The development of oat cultivars with resistance to crown rust caused by Puccinia coronata f. sp. Avenae (Pca) is key for sustainable disease control. This study examined two recombinant inbred line populations, Provena x GS7 and Boyer x GS7, to identify adult plant resistance QTL in Australian fields. Seven distinct QTL associated with rust resistance were identified. KASP markers were developed for single nucleotide polymorphisms (SNPs) tightly linked to the four most significant QTL on chromosomes 4A and 7A. A major QTL named QPc_GS7_4A.2 with a resistance allele derived from line GS7 was mapped to chromosome 4A, overlapping with genomic regions previously associated with both resistance gene Pc61 and adult plant resistance. Genetic mapping for rust resistance at the seedling stage using a subset of Provena x GS7 lines with contrasting alleles at QPc_GS7_4A.2 suggests a role of this locus in seedling resistance, which may be explained by the presence of Pc61. Seedling resistance profiles between GS7 and the Pc61 differential line against 20 Pca isolates, a haplotype analysis of QPc_GS7_4A.2 in the oat crown rust differential set, and a collection of 182 oat lines support this hypothesis, although confirmation needs future research. The KASP markers developed in this study will assist breeders in efficiently integrating the resistance allele for gene combinations in new cultivars.

Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition. Effectors play central roles in this molecular interplay, and zinc-binding folds have been identified in a subset of pathogen effectors, yet the contribution of zinc coordination to effector stability and immune recognition remains unclear. Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites. Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site totally abolishes recognition. Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors.

Accurate gene annotation is crucial for inference of biological knowledge from genomes. However, non-canonical genes such as orphan or single-exon genes as well as those residing in rapidly evolving regions are routinely dismissed in annotation pipelines. In filamentous pathogen genomes, this disproportionately affects the annotation of genes encoding disease-promoting effector proteins. We introduce EffectorGeneP, a machine learning tool that self-trains on transcript data, predicts the most likely coding sequence from transcripts and effectively separates bona fide genes from transcriptional noise. EffectorGeneP annotates over 95% of known effectors correctly, while other state-of-the-art methods annotate 15%-78%. We show that EffectorGeneP expands the predicted secretome of pathogens by over 50% and that high-throughput screening of an effector library in plant protoplasts uncovers the previously poorly annotated AvrSr26 gene family in the wheat stem rust fungus. EffectorGeneP decodes genomes at unprecedented resolution and will enable the study of biological processes in important pathogen species.

Puccinia coronata f. sp. avenae (Pca), a fungal pathogen causing crown rust of oat, demonstrates rapid virulence evolution and adaptation to newly released cultivars. To further capture the genetic variation of Pca, we generated nuclear haplotype-resolved genomes for 10 isolates from Europe, Africa, and the Middle East and compared these with existing references for U.S. and Australian isolates. Of the full collection of 52 haplotypes, 40 were unique. Importantly, the presence of a nearly identical haplotype in a U.K. isolate collected in 1984 and in U.S. isolates from 1990 and 2017 supports the existence of long-lived clonal haplotypes in the global population that have been exchanged between lineages. We identified infrequent recombination between haplotypes from geographically dispersed isolates, hinting toward a globally mobile population of Pca that is mostly composed of persistent clonal lineages with some influence from rare recombination events. One isolate contained an additional scaffold with telomeric and centromeric features, suggesting the presence of a supernumerary chromosome. Whereas the core pan-proteome is enriched for predicted secreted and effector proteins, sequence and expression variation are most prevalent in non-core orthogroups, which also displayed allele-specific expression. We anticipate that this expanded collection of haplotypes will facilitate the development of new surveillance technologies and identification of virulence loci. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Barley leaf rust disease, caused by Puccinia hordei, leads to substantial yield losses and diminished malting quality of barley across temperate growing regions worldwide. To address the paucity of high-resolution genomic resources for this pathogen, we generated haplotype-phased, chromosome-scale assemblies for 10 globally distributed isolates using PacBio HiFi and Hi-C sequencing. Phylogenomic analysis revealed 7 distinct lineages of P. hordei, including evidence of nuclear exchange, with a shared nuclear haplotype detected between 2 US lineages. Nuclear genome sizes ranged from ∼140 to 147 Mbp, with the exception of isolate 90ISR03 from Israel (∼163 Mbp), which also harbored a 6.2-Mbp extra scaffold in one nucleus exhibiting chromosomal characteristics. Consistent with its larger genome, P. hordei had a higher repeat content (∼70%) than related cereal rust fungi, driven primarily by the proliferation of long terminal repeat (LTR) retroelements and DNA transposons. Across the global pan-genome of 13 unique nuclear haplotypes, approximately one-third of all protein orthogroups were conserved across all isolates. Only 18% of predicted effector orthogroups were conserved across all haplotypes, reflecting the highly dynamic and variable nature of the effector repertoire. The long-term propagation of clonal P. hordei lineages is apparent both within the United States and globally, and nuclear exchange plays a role in generating novel diversity. Genome plasticity is evident in extensive structural variation, including large-scale translocations and inversions as well as an extra chromosome. These chromosome-level, haplotype-resolved genomes provide a foundational resource for exploring the evolution, diversity, and avirulence gene repertoire of P. hordei.

