as skeleton markers. A total map length of 2169 cM was obtained with an average distance of 1.5 cM between SNPs. A total of 12 GPC QTLs and 11 TKW QTLs were found under five different environments. No significant correlations were found between GPC and TKW across all environments. Four major GPC QTLs with favorable alleles from WEW were found on chromosomes 4BS, 5AS, 6BS and 7BL. The 6BS GPC QTL coincided with the physical position of the NAC transcription factor TtNAM-B1, underlying the cloned QTL, Gpc-B1. Comparisons of the physical intervals of the GPC QTLs described here with the results previously reported in other durum × WEW RIL population led to the discovery of seven novel GPC QTLs. Therefore, our research emphasizes the importance of GPC QTL dissection in diverse WEW accessions as a source of novel alleles for improvement of GPC in cultivated wheat.

Glycerol-Induced Powdery Mildew Resistance in Wheat by Regulating Plant Fatty Acid Metabolism, Plant Hormones Cross-Talk, and Pathogenesis-Related Genes.

Our previous study indicated that glycerol application induced resistance to powdery mildew (Bgt) in wheat by regulating two important signal molecules, glycerol-3-phosphate (G3P) and oleic acid (OA18:1). Transcriptome analysis of wheat leaves treated by glycerol and inoculated with Bgt was performed to identify the activated immune response pathways. We identified a set of differentially expressed transcripts (e.g., TaGLI1, TaACT1, and TaSSI2) involved in glycerol and fatty acid metabolism that were upregulated in response to Bgt infection and might contribute to G3P and OA18:1 accumulation. Gene Ontology (GO) enrichment analysis revealed GO terms induced by glycerol, such as response to jasmonic acid (JA), defense response to bacterium, lipid oxidation, and growth. In addition, glycerol application induced genes (e.g., LOX, AOS, and OPRs) involved in the metabolism pathway of linolenic and alpha-linolenic acid, which are precursor molecules of JA biosynthesis. Glycerol induced JA and salicylic acid (SA) levels, while glycerol reduced the auxin (IAA) level in wheat. Glycerol treatment also induced pathogenesis related (PR) genes, including PR-1, PR-3, PR-10, callose synthase, PRMS, RPM1, peroxidase, HSP70, HSP90, etc. These results indicate that glycerol treatment regulates fatty acid metabolism and hormones cross-talk and induces the expression of PR genes that together contribute to Bgt resistance in wheat.

A wheat/rye polymorphism affects seminal root length and yield across different irrigation regimes.

The introgression of a small segment of wheat (Triticum aestivum L.) chromosome arm 1BS in the distal region of the rye (Secale cereale L.) 1RS.1BL arm translocation in wheat (henceforth 1RSRW) was previously associated with reduced grain yield, carbon isotope discrimination, and stomatal conductance, suggesting reduced access to soil moisture. Here we show that lines with the normal 1RS arm have longer roots than lines with the 1RSRW arm in both field and hydroponic experiments. In the 1RSRW lines, differences in seminal root length were associated with a developmentally regulated arrest of the root apical meristem (RAM). Approximately 10 d after germination, the seminal roots of the 1RSRW plants showed a gradual reduction in elongation rate, and stopped growing a week later. Seventeen days after germination, the roots of the 1RSRW plants showed altered gradients of reactive oxygen species and emergence of lateral roots close to the RAM, suggesting changes in the root meristem. The 1RSRW lines also showed reduced biomass (estimated by the normalized difference vegetation index) and grain yield relative to the 1RS lines, with larger differences under reduced or excessive irrigation than under normal irrigation. These results suggest that this genetic variation could be useful to modulate root architecture.

Wheat Stripe Rust Resistance Protein WKS1 Reduces the Ability of the Thylakoid-Associated Ascorbate Peroxidase to Detoxify Reactive Oxygen Species.

Stripe rust is a devastating fungal disease of wheat caused by Puccinia striiformis f. sp tritici (Pst). The WHEAT KINASE START1 (WKS1) resistance gene has an unusual combination of serine/threonine kinase and START lipid binding domains and confers partial resistance to Pst. Here, we show that wheat (Triticum aestivum) plants transformed with the complete WKS1 (variant WKS1.1) are resistant to Pst, whereas those transformed with an alternative splice variant with a truncated START domain (WKS1.2) are susceptible. WKS1.1 and WKS1.2 preferentially bind to the same lipids (phosphatidic acid and phosphatidylinositol phosphates) but differ in their protein-protein interactions. WKS1.1 is targeted to the chloroplast where it phosphorylates the thylakoid-associated ascorbate peroxidase (tAPX) and reduces its ability to detoxify peroxides. Increased expression of WKS1.1 in transgenic wheat accelerates leaf senescence in the absence of Pst. Based on these results, we propose that the phosphorylation of tAPX by WKS1.1 reduces the ability of the cells to detoxify reactive oxygen species and contributes to cell death. This response takes several days longer than typical hypersensitive cell death responses, thus allowing the limited pathogen growth and restricted sporulation that is characteristic of the WKS1 partial resistance response to Pst.

Distribution and haplotype diversity of WKS resistance genes in wild emmer wheat natural populations.

