The trichothecene mycotoxin deoxynivalenol facilitates cell-to-cell invasion during wheat-tissue colonization by Fusarium graminearum.

Fusarium head blight disease on small-grain cereals is primarily caused by the ascomycete fungal pathogen Fusarium graminearum. Infection of floral spike tissues is characterized by the biosynthesis and secretion of potent trichothecene mycotoxins, of which deoxynivalenol (DON) is widely reported due to its negative impacts on grain quality and consumer safety. The TRI5 gene encodes an essential enzyme in the DON biosynthesis pathway and the single gene deletion mutant, ΔTri5, is widely reported to restrict disease progression to the inoculated spikelet. In this study, we present novel bioimaging evidence revealing that DON facilitates the traversal of the cell wall through plasmodesmata, a process essential for successful colonization of host tissue. Chemical complementation of ΔTri5 did not restore macro- or microscopic phenotypes, indicating that DON secretion is tightly regulated both spatially and temporally. A comparative qualitative and quantitative morphological cellular analysis revealed infections had no impact on plant cell wall thickness. Immunolabelling of callose at plasmodesmata during infection indicates that DON can increase deposits when applied exogenously but is reduced when F. graminearum hyphae are present. This study highlights the complexity of the interconnected roles of mycotoxin production, cell wall architecture and plasmodesmata in this highly specialized interaction.

Antimicrobial stewardship markers and healthcare-associated pneumonia threshold criteria in UK hospitals: analysis of the MicroGuideTm application.

Background: To address antimicrobial resistance, antimicrobial stewardship (AMS) principles must be implemented and adhered to. Clinical decision aids such as the MicroGuideTM app are an important part of these efforts. We sought to evaluate the consistency of core AMS information and the diversity of classification thresholds for healthcare-associated pneumonia (HAP) in the MicroGuide app. Methods: Guidelines in the MicroGuide app were extracted and analysed for content related to AMS and HAP. Guidelines were characterized according to HAP naming classification; community-acquired pneumonia (CAP) classifications were analysed to serve as a comparator group. Results: In total, 115 trusts (119 hospitals) were included. Nearly all hospitals had developed MicroGuide sections on AMS (n = 112/119, 94%) and sepsis management (n = 117/119, 98%). Other AMS sections were outpatient parenteral antimicrobial therapy (47%), antifungal stewardship (70%), critical care (23%) and IV to oral switch therapy (83%). Only 9% of hospitals included guidance on the maximum six key AMS sections identified. HAP definitions varied widely across hospitals with some classifying by time to onset and some classifying by severity or complexity. The largest proportion of HAP guidelines based classification on severity/complexity (n = 69/119, 58%). By contrast, definitions in CAP guidelines were uniform. Conclusions: The high heterogeneity in HAP classification identified suggests inconsistency of practice in identifying thresholds for HAP in the UK. This complicates HAP management and AMS practices. To address HAP in alignment with AMS principles, a comprehensive strategy that prioritizes uniform clinical definitions and thresholds should be developed.

An Insurance Value Modeling Approach That Captures the Wider Value of a Novel Antimicrobial to Health Systems, Patients, and the Population.

Background: Traditional health economic evaluations of antimicrobials currently underestimate their value to wider society. They can be supplemented by additional value elements including insurance value, which captures the value of an antimicrobial in preventing or mitigating impacts of adverse risk events. Despite being commonplace in other sectors, constituents of the impacts and approaches for estimating insurance value have not been investigated. Objectives: This study assessed the insurance value of a novel gram-negative antimicrobial from operational healthcare, wider population health, productivity, and informal care perspectives. Methods: A novel mixed-methods approach was used to model insurance value in the United Kingdom: (1) literature review and multidisciplinary expert workshops to identify risk events for 4 relevant scenarios: ward closures, unavoidable shortage of conventional antimicrobials, viral respiratory pandemics, and catastrophic antimicrobial resistance (AMR); (2) parameterizing mitigable costs and frequencies of risk events across perspectives and scenarios; (3) estimating insurance value through a Monte Carlo simulation model for extreme events and a dynamic disease transmission model. Results: The mean insurance value across all scenarios and perspectives over 10 years in the UK was £718 million, should AMR remain unchanged, where only £134 million related to operational healthcare costs. It would be 50%-70% higher if AMR steadily increased or if a more risk-averse view (1-in-10 year downside) of future events is taken. Discussion: The overall insurance value if AMR remains at current levels (a conservative projection), is over 5 times greater than insurance value from just the operational healthcare costs perspective, traditionally the sole perspective used in health budgeting. Insurance value was generally larger for nationwide or universal (catastrophic AMR, pandemic, and conventional antimicrobial shortages) rather than localized (ward closure) scenarios, across perspectives. Components of this insurance value match previously published estimates of operational costs and mortality impacts. Conclusions: Insurance value of novel antimicrobials can be systematically modeled and substantially augments their traditional health economic value in normal circumstances. These approaches are generalizable to similar health interventions and form a framework for health systems and governments to capture broader value in health technology assessments, improve healthcare access, and increase resilience by planning for adverse scenarios.