Puccinia graminis f. sp. tritici (Pgt) causes wheat stem rust, a devastating disease of cereals. Recent approaches to examine populations at a genomic level have provided valuable information on the genotypic diversity of Pgt populations in Africa, Europe and North America and evolutionary mechanisms underlying the emergence of new races. However, an in-depth characterisation of Pgt populations in South America has been lacking. To bridge this knowledge gap, 91 Pgt isolates were collected from Argentina and Uruguay in 2020 and 2021 and used to generate transcriptome and whole genome sequence data and pathotype information. Phylogenetic analyses revealed that this South American Pgt population includes three clonal lineages, two of which have not been detected elsewhere. The third lineage is globally dispersed, including isolates from Africa and the Middle East. The predominant lineage, unique to South America, encompassed 90% of the samples and showed related pathotypes differing by virulence on single resistance genes consistent with evolution by stepwise mutation within the clonal lineage. There was no evidence of sexual recombination giving rise to new genetic diversity in this population. These observations urge to routinely incorporate and compare genotypic data from South American Pgt isolates to surveillance data from other geographic regions. .

Dikaryotic rust fungi maintain two distinct haploid nuclei for most of their life cycle, making their large, repeat-rich genomes difficult to assemble and phase. Here we present haplotype-phased, near chromosome-scale genome assemblies for the poplar rust pathogens Melampsora larici-populina 98AG31 and Melampsora allii-populina 12AY07, generated using PacBio HiFi sequencing and Hi-C-guided scaffolding. For each species, we resolved 18 chromosomes per haplotype, providing the first near chromosome-level representations of poplar rust fungal species. M. larici-populina diploid assembly spans ∼203 Mb, while M. allii-populina reaches ∼416 Mb, with high completeness and strong collinearity between haplotypes. Compared with previous fragmented or collapsed references, these assemblies greatly improve contiguity, recover centromeric and telomeric features, and support the transposable element-driven genome size expansion in M. allii-populina. The haplotype-aware annotations of genes and predicted effectors derived from these resources will enable detailed analyses of genome architecture, repeat dynamics, and key loci such as avirulence genes. Together, these assemblies provide a robust genomic resource for investigating host adaptation, virulence evolution, and population diversity in poplar rust fungi.

The fungal pathogen Rhizoctonia solani infects a diverse range of host plants and remains an intractable and economically significant disease for many crops. R. solani is classified into reproductively incompatible anastomosis groups (AGs). In the vegetative stage, most plant-pathogenic R. solani isolates are multinuclear and heterokaryotic, but little was previously known about the diversity between haplotypes due to highly fragmented, collapsed short-read assemblies. We present fully-phased, chromosome-scale genome assemblies of the broad host-range R. solani isolates AG8-1 and AG8-3. We demonstrate that both AG8 isolates have 2 distinct haplotypes, each of which is ∼50 Mbp spread across 16 chromosomes and use PacBio Iso-Seq data to achieve a high-quality gene annotation. We show that the 2 nuclear haplotypes display high heterozygosity and differences in haplotype abundance in vegetative cultures. Using transcriptome sequencing during infection of different host plants for AG8-1 and wheat for AG8-3, we show that the less abundant haplotype in both AG8-1 and AG8-3 might harbor more genes upregulated during infection. Taken together, these findings address some of the observed phylogenetic heterogeneity of AG-8 isolates and provide a platform to further dissect the mechanisms enabling this globally significant agricultural pathogen to inflict losses to a range of crop hosts.

