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This strategic plan summarizes the major accomplishments achieved in the last quinquennial by the soybean [Glycine max (L.) Merr.] genetics and genomics research community and outlines key priorities for the next 5 years (2024-2028). This work is the result of deliberations among over 50 soybean researchers during a 2-day workshop in St Louis, MO, USA, at the end of 2022. The plan is divided into seven traditional areas/disciplines: Breeding, Biotic Interactions, Physiology and Abiotic Stress, Functional Genomics, Biotechnology, Genomic Resources and Datasets, and Computational Resources. One additional section was added, Training the Next Generation of Soybean Researchers, when it was identified as a pressing issue during the workshop. This installment of the soybean genomics strategic plan provides a snapshot of recent progress while looking at future goals that will improve resources and enable innovation among the community of basic and applied soybean researchers. We hope that this work will inform our community and increase support for soybean research., (© 2024 The Author(s). The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Soybean rust is an economically significant disease caused by the fungus Phakopsora pachyrhizi that negatively impacts soybean ( Glycine max [L.] Merr.) production throughout the world. Susceptible plants infected by P. pachyrhizi develop tan-colored lesions on the leaf surface that give rise to funnel-shaped uredinia as the disease progresses. While most soybean germplasm is susceptible, seven genetic loci ( Rpp1 to Rpp7 ) that provide race-specific resistance to P. pachyrhizi ( Rpp ) have been identified. Rpp3 was first discovered and characterized in the soybean accession PI 462312 (Ankur), and it was also determined to be one of two Rpp genes present in PI 506764 (Hyuuga). Genetic crosses with PI 506764 were later used to fine-map the Rpp3 locus to a 371-kb region on chromosome 6. The corresponding region in the susceptible Williams 82 (Wm82) reference genome contains several homologous nucleotide binding site-leucine rich repeat (NBS-LRR) genes. To identify Rpp3 , we designed oligonucleotide primers to amplify Rpp3 candidate ( Rpp3C ) NBS-LRR genes at this locus from PI 462312, PI 506764, and Wm82 using polymerase chain reaction (PCR). Five Rpp3C genes were identified in both Rpp3 -resistant soybean lines, and co-silencing these genes compromised resistance to P. pachyrhizi . Gene expression analysis and sequence comparisons of the Rpp3C genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3 , is responsible for Rpp3 -mediated resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024., Competing Interests: The author(s) declare no conflict of interest.

The fungal pathogen, Magnaporthe oryzae Triticum pathotype, causing wheat blast disease was first identified in South America and recently spread across continents to South Asia and Africa. Here, we studied the genetic relationship among isolates found on the three continents. Magnaporthe oryzae strains closely related to a South American field isolate B71 were found to have caused the wheat blast outbreaks in South Asia and Africa. Genomic variation among isolates from the three continents was examined using an improved B71 reference genome and whole-genome sequences. We found strong evidence to support that the outbreaks in Bangladesh and Zambia were caused by the introductions of genetically separated isolates, although they were all close to B71 and, therefore, collectively referred to as the B71 branch. In addition, B71 branch strains carried at least one supernumerary mini-chromosome. Genome assembly of a Zambian strain revealed that its mini-chromosome was similar to the B71 mini-chromosome but with a high level of structural variation. Our findings show that while core genomes of the multiple introductions are highly similar, the mini-chromosomes have undergone marked diversification. The maintenance of the mini-chromosome and rapid genomic changes suggest the mini-chromosomes may serve important virulence or niche adaptation roles under diverse environmental conditions., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Wheat blast, caused by the Pyricularia oryzae Triticum lineage (PoT), first emerged in Brazil and quickly spread to neighboring countries. Its recent appearance in Bangladesh and Zambia highlights a need to understand the disease's population biology and epidemiology so as to mitigate pandemic outbreaks. Current knowledge is mostly based on characterizations of Brazilian wheat blast isolates and comparison with isolates from non-wheat, endemic grasses. These foregoing studies concluded that the wheat blast population lacks host specificity and, as a result, undergoes extensive gene flow with populations infecting non-wheat hosts. Additionally, based on genetic similarity between wheat blast and isolates infecting Urochloa species, it was proposed that the disease originally emerged via a host jump from this grass and that Urochloa likely plays a central role in wheat blast epidemiology owing to its widespread use as a pasture grass. However, due to inconsistencies with broader phylogenetic studies, we suspected that these seminal studies had not actually sampled the populations normally found on endemic grasses and, instead, had repeatedly isolated members of PoT and the related Lolium pathogen lineage (PoL1). Re-analysis of the Brazilian data as part of a comprehensive, global, phylogenomic dataset that included a small number of South American isolates sampled away from wheat confirmed our suspicion and identified four new P. oryza e lineages on grass hosts. As a result, the conclusions underpinning current understanding in wheat blast's evolution, population biology, and epidemiology are unsubstantiated and could be equivocal., Competing Interests: The author(s) declare no conflict of interest.

