Comparative Analysis of the Avirulence Effectors Produced by the Fungal Stem Rust Pathogen of Wheat.

Crops are constantly exposed to pathogenic microbes. Rust fungi are examples of these harmful microorganisms, which have a major economic impact on wheat production. To protect themselves from pathogens like rust fungi, plants employ a multilayered immune system that includes immunoreceptors encoded by resistance genes. Significant efforts have led to the isolation of numerous resistance genes against rust fungi in cereals, especially in wheat. However, the evolution of virulence of rust fungi hinders the durability of resistance genes as a strategy for crop protection. Rust fungi, like other biotrophic pathogens, secrete an arsenal of effectors to facilitate infection, and these are the molecules that plant immunoreceptors target for pathogen recognition and mounting defense responses. When recognized, these effector proteins are referred to as avirulence (Avr) effectors. Despite the many predicted effectors in wheat rust fungi, only five Avr genes have been identified, all from wheat stem rust. Knowledge of the Avr genes and their variation in the fungal population will inform deployment of the most appropriate wheat disease-resistance genes for breeding and farming. The review provides an overview of methodologies as well as the validation techniques that have been used to characterize Avr effectors from wheat stem rust. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genome-Enabled Analysis of Population Dynamics and Virulence-Associated Loci in the Oat Crown Rust Fungus Puccinia coronata f. sp. avenae.

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the United States and South Africa. Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the United States and South Africa. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the United States and contributions from clonality and sexuality. The population from South Africa is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define 11 virulence-associated loci corresponding to 25 oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the United States to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines, suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognizing genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

AvrSr27 is a zinc-bound effector with a modular structure important for immune recognition.

Effector proteins are central to the success of plant pathogens, while immunity in host plants is driven by receptor-mediated recognition of these effectors. Understanding the molecular details of effector-receptor interactions is key for the engineering of novel immune receptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tritici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We characterised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assays and examined Sr27-mediated recognition using transient expression in Nicotiana spp. and wheat protoplasts. The AvrSr27 structure contains a novel ß-strand rich modular fold consisting of two structurally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufficient for interaction with Sr27 and triggering cell death. We identified two Pgt proteins structurally related to AvrSr27 but with low sequence identity that can also associate with Sr27, albeit more weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in transient expression systems. Collectively, our findings have important implications for utilising protein prediction platforms for effector proteins, and those embarking on bespoke engineering of immunity receptors as solutions to plant disease.

Genotypic and Resistance Profile Analysis of Two Oat Crown Rust Differential Sets Urge Coordination and Standardization.

Puccinia coronata f. sp. avenae is the causal agent of the disease known as crown rust, which represents a bottleneck in oat production worldwide. Characterization of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared the virulence profiles of 25 P. coronata f. sp. avenae isolates from Australia using two host differential sets, one from Australia and one from the United States. These differential sets were also genotyped using diversity arrays technology sequencing technology. Phenotypic and genotypic discrepancies were detected on 8 out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterize materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Virulence Patterns of Oat Crown Rust in Australia - Season 2022.

Puccinia coronata f. sp. avenae (Pca) is an important foliar pathogen of oat which causes crown rust disease. The virulence profile of 48 Pca isolates derived from different locations in Australia was characterized using a collection of oat lines often utilized in rust surveys in the United States and Australia. This analysis indicates that Pca populations in Eastern Australia are broadly virulent, which contrasts with the population in Western Australia (WA). Several oat lines/Pc genes are effective against all rust samples collected from WA, suggesting they may provide useful resistance in this region if deployed in combination. We identified 19 lines from the United States oat differential set that display disease resistance to Pca in WA, with some in agreement with previous rust survey reports. We adopted the 10-letter nomenclature system to define oat crown rust races in Australia and compare the frequency of those virulence traits to published data from the United States. Based on this nomenclature, 42 unique races were detected among the 48 isolates, reflecting the high diversity of virulence phenotypes for Pca in Australia. Nevertheless, the Pca population in the United States is substantially more broadly virulent than that of Australia. Close examination of resistance profiles for the oat differential set lines after infection with Pca supports hypotheses of allelism or redundancy among Pc genes or the presence of several resistance genes in some oat differential lines. These findings illustrate the need to deconvolute the oat differential set using molecular tools.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic Analysis and Physical Mapping of Oat Adult Plant Resistance Loci Against Puccinia coronata f. sp. avenae.

Six quantitative trait loci (QTLs) for adult plant resistance against oat crown rust (Puccinia coronata f. sp. avenae) were identified from mapping three recombinant inbred populations. Using genotyping-by-sequencing with markers called against the OT3098 v1 reference genome, the QTLs were mapped on six different chromosomes: Chr1D, Chr4D, Chr5A, Chr5D, Chr7A, and Chr7C. Composite interval mapping with marker cofactor selection showed that the phenotypic variance explained by all identified QTLs for coefficient of infection range from 12.2 to 46.9%, whereas heritability estimates ranged from 0.11 to 0.38. The significant regions were narrowed down to intervals of 3.9 to 25 cM, equivalent to physical distances of 11 to 133 Mb. At least two flanking single-nucleotide polymorphism markers were identified within 10 cM of each QTL that could be used in marker-assisted introgression, pyramiding, and selection. The additive effects of the QTLs in each population were determined using single-nucleotide polymorphism haplotype data, which showed a significantly lower coefficient of infection in lines homozygous for the resistant alleles. Analysis of pairwise linkage disequilibrium also revealed high correlation of markers and presence of linkage blocks in the significant regions. To further facilitate marker-assisted breeding, polymerase chain reaction allelic competitive extension (PACE) markers for the adult plant resistance loci were developed. Putative candidate genes were also identified in each of the significant regions, which include resistance gene analogs that encode for kinases, ligases, and predicted receptors of avirulence proteins from pathogens.

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci.

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.

A reference-anchored oat linkage map reveals quantitative trait loci conferring adult plant resistance to crown rust (Puccinia coronata f. sp. avenae).

KEY MESSAGE: We mapped three adult plant resistance (APR) loci on oat chromosomes 4D and 6C and developed flanking KASP/PACE markers for marker-assisted selection and gene pyramiding. Using sequence orthology search and the available oat genomic and transcriptomic data, we surveyed these genomic regions for genes that may control disease resistance. Sources of durable disease resistance are needed to minimize yield losses in cultivated oat caused by crown rust (Puccinia coronata f. sp. avenae). In this study, we developed five oat recombinant inbred line mapping populations to identify sources of adult plant resistance from crosses between five APR donors and Otana, a susceptible variety. The preliminary bulk segregant mapping based on allele frequencies showed two regions in linkage group Mrg21 (Chr4D) that are associated with the APR phenotype in all five populations. Six markers from these regions in Chr4D were converted to high-throughput allele specific PCR assays and were used to genotype all individuals in each population. Simple interval mapping showed two peaks in Chr4D, named QPc.APR-4D.1 and QPc.APR-4D.2, which were detected in the OtanaA/CI4706-2 and OtanaA/CI9416-2 and in the Otana/PI189733, OtanaD/PI260616, and OtanaA/CI8000-4 populations, respectively. These results were validated by mapping two entire populations, Otana/PI189733 and OtanaA/CI9416, genotyped using Illumina HiSeq, in which polymorphisms were called against the OT3098 oat reference genome. Composite interval mapping results confirmed the presence of the two quantitative trait loci (QTL) located on oat chromosome 4D and an additional QTL with a smaller effect located on chromosome 6C. This mapping approach also narrowed down the physical intervals to between 5 and 19 Mb, and indicated that QPc.APR-4D.1, QPc.APR-4D.2, and QPc.APR-6C explained 43.4%, 38.5%, and 21.5% of the phenotypic variation, respectively. In a survey of the gene content of each QTL, several clusters of disease resistance genes that may contribute to APR were found. The allele specific PCR markers developed for these QTL regions would be beneficial for marker-assisted breeding, gene pyramiding, and future cloning of resistance genes from oat.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Rpg7: A New Gene for Stem Rust Resistance from Hordeum vulgare ssp. spontaneum.

Wheat stem rust (causal organism: Puccinia graminis f. sp. tritici) is an important fungal disease that causes significant yield losses in barley. The deployment of resistant cultivars is the most effective means of controlling this disease. Stem rust evaluations of a diverse collection of wild barley (Hordeum vulgare ssp. spontaneum) identified two Jordanian accessions (WBDC094 and WBDC238) with resistance to a virulent pathotype (P. graminis f. sp. tritici HKHJC) from the United States. To elucidate the genetics of stem rust resistance, both accessions were crossed to the susceptible landrace Hiproly. Segregation ratios of F2 and F3 progeny indicated that a single dominant gene confers resistance to P. graminis f. sp. tritici HKHJC. Molecular mapping of the resistance locus was performed in the Hiproly/WBDC238 F2 population based on 3,329 single-nucleotide polymorphism markers generated by genotyping-by-sequencing. Quantitative trait locus analysis positioned the resistance gene to the long arm of chromosome 3H between the physical/genetic positions of 683.8 Mbp/172.9 cM and 693.7 Mbp/176.0 cM. Because this resistance gene is novel, it was assigned the new gene locus symbol of Rpg7 with a corresponding allele symbol of Rpg7.i. At the seedling stage, Rpg7 confers resistance against a number of other important P. graminis f. sp. tritici pathotypes from the United States (MCCFC, QCCJB, and TTTTF) and Africa (TTKSK) as well as an isolate (92-MN-90) of the rye stem rust pathogen (P. graminis f. sp. secalis) from Minnesota. The resistance conferred by Rpg7 can be readily transferred into breeding programs because of its simple inheritance and clear phenotypic expression.

Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta.

Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.

Detection of Race-Specific Resistance Against Puccinia coronata f. sp. avenae in Brachypodium Species.

Oat crown rust caused by Puccinia coronata f. sp. avenae is the most destructive foliar disease of cultivated oat. Characterization of genetic factors controlling resistance responses to Puccinia coronata f. sp. avenae in nonhost species could provide new resources for developing disease protection strategies in oat. We examined symptom development and fungal colonization levels of a collection of Brachypodium distachyon and B. hybridum accessions infected with three North American P. coronata f. sp. avenae isolates. Our results demonstrated that colonization phenotypes are dependent on both host and pathogen genotypes, indicating a role for race-specific responses in these interactions. These responses were independent of the accumulation of reactive oxygen species. Expression analysis of several defense-related genes suggested that salicylic acid and ethylene-mediated signaling but not jasmonic acid are components of resistance reaction to P. coronata f. sp. avenae. Our findings provide the basis to conduct a genetic inheritance study to examine whether effector-triggered immunity contributes to nonhost resistance to P. coronata f. sp. avenae in Brachypodium spp.

Solution NMR structures of Pyrenophora tritici-repentis ToxB and its inactive homolog reveal potential determinants of toxin activity.