Abstract Rust fungi are significant threats to global food security, causing substantial damage to crops through their ability to adapt and evolve new strains that overcome resistance. These obligate biotrophs infect host plants by secreting effector proteins that manipulate host physiology to promote infection and colonisation. We used AlphaFold2 to investigate structural conservation among effector proteins for the secretomes of Melampsora lini and four Puccinia species. AlphaFold2 yielded high-confidence predictions for 45.7% of the 27,090 secreted proteins, while 19% were poor quality. Comparative analysis revealed extensive structural diversity across the rust secretomes, with all thirteen known rust Avr proteins belonging to different clusters apart from AvrSr13 and AvrSr33. Nevertheless, there were still numerous large clusters of structurally-related proteins, including 59 clusters with over 50 members each, three of which contained known Avr proteins. Of the major structural families defined in other fungi, the rust species studied here only contained FOLD and ToxA-like families. Structural analysis of cysteine-rich proteins revealed over a thousand effector candidates featuring zinc-binding sites, with approximately 75% predicted to be cytoplasmic effectors. In contrast, cysteine-rich apoplastic effector candidates were characterized by a high frequency of disulfide bonds. One family of predicted metal-binding proteins was greatly expanded in P. graminis f. sp. tritici and includes AvrSr13 and AvrSr33. We confirmed that purified AvrSr13 and AvrSr22 proteins bind to zinc in vitro using biochemical assays. Taken together, structural modeling provides new avenues to study sequence-unrelated effectors and highlights the high degree of diversity in the effector repertoires of rust species.

Crown rust, caused by Puccinia coronata f. sp. avenae ( Pca ), remains a persistent threat to oat production in Australia. To monitor recent shifts in virulence and population structure, 30 Pca isolates collected during the 2024 growing season across major Australian oat-producing regions were analysed. Virulence analysis of 30 isolates using 52 oat differential lines identified 25 unique races that were not detected in previous years. Whole-genome sequencing of 28 of these isolates were analysed in the context of a broader historical Australian and international genomic datasets including isolates from Taiwan, South Africa, USA. Results confirmed the uniqueness of the Australian Pca population and revealed well-established genotypic lineages persisting over multiple years, with L18 and L16 being dominant. Notably, L16 was again present in Western Australia after being undetected in 2023, while L18 maintained its prevalence for a third consecutive season. Beyond these dominant groups, phylogenetic analysis and a k -mer containment analysis also identified a novel and genetically distinct lineage, designated as L19, represented by one isolate collected in WA. To add to the characterisation of lineage L19, we recorded virulence phenotypes on a small collection of current commercial cultivars. These findings enhance understanding of Pca diversity and emphasise the importance of surveillance approaches that integrate phenotypic and genomic surveillance.

Wheat stem rust is a disease of global importance caused by the fungal pathogen Puccinia graminis f. sp. tritici (Pgt). Here we generate chromosome-level, nuclear-phased genome references for Pgt isolates ETH2013-1 and ITA2018-1, representing races TKTTF and TTRTF respectively, that have caused major epidemics in Africa and Europe. The nuclear haplotypes of ETH2013-1 and ITA2018-1 are unique and unrelated to those of Ug99 and Pgt21. Pgt nuclear haplotypes show extensive variation in sequence and copy number of six known Avr genes and AvrSr33, which we identify through an effector gene library screen. Recognition properties of 22 novel Avr gene variants explain the race virulence phenotypes and the outbreak of TTRTF on durum cultivars containing Sr13b, since ITA2018-1 carries a homozygous deletion of AvrSr13. This work establishes an Avr gene atlas for Pgt that can inform wheat breeding and enable development of sequence-based virulence diagnostic tools for pathogen surveillance.

Crown rust, caused by Puccinia coronata f. sp. avenae ( Pca ), remains a persistent threat to oat production in Australia. To monitor recent shifts in virulence and population structure, 30 Pca isolates collected during the 2024 growing season across major Australian oat-producing regions were analysed. Phenotypic analysis of 30 isolates using 52 oat differential lines identified 27 unique races. Whole-genome sequencing of 28 of these isolates revealed that most of them belonged to previously established lineages, with L18 and L16 being dominant. Notably, L16 was again present in Western Australia after being undetected in 2023, while L18 maintained its prevalence for a third consecutive season. Beyond these dominant groups, phylogenetic analysis also identified a novel and genetically distinct lineage, designated as L19, and represented by one isolate collected in WA. Although L19 exhibited a virulence profile similar to other WA isolates, its unique genomic signature underscores the need to integrate phenotypic and genomic surveillance. These findings enhance understanding of Pca diversity and inform resistance breeding and disease management strategies in Australian oats. ### Competing Interest Statement The authors have declared no competing interest. GRDC, CSP2204-007RTX CSIRO