Most plant pathogens exhibit host specificity but when former barriers to infection break down, new diseases can rapidly emerge. For a number of fungal diseases, there is increasing evidence that hybridization plays a major role in driving host jumps. However, the relative contributions of existing variation versus new mutations in adapting to new host(s) is unclear. Here we reconstruct the evolutionary history of two recently emerged populations of the fungus Pyricularia oryzae that are responsible for two new plant diseases: wheat blast and grey leaf spot of ryegrasses. We provide evidence that wheat blast/grey leaf spot evolved through two distinct mating episodes: the first occurred ~60 years ago, when a fungal individual adapted to Eleusine mated with another individual from Urochloa. Then, about 10 years later, a single progeny from this cross underwent a series of matings with a small number of individuals from three additional host-specialized populations. These matings introduced non-functional alleles of two key host-specificity factors, whose recombination in a multi-hybrid swarm probably facilitated the host jump. We show that very few mutations have arisen since the founding event and a majority are private to individual isolates. Thus, adaptation to the wheat or Lolium hosts appears to have been instantaneous, and driven entirely by selection on repartitioned standing variation, with no obvious role for newly formed mutations., (© 2023. The Author(s).)

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT) is a threat to wheat production especially in the warmer-humid environments. In Zambia, wheat blast symptoms were observed for the first time on wheat (Triticum aestivum L.) grown in experimental plots and five farmers' fields in Mpika district of Muchinga Province during the 2017-18 rainy season. Infected plants showed the typical wheat blast symptoms with the spike becoming partially or completely bleached with the blackening of the rachis in a short span of time. Incidence of blast symptoms on nearly all wheat heads was high and ranged from 50 to 100%. Examination of diseased plant leaves showed the presence of elliptical, grayish to tan necrotic lesions with dark borders on the leaf often mixed with other foliar diseases. A study was conducted to isolate and identify the causal pathogen(s) using classical and molecular methods and determine the pathogenicity of the detected disease causal agent. Morphobiometrical determination of causal pathogen revealed conidia with characteristic pear shaped 2-septate hyaline spores associated with M. oryzae species. Preliminary polymerase chain reaction screening of six isolates obtained from wheat blast infected samples with diagnostic primers (MoT3F/R) was conducted at ZARI, Zambia, and subsequent analysis of two isolates with MoT3F/R and C17F/R was performed at USDA-ARS, USA. Both experiments confirmed that MoT is the causal agent of wheat blast in Zambia. Further, pathogenicity tests performed with pure culture isolates from samples WS4 and WS5 produced typical blast symptoms on all the six inoculated wheat genotypes. Results of this study indicate that MoT is causing wheat blast in rain-fed wheat grown in Zambia, thus making it the first report of MoT in Zambia and Africa. This inter-continental movement of the pathogen (disease) has serious implication for wheat production and trade that needs to be urgently addressed., Competing Interests: The authors have declared that no competing interest exist.

Uromyces transversalis , the causal agent of Gladiolus rust, is an invasive plant pathogen in the United States and is regulated as a quarantine pathogen in Europe. The aim of this research was to: (i) determine the origin of introductions of U. transversalis to the United States, (ii) track the movement of genotypes, and (iii) understand the worldwide genetic diversity of the species. To develop molecular markers for genotyping, whole genome sequencing was performed on three isolates collected in the United States. Genomes were assembled de novo and searched for microsatellite regions. Primers were developed and tested on ten isolates from the United States resulting in the identification of 24 polymorphic markers. Among 92 isolates collected from Costa Rica, Mexico, New Zealand, Australia, and the United States there were polymorphisms within isolates with no genotypic diversity detected among isolates; however, missing data among the New Zealand and Australia isolates due to either poor amplification of degraded DNA or null alleles as a result of genetic differences made it difficult to generate conclusions about these populations. The microsatellite loci and flanking regions showed high diversity and two divergent genomes within dikaryotic individuals, yet no diversity among individuals, suggesting that the invasive U. transversalis populations from North America are strictly clonal., Competing Interests: The authors declare there are no competing interests., (©2019 DeLong et al.)

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1 -silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.

This is a response to a recent Letter to the Editor of Phytopathology, in which Gupta et al. (2019) caution against the indiscriminate use of the MoT3 diagnostic assay that distinguishes isolates of Magnaporthe oryzae in the Triticum lineage from those that do not cause aggressive wheat blast. We confirm that the assay does reliably distinguish between wheat and rice isolates from Bangladesh and worldwide, as described in the original paper by Pieck et al. (2017) . We have been unable to reproduce the equally intense amplification of WB12 and WB12-like sequences reported in Figure 1 of the Letter. Other data presented by Gupta et al. (2019) support the specificity of the MoT3 assay. Therefore, cautions beyond those always associated with accurate reproduction of diagnostic assays are unwarranted.

Phakopsora pachyrhizi is the causal agent of Asian soybean rust. Susceptible soybean plants infected by virulent isolates of P. pachyrhizi are characterized by tan-colored lesions and erumpent uredinia on the leaf surface. Germplasm screening and genetic analyses have led to the identification of seven loci, Rpp1 to Rpp7, that provide varying degrees of resistance to P. pachyrhizi (Rpp). Two genes, Rpp1 and Rpp1b, map to the same region on soybean chromosome 18. Rpp1 is unique among the Rpp genes in that it confers an immune response (IR) to avirulent P. pachyrhizi isolates. The IR is characterized by a lack of visible symptoms, whereas resistance provided by Rpp1b to Rpp7 results in red-brown foliar lesions. Rpp1 maps to a region spanning approximately 150 kb on chromosome 18 between markers Sct_187 and Sat_064 in L85-2378 (Rpp1), an isoline developed from Williams 82 and PI 200492 (Rpp1). To identify Rpp1, we constructed a bacterial artificial chromosome library from soybean accession PI 200492. Sequencing of the Rpp1 locus identified three homologous nucleotide binding site-leucine rich repeat (NBS-LRR) candidate resistance genes between Sct_187 and Sat_064. Each candidate gene is also predicted to encode an N-terminal ubiquitin-like protease 1 (ULP1) domain. Cosilencing of the Rpp1 candidates abrogated the immune response in the Rpp1 resistant soybean accession PI 200492, indicating that Rpp1 is a ULP1-NBS-LRR protein and plays a key role in the IR.

Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields.

Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae ) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae , including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species ( Pyricularia graminis-tritici ). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae , each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen. IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.

Rust fungi, such as the soybean rust pathogen Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are hyphal structures intimately associated with host-plant cell membranes. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characterization of effector proteins of rust fungi is important for understanding mechanisms that underlie their virulence and pathogenicity. Hundreds of candidate effector proteins have been predicted for rust pathogens, but it is not clear how to prioritize these effector candidates for further characterization. There is a need for high-throughput approaches for screening effector candidates to obtain experimental evidence for effector-like functions, such as the manipulation of host immune systems. We have focused on identifying effector candidates with immune-related functions in the soybean rust fungus P. pachyrhizi. To facilitate the screening of many P. pachyrhizi effector candidates (named PpECs), we used heterologous expression systems, including the bacterial type III secretion system, Agrobacterium infiltration, a plant virus, and a yeast strain, to establish an experimental pipeline for identifying PpECs with immune-related functions and establishing their subcellular localizations. Several PpECs were identified that could suppress or activate immune responses in nonhost Nicotiana benthamiana, N. tabacum, Arabidopsis, tomato, or pepper plants.

Key Message: A novel Rpp gene from PI 605823 for resistance to Phakopsora pachyrhizi was mapped on chromosome 19. Soybean rust, caused by the obligate biotrophic fungal pathogen Phakopsora pachyrhizi Syd. & P. Syd, is a disease threat to soybean production in regions of the world with mild winters. Host plant resistance conditioned by resistance to P. pachyrhizi (Rpp) genes has been found in numerous soybean accessions, and at least 10 Rpp genes or alleles have been mapped to six genetic loci. Identifying additional disease-resistance genes will facilitate development of soybean cultivars with durable resistance. PI 605823, a plant introduction from Vietnam, was previously identified as resistant to US populations of P. pachyrhizi in greenhouse and field trials. In this study, bulked segregant analysis using an F 2 population derived from 'Williams 82' × PI 605823 identified a genomic region associated with resistance to P. pachyrhizi isolate GA12, which had been collected in the US State of Georgia in 2012. To further map the resistance locus, linkage mapping was carried out using single-nucleotide polymorphism markers and phenotypic data from greenhouse assays with an F 2:3 population derived from Williams 82 × PI 605823 and an F 4:5 population derived from '5601T' × PI 605823. A novel resistance gene, Rpp7, was mapped to a 154-kb interval (Gm19: 39,462,291-39,616,643 Glyma.Wm82.a2) on chromosome 19 that is different from the genomic locations of any previously reported Rpp genes. This new gene could be incorporated into elite breeding lines to help provide more durable resistance to soybean rust.

Wheat blast has emerged as a major threat to wheat production in South America. Although originally restricted to Brazil, the disease has since been observed in the neighboring countries of Argentina, Bolivia, and Paraguay and recently the pathogen, Magnaporthe oryzae Triticum pathotype, was isolated from infected wheat in Bangladesh. There is growing concern that the pathogen may continue to spread to other parts of the world, including the United States, where several M. oryzae pathotypes are endemic. M. oryzae pathotypes are morphologically indistinguishable and, therefore, must be characterized genotypically. Symptoms of wheat blast include bleaching of the head, which closely resembles the symptoms of Fusarium head blight, further complicating efforts to monitor for the presence of the pathogen in the field. We used a genomics-based approach to identify molecular markers unique to the Triticum pathotype of M. oryzae. One of these markers, MoT3, was selected for the development of a polymerase chain reaction (PCR)-based diagnostic assay that was evaluated for specificity using DNA from 284 M. oryzae isolates collected from a diverse array of host species. Conventional PCR primers were designed to amplify a 361-bp product, and the protocol consistently amplified from as little as 0.1 ng of purified DNA. The specificity of the MoT3-based assay was also evaluated using Fusarium spp. DNA, from which no amplicons were detected.

Gray leaf spot (GLS) is a destructive disease of perennial ryegrass caused by a host specific pathotype of the ascomycete Magnaporthe oryzae. Early diagnosis is crucial for effective disease management and the implementation of Integrated Pest Management practices. However, a rapid protocol for the detection of low levels of airborne inoculum is still missing. We developed a pathogen-specific quantitative loop-mediated isothermal amplification (qLAMP) assay coupled with a spore trap system for rapid detection and quantification of airborne inoculum of the M. oryzae perennial ryegrass pathotype, and tested its suitability for implementation in GLS-infected turfgrass fields. In summer 2015, two perennial ryegrass plots were artificially inoculated with the pathogen, with four continuously running custom impaction spore traps placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay, while plots were monitored for symptom development. Results confirmed that the qLAMP assay-trap system was able to detect as few as 10 conidia up to 12 days before symptoms developed in the field. LAMP technology is particularly appropriate for field implementation by nontechnical users, and has the potential to be a powerful decision support tool to guide timing of fungicide applications for GLS management.

The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate biotrophic pathogen causing severe soybean disease epidemics. Molecular mechanisms by which P. pachyrhizi and other rust fungi interact with their host plants are poorly understood. The genomes of all rust fungi encode many small, secreted cysteine-rich proteins (SSCRP). While these proteins are thought to function within the host, their roles are completely unknown. Here, we present the characterization of P. pachyrhizi effector candidate 23 (PpEC23), a SSCRP that we show to suppress plant immunity. Furthermore, we show that PpEC23 interacts with soybean transcription factor GmSPL12l and that soybean plants in which GmSPL12l is silenced have constitutively active immunity, thereby identifying GmSPL12l as a negative regulator of soybean defenses. Collectively, our data present evidence for a virulence function of a rust SSCRP and suggest that PpEC23 is able to suppress soybean immune responses and physically interact with soybean transcription factor GmSPL12l, a negative immune regulator., Competing Interests: The authors have declared that no competing interests exist.

Key Message: The Rpp6 locus of PI 567102B was mapped from 5,953,237 to 5,998,461 bp (chromosome 18); and a novel allele at the Rpp6 locus or tightly linked gene Rpp[PI567068A] of PI 567068A was mapped from 5,998,461 to 6,160,481 bp. Soybean rust (SBR), caused by the obligate, fungal pathogen Phakopsora pachyrhizi is an economic threat to soybean production, especially in the Americas. Host plant resistance is an important management strategy for SBR. The most recently described resistance to P. pachyrhizi (Rpp) gene is Rpp6 contributed by PI 567102B. Rpp6 was previously mapped to an interval of over four million base pairs on chromosome 18. PI 567068A was recently demonstrated to possess a resistance gene near the Rpp6 locus, yet PI 567068A gave a differential isolate reaction to several international isolates of P. pachyrhizi. The goals of this research were to fine map the Rpp6 locus of PI 567102B and PI 567068A and determine whether or not PI 567068A harbors a novel Rpp6 allele or another allele at a tightly linked resistance locus. Linkage mapping in this study mapped Rpp6 from 5,953,237 to 5,998,461 bp (LOD score of 58.3) and the resistance from PI 567068A from 5,998,461 to 6,160,481 bp (LOD score of 4.4) (Wm82.a1 genome sequence). QTL peaks were 139,033 bp apart from one another as determined by the most significant SNPs in QTL mapping. The results of haplotype analysis demonstrated that PI 567102B and PI 567068A share the same haplotype in the resistance locus containing both Rpp alleles, which was designated as the Rpp6/Rpp[PI567068A] haplotype. The Rpp6/Rpp[PI567068A] haplotype identified in this study can be used as a tool to rapidly screen other genotypes that possess a Rpp gene(s) and detect resistance at the Rpp6 locus in diverse germplasm.