Pyrenophora tritici-repentis Ptr ToxB (ToxB) is a proteinaceous host-selective toxin produced by Pyrenophora tritici-repentis (P. tritici-repentis), a plant pathogenic fungus that causes the disease tan spot of wheat. One feature that distinguishes ToxB from other host-selective toxins is that it has naturally occurring homologs in non-pathogenic P. tritici-repentis isolates that lack toxic activity. There are no high-resolution structures for any of the ToxB homologs, or for any protein with >30% sequence identity, and therefore what underlies activity remains an open question. Here, we present the NMR structures of ToxB and its inactive homolog Ptr toxb. Both proteins adopt a ß-sandwich fold comprising three strands in each half that are bridged together by two disulfide bonds. The inactive toxb, however, shows higher flexibility localized to the sequence-divergent ß-sandwich half. The absence of toxic activity is attributed to a more open structure in the vicinity of one disulfide bond, higher flexibility, and residue differences in an exposed loop that likely impacts interaction with putative targets. We propose that activity is regulated by perturbations in a putative active site loop and changes in dynamics distant from the site of activity. Interestingly, the new structures identify AvrPiz-t, a secreted avirulence protein produced by the rice blast fungus, as a structural homolog to ToxB. This homology suggests that fungal proteins involved in either disease susceptibility such as ToxB or resistance such as AvrPiz-t may have a common evolutionary origin.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

Persistence of the Host-Selective Toxin Ptr ToxB in the Apoplast.

The necrotrophic fungus Pyrenophora tritici-repentis is responsible for the disease tan spot of wheat. Ptr ToxB (ToxB), a proteinaceous host-selective toxin, is one of the effectors secreted by P. tritici-repentis. ToxB induces chlorosis in toxin-sensitive wheat cultivars and displays characteristics common to apoplastic effectors. We addressed the hypothesis that ToxB exerts its activity extracellularly. Our data indicate that hydraulic pressure applied in the apoplast following ToxB infiltration can displace ToxB-induced symptoms. In addition, treatment with a proteolytic cocktail following toxin infiltration results in reduction of symptom development and indicates that ToxB requires at least 8 h in planta to induce maximum symptom development. In vitro assays demonstrate that apoplastic fluids extracted from toxin-sensitive and -insensitive wheat cultivars cannot degrade ToxB. Additionally, ToxB can be reisolated from apoplastic fluid after toxin infiltration. Furthermore, localization studies of fluorescently labeled ToxB indicate that the toxin remains in the apoplast in toxin-sensitive and -insensitive wheat cultivars. Our findings support the hypothesis that ToxB acts as an extracellular effector.

Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1-3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.

Nuclear exchange generates population diversity in the wheat leaf rust pathogen Puccinia triticina.

In clonally reproducing dikaryotic rust fungi, non-sexual processes such as somatic nuclear exchange are postulated to play a role in diversity but have been difficult to detect due to the lack of genome resolution between the two haploid nuclei. We examined three nuclear-phased genome assemblies of Puccinia triticina, which causes wheat leaf rust disease. We found that the most recently emerged Australian lineage was derived by nuclear exchange between two pre-existing lineages, which originated in Europe and North America. Haplotype-specific phylogenetic analysis reveals that repeated somatic exchange events have shuffled haploid nuclei between long-term clonal lineages, leading to a global P. triticina population representing different combinations of a limited number of haploid genomes. Thus, nuclear exchange seems to be the predominant mechanism generating diversity and the emergence of new strains in this otherwise clonal pathogen. Such genomics-accelerated surveillance of pathogen evolution paves the way for more accurate global disease monitoring.

Haplotype-phased and chromosome-level genome assembly of Puccinia polysora, a giga-scale fungal pathogen causing southern corn rust.

Rust fungi are characterized by large genomes with high repeat content and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we described a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently one of the two giga-scale fungi assembled to chromosome level. Transcript-based annotation identified 47,512 genes for the dikaryotic genome with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P. polysora genome displayed over 85% repeat contents, with genome-size expansion and copy number increasing of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species having smaller genomes. Fine-time-point transcriptomics revealed seven clusters of coexpressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a "two-speed genome" evolution in P. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P. polysora in China with low geographic differentiation. Nevertheless, a minor population differentiated from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing virulence to evade recognition by RppC, a major resistance gene in Chinese corn cultivars. The high-quality assembly provides valuable genomic resources for future studies on disease management and the evolution of P. polysora.