Hiding in plain sight: Genome-wide recombination and a dynamic accessory genome drive diversity in Fusarium oxysporum f.sp. ciceris.

Understanding the origins of variation in agricultural pathogens is of fundamental interest and practical importance, especially for diseases that threaten food security. Fusarium oxysporum is among the most important of soil-borne pathogens, with a global distribution and an extensive host range. The pathogen is considered to be asexual, with horizontal transfer of chromosomes providing an analog of assortment by meiotic recombination. Here, we challenge those assumptions based on the results of population genomic analyses, describing the pathogen's diversity and inferring its origins and functional consequences in the context of a single, long-standing agricultural system. We identify simultaneously low nucleotide distance among strains, and unexpectedly high levels of genetic and genomic variability. We determine that these features arise from a combination of genome-scale recombination, best explained by widespread sexual reproduction, and presence-absence variation consistent with chromosomal rearrangement. Pangenome analyses document an accessory genome more than twice the size of the core genome, with contrasting evolutionary dynamics. The core genome is stable, with low diversity and high genetic differentiation across geographic space, while the accessory genome is paradoxically more diverse and unstable but with lower genetic differentiation and hallmarks of contemporary gene flow at local scales. We suggest a model in which episodic sexual reproduction generates haplotypes that are selected and then maintained through clone-like dynamics, followed by contemporary genomic rearrangements that reassort the accessory genome among sympatric strains. Taken together, these processes contribute unique genome content, including reassortment of virulence determinants that may explain observed variation in pathogenic potential.

Migration through a Major Andean Ecogeographic Disruption as a Driver of Genetic and Phenotypic Diversity in a Wild Tomato Species.

Evolutionary dynamics at the population level play a central role in creating the diversity of life on our planet. In this study, we sought to understand the origins of such population-level variation in mating systems and defensive acylsugar chemistry in Solanum habrochaites-a wild tomato species found in diverse Andean habitats in Ecuador and Peru. Using Restriction-site-Associated-DNA-Sequencing (RAD-seq) of 50 S. habrochaites accessions, we identified eight population clusters generated via isolation and hybridization dynamics of 4-6 ancestral populations. Detailed characterization of mating systems of these clusters revealed emergence of multiple self-compatible (SC) groups from progenitor self-incompatible populations in the northern part of the species range. Emergence of these SC groups was also associated with fixation of deleterious alleles inactivating acylsugar acetylation. The Amotape-Huancabamba Zone-a geographical landmark in the Andes with high endemism and isolated microhabitats-was identified as a major driver of differentiation in the northern species range, whereas large geographical distances contributed to population structure and evolution of a novel SC group in the central and southern parts of the range, where the species was also inferred to have originated. Findings presented here highlight the role of the diverse ecogeography of Peru and Ecuador in generating population differentiation, and enhance our understanding of the microevolutionary processes that create biological diversity.

Global-level population genomics reveals differential effects of geography and phylogeny on horizontal gene transfer in soil bacteria.

Although microorganisms are known to dominate Earth's biospheres and drive biogeochemical cycling, little is known about the geographic distributions of microbial populations or the environmental factors that pattern those distributions. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of the nitrogen-fixing bacterial symbionts of the crop chickpea, generating 1,027 draft whole-genome sequences at the level of bacterial populations, including 14 high-quality PacBio genomes from a phylogenetically representative subset. We find that diverse Mesorhizobium taxa perform symbiosis with chickpea and have largely overlapping global distributions. However, sampled locations cluster based on the phylogenetic diversity of Mesorhizobium populations, and diversity clusters correspond to edaphic and environmental factors, primarily soil type and latitude. Despite long-standing evolutionary divergence and geographic isolation, the diverse taxa observed to nodulate chickpea share a set of integrative conjugative elements (ICEs) that encode the major functions of the symbiosis. This symbiosis ICE takes 2 forms in the bacterial chromosome-tripartite and monopartite-with tripartite ICEs confined to a broadly distributed superspecies clade. The pairwise evolutionary relatedness of these elements is controlled as much by geographic distance as by the evolutionary relatedness of the background genome. In contrast, diversity in the broader gene content of Mesorhizobium genomes follows a tight linear relationship with core genome phylogenetic distance, with little detectable effect of geography. These results illustrate how geography and demography can operate differentially on the evolution of bacterial genomes and offer useful insights for the development of improved technologies for sustainable agriculture.