Soybean pathogens significantly impact yield, resulting in over $4 billion dollars in lost revenue annually in the United States. Despite the deployment of improved soybean cultivars, pathogens continue to evolve to evade plant defense responses. Thus, there is an urgent need to identify and characterize gene networks controlling defense responses to harmful pathogens. In this review, we focus on major advances that have been made in identifying the genes and gene networks regulating defense responses with an emphasis on soybean-pathogen interactions that have been amenable to gene function analyses using gene silencing technologies. Further we describe new research striving to identify genes involved in durable broad-spectrum resistance. Finally, we consider future prospects for functional genomic studies in soybean and demonstrate that understanding soybean disease and stress tolerance will be expedited at an unprecedented pace., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The obligate biotrophic pathogen Puccinia horiana is the causal agent of chrysanthemum white rust. Although P. horiana is a quarantine organism, it has been able to spread to most chrysanthemum-producing regions in the world since the 1960s; however, the transfer routes are largely obscure. An extremely low level of allelic diversity was observed in a geographically diverse set of eight isolates using complexity reduction of polymorphic sequences (CRoPS) technology. Only 184 of the 16,196 contigs (1.1%) showed one or more single-nucleotide polymorphisms (SNPs). Thirty-two SNPs and one simple-sequence repeat were translated into molecular markers and used to genotype 45 isolates originating from North and South America, Asia, and Europe. In most cases, phylogenetic clustering was related to geographic origin, indicating local establishment. The European isolates mostly grouped in two major populations that may relate to the two historic introductions previously reported. However, evidence of recent geographic transfer was also observed, including transfer events between Europe and South America and between Southeast Asia and Europe. In contrast with the presumed clonal propagation of this microcyclic rust, strong indications of marker recombination were observed, presumably as a result of anastomosis, karyogamy, and somatic meiosis. Recombination and transfer also explain the geographic dispersal of specific markers. A near-to-significant correlation between the genotypic data and previously obtained pathotype data was observed and one marker was associated with the most virulent pathotype group. In combination with a fast SNP detection method, the markers presented here will be helpful tools to further elucidate the transfer pathways and local survival of this pathogen.

Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research.

Magnaporthe oryzae is the causal agent of blast disease on several graminaceous plants. The M. oryzae population causing wheat blast has not been officially reported outside South America. Wheat production in the United States is at risk to this pathogen if it is introduced and established. Proactive testing of U.S. wheat cultivars for their reaction to blast and identification of resistance resources is crucial due to the national and global importance of the U.S. wheat industry. In this preliminary study, the phenotypic reaction of 85 U.S. wheat cultivars to M. oryzae (Triticum pathotype) was determined. Although there was a significant correlation in the reaction to blast at the seedling and adult plant stages, only 57% of the head reaction was explained by the seedling reaction. Because of the importance of disease development at the head stage in the field, assessment of all 85 cultivars occurred at the head stage. Among cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars Postrock, JackPot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection. No evidence of the presence of physiological races among isolates T-7, T-12, T-22, and T-25 was found.

ABSTRACT Asian soybean rust (ASR) is an economically significant disease caused by the fungus Phakopsora pachyrhizi. The soybean genes Rpp3 and Rpp?(Hyuuga) confer resistance to specific isolates of the pathogen. Both genes map to chromosome 6 (Gm06) (linkage group [LG] C2). We recently identified 12 additional soybean accessions that harbor ASR resistance mapping to Gm06, within 5 centimorgans of Rpp3 and Rpp?(Hyuuga). To further characterize genotypes with resistance on Gm06, we used a set of eight P. pachyrhizi isolates collected from geographically diverse areas to inoculate plants and evaluate them for differential phenotypic responses. Three isolates elicited different responses from soybean accessions PI 462312 (Ankur) (Rpp3) and PI 506764 (Hyuuga) (Rpp?[Hyuuga]). In all, 11 of the new accessions yielded responses identical to either PI 462312 or Hyuuga and 1 of the new accessions, PI 417089B (Kuro daizu), differed from all others. Additional screening of Hyuuga-derived recombinant inbred lines indicated that Hyuuga carries two resistance genes, one at the Rpp3 locus on Gm06 and a second, unlinked ASR resistance gene mapping to Gm03 (LG-N) near Rpp5. These findings reveal a natural case of gene pyramiding for ASR resistance in Hyuuga and underscore the importance of utilizing multiple isolates of P. pachyrhizi when screening for ASR resistance.