Rust fungi (order Pucciniales) form the largest group of plant pathogens. Early studies addressed infection processes and heritability of virulence but were limited by the complexity of these organisms. We present six lessons highlighting major discoveries about rust genome biology. First, during their main infectious stage, rusts carry two haploid genomes that can be highly heterozygous, and each genome is packaged in its own nucleus. Remarkably, this allows for the exchange of whole nuclei. Second, haploid genome size is extremely variable, ranging from ∼75 Mb to 1 GB. Third, genome expansions are driven by repetitive elements, but genomes are not compartmentalized. Fourth, although chromosome number and biotrophy-related gene content are conserved across the order, effectors are highly divergent in sequence. Fifth, resolution of the mating-type locus highlights its role in genetic exchange in populations. Sixth, individual haplotypes can now be tracked to reveal nuclear exchange events, delivering unprecedented insights into rust epidemiology.

Crown rust disease, caused by Puccinia coronata f. sp. avenae , poses a significant threat to global oat ( Avena sativa L.) production. Molecular markers are essential to assist in the integration of multiple resistance genes into a single oat cultivar to achieve genetic resistance durability. Here, we validated previously reported markers for the race‐specific resistance genes Pc39 , Pc45 / PcKM , Pc54 , and Pc68 and developed new kompetitive allele‐specific PCR markers closely linked to these loci. These markers were subsequently screened across a collection of 150 oat cultivars. Analysis of molecular marker data, pedigree information, and disease resistance profiles identified several oat cultivars likely carrying Pc39 and Pc68 . Newly identified carriers of Pc68 include the cultivars Galileo and Graza80, while Warrego, Kowari, Possum, Forester, Drummond, Hokonui, Glider, and Culgoa were identified as potential carriers of Pc39 . Discrepancies between the previous postulation of Pc gene carriers and our phenotyping and genotyping analysis were also found. For example, the oat line Glider was previously postulated to carry Pc58 or Pc59 , but it was positive for the Pc39 ‐associated marker and had a similar resistance profile to Pc39 carriers. These findings underscore the importance of the utilization of molecular markers in tracking the resistance genes in breeding germplasm.

Crown rust caused by the basidiomycete fungus Puccinia coronata f. sp. avenae (Pca) results in significant crop losses worldwide. Genetic solutions to protect against this disease require disease resistance gene discovery and introduction of resistance genes into elite germplasm by breeders. To inform disease resistance breeding activities, it is paramount to monitor changes of virulence in the Pca population and link those to genotypes of the pathogen. In 2023, a collection of 37 Pca isolates from diverse regions in Australia were gathered and their infection types across a commonly used set of oat differential lines determined to assign virulence pathotypes. We compared those virulence phenotypes to data collected in previous collections. While some of the virulence phenotypes had been reported in 2022, our analysis detected new virulence or an increase in frequency for virulence on some resistance genes. Notably, some of the frequency increases in virulence were recorded in Western Australia (WA), a region of interest due to its role in oat production for milling.

The role of nucleotide-binding leucine-rich repeat (NLR) receptors in plant immunity is well studied, but the function of a class of tandem kinases (TKs) that confer disease resistance in wheat and barley remains unclear. In this study, we show that the SR62 locus is a digenic module encoding the Sr62TK TK and an NLR (Sr62NLR), and we identify the corresponding AvrSr62 effector. AvrSr62 binds to the N-terminal kinase 1 of Sr62TK, triggering displacement of kinase 2, which activates Sr62NLR. Modeling and mutation analysis indicated that this is mediated by overlapping binding sites (i) on kinase 1 for binding AvrSr62 and kinase 2 and (ii) on kinase 2 for binding kinase 1 and Sr62NLR. Understanding this two-component resistance complex may help engineering and breeding plants for durable resistance.

Genetic diversity of pathogen populations plays an important role in host adaptation. While single high quality genome references are a valuable resource, a compilation of genome references from individuals increases the breadth of genetic variation within a species that is captured. Puccinia coronata f. sp. avenae ( Pca ), a fungal pathogen causing crown rust of oat, demonstrates rapid virulence evolution and adaptation to newly released cultivars. To broaden the geographic and temporal distribution of available Pca genomes, we generated nuclear haplotype-resolved genome references for ten isolates from Europe, Africa and the Middle East and compared these with existing references for USA and Australian isolates. Of the full collection of 52 haplotypes, 40 were unique. Importantly, the presence of a nearly identical haplotype in a UK isolate collected in 1984 and in USA isolates from 1990 and 2017 supports the existence of long-lived clonal haplotypes in the global population that have been exchanged between lineages. Taken together with the identification of infrequent recombination between haplotypes from geographically dispersed isolates, this evidence reflects a globally mobile population of Pca that is mostly comprised of persistent clonal lineages with some influence from rare recombination events. Analysis of the core and non-core proteome suggests that while the core proteome is enriched for predicted secreted and effector proteins, sequence and expression variation are most prevalent in non-core orthogroups. We anticipate that this expanded collection of haplotypes will facilitate the development of new surveillance technologies and identification of virulence loci. ### Competing Interest Statement The authors have declared no competing interest. USDA-NIFA BBSRC, 2022-67013-36505 GRDC, CSP2204-007RTX