Fungal plant pathogen "mutagenomics" reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor.

"Mutagenomics" is the combination of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated "random" T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have a distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the predicted protein's N-terminus, while the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains' wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines.

Combined pangenomics and transcriptomics reveals core and redundant virulence processes in a rapidly evolving fungal plant pathogen.

BACKGROUND: Studying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their "core biology", being present, functional and expressed by all strains or "flexible biology", varying between strains. Genes supporting flexible biology may be considered to be "accessory", whilst the "core" gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study. RESULTS: We constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a "core" gene set comprising 9807 sequences which were (1) present in all isolates, (2) lacking inactivating polymorphisms and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes, was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression. CONCLUSIONS: These data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens.

Reducing Dietary Acrylamide Exposure from Wheat Products through Crop Management and Imaging.

The nutritional safety of wheat-based food products is compromised by the presence of the processing contaminant acrylamide. Reduction of the key acrylamide precursor, free (soluble, non-protein) asparagine, in wheat grain can be achieved through crop management strategies, but such strategies have not been fully developed. We ran two field trials with 12 soft (biscuit) wheat varieties and different nitrogen, sulfur, potassium, and phosphorus fertilizer combinations. Our results indicated that a nitrogen-to-sulfur ratio of 10:1 kg/ha was sufficient to prevent large increases in free asparagine, whereas withholding potassium or phosphorus alone did not cause increases in free asparagine when sulfur was applied. Multispectral measurements of plants in the field were able to predict the free asparagine content of grain with an accuracy of 71%, while a combination of multispectral, fluorescence, and morphological measurements of seeds could distinguish high free asparagine grain from low free asparagine grain with an accuracy of 86%. The acrylamide content of biscuits correlated strongly with free asparagine content and with color measurements, indicating that agronomic strategies to decrease free asparagine would be effective and that quality control checks based on product color could eliminate high acrylamide biscuit products.

The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection.

Plants have developed intricate defense mechanisms, referred to as innate immunity, to defend themselves against a wide range of pathogens. Plants often respond rapidly to pathogen attack by the synthesis and delivery to the primary infection sites of various antimicrobial compounds, proteins, and small RNA in membrane vesicles. Much of the evidence regarding the importance of vesicular trafficking in plant-pathogen interactions comes from studies involving model plants whereas this process is relatively understudied in crop plants. Here we assessed whether the vesicular trafficking system components previously implicated in immunity in Arabidopsis play a role in the interaction with Fusarium graminearum, a fungal pathogen well-known for its ability to cause Fusarium head blight disease in wheat. Among the analysed vesicular trafficking mutants, two independent T-DNA insertion mutants in the AtMin7 gene displayed a markedly enhanced susceptibility to F. graminearum. Earlier studies identified this gene, encoding an ARF-GEF protein, as a target for the HopM1 effector of the bacterial pathogen Pseudomonas syringae pv. tomato, which destabilizes MIN7 leading to its degradation and weakening host defenses. To test whether this key vesicular trafficking component may also contribute to defense in crop plants, we identified the candidate TaMin7 genes in wheat and knocked-down their expression through virus-induced gene silencing. Wheat plants in which TaMin7 genes were silenced displayed significantly more Fusarium head blight disease. This suggests that disruption of MIN7 function in both model and crop plants compromises the trafficking of innate immunity signals or products resulting in hypersusceptibility to various pathogens.

Common Bean Subtelomeres Are Hot Spots of Recombination and Favor Resistance Gene Evolution.