Asian soybean rust is an aggressive foliar disease caused by the obligate biotrophic fungus Phakopsora pachyrhizi. On susceptible plants, the pathogen penetrates and colonizes leaf tissue, resulting in the formation of necrotic lesions and the development of numerous uredinia. The soybean Rpp2 gene confers resistance to specific isolates of P. pachyrhizi. Rpp2-mediated resistance limits the growth of the pathogen and is characterized by the formation of reddish-brown lesions and few uredinia. Using virus-induced gene silencing, we screened 140 candidate genes to identify those that play a role in Rpp2 resistance toward P. pachyrhizi. Candidate genes included putative orthologs to known defense-signaling genes, transcription factors, and genes previously found to be upregulated during the Rpp2 resistance response. We identified 11 genes that compromised Rpp2-mediated resistance when silenced, including GmEDS1, GmNPR1, GmPAD4, GmPAL1, five predicted transcription factors, an O-methyl transferase, and a cytochrome P450 monooxygenase. Together, our results provide new insight into the signaling and biochemical pathways required for resistance against P. pachyrhizi.

Programmed cell death (PCD) is triggered when Pto, a Ser-Thr protein kinase, recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv tomato. This PCD requires mitogen-activated protein kinase kinase kinase (MAPKKK alpha ) as a positive regulator in tomato (Solanum lycopersicum) and Nicotiana benthamiana. To examine how PCD-eliciting activity of the tomato MAPKKK alpha protein is regulated, we screened for MAPKKK alpha -interacting proteins in tomato and identified a 14-3-3 protein, TFT7. Virus-induced gene silencing using the TFT7 gene in N. benthamiana compromised both Pto- and MAPKKK alpha -mediated PCD, and coexpression of TFT7 with tomato MAPKKK alpha enhanced MAPKKK alpha -mediated PCD. TFT7 was also required for PCD associated with several other disease resistance proteins and contributed to resistance against P. syringae pv tomato. Coexpression of TFT7 with MAPKKK alpha in vivo caused increased accumulation of the kinase and enhanced phosphorylation of two MAP kinases. TFT7 protein contains a phosphopeptide binding motif that is present in human 14-3-3 epsilon, and substitutions in this motif abolished interaction with MAPKKK alpha in vivo and also the PCD-enhancing activity of TFT7. A 14-3-3 binding motif, including a putative phosphorylated Ser-535, is present in the C-terminal region of MAPKKK alpha. An S535A substitution in MAPKKK alpha reduced interaction with TFT7 and both PCD-eliciting ability and stability of MAPKKK alpha. Our results provide new insights into a role for 14-3-3 proteins in regulating immunity-associated PCD pathways in plants.

Puccinia horiana, the causal agent of chrysanthemum white rust, is a pathogen of quarantine status in many countries where Chrysanthemum × morifolium cultivars are grown. Historically, identification protocols for white rust relied upon macroscopic symptom development and microscopic examination of infected leaves for teliospores. Symptoms become visible 7 to 10 days after initial infection under favorable conditions followed by the production of telia. Infected plants can therefore evade detection before symptoms and fruiting bodies are evident. Conventional and real-time polymerase chain reaction (PCR) assays were developed to detect P. horiana using primers designed to amplify portions of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (rDNA). The species-specific primers could detect the pathogen from 1 ng of DNA isolated from infected leaf tissue in conventional PCR assays and from 1 pg in real-time PCR assays. While both assays were capable of detecting P. horiana in symptomatic tissue, the greater sensitivity offered by the real-time PCR assay makes it more reliable for detecting the pathogen during the latent stage of infection. The P. horiana primers did not amplify the rDNA target using DNA isolated from leaf tissue infected with P. chrysanthemi.

Asian soybean rust is a formidable threat to soybean (Glycine max) production in many areas of the world, including the United States. Only five sources of resistance have been identified (Resistance to Phakopsora pachyrhizi1 [Rpp1], Rpp2, Rpp3, Rpp4, and Rpp5). Rpp4 was previously identified in the resistant genotype PI459025B and mapped within 2 centimorgans of Satt288 on soybean chromosome 18 (linkage group G). Using simple sequence repeat markers, we developed a bacterial artificial chromosome contig for the Rpp4 locus in the susceptible cv Williams82 (Wm82). Sequencing within this region identified three Rpp4 candidate disease resistance genes (Rpp4C1-Rpp4C3 [Wm82]) with greatest similarity to the lettuce (Lactuca sativa) RGC2 family of coiled coil-nucleotide binding site-leucine rich repeat disease resistance genes. Constructs containing regions of the Wm82 Rpp4 candidate genes were used for virus-induced gene silencing experiments to silence resistance in PI459025B, confirming that orthologous genes confer resistance. Using primers developed from conserved sequences in the Wm82 Rpp4 candidate genes, we identified five Rpp4 candidate genes (Rpp4C1-Rpp4C5 [PI459025B]) from the resistant genotype. Additional markers developed from the Wm82 Rpp4 bacterial artificial chromosome contig further defined the region containing Rpp4 and eliminated Rpp4C1 (PI459025B) and Rpp4C3 (PI459025B) as candidate genes. Sequencing of reverse transcription-polymerase chain reaction products revealed that Rpp4C4 (PI459025B) was highly expressed in the resistant genotype, while expression of the other candidate genes was nearly undetectable. These data support Rpp4C4 (PI459025B) as the single candidate gene for Rpp4-mediated resistance to Asian soybean rust.