KEY MESSAGE: The wheat stripe rust resistance gene Yr36 ( WKS1 ) with a unique kinase-START domain architecture is highly conserved in wild emmer wheat natural populations. Wild emmer wheat (Triticum dicoccoides) populations have developed various resistance strategies against the stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst). The wild emmer gene, Yr36 (WKS1), which confers partial resistance to a broad spectrum of Pst races, is composed of a kinase and a START lipid-binding domain, a unique gene architecture found only in the Triticeae tribe. The analysis of 435 wild emmer accessions from a broad range of natural habitats revealed that WKS1 and its paralogue WKS2 are present only in the southern distribution range of wild emmer in the Fertile Crescent, supporting the idea that wheat domestication occurred in the northern populations. An analysis of full-length WKS1 sequence from 54 accessions identified 15 different haplotypes and very low-nucleotide diversity (π = 0.00019). The high level of WKS1 sequence conservation among wild emmer populations is in contrast to the high level of diversity previously observed in NB-LRR genes (e.g., Lr10 and Pm3). This phenomenon may reflect the different resistance mechanisms and different evolutionary pathways that shaped these genes, and may shed light on the evolution of genes that confer partial resistance to stripe rust. Only five WKS1 coding sequence haplotypes were revealed among all tested accessions, encoding four different putative WKS1 proteins (designated P0, P1, P2, and P3). Infection tests showed that P0, P1, and P3 haplotypes display a resistance response, while P2 displayed a susceptible response. These results show that the WKS1 proteins (P0, P1, and P3) can be useful to improve wheat resistance to stripe rust.

Transcriptome profiling of wheat glumes in wild emmer, hulled landraces and modern cultivars.

BACKGROUND: Wheat domestication is considered as one of the most important events in the development of human civilization. Wheat spikelets have undergone significant changes during evolution under domestication, resulting in soft glumes and larger kernels that are released easily upon threshing. Our main goal was to explore changes in transcriptome expression in glumes that accompanied wheat evolution under domestication. METHODS: A total of six tetraploid wheat accessions were selected for transcriptome profiling based on their rachis brittleness and glumes toughness. RNA pools from glumes of the central spikelet at heading time were used to construct cDNA libraries for sequencing. The trimmed reads from each library were separately aligned to the reference sub-genomes A and B, which were extracted from wheat survey sequence. Differentially expression analysis and functional annotation were performed between wild and domesticated wheat, to identity candidate genes associated with evolution under domestication. Selected candidate genes were validated using real time PCR. RESULTS: Transcriptome profiles of wild emmer wheat, wheat landraces, and wheat cultivars were compared using next generation sequencing (RNA-seq). We have found a total of 194,893 transcripts, of which 73,150 were shared between wild, landraces, and cultivars. From 781 differentially expressed genes (DEGs), 336 were down-regulated and 445 were up-regulated in the domesticated compared to wild wheat genotypes. Gene Ontology (GO) annotation assigned 293 DEGs (37.5 %) to GO term groups, of which 134 (17.1 %) were down-regulated and 159 (20.4 %) up-regulated in the domesticated wheat. Some of the down-regulated DEGs in domesticated wheat are related to the biosynthetic pathways that eventually define the mechanical strength of the glumes, such as cell wall, lignin, pectin and wax biosynthesis. The reduction in gene expression of such genes, may explain the softness of the glumes in the domesticated forms. In addition, we have identified genes involved in nutrient remobilization that may affect grain size and other agronomic traits evolved under domestication. CONCLUSIONS: The comparison of RNA-seq profiles between glumes of wheat groups differing in glumes toughness and rachis brittleness revealed a few DEGs that may be involved in glumes toughness and nutrient remobilization. These genes may be involved in processes of wheat improvement under domestication.

A high-density, SNP-based consensus map of tetraploid wheat as a bridge to integrate durum and bread wheat genomics and breeding.

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor (wild emmer, T. turgidum ssp. dicoccoides). The consensus map harboured 30 144 markers (including 26 626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631 cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes.

Wheat syntenome unveils new evidences of contrasted evolutionary plasticity between paleo- and neoduplicated subgenomes.

Bread wheat derives from a grass ancestor structured in seven protochromosomes followed by a paleotetraploidization to reach a 12 chromosomes intermediate and a neohexaploidization (involving subgenomes A, B and D) event that finally shaped the 21 modern chromosomes. Insights into wheat syntenome in sequencing conserved orthologous set (COS) genes unravelled differences in genomic structure (such as gene conservation and diversity) and genetical landscape (such as recombination pattern) between ancestral as well as recent duplicated blocks. Contrasted evolutionary plasticity is observed where the B subgenome appears more sensitive (i.e. plastic) in contrast to A as dominant (i.e. stable) in response to the neotetraploidization and D subgenome as supra-dominant (i.e. pivotal) in response to the neohexaploidization event. Finally, the wheat syntenome, delivered through a public web interface PlantSyntenyViewer at http://urgi.versailles.inra.fr/synteny-wheat, can be considered as a guide for accelerated dissection of major agronomical traits in wheat.

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes.

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen.