Regenerating plants from immature embryos poses a significant obstacle for achieving genotype-independent transformation in cereals due to both constraints in the starting material and the recalcitrance of most elite varieties to transformation and regeneration. We tackled this issue by exploring the genetic basis of regeneration ability (RA) from mature embryos of barley ( Hordeum vulgare ) through analysis of a bi-parental population derived from a cross between the highly regenerative cultivar Golden Promise (GP) and the poorly regenerative Noga cultivar. By conducting QTL mapping on 122 F 2:3 progenies genotyped with a barley 50K SNP array, we pinpointed a locus on chromosome 2H, named Barley regeneration ability 1 ( Bra1 ), accounting for 12.6% of the phenotypic variance in mature embryo regeneration. Single-point analysis indicated a recessive mode of inheritance ( bra1 ), with individuals homozygous for GP allele showed on average 134.8% greater shooting percentage compared to Noga homozygotes or heterozygotes. Time-course transcriptomics spanning four phases of tissue culture identified eight candidate genes located within the 8.7 Mbp Bra1 region that exhibited expression profile consistent with the recessive mode of inheritance. This short list of candidate genes includes auxin signaling elements (like ARG7-like) along with additional regulatory proteins (G-type lectin S-receptor-like kinase, norbelladine synthase-like), all markedly downregulated in the homozygous carriers for the GP allele. High-resolution mapping with 63 recombinants derived from an additional 210 F 2 population between GP and Noga verified the QTL effect. However, regeneration and shooting analysis of selected recombinant families suggested that, although certain haplotype had on average ~ 107% increase in RA, there could be more than one factor in Bra1 that modulates this complex trait. Our findings imply that Bra1 functions as a suppressor of regeneration capacity in mature barley embryos, with dominant inhibitory alleles from recalcitrant genotypes limiting shoot organogenesis. This work provides both a genetic marker system for breeding programs and a foundation for developing dry-seed-based transformation protocols through targeted manipulation of the identified regulatory network.

Seed weight (SW) and nutrient allocation are key determinants of yield and grain quality in wheat, yet the regulatory basis of naturally occurring variation in these traits remains poorly resolved. Wild emmer wheat (Triticum dicoccoides), the progenitor of modern wheat, retains extensive eco-geographically structured genetic diversity that was largely eroded during domestication. Here, we identify a B3-domain transcription factor (B3TF) as a key regulator of seed growth and metabolic partitioning in wheat. Genome-wide association analysis of ~460 wild emmer accessions reveals a major locus on chromosome 2BL associated with SW, seed area and nitrogen (N) content, displaying pronounced climatic differentiation across environmental gradients. Introgression of the 2BL wild segment into the hexaploid wheat cultivars Chinese Spring and Bethlehem increases SW in cultivated backgrounds. Independent loss-of-function alleles generated by EMS mutagenesis in the tetraploid wheat cultivar Kronos produce larger seeds. Further, RNA-seq of EMS mutants revealed metabolic reprogramming with upregulated fatty acid, nitrogen and phenylpropanoid pathways and downregulated carbohydrate metabolism and sugar transport. Metabolomic, lipidomic and ICP-MS data showed increased essential amino acids, sugars, lipids, N content and minerals (Zn, Fe, Mo). Furthermore, CRISPR/Cas9-mediated editing in the hexaploid wheat cultivar Fielder produced similar increases in SW and N content as observed in the EMS mutants, establishing this gene as a negative regulator of seed growth across ploidy levels. In addition, natural haplotypes show reciprocal climatic distributions, linking regulatory variation to environmental adaptation. Our findings uncover a TF underlying natural seed trait variation in wild wheat, providing a framework for exploiting regulatory alleles to enhance yield and nutritional quality in modern wheat.

Plant responses to pathogens often rely on receptor-like cytoplasmic kinases (RLCKs) that mediate signaling through interactions with receptor kinases and downstream components. Here, we studied the tomato RLCK, TPK1b Related Protein Kinase (TPK09), and demonstrate its function in integrating defense with light stress responses. Tomato tpk09 mutants exhibited increased susceptibility to Botrytis cinerea and the vascular pathogen Fusarium oxysporum, whereas transgenic expression of TPK09 enhanced resistance to Botrytis. Disease severity in tpk09 mutants was elevated under light-emitting diode (LED) compared to fluorescent light (FL). In the absence of infection, mutants displayed severe necrosis and elevated H2O2 accumulation under LED lighting. TPK09 interacts with the cell death-inducing transglycosylase BcCrh1 from Botrytis. Consistently, TPK09 suppresses cell death triggered by Botrytis infection as well as by BcCrh1 expression. Loss of TPK09 abolished pathogen-induced expression of the tomato suppressor of cell death BAX INHIBITOR-LIKE1 and compromised chitin- and flg22-triggered reactive oxygen species (ROS) accumulation and immune gene activation. Further, TPK09 mitigates damage to the photosynthetic system under elevated light stress, demonstrated by a decrease in the effective photochemical quantum yield of PSII and electron transport rate in the mutant plants. In addition, TPK09 expression is induced by light but suppressed under dark conditions, and the mutant seedlings were insensitive to hypocotyl growth responses to light. RNA sequencing (RNA-seq) studies suggest TPK09 is required for expression of genes involved in light harvesting, photosynthesis, and stress response functions. Collectively, TPK09 plays a key role in enhancing fungal resistance, maintaining the homeostasis of the photosynthetic apparatus and ROS levels, and mitigating photooxidative damage.

Mycoviruses are ubiquitous in fungi, and some of these viruses induce hypovirulence, making them potential biocontrol agents. However, the effect of host genetic background at the pathogen population level on the performance of mycoviruses has not been fully investigated. Here, five diverse species of mycoviruses in Botrytis cinerea were used as a model to investigate the effect of population variation on mycovirus-induced phenotypes. The five mycoviruses were fully sequenced, including two hypoviruses (dsRNA1 and dsRNA2), one victorivirus (dsRNA3), and one partitivirus (dsRNA7 and dsRNA8), while one virus (dsRNA4, dsRNA5, and dsRNA6) remained unclassified. These viruses were readily transmitted to three B. cinerea strains, resulting in several derivative strains infected by different combinations of the five viruses. Notably, infection by some of these five viruses generally increased sporulation. However, the symptoms induced by the viruses varied, ranging from no significant effects to reduced virulence among different B. cinerea strains with the same viral profile. Therefore, we propose that the B. cinerea population can be broadly categorized into sensitive and insensitive groups regarding viral infection. These findings suggest that variation within the B. cinerea population may affect mycovirus-mediated phenotypic changes in their hosts. The presence of the insensitive group within the fungal population could also complicate the application of mycoviruses for fungal disease control.

Heat adaptation is a multilayered universal process involving a coordinated response of general and heat-specific cellular systems and processes. Here, we demonstrate that adaptation of the plant pathogenic fungus Botrytis cinerea to mild heat stress requires both autophagy and the mitochondrial Lon1 protease. Deleting bclon1 or blocking autophagy by deleting the bcatg1 autophagy-regulating gene did not affect fungal survival at optimal temperature. Under heat stress, deletion of bclon1 induced earlier and more intense autophagy, mitochondrial malfunction, and accelerated fungal cell death. These phenomena were intensified in a bcatg1/lon1 double mutant, indicating coordinated activity of both pathways in heat adaptation. Blocking autophagy, but not bclon1, also affected mycelia growth, spore germination, as well as nuclei division and spore morphology. Our results support a cytoprotective role for autophagy downstream of mitochondria-driven death signals, possibly as a mechanism that promotes growth arrest and helps remove damaged cellular components.

Viruses, ubiquitous non-cellular organisms, pose significant threats to human health and to the agricultural productivity of both livestock and crops. Emerging evidence indicates that multiple viruses can infect a single host, and viral co-infection can exert a profound influence on host physiology. However, our understanding of the prevalence of co-infection and the compatibility of phylogenetically distant viruses is still limited. In this study, we surveyed 406 field strains of the plant fungal pathogen Botrytis cinerea and identified 76 mycoviruses. Strikingly, 404 strains were co-infected with two or more viruses, with some harboring up to 25 viruses simultaneously. We discerned significant preference patterns among viruses in their host. Specifically, we identified “one-to-one” and “two-to-one” rules, wherein one or two viruses could be used to reliably predict the presence or absence of other viruses in the same host, and validated these predicted rules by using five B. cinerea strains. Furthermore, through the RNA-sequencing approach, we uncovered B. cinerea genes associated with the differences caused by different sets of co-infecting viruses. These are implicated in integral components of membrane, transmembrane transporter activity, autophagy pathways, mitophagy pathway, fatty acid biosynthetic process, sphingolipid metabolism, and glycosphingolipid biosynthesis. Our findings underscore the high prevalence of co-infection by multiple viruses in a fungal host within a population and highlight compatibility dynamics among phylogenetically diverse viruses. These insights contribute to our understanding of viral ecology and hold promise for informing strategies to manage viral diseases effectively. IMPORTANCE Viruses, pervasive threats to both humans and agriculture, often infect hosts concurrently, profoundly impacting physiology. Despite this, the prevalence and compatibility of co-infecting viruses remain poorly understood. In the study of 406 Botrytis cinerea strains, we discovered a striking phenomenon: 404 out of the 406 strains hosted multiple viruses, some with up to 25 at once. Through rigorous analysis, we unveiled distinct preference patterns among these viruses within hosts, identifying predictive co-infection rules validated by experimentation. Furthermore, we identified genes linked to these dynamics, shedding light on critical cellular processes involved in the regulation of the co-infection rules. These findings highlight the widespread nature of viral co-infection and offer insights crucial for effectively managing viral diseases.

Wild plants can contribute valuable genes to their domesticated relatives1. Fertility barriers and a lack of genomic resources have hindered the effective use of crop-wild introgressions. Decades of research into barley's closest wild relative, Hordeum bulbosum, a grass native to the Mediterranean basin and Western Asia, have yet to manifest themselves in the release of a cultivar bearing alien genes2. Here we construct a pangenome of bulbous barley comprising 10 phased genome sequence assemblies amounting to 32 distinct haplotypes. Autotetraploid cytotypes, among which the donors of resistance-conferring introgressions are found, arose at least twice, and are connected among each other and to diploid forms through gene flow. The differential amplification of transposable elements after barley and H. bulbosum diverged from each other is responsible for genome size differences between them. We illustrate the translational value of our resource by mapping non-host resistance to a viral pathogen to a structurally diverse multigene cluster that has been implicated in diverse immune responses in wheat and barley.

Botrytis cinerea is a major necrotrophic pathogen responsible for significant crop losses worldwide. Alternative strategies to control B. cinerea are urgently needed to reduce dependence on chemical fungicides, which are increasingly ineffective due to resistance and pose environmental risks. In this study, we identified two immunogenic epitopes derived from the B. cinerea cell death-inducing protein BcCrh1 and used them to engineer disease-resistant plants through a novel, spatially compartmentalized dual-epitope immune activation strategy. The first epitope is derived from a 35-amino acid intracellular peptide that exhibits both immunogenicity and cell death-inducing activity, which was mutated to separate these two properties. The second peptide represents an immunogenic portion of the protein that activates extracellular plant immunity. Transcriptomic and metabolomic analyses revealed that these epitopes trigger complementary defense pathways, and their co-expression integrates these responses into a robust, multilayered immunity, providing significantly enhanced protection compared with individual expression. Although constitutive expression of two epitopes conferred resistance, it also led to growth penalties. In contrast, pathogen-inducible expression of two epitopes preserved normal plant development while maintaining strong resistance to both B. cinerea and Pseudomonas syringae in Arabidopsis and tomato. This inducible strategy offers a major advantage by minimizing fitness costs while maximizing protection, highlighting the potential of spatially and temporally targeted epitope-based immune activation for durable and sustainable crop protection.

Genetic engineering of wheat is complex due to its large genome size, the presence of numerous genes with high sequence similarities, and a multitude of repetitive elements. In addition, genetic transformation of wheat has been difficult, mainly due to poor regeneration in tissue cultures. Recent advances in plant biotechnology, particularly the use of the regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), have provided new tools for wheat transformation and regeneration. Another transformative tool is the RUBY system that involves genetic engineering of three betalain biosynthesis genes, providing a noninvasive, visually detectable red pigment. In this study, we used the GRF4-GIF1 chimera along with the RUBY system to advance transformation and gene editing in wheat and barley. The GRF4-GIF1 chimera significantly aided wheat regeneration; however, it had an opposite effect in barley, where it inhibited the regeneration process. Therefore, we generated RUBY transgenic barley lines using constructs that did not include the GRF4-GIF1 chimera. Additionally, we used the RUBY cassette for fast assessment of gene editing by knockingout the first betalain biosynthetic gene in RUBY- positive transgenic wheat plants, resulting in a change of leaf color from red to green. The edited RUBY wheat lines lost more than just the red color. They also lost betalain-related traits, such as being less likely to get leaf rust (Puccinia triticina) and salt stress. Importantly, the loss of RUBY did not affect plant viability, making it a useful tool for genome editing and a viable alternative to destructive methods.

Background/Objective: Transcatheter aortic valve replacement (TAVR) is indicated for severe aortic stenosis patients with a prohibitive surgical risk. However, its use has been expanding in recent years to include intermediate- and low-risk patients. Thus, registry data describing changes in patient characteristics and outcomes are needed. The aim of this study was to analyse the temporal changes in patient profiles and clinical outcomes of all-comer TAVR. Methods: Baseline characteristics and VARC-3 outcomes of 1632 consecutive patients undergoing TAVR between 2008 and 2021 were analysed. Results: The annual rate of TAVR increased from 30 procedures in 2008–2009 to 398 in 2020–2021. Over the follow-up period, patient age decreased from 85 ± 4 to 80 ± 6.8 (p < 0.001) and the STS score decreased from 5.9% to 2.8% (p < 0.001). Procedural characteristics significantly changed, representing a shift into a minimally invasive approach: adoption of local anaesthesia (none to 48%, p < 0.001) and preference of transfemoral access (74% in 2011–2012 vs. 94.5% in 2020–2021, p < 0.001). The rates of almost all procedural complications decreased, including major vascular and bleeding complications, acute kidney injury (AKI) and in-hospital heart failure. There was a striking decline in rates of complete atrioventricular block (CAVB) and the need for a permanent pacemaker (PPM). PPM rates, however, remain high (17.8%). Thirty-day and one-year mortality significantly declined to 1.8% and 8.3%, respectively. Multivariable analysis shows that AKI, bleeding and stroke are strong predictors of one-year mortality (p < 0.001). Conclusions: The TAVR procedure has changed dramatically during the last 14 years in terms of patient characteristics, procedural aspects and device maturity. These shifts have led to improved procedural safety, contributing to improved short- and long-term patient outcomes.

Bread wheat (Triticum aestivum) is a globally dominant crop and major source of calories and proteins for the human diet. Compared with its wild ancestors, modern bread wheat shows lower genetic diversity, caused by polyploidisation, domestication and breeding bottlenecks1,2. Wild wheat relatives represent genetic reservoirs, and harbour diversity and beneficial alleles that have not been incorporated into bread wheat. Here we establish and analyse extensive genome resources for Tausch’s goatgrass (Aegilops tauschii), the donor of the bread wheat D genome. Our analysis of 46 Ae. tauschii genomes enabled us to clone a disease resistance gene and perform haplotype analysis across a complex disease resistance locus, allowing us to discern alleles from paralogous gene copies. We also reveal the complex genetic composition and history of the bread wheat D genome, which involves contributions from genetically and geographically discrete Ae. tauschii subpopulations. Together, our results reveal the complex history of the bread wheat D genome and demonstrate the potential of wild relatives in crop improvement. Analysis of 46 newly sequenced or re-sequenced Tausch’s goatgrass (Aegilops tauschii) accessions establishes the origin of the bread wheat (Triticum aestivum) D genome from genetically and geographically discrete Ae. tauschii subpopulations.

Nucleotide-binding leucine-rich repeat (NLR) disease resistance genes typically confer resistance against races of a single pathogen. Here, we report that Yr87/Lr85, an NLR gene from Aegilops sharonensis and Aegilops longissima, confers resistance against both P. striiformis tritici (Pst) and Puccinia triticina (Pt) that cause stripe and leaf rust, respectively. Yr87/Lr85 confers resistance against Pst and Pt in wheat introgression as well as transgenic lines. Comparative analysis of Yr87/Lr85 and the cloned Triticeae NLR disease resistance genes shows that Yr87/Lr85 contains two distinct LRR domains and that the gene is only found in Ae. sharonensis and Ae. longissima. Allele mining and phylogenetic analysis indicate multiple events of Yr87/Lr85 gene flow between the two species and presence/absence variation explaining the majority of resistance to wheat leaf rust in both species. The confinement of Yr87/Lr85 to Ae. sharonensis and Ae. longissima and the resistance in wheat against Pst and Pt highlight the potential of these species as valuable sources of disease resistance genes for wheat improvement. Leaf rust and stripe rust of wheat are two important fungal diseases of cultivated wheat and they are caused by infection of different pathogens. Here, the authors report the nucleotide-binding leucine-rich repeat (NLR) protein encoding gene Yr87/Lr85 confers resistance to both diseases.

Genetic engineering of wheat is complex due to its large genome size, the presence of numerous genes with high sequence similarities, and a multitude of repetitive elements. In addition, genetic transformation of wheat has been difficult, mainly due to poor regeneration in tissue cultures. Recent advances in plant biotechnology, particularly the use of the regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), have provided new tools for wheat transformation and regeneration. Another transformative tool is the RUBY system, that involves genetic engineering of three betalain biosynthesis genes, providing a noninvasive, visually detectable red pigment. In this study, we used the GRF4-GIF1 chimera along with the RUBY system to advance transformation and gene editing in wheat and barley. The GRF4-GIF1 chimera significantly aided wheat regeneration; however, it had an opposite effect in barley, where it inhibited the regeneration process. Therefore, we primarily generated RUBY transgenic barley lines using constructs that did not include the GRF4-GIF1 chimera. Additionally, we used the RUBY cassette for fast assessment of gene editing by knocking out the first betalain biosynthetic gene in RUBY- positive transgenic wheat plants, resulting in a change of leaf color from red to green. The edited RUBY wheat lines lost more than just the red color. They also lost betalain-related traits, such as being less likely to get leaf rust (Puccinia triticina) and salt stress. Importantly, the loss of RUBY did not affect plant viability, making it a useful tool for genome editing and a viable alternative to destructive methods. ### Competing Interest Statement The authors have declared no competing interest.

Necrotrophic plant pathogens are assumed to exploit the plant hypersensitive response (HR), but the molecular mechanism underlying this exploitation remains largely unclear. Here, we report the discovery and characterization of BcCELP1, an early infection-specific, cell death-inducing effector required for plant colonization by the phytopathogenic fungus Botrytis cinerea. We demonstrate that BcCELP1 is necessary during the initial stage of plant colonization, and that it interacts with the host scaffold protein NbRACK1, promoting NbRACK1’s interaction with the reduced nicotinamide adenine dinucleotide phosphate oxidase NbRBOHB, and thereby contributing to excessive ROS production. We further show that BcCELP1 is produced and specifically leveraged during plant invasion to facilitate the formation of necrotic tissue patches, which serve as foci for subsequent fungal spread. Misregulation of bccelp1 disrupts pathogen development, resulting in reduced disease symptoms. Collectively, these findings reveal an unsuspected sophisticated strategy employed by a necrotrophic pathogen, whereby a fungal effector activates the host ROS-generating machinery in a stage-specific manner to promote effective invasion.

Seeds acquire fungal endophytes either from the environment or from their progenitors. These transmission modes are central in shaping the microbiome because they affect species composition and balance. We studied fungal endophyte communities (FECs) in three plant species: bread wheat ( Triticum aestivum), wild emmer wheat ( Triticum turgidum dicoccoides), and wild barley ( Hordeum spontaneum). We conducted two experiments to test seed-to-seed transmission: (i) we compared FECs in stems and seeds collected from agricultural and natural habitats and (ii) we grew plants under greenhouse conditions to isolate the effect of vertical transmission on the plant FECs. The analysis of seed communities revealed differences in FEC composition and diversity among plant species; however, Alternaria infectoria dominated FECs in all plant species. In field-collected plants, the number of taxa in the seeds was less than half the number in stems, and close to 90% of the seed taxa were found in stems. The FECs from stems and seeds of greenhouse-grown plants were more diverse than the FECs of original seeds; they lacked a single dominant taxon, and the FECs in the new seeds had a similar richness and diversity to stem FECs, with only 40% overlap. The controlled-environment experiment confirmed vertical transmission of certain species but also showed that external infection of the seeds is the main source for specific taxa. Our results show that many taxa can reach the seeds internally, albeit in different abundance, that both infection sources affect seed FEC composition, and that external conditions affect the balance between FECs within the plant.

Plants harbor complex and highly diverse fungal endophyte communities (FECs), making it difficult to evaluate the functional role of individual taxa, subsets of the community, or the FEC as a whole. To reduce the complexity of this system, we aimed to produce fungi-null wheat (Triticum aestivum) plants. To this end, we treated seeds with heat and fungicides and generated plants from rescued embryos and callus tissue. A culture-based approach and reverse transcription PCR analysis were negative, indicating that all treatments produced plants apparently free of fungi. However, the analysis of DNA using digital droplet PCR and next-generation sequencing revealed that tissues from all treatments retained low levels but diversity-rich FECs. While the FECs varied in composition across treatments and tissues, they all included core taxa of the mycobiome. The reduced fungal biomass, along with the changes in FEC composition, negatively affected plant development, supporting a FEC contribution to proper plant development and fitness. Our discovery that a large part of the FEC cannot be separated from plants and can be transmitted through seeds and tissue culture calls for reevaluation of particular microbiome paradigms, such as core taxa concepts, transmission modes, and functional species. IMPORTANCE The native microbiome in a given plant must be considered when evaluating the effect of a single taxon or synthetic community. The pre-existing microbiome can interact with artificially added microbial cargo, which affects the final outcome. Such issues can be at least partially solved by the use of endophyte-free plants, which provide a clean background that should be useful in determining the effect of a single taxon, taxa combinations, or the entire microbiome on plant performance. Previous reports regarded plants as endophyte-free or axenic by the lack of fungal growth on culture media or the generation of plants from tissue cultures. We showed here that while fungi could not be isolated from fungicide-treated or tissue culture-regenerated plants, nevertheless, all plants contained rich fungal endophyte communities; namely, it was impossible to create fungi-free wheat plants. Our results call for rethinking fundamental microbiome-related concepts, such as core taxa, transmission mode, and functional species.

This study proposes a novel method for diagnosing core microbiotas based on prevalence of community members in a meta-community, which could be determined and supported statistically. Using this approach, the study found stratification in community assembly processes within fungal endophyte communities (FECs) in the stems of wheat and cereal-related wild species.

Botrytis cinerea causes gray mold disease in leading crop plants. The disease develops only at cool temperatures, but the fungus remains viable in warm climates and can survive periods of extreme heat. We discovered a strong heat priming effect in which the exposure of B. cinerea to moderately high temperatures greatly improves its ability to cope with subsequent, potentially lethal temperature conditions. We showed that priming promotes protein solubility during heat stress and discovered a group of priming-induced serine-type peptidases. Several lines of evidence, including transcriptomics, proteomics, pharmacology, and mutagenesis data, link these peptidases to the B. cinerea priming response, highlighting their important roles in regulating priming-mediated heat adaptation. By imposing a series of sub-lethal temperature pulses that subverted the priming effect, we managed to eliminate the fungus and prevent disease development, demonstrating the potential for developing temperature-based plant protection methods by targeting the fungal heat priming response. IMPORTANCE Priming is a general and important stress adaptation mechanism. Our work highlights the importance of priming in fungal heat adaptation, reveals novel regulators and aspects of heat adaptation mechanisms, and demonstrates the potential of affecting microorganisms, including pathogens through manipulations of the heat adaptation response.

To safeguard bread wheat against pests and diseases, breeders have introduced over 200 resistance genes into its genome, thus nearly doubling the number of designated resistance genes in the wheat gene pool 1 . Isolating these genes facilitates their fast-tracking in breeding programs and incorporation into polygene stacks for more durable resistance. We cloned the stem rust resistance gene Sr43 , which was crossed into bread wheat from the wild grass Thinopyrum elongatum 2 , 3 . Sr43 encodes an active protein kinase fused to two domains of unknown function. The gene, which is unique to the Triticeae, appears to have arisen through a gene fusion event 6.7 to 11.6 million years ago. Transgenic expression of Sr43 in wheat conferred high levels of resistance to a wide range of isolates of the pathogen causing stem rust, highlighting the potential value of Sr43 in resistance breeding and engineering.

The rust diseases, including leaf rust caused by Puccinia triticina (Pt), stem rust caused by P. graminis f. sp. tritici (Pgt), and stripe rust caused by P. striiformis f. sp. tritici (Pst), are major limiting factors in wheat production worldwide. Identification of novel sources of rust resistance genes is key to developing cultivars resistant to rapidly evolving pathogen populations. Aegilops longissima is a diploid wild grass native to the Levant and closely related to the modern bread wheat D subgenome. To explore resistance genes in the species, we evaluated a large panel of Ae. longissima for resistance to several races of Pt, Pgt, and Pst, and conducted a genome-wide association study (GWAS) to map rust resistance loci in the species. A panel of 404 Ae. longissima accessions, mostly collected from Israel, were screened for seedling-stage resistance to four races of Pt, four races of Pgt, and three races of Pst. Out of the 404 accessions screened, two were found that were resistant to all 11 races of the three rust pathogens screened. The percentage of all accessions screened that were resistant to a given rust pathogen race ranged from 18.5% to 99.7%. Genotyping-by-sequencing (GBS) was performed on 381 accessions of the Ae. longissima panel, wherein 125,343 single nucleotide polymorphisms (SNPs) were obtained after alignment to the Ae. longissima reference genome assembly and quality control filtering. Genetic diversity analysis revealed the presence of two distinct subpopulations, which followed a geographic pattern of a northern and a southern subpopulation. Association mapping was performed in the genotyped portion of the collection (n = 381) and in each subpopulation (n = 204 and 174) independently via a single-locus mixed-linear model, and two multi-locus models, FarmCPU, and BLINK. A large number (195) of markers were significantly associated with resistance to at least one of 10 rust pathogen races evaluated, nine of which are key candidate markers for further investigation due to their detection via multiple models and/or their association with resistance to more than one pathogen race. The novel resistance loci identified will provide additional diversity available for use in wheat breeding.