Rust fungi comprise thousands of species, many of which cause disease on important crop plants. The flax rust fungus Melampsora lini has been a model species for the genetic dissection of plant immunity since the 1940s; however, the highly fragmented and incomplete reference genome has so far hindered progress in effector gene discovery. Here, we generated a fully phased, chromosome-scale assembly of the two nuclear genomes of M. lini strain CH5, resolving an additional 320 Mbp of the sequence. The 482-Mbp dikaryotic genome is at least 79% repetitive, with a large proportion (approximately 40%) of the genome comprising young, highly similar transposable elements. The assembly resolves the known effector gene loci, some of which carry complex duplications that were collapsed in the previous assembly. Using a genetic map followed by manual correction of gene models, we identified the AvrM3 and AvrN genes, which encode unusually large fungal effector proteins and trigger defense responses when co-expressed with the corresponding resistance genes. We located the genes linked to the tetrapolar mating system on chromosomes 4 and 9, but in contrast to the cereal rusts that have one pheromone receptor gene per haplotype, in flax rust, three pheromone receptor genes were found, with two of them closely linked on one haplotype. Taken together, we show that a high-quality assembly is crucial for resolving complex gene loci, and given the increasing number of fungal effectors of large size, the commonly applied criterion for effector candidates of being small proteins needs to be reconsidered. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Oat is a minor forage crop grown in Taiwan. Only a few historical records of oat rust disease have been reported in the country. Therefore, the pathogen population remains poorly characterized. A rust-like disease outbreak was detected at the Experimental Farm of National Taiwan University in 2019, which caused significant damage to field experiments. To determine the identity of the pathogen responsible for this disease outbreak, we collected infected foliar material. Disease signs suggested infection by the oat crown rust fungus. Hence, common procedures in rust pathology were applied to confirm the identity of the pathogen with phenotypic and molecular diagnostic techniques. A total of 50 field pathogen samples from infected oat cultivars were collected in 2019 and five single pustule rust isolates were obtained in 2020 and 2021. These isolates were initially identified as Puccinia coronata var. avenae f. sp. avenae (Pca) based on the phylogenetic analysis of nrITS sequence data. This identification was subsequently confirmed through whole-genome phylogeny, which showed that the representative Taiwanese isolate NTU1 clustered with other Pca representative strains in Basidiomycota. Phenotyping assays across 36 oat differential lines demonstrated that Taiwanese isolates are phenotypically similar with relatively low virulence. This study presents the first molecular confirmation of Pca in Taiwan and reports the virulence profiles of Taiwanese Pca population.

The population structure and evolution of basidiomycetes like rust fungi are influenced by complex reproductive cycles and dikaryotic life stages where two independent nuclear haplotypes are present in the cell. The ability to alternate between asexual (clonal) and sexual reproduction increases the evolutionary capacity in these species. Furthermore, exchange of intact nuclei (somatic hybridization) in rust fungi can allow for rapid generation of genetic variability outside of the sexual cycle. Puccinia coronata f. sp. avenae (Pca), the causal agent of oat crown rust, is a pathogen of global economic importance that is difficult to control due to rapid breakdown of host genetic resistance. The contribution of sexuality, clonality, and migration to virulence evolution varies across Pca populations. As such, the Pca pathosystem is ideal to address the role of mating type, recombination, mutation, and somatic hybridization in host adaptation. We expanded the existing resources for USA and South African populations by generating whole genome sequencing data of Taiwanese and Australian isolates. An atlas of 30 chromosome-level, fully-phased nuclear haplotypes from six USA isolates and nine Australian isolates was created to capture the genomic composition of key Pca lineages. At the haplotype level, we confirmed previous reports of genetic recombination in the USA population and additionally detected either sexual or cryptic recombination between Australian isolates, contrasting previous evaluations that suggested Pca populations in Australia to be purely clonal. We also identified somatic hybridization events in Pca that are not only associated with significant changes in fitness but also imply intercontinental migration of haplotypes, which provides further impetus for molecular monitoring of rust pathogen populations on a global scale.