Gene co-expression analysis reveals transcriptome divergence between wild and cultivated chickpea under drought stress.

Ancestral adaptations in crop wild relatives can provide a genetic reservoir for crop improvement. Here we document physiological changes to mild and severe drought stress, and the associated transcriptome dynamics in both wild and cultivated chickpea. Over 60% of transcriptional changes were related to metabolism, indicating that metabolic plasticity is a core and conserved drought response. In addition, changes in RNA processing and protein turnover were predominant in the data, suggestive of broad restructuring of the chickpea proteome in response to drought. While 12% of the drought-responsive transcripts have similar dynamics in cultivated and wild accessions, numerous transcripts had expression patterns unique to particular genotypes, or that distinguished wild from cultivated genotypes and whose divergence may be a consequence of domestication. These and other comparisons provide a transcriptional correlate of previously described species' genetic diversity, with wild accessions well differentiated from each other and from cultivars, and cultivars essentially indistinguishable at the broad transcriptome level. We identified metabolic pathways such as phenylpropanoid metabolism, and biological processes such as stomatal development, which are differentially regulated across genotypes with potential consequences on drought tolerance. These data indicate that wild Cicer reticulatum may provide both conserved and divergent mechanisms as a resource in breeding for drought tolerance in cultivated chickpea.

Phosphate solubilization and multiple plant growth promoting properties of Mesorhizobium species nodulating chickpea from acidic soils of Ethiopia.

The main purpose of this study was to screen and select strains from seven Mesorhizobium spp. for efficient phosphate solubilizing and other plant growth-promoting traits. Mesorhizobium species were tested for their ability to dissolve inorganic phosphate sources and multiple plant growth-promoting attributes. From a total of 62 Mesorhizobium strains, 47(76%) strains formed clear zones with an average PSI of 1.9-2.7 on Pikovskaya's agar plate. The selected strains also released soluble phosphorus [125-150 P (µgml-1)] from tri-calcium phosphate and low level of phosphorous i.e., 15.4 µg/ml and 14.5 µg/ml from inorganic ferrous and aluminum phosphates, respectively, in a liquid medium after 4 days of incubation. The release of soluble P was significantly (P < 0.01) correlated with a drop in pH of the medium. Moreover, screening for multiple plant growth-promoting attributes showed that 40, 28, 26, 21, and 38% of the strains were capable of producing indole-3-acetic acid, hydrogen cyanide, siderophores, ACC deaminase, and antagonism against Fusarium oxysporum f.sp. ciceris under in vitro conditions. The Mesorhizobium strains were endowed with the presence of ACC deaminase which was rarely reported elsewhere. All taken together, the acidic soils harbor numerous and more diverse phosphate solubilizing and plant growth-promoting Mesorhizobium spp. However, greenhouse and field conditions can be further studied within the context of improving chickpea production in Ethiopia.

Genomic Analysis of Vavilov's Historic Chickpea Landraces Reveals Footprints of Environmental and Human Selection.

A defining challenge of the 21st century is meeting the nutritional demands of the growing human population, under a scenario of limited land and water resources and under the specter of climate change. The Vavilov seed bank contains numerous landraces collected nearly a hundred years ago, and thus may contain 'genetic gems' with the potential to enhance modern breeding efforts. Here, we analyze 407 landraces, sampled from major historic centers of chickpea cultivation and secondary diversification. Genome-Wide Association Studies (GWAS) conducted on both phenotypic traits and bioclimatic variables at landraces sampling sites as extended phenotypes resulted in 84 GWAS hits associated to various regions. The novel haploblock-based test identified haploblocks enriched for single nucleotide polymorphisms (SNPs) associated with phenotypes and bioclimatic variables. Subsequent bi-clustering of traits sharing enriched haploblocks underscored both non-random distribution of SNPs among several haploblocks and their association with multiple traits. We hypothesize that these clusters of pleiotropic SNPs represent co-adapted genetic complexes to a range of environmental conditions that chickpea experienced during domestication and subsequent geographic radiation. Linking genetic variation to phenotypic data and a wealth of historic information preserved in historic seed banks are the keys for genome-based and environment-informed breeding intensification.

MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs.

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

Functional Dissection of the Chickpea (Cicer arietinum L.) Stay-Green Phenotype Associated with Molecular Variation at an Ortholog of Mendel's I Gene for Cotyledon Color: Implications for Crop Production and Carotenoid Biofortification.

"Stay-green" crop phenotypes have been shown to impact drought tolerance and nutritional content of several crops. We aimed to genetically describe and functionally dissect the particular stay-green phenomenon found in chickpeas with a green cotyledon color of mature dry seed and investigate its potential use for improvement of chickpea environmental adaptations and nutritional value. We examined 40 stay-green accessions and a set of 29 BC2F4-5 stay-green introgression lines using a stay-green donor parent ICC 16340 and two Indian elite cultivars (KAK2, JGK1) as recurrent parents. Genetic studies of segregating populations indicated that the green cotyledon trait is controlled by a single recessive gene that is invariantly associated with the delayed degreening (extended chlorophyll retention). We found that the chickpea ortholog of Mendel's I locus of garden pea, encoding a SGR protein as very likely to underlie the persistently green cotyledon color phenotype of chickpea. Further sequence characterization of this chickpea ortholog CaStGR1 (CaStGR1, for carietinum stay-green gene 1) revealed the presence of five different molecular variants (alleles), each of which is likely a loss-of-function of the chickpea protein (CaStGR1) involved in chlorophyll catabolism. We tested the wild type and green cotyledon lines for components of adaptations to dry environments and traits linked to agronomic performance in different experimental systems and different levels of water availability. We found that the plant processes linked to disrupted CaStGR1 gene did not functionality affect transpiration efficiency or water usage. Photosynthetic pigments in grains, including provitaminogenic carotenoids important for human nutrition, were 2-3-fold higher in the stay-green type. Agronomic performance did not appear to be correlated with the presence/absence of the stay-green allele. We conclude that allelic variation in chickpea CaStGR1 does not compromise traits linked to environmental adaptation and agronomic performance, and is a promising genetic technology for biofortification of provitaminogenic carotenoids in chickpea.

The Medicago genome provides insight into the evolution of rhizobial symbioses.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Races of the Celery Pathogen Fusarium oxysporum f. sp. apii Are Polyphyletic.

Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. sp. apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. sp. apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. sp. apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.

Multiple post-domestication origins of kabuli chickpea through allelic variation in a diversification-associated transcription factor.

Chickpea (Cicer arietinum) is among the founder crops domesticated in the Fertile Crescent. One of two major forms of chickpea, the so-called kabuli type, has white flowers and light-colored seed coats, properties not known to exist in the wild progenitor. The origin of the kabuli form has been enigmatic. We genotyped a collection of wild and cultivated chickpea genotypes with 538 single nucleotide polymorphisms (SNPs) and examined patterns of molecular diversity relative to geographical sources and market types. In addition, we examined sequence and expression variation in candidate anthocyanin biosynthetic pathway genes. A reduction in genetic diversity and extensive genetic admixture distinguish cultivated chickpea from its wild progenitor species. Among germplasm, the kabuli form is polyphyletic. We identified a basic helix-loop-helix (bHLH) transcription factor at chickpea's B locus that conditions flower and seed colors, orthologous to Mendel's A gene of garden pea, whose loss of function is associated invariantly with the kabuli type of chickpea. From the polyphyletic distribution of the kabuli form in germplasm, an absence of nested variation within the bHLH gene and invariant association of loss of function of bHLH among the kabuli type, we conclude that the kabuli form arose multiple times during the phase of phenotypic diversification after initial domestication of cultivated chickpea.

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals.

The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:ß-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

The ecological genomic basis of salinity adaptation in Tunisian Medicago truncatula.