Subtelomeres of most eukaryotes contain fast-evolving genes usually involved in adaptive processes. In common bean (Phaseolus vulgaris), the Co-2 anthracnose resistance (R) locus corresponds to a cluster of nucleotide-binding-site leucine-rich-repeat (NL) encoding sequences, the prevalent class of plant R genes. To study the recent evolution of this R gene cluster, we used a combination of sequence, genetic and cytogenetic comparative analyses between common bean genotypes from two distinct gene pools (Andean and Mesoamerican) that diverged 0.165 million years ago. Co-2 is a large subtelomeric cluster on chromosome 11 comprising from 32 (Mesoamerican) to 52 (Andean) NL sequences embedded within khipu satellite repeats. Since the recent split between Andean and Mesoamerican gene pools, the Co-2 cluster has experienced numerous gene-pool specific NL losses, leading to distinct NL repertoires. The high proportion of solo-LTR retrotransposons indicates that the Co-2 cluster is located in a hot spot of unequal intra-strand homologous recombination. Furthermore, we observe large segmental duplications involving both Non-Homologous End Joining and Homologous Recombination double-strand break repair pathways. Finally, the identification of a Mesoamerican-specific subtelomeric sequence reveals frequent interchromosomal recombinations between common bean subtelomeres. Altogether, our results highlight that common bean subtelomeres are hot spots of recombination and favor the rapid evolution of R genes. We propose that chromosome ends could act as R gene incubators in many plant genomes.

Using decoys to expand the recognition specificity of a plant disease resistance protein.

Maintaining high crop yields in an environmentally sustainable manner requires the development of disease-resistant crop varieties. We describe a method to engineer disease resistance in plants by means of an endogenous disease resistance gene from Arabidopsis thaliana named RPS5, which encodes a nucleotide-binding leucine-rich repeat (NLR) protein. RPS5 is normally activated when a second host protein, PBS1, is cleaved by the pathogen-secreted protease AvrPphB. We show that the AvrPphB cleavage site within PBS1 can be substituted with cleavage sites for other pathogen proteases, which then enables RPS5 to be activated by these proteases, thereby conferring resistance to new pathogens. This "decoy" approach may be applicable to other NLR proteins and should enable engineering of resistance in plants to diseases for which we currently lack robust genetic resistance.

Pseudomonas syringae Effector AvrPphB Suppresses AvrB-Induced Activation of RPM1 but Not AvrRpm1-Induced Activation.

The Pseudomonas syringae effector AvrB triggers a hypersensitive resistance response in Arabidopsis and soybean plants expressing the disease resistance (R) proteins RPM1 and Rpg1b, respectively. In Arabidopsis, AvrB induces RPM1-interacting protein kinase (RIPK) to phosphorylate a disease regulator known as RIN4, which subsequently activates RPM1-mediated defenses. Here, we show that AvrPphB can suppress activation of RPM1 by AvrB and this suppression is correlated with the cleavage of RIPK by AvrPphB. Significantly, AvrPphB does not suppress activation of RPM1 by AvrRpm1, suggesting that RIPK is not required for AvrRpm1-induced modification of RIN4. This observation indicates that AvrB and AvrRpm1 recognition is mediated by different mechanisms in Arabidopsis, despite their recognition being determined by a single R protein. Moreover, AvrB recognition but not AvrRpm1 recognition is suppressed by AvrPphB in soybean, suggesting that AvrB recognition requires a similar molecular mechanism in soybean and Arabidopsis. In support of this, we found that phosphodeficient mutations in the soybean GmRIN4a and GmRIN4b proteins are sufficient to block Rpg1b-mediated hypersensitive response in transient assays in Nicotiana glutinosa. Taken together, our results indicate that AvrB and AvrPphB target a conserved defense signaling pathway in Arabidopsis and soybean that includes RIPK and RIN4.

Determining the GmRIN4 requirements of the soybean disease resistance proteins Rpg1b and Rpg1r using a nicotiana glutinosa-based agroinfiltration system.