Twenty-four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty-one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi., (Journal compilation © 2008 Blackwell Publishing Ltd. No claim to original US government works.)

Nigeria (1) and Uganda (3) are the closest countries to the Democratic Republic of Congo (DRC) where soybean rust caused by Phakopsora pachyrhizi has been reported. In February 2007, during a disease survey in DRC, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 10 fields in the following areas in Bas Congo Province: Bangu, Kimpese, Kolo-Fuma, Lukala, Mbanza-Ngungu, Mpalukide, Mvuazi, and Ntemo. Rust incidence in these fields ranged from 85 to 100%, while severity ranged between 3 and 35% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 16 to 20 μm. Within a week of collection, infected leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit (FDWSRU) for pathogen identification. DNA was extracted from sections of leaves containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 10 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Infected leaves of cultivar Vuangi collected from one field each in the INERA Research Station, Kimpese-Crawford, and Kimpese-Ceco were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves of susceptible cultivar TGx 1485-1D (4). Lesions on inoculated leaves developed 5 days after inoculation (DAI), and pustules (110 to 130 μm) formed 7 DAI and erupted 2 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculation of another set of detached leaves with a spore suspension (1 × 10 6 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. Seedlings of cultivar Williams also showed typical rust symptoms when inoculated separately with urediniospores collected from nine fields (i.e., all except Kimpese-Ceco, which was infective in the detached leaf assay). Inoculation and incubation were carried out at the FDWSRU Plant Pathogen Containment Facility at Fort Detrick as described earlier (2). The PCR assay, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in DRC. To our knowledge, this is the first report of P. pachyrhizi infecting soybean in DRC. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) E. Kawuki et al. J. Phytopathol. 151:7, 2003. (4) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

Nigeria is the only country in West Africa where soybean rust, caused by Phakopsora pachyrhizi, has been officially reported (1). During a disease survey in Ghana during October 2006, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 11 fields in the following districts: Kassena Nankana in the Upper East Region; East Gonja, Central Gonja, and Tolon-Kumbungu in the Northern Region; and Ejisu-Juabeng in the Ashanti Region. Disease incidence in these fields ranged from 50 to 100% and disease severity ranged between 3 and 40% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 14 to 18 μm. Within a week of collection, leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit for verification of pathogen identity. DNA was extracted from leaf pieces containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 11 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Sequence alignment of the internal transcribed spacer (ITS) region 2 further confirmed the identification as P. pachyrhizi (2). Infected leaves from three fields were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves (for each isolate) of susceptible cultivar TGx 1485-1D (3). The abaxial surface of detached leaves was sprayed with 400 μl of spore suspension (1 × 10 6 spores per ml). A single leaf piece was placed in a 9-cm-diameter petri dish with adaxial side appressed on 1% technical agar amended with 10 μg/ml of kinetin. Lactic acid (1.5 ml/liter) and benomyl (12.5 mg/liter) were added to the agar medium to inhibit growth of saprophytic fungi and bacteria. Petri dishes were incubated at 20°C with a 12-h light/12-h dark cycle. Lesions on inoculated leaves developed 5 to 6 days after inoculation (DAI), and pustules (105 to 120 μm) formed 7 to 8 DAI and erupted 3 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculating another set of detached leaves with a spore suspension (1 × 10 6 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. The PCR assay, alignment of ITS region 2, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in Ghana. To our knowledge, this is the first report of P. pachyrhizi in Ghana. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

Bacterial speck disease in tomato is caused by Pseudomonas syringae pv. tomato. Resistance to this disease is conferred by the host Pto kinase, which recognizes P. s. pv. tomato strains that express the effector AvrPto. We report here that an AvrPto-dependent Pto-interacting protein 3 (Adi3) is a member of the AGC family of protein kinases. In mammals, AGC kinases are regulated by 3-phosphoinositide-dependent protein kinase-1 (Pdk1). We characterized tomato Pdk1 and showed that Pdk1 and Pto phosphorylate Adi3. Gene silencing of Adi3 in tomato causes MAPKKKalpha-dependent formation of necrotic lesions. Use of a chemical inhibitor of Pdk1, OSU-03012, also implicates Pdk1 and Adi3 in plant cell death regulation. Adi3 thus appears to function analogously to the mammalian AGC kinase protein kinase B/Akt by negatively regulating cell death via Pdk1 phosphorylation. We speculate that the negative regulatory function of Adi3 might be subverted by interaction with Pto/AvrPto, leading to host cell death that is associated with pathogen attack.

