Puccinia coronata var. coronata, a Crown Rust Pathogen of Two Highly Invasive Species, Is Detected Across the Midwest and Northeastern United States.

Puccinia coronata var. coronata (Pcc) causes crown rust disease of glossy buckthorn (Frangula alnus) and reed canarygrass (Phalaris arundinacea), two highly invasive plant species in North America. Pcc is closely related to major pathogens of cereals, turfgrasses, and forage grasses. It occurs throughout Europe but was first recorded in North America in 2013. Where its hosts co-occur, such as in wetlands in the Twin Cities metro area in Minnesota, we have observed Pcc causing significant infection that results in defoliation and fruit loss in glossy buckthorns and premature leaf senescence in reed canarygrass. In this research, we mapped the distribution of this likely recently introduced rust fungus and provided a description of disease signs and symptoms and pathogen morphology. Samples were acquired by two primary means: by surveys in Minnesota and by correspondence with users of iNaturalist.org, a social network for nature enthusiasts and community scientists. With an Oxford Nanopore MinION, we sequenced two to four loci from 22 samples across 13 states and identified samples by phylogenetic analysis and sequence similarity. Notably, four pure isolates appear to have intragenomic variation of the ITS region. We found that Pcc is present throughout the range of glossy buckthorn in the eastern United States. In Minnesota, Pcc is not common outside the range of glossy buckthorn despite the presence of susceptible grass hosts.

Population genomics of Puccinia graminis f.sp. tritici highlights the role of admixture in the origin of virulent wheat rust races.

Puccinia graminis f.sp. tritici (Pgt) causes stem rust disease in wheat that can result in severe yield losses. The factors driving the evolution of its virulence and adaptation remain poorly characterized. We utilize long-read sequencing to develop a haplotype-resolved genome assembly of a U.S. isolate of Pgt. Using Pgt haplotypes as a reference, we characterize the structural variants (SVs) and single nucleotide polymorphisms in a diverse panel of isolates. SVs impact the repertoire of predicted effectors, secreted proteins involved in host-pathogen interaction, and show evidence of purifying selection. By analyzing global and local genomic ancestry we demonstrate that the origin of 8 out of 12 Pgt clades is linked with either somatic hybridization or sexual recombination between the diverged donor populations. Our study shows that SVs and admixture events appear to play an important role in broadening Pgt virulence and the origin of highly virulent races, creating a resource for studying the evolution of Pgt virulence and preventing future epidemic outbreaks.

Development of a Diagnostic Assay for Differentiation Between Genetic Groups in Clades I, II, III, and IV of Puccinia graminis f. sp. tritici.

Wheat stem rust has reemerged as a serious disease caused by new variants of Puccinia graminis f. sp. tritici. Variants with significant virulence and broad geographic distribution (Africa, Central Asia, and Europe) include the Ug99 race group, race TTRTF, and TKTTF race group. Genetic analysis has placed isolates representing these critical new virulent races into 12 genetic groups that make up clades I to IV. Development of molecular diagnostic assays for these 12 genetic groups will be an important component of global surveillance efforts. A single-nucleotide polymorphism database was mined for candidate markers that would differentiate between these 12 genetic groups. Thirty-five candidate markers were screened, and a core set of 17 markers was tested against a set of 94 isolates representing a broad range of genotypes and race phenotypes. These core markers were 100% accurate in identifying the 12 genetic groups for 52 isolates in clades I to IV, and no false positives were observed with nontarget isolates. The assay has built-in redundancy so that minor genetic changes or errors in genotyping calling will not affect the accuracy of the results. This assay is also effective in identifying the genetic groups in clade V from Germany and Georgia, the three main subgroups in North American clade VI, and clade VII consisting of race TTTTF found in North and South America. This assay provides a rapid diagnostic tool for both living and nonliving samples to detect these critical new races or race groups of P. graminis f. sp. tritici.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genotyping-by-Sequencing for the Study of Genetic Diversity in Puccinia triticina.

Leaf rust, caused by Puccinia triticina Erikss., is globally the most widespread rust of wheat. Populations of P. triticina are highly diverse for virulence, with many different races found annually. The genetic diversity of P. triticina populations has been previously assessed using different types of DNA markers. Genotyping technologies that provide a higher density of markers distributed across the genome will be more powerful for analysis of genetic and phylogenetic relationships in P. triticina populations. In this study, we utilized restriction-associated DNA (RAD) genotyping-by-sequencing (GBS) adapted for the Ion Torrent sequencing platform for the study of population diversity in P. triticina. A collection of 102 isolates, collected mainly from tetraploid and hexaploid wheat, was used. The virulence phenotypes of the isolates were determined on 20 lines of Thatcher wheat near isogenic for leaf rust resistance genes. Seven races were found among 57 isolates collected from tetraploid wheat, and 21 races were observed among 40 hexaploid wheat type isolates. This is the first study to report durum wheat virulent races to Lr3bg in Tunisia, Lr14a in Morocco, and Lr3bg and Lr28 in Mexico. Ethiopian isolates with high virulence to durum wheat but avirulent on Thatcher (hexaploid wheat) were tested for virulence on a set of durum (tetraploid) differentials. A subset of 30 isolates representing most of the virulence phenotypes in the 102 isolates were genotyped using RAD-GBS. Phylogenetic analysis of 30 isolates using 2,125 single nucleotide polymorphism (SNP) markers showed nine distinct clusters. There was a general correlation between virulence phenotypes and SNP genotypes. The high bootstrap values between clusters of isolates in the phylogenetic tree indicated that RAD-GBS can be used as a new genotyping tool that is fast, simple, high throughput, cost effective, and provides a sufficient number of markers for the study of genetic diversity in P. triticina.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Progress on Molecular Genetics and Manipulation of Rust Fungi.

Among the thousands of rust species described, many are known for their devastating effects on their hosts, which include major agriculture crops and trees. Hence, for over a century, these basidiomycete pathogenic fungi have been researched and experimented with. However, due to their biotrophic nature, they are challenging organisms to work with and, needing their hosts for propagation, represent pathosystems that are not easily experimentally accessible. Indeed, efforts to perform genetics have been few and far apart for the rust fungi, though one study performed in the 1940s was famously instrumental in formulating the gene-for-gene hypothesis describing pathogen-host interactions. By taking full advantage of the molecular genetic tools developed in the 1980s, research on many plant pathogenic microbes thrived, yet similar work on the rusts remained very challenging though not without some successes. However, the genomics era brought real breakthrough research for the biotrophic fungi and with innovative experimentation and the use of heterologous systems, molecular genetic analyses over the last 2 decades have significantly advanced our insight into the function of many rust fungus genes and their role in the interaction with their hosts. This has allowed optimizing efforts for resistance breeding and the design and testing of various novel strategies to reduce the devastating diseases they cause.

Presence of a Sexual Population of Puccinia graminis f. sp. tritici in Georgia Provides a Hotspot for Genotypic and Phenotypic Diversity.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re-emerging disease exemplified by recent epidemics caused by new virulent races. Understanding the sources and origins of genetic variations in the pathogen populations globally can facilitate the development of better strategies in disease management. We analyzed 68 wheat stem rust samples collected between 2013 and 2015 from Georgia where stem rust incidences are frequent and the alternate host, common barberry, is present. A total of 116 single-pustule isolates were derived and evaluated on stem rust differential lines to determine the virulence phenotypes and 23 races were identified, many of which were detected for the first time. Unique virulence combinations including, Sr22+Sr24 and Sr13b+Sr35+Sr37 were detected. These virulence combinations pose new challenges to breeding programs because many of these genes are used in breeding for resistance to the Ug99 race group. Sixty-one isolates were genotyped using a custom single-nucleotide polymorphism chip and 17 genotypes were identified. The 2013 isolates contained 11 multilocus genotypes compared with isolates of 2014 and 2015, with five and three genotypes, respectively. The higher levels of virulence and genotypic diversity observed in the 2013 samples strongly indicated that sexual recombination occurs in the Georgian P. graminis f. sp. tritici population, and that the Caucasus region of Eurasia may be an important source of new races.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Resistance gene cloning from a wild crop relative by sequence capture and association genetics.

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel.

Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci.

Three members of the Puccinia genus, Pucciniatriticina (Pt), Pstriiformis f.sp. tritici (Pst), and Pgraminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

Changing the Game: Using Integrative Genomics to Probe Virulence Mechanisms of the Stem Rust Pathogen Puccinia graminis f. sp. tritici.

The recent resurgence of wheat stem rust caused by new virulent races of Puccinia graminis f. sp. tritici (Pgt) poses a threat to food security. These concerns have catalyzed an extensive global effort toward controlling this disease. Substantial research and breeding programs target the identification and introduction of new stem rust resistance (Sr) genes in cultivars for genetic protection against the disease. Such resistance genes typically encode immune receptor proteins that recognize specific components of the pathogen, known as avirulence (Avr) proteins. A significant drawback to deploying cultivars with single Sr genes is that they are often overcome by evolution of the pathogen to escape recognition through alterations in Avr genes. Thus, a key element in achieving durable rust control is the deployment of multiple effective Sr genes in combination, either through conventional breeding or transgenic approaches, to minimize the risk of resistance breakdown. In this situation, evolution of pathogen virulence would require changes in multiple Avr genes in order to bypass recognition. However, choosing the optimal Sr gene combinations to deploy is a challenge that requires detailed knowledge of the pathogen Avr genes with which they interact and the virulence phenotypes of Pgt existing in nature. Identifying specific Avr genes from Pgt will provide screening tools to enhance pathogen virulence monitoring, assess heterozygosity and propensity for mutation in pathogen populations, and confirm individual Sr gene functions in crop varieties carrying multiple effective resistance genes. Toward this goal, much progress has been made in assembling a high quality reference genome sequence for Pgt, as well as a Pan-genome encompassing variation between multiple field isolates with diverse virulence spectra. In turn this has allowed prediction of Pgt effector gene candidates based on known features of Avr genes in other plant pathogens, including the related flax rust fungus. Upregulation of gene expression in haustoria and evidence for diversifying selection are two useful parameters to identify candidate Avr genes. Recently, we have also applied machine learning approaches to agnostically predict candidate effectors. Here, we review progress in stem rust pathogenomics and approaches currently underway to identify Avr genes recognized by wheat Sr genes.

Kenyan Isolates of Puccinia graminis f. sp. tritici from 2008 to 2014: Virulence to SrTmp in the Ug99 Race Group and Implications for Breeding Programs.

Frequent emergence of new variants in the Puccinia graminis f. sp. tritici Ug99 race group in Kenya has made pathogen survey a priority. We analyzed 140 isolates from 78 P. graminis f. sp. tritici samples collected in Kenya between 2008 and 2014 and identified six races, including three not detected prior to 2013. Genotypic analysis of 20 isolates from 2013 and 2014 collections showed that the new races TTHST, TTKTK, and TTKTT belong to the Ug99 race group. International advanced breeding lines were evaluated against an isolate of TTKTT (Sr31, Sr24, and SrTmp virulence) at the seedling stage. From 169 advanced lines from Kenya, 23% of lines with resistance to races TTKSK and TTKST were susceptible to TTKTT and, from two North American regional nurseries, 44 and 91% of resistant lines were susceptible. Three lines with combined resistance genes were developed to facilitate pathogen monitoring and race identification. These results indicate the increasing virulence and variability in the Kenyan P. graminis f. sp. tritici population and reveal vulnerabilities of elite germplasm to new races.

A New Rust Disease on Wild Coffee (Psychotria nervosa) Caused by Puccinia mysuruensis sp. nov.

Psychotria nervosa, commonly called "wild coffee" (Rubiaceae), is an important ethno-medicinal plant in India. In 2010, a new rust disease of P. nervosa was observed in three regions of Mysore District, Karnataka (India), with disease incidence ranging from 58 to 63%.Typical symptoms of the rust disease on wild coffee were prominently visible during the early monsoon season (May to June), with chlorotic spots on the adaxial and black pustules (telia) on the abaxial leaf surface. Telia produced abundant teliospores, which were bicelled, pedicillate, and measured 33 to 45 by 19 to 30 µm. The germination of teliospores produced a typical metabasidium bearing four basidiospores, each containing two haploid nuclei. Spore stages of the wild coffee rust pathogen were studied using artificially inoculated healthy wild coffee plants with germinated teliospores. Only telia were observed on the inoculated plants, indicating that this rust fungus has an abbreviated microcyclic life cycle that includes only teliospores and basidiospores. Phylogenetic analysis based on internal transcribed spacer and partial large subunit (LSU) sequence data showed that the wild coffee rust pathogen is related to Macruropyxis fraxini, Puccinia bartholomaei, P. choridis, and P. sparganioidis. The herbarium sample of P. psychotriae was examined and was shown to be different with respect to telium size and teliospore dimensions (24 to 32 by 13 to 18 µm). Therefore, the rust pathogen causing wild coffee rust is a new species, P. mysuruensis sp. nov.

Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia.

BACKGROUND: The cereal rust fungi are destructive pathogens that affect grain production worldwide. Although the genomic and transcript sequences for three Puccinia species that attack wheat have been released, the functions of large repertories of genes from Puccinia still need to be addressed to understand the infection process of these obligate parasites. Host-induced gene silencing (HIGS) has emerged a useful tool to examine the importance of rust fungus genes while growing within host plants. In this study, HIGS was used to test genes from Puccinia with transcripts enriched in haustoria for their ability to interfere with full development of the rust fungi. RESULTS: Approximately 1200 haustoria enriched genes from Puccinia graminis f. sp. tritici (Pgt) were identified by comparative RNA sequencing. Virus-induced gene silencing (VIGS) constructs with fragments of 86 Puccinia genes, were tested for their ability to interfere with full development of these rust fungi. Most of the genes tested had no noticeable effects, but 10 reduced Pgt development after co-inoculation with the gene VIGS constructs and Pgt. These included a predicted glycolytic enzyme, two other proteins that are probably secreted and involved in carbohydrate or sugar metabolism, a protein involved in thiazol biosynthesis, a protein involved in auxin biosynthesis, an amino acid permease, two hypothetical proteins with no conserved domains, a predicted small secreted protein and another protein predicted to be secreted with similarity to bacterial proteins involved in membrane transport. Transient silencing of four of these genes reduced development of P. striiformis (Pst), and three of also caused reduction of P. triticina (Pt) development. CONCLUSIONS: Partial suppression of transcripts involved in a large variety of biological processes in haustoria cells of Puccinia rusts can disrupt their development. Silencing of three genes resulted in suppression of all three rust diseases indicating that it may be possible to engineer durable resistance to multiple rust pathogens with a single gene in transgenic wheat plants for sustainable control of cereal rusts.

Emergence and Spread of New Races of Wheat Stem Rust Fungus: Continued Threat to Food Security and Prospects of Genetic Control.

Race Ug99 (TTKSK) of Puccinia graminis f. sp. tritici, detected in Uganda in 1998, has been recognized as a serious threat to food security because it possesses combined virulence to a large number of resistance genes found in current widely grown wheat (Triticum aestivum) varieties and germplasm, leading to its potential for rapid spread and evolution. Since its initial detection, variants of the Ug99 lineage of stem rust have been discovered in Eastern and Southern African countries, Yemen, Iran, and Egypt. To date, eight races belonging to the Ug99 lineage are known. Increased pathogen monitoring activities have led to the identification of other races in Africa and Asia with additional virulence to commercially important resistance genes. This has led to localized but severe stem rust epidemics becoming common once again in East Africa due to the breakdown of race-specific resistance gene SrTmp, which was deployed recently in the 'Digalu' and 'Robin' varieties in Ethiopia and Kenya, respectively. Enhanced research in the last decade under the umbrella of the Borlaug Global Rust Initiative has identified various race-specific resistance genes that can be utilized, preferably in combinations, to develop resistant varieties. Research and development of improved wheat germplasm with complex adult plant resistance (APR) based on multiple slow-rusting genes has also progressed. Once only the Sr2 gene was known to confer slow rusting APR; now, four more genes-Sr55, Sr56, Sr57, and Sr58-have been characterized and additional quantitative trait loci identified. Cloning of some rust resistance genes opens new perspectives on rust control in the future through the development of multiple resistance gene cassettes. However, at present, disease-surveillance-based chemical control, large-scale deployment of new varieties with multiple race-specific genes or adequate levels of APR, and reducing the cultivation of susceptible varieties in rust hot-spot areas remains the best stem rust management strategy.

Phenotypic and Genotypic Characterization of Race TKTTF of Puccinia graminis f. sp. tritici that Caused a Wheat Stem Rust Epidemic in Southern Ethiopia in 2013-14.

A severe stem rust epidemic occurred in southern Ethiopia during November 2013 to January 2014, with yield losses close to 100% on the most widely grown wheat cultivar, 'Digalu'. Sixty-four stem rust samples collected from the regions were analyzed. A meteorological model for airborne spore dispersal was used to identify which regions were most likely to have been infected from postulated sites of initial infection. Based on the analyses of 106 single-pustule isolates derived from these samples, four races of Puccinia graminis f. sp. tritici were identified: TKTTF, TTKSK, RRTTF, and JRCQC. Race TKTTF was found to be the primary cause of the epidemic in the southeastern zones of Bale and Arsi. Isolates of race TKTTF were first identified in samples collected in early October 2013 from West Arsi. It was the sole or predominant race in 31 samples collected from Bale and Arsi zones after the stem rust epidemic was established. Race TTKSK was recovered from 15 samples from Bale and Arsi zones at low frequencies. Genotyping indicated that isolates of race TKTTF belongs to a genetic lineage that is different from the Ug99 race group and is composed of two distinct genetic types. Results from evaluation of selected germplasm indicated that some cultivars and breeding lines resistant to the Ug99 race group are susceptible to race TKTTF. Appearance of race TKTTF and the ensuing epidemic underlines the continuing threats and challenges posed by stem rust not only in East Africa but also to wider-scale wheat production.

Conserved loci of leaf and stem rust fungi of wheat share synteny interrupted by lineage-specific influx of repeat elements.

BACKGROUND: Wheat leaf rust (Puccinia triticina Eriks; Pt) and stem rust fungi (P. graminis f.sp. tritici; Pgt) are significant economic pathogens having similar host ranges and life cycles, but different alternate hosts. The Pt genome, currently estimated at 135 Mb, is significantly larger than Pgt, at 88 Mb, but the reason for the expansion is unknown. Three genomic loci of Pt conserved proteins were characterized to gain insight into gene content, genome complexity and expansion. RESULTS: A bacterial artificial chromosome (BAC) library was made from P. triticina race 1, BBBD and probed with Pt homologs of genes encoding two predicted Pgt secreted effectors and a DNA marker mapping to a region of avirulence. Three BACs, 103 Kb, 112 Kb, and 166 Kb, were sequenced, assembled, and open reading frames were identified. Orthologous genes were identified in Pgt and local conservation of gene order (microsynteny) was observed. Pairwise protein identities ranged from 26 to 99%. One Pt BAC, containing a RAD18 ortholog, shares syntenic regions with two Pgt scaffolds, which could represent both haplotypes of Pgt. Gene sequence is diverged between the species as well as within the two haplotypes. In all three BAC clones, gene order is locally conserved, however, gene shuffling has occurred relative to Pgt. These regions are further diverged by differing insertion loci of LTR-retrotransposon, Gypsy, Copia, Mutator, and Harbinger mobile elements. Uncharacterized Pt open reading frames were also found; these proteins are high in lysine and similar to multiple proteins in Pgt. CONCLUSIONS: The three Pt loci are conserved in gene order, with a range of gene sequence divergence. Conservation of predicted haustoria expressed secreted protein genes between Pt and Pgt is extended to the more distant poplar rust, Melampsora larici-populina. The loci also reveal that genome expansion in Pt is in part due to higher occurrence of repeat-elements in this species.

Molecular Phylogenetic Relationships of the Brown Leaf Rust Fungi on Wheat, Rye, and Other Grasses.

The classification of brown leaf rust fungi (Puccinia recondita complex and allied species) on wheat (Triticum aestivum), rye (Secale cereale), and other grasses in the family Poaceae has experienced a long history of controversy and uncertainty due to the reduced morphological characteristics available for taxonomy and difficulty of conducting interfertility experiments. However, because these are pathogens on important crops, it is important to clarify the species delimitations reflecting the natural lineages. In this study, phylogenetic analyses were conducted with DNA sequence data from the ribosomal DNA internal transcribed spacer region and elongation factor 1-α to elucidate this species complex. Three phylogenetic lineages were recovered within the complex of rye leaf rust fungi, P. recondita sensu stricto, which is congruent with existing classifications based on DNA content, sexual compatibility, and morphological studies. The brown leaf rust fungus on wheat (P. triticina) grouped with the related species P. persistens on Elymus repens and E. intermedia as a strongly supported clade. Collections on other Elymus spp. were separated into six clades. Based on the phylogenetic affinities of nine type specimens and aecial host associations, potential taxonomic names were evaluated for selected lineages.

Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi.

Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance.

The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.

Obligate biotrophy features unraveled by the genomic analysis of rust fungi.

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Real-Time PCR Detection and Discrimination of the Southern and Common Corn Rust Pathogens Puccinia polysora and Puccinia sorghi.

Over the past several years, southern corn rust (SCR) outbreaks caused by the fungus Puccinia polysora have become increasingly problematic for corn growers in the United States. SCR is currently diagnosed through the visual examination of disease symptoms and pathogen morphology, including pigmentation, size, shape, and location of fruiting structures. However, these characteristics are similar to those produced by the common corn rust fungus P. sorghi, confounding accurate visual diagnosis of SCR. Here we report the development of a real-time polymerase chain reaction assay that discriminates between P. polysora and P. sorghi. Sequences of the rDNA internal transcribed spacer region were determined for P. polysora and P. sorghi. 5-Carboxyfluorescein fluorophore-labeled hydrolysis probes that differed at 14 nucleotide positions between the species were developed from these data and used to screen DNA extracted directly from rust-infected corn leaves. Species-specific, reproducible identifications of the pathogens were made from as little as 50 pg of DNA within 30 min, and were reliably performed from both recent collections and herbarium specimens. This assay will be useful for rapid and accurate diagnosis of SCR, and could serve as a tool to monitor the distribution and incidence of the disease in the United States.