BACKGROUND: As our world becomes warmer, agriculture is increasingly impacted by rising soil salinity and understanding plant adaptation to salt stress can help enable effective crop breeding. Salt tolerance is a complex plant phenotype and we know little about the pathways utilized by naturally tolerant plants. Legumes are important species in agricultural and natural ecosystems, since they engage in symbiotic nitrogen-fixation, but are especially vulnerable to salinity stress. RESULTS: Our studies of the model legume Medicago truncatula in field and greenhouse settings demonstrate that Tunisian populations are locally adapted to saline soils at the metapopulation level and that saline origin genotypes are less impacted by salt than non-saline origin genotypes; these populations thus likely contain adaptively diverged alleles. Whole genome resequencing of 39 wild accessions reveals ongoing migration and candidate genomic regions that assort non-randomly with soil salinity. Consistent with natural selection acting at these sites, saline alleles are typically rare in the range-wide species' gene pool and are also typically derived relative to the sister species M. littoralis. Candidate regions for adaptation contain genes that regulate physiological acclimation to salt stress, such as abscisic acid and jasmonic acid signaling, including a novel salt-tolerance candidate orthologous to the uncharacterized gene AtCIPK21. Unexpectedly, these regions also contain biotic stress genes and flowering time pathway genes. We show that flowering time is differentiated between saline and non-saline populations and may allow salt stress escape. CONCLUSIONS: This work nominates multiple potential pathways of adaptation to naturally stressful environments in a model legume. These candidates point to the importance of both tolerance and avoidance in natural legume populations. We have uncovered several promising targets that could be used to breed for enhanced salt tolerance in crop legumes to enhance food security in an era of increasing soil salinization.

The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection.

Symbiosis between legume plants and soil rhizobia culminates in the formation of a novel root organ, the 'nodule', containing bacteria differentiated as facultative nitrogen-fixing organelles. MtNF-YA1 is a Medicago truncatula CCAAT box-binding transcription factor (TF), formerly called HAP2-1, highly expressed in mature nodules and required for nodule meristem function and persistence. Here a role for MtNF-YA1 during early nodule development is demonstrated. Detailed expression analysis based on RNA sequencing, quantitiative real-time PCR (qRT-PCR), as well as promoter-ß-glucuronidase (GUS) fusions reveal that MtNF-YA1 is first induced at the onset of symbiotic development during preparation for, and initiation and progression of, symbiotic infection. Moreover, using a new knock-out mutant, Mtnf-ya1-1, it is shown that MtNF-YA1 controls infection thread (IT) progression from initial root infection through colonization of nodule tissues. Extensive confocal and electronic microscopic observations suggest that the bulbous and erratic IT growth phenotypes observed in Mtnf-ya1-1 could be a consequence of the fact that walls of ITs in this mutant are thinner and less coherent than in the wild type. It is proposed that MtNF-YA1 controls rhizobial infection progression by regulating the formation and the wall of ITs.

Back to the wilds: tapping evolutionary adaptations for resilient crops through systematic hybridization with crop wild relatives.

The genetic diversity of our crop plants has been substantially reduced during the process of domestication and breeding. This reduction in diversity necessarily constrains our ability to expand a crop's range of cultivation into environments that are more extreme than those in which it was domesticated, including into "sustainable" agricultural systems with reduced inputs of pesticides, water, and fertilizers. Conversely, the wild progenitors of crop plants typically possess high levels of genetic diversity, which underlie an expanded (relative to domesticates) range of adaptive traits that may be of agricultural relevance, including resistance to pests and pathogens, tolerance to abiotic extremes, and reduced dependence on inputs. Despite their clear potential for crop improvement, wild relatives have rarely been used systematically for crop improvement, and in no cases, have full sets of wild diversity been introgressed into a crop. Instead, most breeding efforts have focused on specific traits and dealt with wild species in a limited and typically ad hoc manner. Although expedient, this approach misses the opportunity to test a large suite of traits and deploy the full potential of crop wild relatives in breeding for the looming challenges of the 21st century. Here we review examples of hybridization in several species, both intentionally produced and naturally occurring, to illustrate the gains that are possible. We start with naturally occurring hybrids, and then examine a range of examples of hybridization in agricultural settings.