Studies of the early events that follow pathogen recognition have established the importance of mitogen-activated protein kinase (MAPK) cascades in plant defense signaling. Specific MAPK cascade components have now been identified in several species, and so it has become possible to compare defense responses to diverse pathogens and to elucidate downstream events that are initiated by these key signaling molecules. Recent work utilizing post-transcriptional gene silencing techniques and the development of stable transgenic lines for both gain- and loss-of-function analysis have further advanced our understanding of the role of MAPK cascades in plant immunity.

The Pto kinase mediates resistance to bacterial speck disease in tomato by activating host defenses upon recognition of Pseudomonas syringae pv. tomato strains expressing the AvrPto or AvrPtoB proteins. Previous gene-silencing experiments have indicated that mitogen activated protein kinase (MAPK) cascades play a key role downstream of the Pto kinase to activate host defense responses. Here we use biochemical methods to demonstrate that two tomato MAPKs, LeMPK2 and LeMPK3, are activated in leaves in a Pto-specific manner upon expression of AvrPto and AvrPtoB. We show that these same MAPKs are activated upon overexpression of LeMAPKKKalpha, a protein previously demonstrated to be involved in Pto-mediated immunity. We identified two phylogenetically unrelated MAPK kinases (LeMKK2 and LeMKK4) that when overexpressed in leaves elicit cell death and activate LeMPK2 and LeMPK3. In vitro analysis demonstrated that LeMKK2 and LeMKK4 each phosphorylate the same subset of three MAPKs. Together these data provide biochemical evidence for the involvement of MAPK cascades in Pto-mediated resistance.

Many plant pathogens cause disease symptoms that manifest over days as regions of localized cell death. Localized cell death (the hypersensitive response; HR) also occurs in disease-resistant plants, but this response appears within hours of attempted infection and may restrict further pathogen growth. We identified a MAP kinase kinase kinase gene (MAPKKKalpha) that is required for the HR and resistance against Pseudomonas syringae. Significantly, we found that MAPKKKalpha also regulates cell death in susceptible leaves undergoing P. syringae infection. Overexpression of MAPKKKalpha in leaves activated MAPKs and caused pathogen-independent cell death. By overexpressing MAPKKKalpha in leaves and suppressing expression of various MAPKK and MAPK genes by virus-induced gene silencing, we identified two distinct MAPK cascades that act downstream of MAPKKKalpha. These results demonstrate that signal transduction pathways associated with both plant immunity and disease susceptibility share a common molecular switch.

The Pto gene in tomato confers gene-for-gene resistance to Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease. Pto was first introgressed from a wild species of tomato into cultivated tomato varieties over 60 years ago and is now widely used to control speck disease. Cloning of the Pto gene revealed that it encodes a cytoplasmically localized serine-threonine protein kinase. The molecular basis of gene-for-gene recognition in this pathosystem is the direct physical interaction of the Pto kinase with either of two Pseudomonas effector proteins, AvrPto and AvrPtoB. Upon recognition of AvrPto or AvrPtoB, the Pto kinase acts in concert with Prf, a leucine-rich repeat-containing protein, to activate multiple signal transduction pathways. There has been much progress in understanding the evolutionary origin of the Pto gene, structural details about how the Pto kinase interacts with AvrPto and AvrPtoB, signaling steps downstream of Pto, and defense responses activated by the Pto pathway. Future work on this model system will focus on how the interaction of the Pto kinase with bacterial effector proteins activates signal transduction, defining the specific role of signaling components, and ultimately, determining which host defense responses are most responsible for inhibiting growth of the pathogen and suppressing symptoms of bacterial speck disease.

Strains of the filamentous fungus Cochliobolus carbonum that produce the host-selective compound HC-toxin, a cyclic tetrapeptide, are highly virulent on certain genotypes of maize (Zea mays L.). Production of HC-toxin is under the control of a complex locus, TOX2, which is composed of at least seven linked and duplicated genes that are present only in toxin-producing strains of C. carbonum. One of these genes, TOXE, was earlier shown to be required for the expression of the other TOX2 genes. TOXE has four ankyrin repeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, but lacks any apparent leucine zipper. Here we show that TOXE is a DNA-binding protein that recognizes a ten-base motif (the "tox-box") without dyad symmetry that is present in the promoters of all of the known TOX2 genes. Both the basic region and the ankyrin repeats are involved in DNA binding. A region of TOXE that includes the first ankyrin repeat is necessary and sufficient for transcriptional activation in yeast. The data indicate that TOXE is the prototype of a new family of transcription factor, so far found only in plant-pathogenic fungi. TOXE plays a specific regulatory role in HC-toxin production and, therefore, pathogenicity by C. carbonum.