Rpg1b and Rpg1r are soybean disease resistance (R) genes responsible for conferring resistance to Pseudomonas syringae strains expressing the effectors AvrB and AvrRpm1, respectively. The study of these cloned genes would be greatly facilitated by the availability of a suitable transient expression system. The commonly used Niciotiana benthamiana-based system is not suitable for studying Rpg1b and Rpg1r function, however, because expression of AvrB or AvrRpm1 alone induces a hypersensitive response (HR), indicating that N. benthamiana contains endogenous R genes that recognize these effectors. To identify a suitable alternative host for transient expression assays, we screened 13 species of Nicotiana along with 11 accessions of N. tabacum for lack of response to transient expression of AvrB and AvrRpm1. We found that N. glutinosa did not respond to either effector and was readily transformable as determined by transient expression of ß-glucuronidase. Using this system, we determined that Rpg1b-mediated HR in N. glutinosa required co-expression of avrB and a soybean ortholog of the Arabidopsis RIN4 gene. All four soybean RIN4 orthologs tested worked in the assay. In contrast, Rpg1r did not require co-expression of a soybean RIN4 ortholog to recognize AvrRpm1, but recognition was suppressed by co-expression with AvrRpt2. These observations suggest that an endogenous RIN4 gene in N. glutinosa can substitute for the soybean RIN4 ortholog in the recognition of AvrRpm1 by Rpg1r.

Evolutionary relationship of disease resistance genes in soybean and Arabidopsis specific for the Pseudomonas syringae effectors AvrB and AvrRpm1.

In Arabidopsis (Arabidopsis thaliana), the Pseudomonas syringae effector proteins AvrB and AvrRpm1 are both detected by the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) disease resistance (R) protein. By contrast, soybean (Glycine max) can distinguish between these effectors, with AvrB and AvrRpm1 being detected by the Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r R proteins, respectively. We have been using these genes to investigate the evolution of R gene specificity and have previously identified RPM1 and Rpg1b. Here, we report the cloning of Rpg1r, which, like RPM1 and Rpg1b, encodes a coiled-coil (CC)-nucleotide-binding (NB)-leucine-rich repeat (LRR) protein. As previously found for Rpg1b, we determined that Rpg1r is not orthologous with RPM1, indicating that the ability to detect both AvrB and AvrRpm1 evolved independently in soybean and Arabidopsis. The tightly linked soybean Rpg1b and Rpg1r genes share a close evolutionary relationship, with Rpg1b containing a recombination event that combined a NB domain closely related to Rpg1r with CC and LRR domains from a more distantly related CC-NB-LRR gene. Using structural modeling, we mapped polymorphisms between Rpg1b and Rpg1r onto the predicted tertiary structure of Rpg1b, which revealed highly polymorphic surfaces within both the CC and LRR domains. Assessment of chimeras between Rpg1b and Rpg1r using a transient expression system revealed that AvrB versus AvrRpm1 specificity is determined by the C-terminal portion of the LRR domain. The P. syringae effector AvrRpt2, which targets RPM1 INTERACTOR4 (RIN4) proteins in both Arabidopsis and soybean, partially blocked recognition of both AvrB and AvrRpm1 in soybean, suggesting that both Rpg1b and Rpg1r may detect these effectors via modification of a RIN4 homolog.

Evolution of a complex disease resistance gene cluster in diploid Phaseolus and tetraploid Glycine.

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.

Specific resistances against Pseudomonas syringae effectors AvrB and AvrRpm1 have evolved differently in common bean (Phaseolus vulgaris), soybean (Glycine max), and Arabidopsis thaliana.

*In plants, the evolution of specific resistance is poorly understood. Pseudomonas syringae effectors AvrB and AvrRpm1 are recognized by phylogenetically distinct resistance (R) proteins in Arabidopsis thaliana (Brassicaceae) and soybean (Glycine max, Fabaceae). In soybean, these resistances are encoded by two tightly linked R genes, Rpg1-b and Rpg1-r. To study the evolution of these specific resistances, we investigated AvrB- and AvrRpm1-induced responses in common bean (Phaseolus vulgaris, Fabaceae). *Common bean genotypes of various geographical origins were inoculated with P. syringae strains expressing AvrB or AvrRpm1. A common bean recombinant inbred line (RIL) population was used to map R genes to AvrRpm1. *No common bean genotypes recognized AvrB. By contrast, multiple genotypes responded to AvrRpm1, and two independent R genes conferring AvrRpm1-specific resistance were mapped to the ends of linkage group B11 (Rpsar-1, for resistance to Pseudomonas syringae effector AvrRpm1 number 1) and B8 (Rpsar-2). Rpsar-1 is located in a region syntenic with the soybean Rpg1 cluster. However, mapping of specific Rpg1 homologous genes suggests that AvrRpm1 recognition evolved independently in common bean and soybean. *The conservation of the genomic position of AvrRpm1-specific genes between soybean and common bean suggests a model whereby specific clusters of R genes are predisposed to evolve recognition of the same effector molecules.