Population differentiation for germination and early seedling root growth traits under saline conditions in the annual legume Medicago truncatula (Fabaceae).

PREMISE OF THE STUDY: Seedling establishment and survival are highly sensitive to soil salinity and plants that evolved in saline environments are likely to express traits that increase fitness in those environments. Such traits are of ecological interest and they may have practical value for improving salt tolerance in cultivated species. We examined responses to soil salinity and tested potential mechanisms of salt tolerance in Medicago truncatula, using genotypes that originated from natural populations occurring on saline and nonsaline soils. METHODS: Germination and seedling responses were quantified and compared between saline and nonsaline origin genotypes. Germination treatments included a range of sodium chloride (NaCl) concentrations in both offspring and parental environments. Seedling treatments included NaCl, abscisic acid (ABA), and potassium chloride (KCl). KEY RESULTS: Saline origin genotypes displayed greater salinity tolerance for germination and seedling traits relative to nonsaline origin genotypes. We observed population specific differences for the effects of salinity on time to germination and for the impact of parental environment on germination rates. ABA and NaCl treatments had similar negative effects on root growth, although relative sensitivities differed, with saline population less sensitive to NaCl and more sensitive to ABA compared to their nonsaline counterparts. CONCLUSIONS: We report population differentiation for germination and seedling growth traits under saline conditions among populations derived from saline and nonsaline environments. These observations are consistent with a syndrome of adaptations for salinity tolerance during early plant development, including traits that are common among saline environments and those that are idiosyncratic to local populations.

Water deficit modulates the response of Vitis vinifera to the Pierce's disease pathogen Xylella fastidiosa.

Pierce's disease, caused by the bacterium Xylella fastidiosa, is one of the most devastating diseases of cultivated grape, currently restricted to the Americas. To test the long-standing hypothesis that Pierce's disease results from pathogen-induced drought stress, we used the Affymetrix Vitis GeneChip to compare the transcriptional response of Vitis vinifera to Xylella infection, water deficit, or a combination of the two stresses. The results reveal a redirection of gene transcription involving 822 genes with a minimum twofold change (P < 0.05), including the upregulation of transcripts for phenylpropanoid and flavonoid biosynthesis, pathogenesis-related proteins, abscisic acid- and jasmonic acid-responsive biosynthesis, and downregulation of transcripts related to photosynthesis, growth, and nutrition. Although the transcriptional response of plants to Xylella infection was largely distinct from the response of healthy plants to water stress, we find that 138 of the pathogen-induced genes exhibited a significantly stronger transcriptional response when plants were simultaneously exposed to infection and drought stress, suggesting a strong interaction between disease and water deficit. This interaction between drought stress and disease was mirrored in planta at the physiological level for aspects of water relations and photosynthesis and in terms of the severity of disease symptoms and the extent of pathogen colonization, providing a molecular correlate of the classical concept of the disease triangle in which environment impacts disease severity.

Development of tools for the biochemical characterization of the symbiotic receptor-like kinase DMI2.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Isolation and characterization of nucleotide-binding site resistance gene homologues in common bean (Phaseolus vulgaris).

Common bean production is constrained by many fungal, viral, and bacterial pathogens. Thus, the identification of resistance (R) genes is an important focal point of common bean research. The main goal of our study was to identify resistance gene homologues (RGH) in the crop, using degenerate primers designed from conserved sequences in the nucleotide-binding site (NBS) domains of R-genes from the model legume Medicago truncatula. Total DNA of the Andean common bean genotype G19833 was used for amplification of over 500 primer combinations. Sequencing of amplicons showed that 403 cloned fragments had uninterrupted open reading frames and were considered representative of functional RGH genes. The sequences were grouped at two levels of nucleotide identity (90 and 80%) and representative sequences of each group were used for phylogenetic analyses. The RGH sequence diversity of common bean was divided into TIR and non-TIR families, each with different clusters. The TIR sequences grouped into 14 clades while non-TIR sequences grouped into seven clades. Pairwise comparisons showed purifying selection, although some sequences may have been the result of diversifying selection. Knowledge about RGH genes in common bean can allow the design of molecular markers for pyramiding of resistance genes against various pathogens.

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs.

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.