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean.

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.

Replication of nonautonomous retroelements in soybean appears to be both recent and common.

Retrotransposons and their remnants often constitute more than 50% of higher plant genomes. Although extensively studied in monocot crops such as maize (Zea mays) and rice (Oryza sativa), the impact of retrotransposons on dicot crop genomes is not well documented. Here, we present an analysis of retrotransposons in soybean (Glycine max). Analysis of approximately 3.7 megabases (Mb) of genomic sequence, including 0.87 Mb of pericentromeric sequence, uncovered 45 intact long terminal repeat (LTR)-retrotransposons. The ratio of intact elements to solo LTRs was 8:1, one of the highest reported to date in plants, suggesting that removal of retrotransposons by homologous recombination between LTRs is occurring more slowly in soybean than in previously characterized plant species. Analysis of paired LTR sequences uncovered a low frequency of deletions relative to base substitutions, indicating that removal of retrotransposon sequences by illegitimate recombination is also operating more slowly. Significantly, we identified three subfamilies of nonautonomous elements that have replicated in the recent past, suggesting that retrotransposition can be catalyzed in trans by autonomous elements elsewhere in the genome. Analysis of 1.6 Mb of sequence from Glycine tomentella, a wild perennial relative of soybean, uncovered 23 intact retroelements, two of which had accumulated no mutations in their LTRs, indicating very recent insertion. A similar pattern was found in 0.94 Mb of sequence from Phaseolus vulgaris (common bean). Thus, autonomous and nonautonomous retrotransposons appear to be both abundant and active in Glycine and Phaseolus. The impact of nonautonomous retrotransposon replication on genome size appears to be much greater than previously appreciated.

Diversification of non-TIR class NB-LRR genes in relation to whole-genome duplication events in Arabidopsis.

Arabidopsis thaliana is believed to have experienced at least two and possibly three whole-genome duplication events in its evolutionary history. In order to investigate the evolutionary relationships between these duplication events and diversification of disease resistance (R) genes, segmental-duplication events containing R genes belonging to the nucleotide binding-leucine rich repeat (NB-LRR) class were identified. Of 153 segmental-duplication events containing NB-LRR genes, only 22 contained NB-LRR genes in both members of the duplication pair, indicating a high frequency of NB-LRR gene loss after whole-genome duplication. The relative age of the duplication events was estimated based on the average synonymous substitution rate of the duplicated gene pairs in the segments. These data were combined with phylogenetic analyses. NB-LRR genes present in segment pairs derived from the most recent whole-genome duplication event, estimated to have occurred only 20 to 40 million years ago, occupy very distant branches of the NB-LRR phylogenetic tree. These data suggest that when NB-LRR clusters are duplicated as part of a whole-genome duplication, homoeologous NB-LRR genes are preferentially lost, either by eliminating one copy of the cluster or by eliminating individual genes such that only paralogous NB-LRR genes are maintained.

Convergent evolution of disease resistance gene specificity in two flowering plant families.

Plant disease resistance (R) genes that mediate recognition of the same pathogen determinant sometimes can be found in distantly related plant families. This observation implies that some R gene alleles may have been conserved throughout the diversification of land plants. To address this question, we have compared R genes from Glycine max (soybean), Rpg1-b, and Arabidopsis thaliana, RPM1, that mediate recognition of the same type III effector protein from Pseudomonas syringae, AvrB. RPM1 has been cloned previously, and here, we describe the isolation of Rpg1-b. Although RPM1 and Rpg1-b both belong to the coiled-coil nucleotide binding site (NBS) Leu-rich repeat (LRR) class of R genes, they share only limited sequence similarity outside the conserved domains characteristic of this class. Phylogenetic analyses of A. thaliana and legume NBS-LRR sequences demonstrate that Rpg1-b and RPM1 are not orthologous. We conclude that convergent evolution, rather than the conservation of an ancient specificity, is responsible for the generation of these AvrB-specific genes.

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes.

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.