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Leguminous plants can enter into root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. An intriguing but still poorly understood property of the symbiosis is its host specificity, which is controlled at multiple levels involving both rhizobial and host genes. It is widely believed that the host specificity is determined by specific recognition of bacterially derived Nod factors by the cognate host receptor(s), Here we describe the positional cloning of two soybean genes Rj2 and Rfg1 that restrict nodulation with specific strains of Bradyrhizobium japonicum and Sinorhizobium fredii, respectively. We show that Rj2 and Rfg1 are allelic genes encoding a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant resistance (R) proteins. The involvement of host R genes in the control of genotype-specific infection and nodulation reveals a common recognition mechanism underlying symbiotic and pathogenic host-bacteria interactions and suggests the existence of their cognate avirulence genes derived from rhizobia. This study suggests that establishment of a root nodule symbiosis requires the evasion of plant immune responses triggered by rhizobial effectors. soybean | nodulation | nitrogen fixation | defense doi/ 10.1073/pnas.1011957107

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the scarlet runner bean (Phaseolus coccineus) G564 gene to understand how genes are activated specifically within the suspensor during early embryo development. Previously, we showed that the G564 upstream region has a block of tandem repeats, which contain a conserved 10-bp motif ([GAAAAG.sup.C]/[sub.T]GAA), and that deletion of these repeats results in a loss of suspensor transcription. Here, we use gain-of-function (GOF) experiments with transgenic globular-stage tobacco embryos to show that only 1 of the 5 tandem repeats is required to drive suspensor-specific transcription. Fine-scale deletion and scanning mutagenesis experiments with I tandem repeat uncovered a 54-bp region that contains all of the sequences required to activate transcription in the suspensor, including the 10-bp motif (GAAAAGCGAA) and a similar 10-bp-like motif (GAAAAACGAA). Site-directed mutagenesis and GOF experiments indicated that both the 10-bp and 10-bp-like motifs are necessary, but not sufficient to activate transcription in the suspensor, and that a sequence (TTGGT) between the 10-bp and the 10-bp-like motifs is also necessary for suspensor transcription. Together, these data identify sequences that are required to activate transcription in the suspensor of a plant embryo after fertilization. promoter analysis | scarlet runner bean

Expression of the Arabidopsis TRANSPARENT TESTA 2 (TT2) MYB family transcription factor leads to massive accumulation of proanthocyanidins (PAs) in hairy roots of Medicago truncatula. Microarray analysis showed that TT2 induces genes for flavonoid/PA biosynthesis, transcription factors, and a large number of genes of unknown function. A second microarray dataset identified genes that were preferentially expressed in the M. truncatula seed coat. Comparison of the two datasets defines target genes for steps that are yet unidentified in PA biosynthesis and accumulation. Of these genes, a glycosyltransferase, UGT72L1, was active specifically toward the PA precursor (-)--epicatechin, and its expression pattern in developing seeds correlated with the presence of epicatechin glucoside and accumulation of PA5. UGT72L1 may be involved in the production of epicatechin 3'-O-glucoside in the seed coat as a key step in PA biosynthesis or its regulation. glucosyltransferase | model legume | proanthocyanidin

Rhizobia can infect roots of host legume plants and induce new organs called nodules, in which they fix atmospheric nitrogen. Infection generally starts with root hair curling, then proceeds inside newly formed, intracellular tubular structures called infection threads. A successful symbiotic interaction relies on infection threads advancing rapidly at their tips by polar growth through successive cell layers of the root toward developing nodule primordia. To identify a plant component that controls this tip growth process, we characterized a symbiotic mutant of Medicago truncatula, called rpg for rhizobium-directed polar growth. In this mutant, nitrogen-fixing nodules were rarely formed due to abnormally thick and slowly progressing infection threads. Root hair curling was also abnormal, indicating that the RPG gene fulfils an essential function in the process whereby rhizobia manage to dominate the process of induced tip growth for root hair infection. Map-based cloning of RPG revealed a member of a previously unknown plant-specific gene family encoding putative Iong coiled-coil proteins we have called RRPs (RPG-related proteins) and characterized by an 'RRP domain' specific to this family. RPG expression was strongly associated with rhizobial infection, and the RPG protein showed a nuclear localization, indicating that this symbiotic gene constitutes an important component of symbiotic signaling. genetics | symbiosis | coiled-coil

Sinorhizobium meliloti forms symbiotic, nitrogen-fixing nodules on the roots of Medicago truncatula. The bacteria invade and colonize the roots through structures called infection threads. S. meliloti unable to produce the exopolysaccharide succinoglycan are unable to establish a symbiosis because they are defective in initiating the production of infection threads and in invading the plant. Here, we use microarrays representing 16,000 M. truncatula genes to compare the differential transcriptional responses of this host plant to wild-type and succinoglycan-deficient S. meliloti at the early time point of 3 days postinoculation. This report describes an early divergence in global plant gene expression responses caused by a rhizobial defect in succinoglycan production, rather than in Nod factor production. The microarray data show that M. truncatula inoculated with wild-type, succinoglycan-producing S. meliloti more strongly express genes encoding translation components, protein degradation machinery, and some nodulins than plants inoculated with succinoglycan-deficient bacteria. This finding is consistent with wild-type-inoculated plants having received a signal, distinct from the well characterized Nod factor, to alter their metabolic activity and prepare for invasion. In contrast, M. truncatula inoculated with succinoglycan-deficient S. meliloti more strongly express an unexpectedly large number of genes in two categories: plant defense responses and unknown functions. One model consistent with our results is that appropriate symbiotically active exopolysaccharides act as signals to plant hosts to initiate infection thread formation and that, in the absence of this signal, plants terminate the infection process, perhaps via a defense response. nitrogen fixation | nodule | succinoglycan | rnicroarray | legume

Cell suspensions of the model legume Medicago truncatula accumulated the isoflavonoid phytoalexin medicarpin in response to yeast elicitor or methyl jasmonate (MJ), accompanied by decreased levels of isoflavone glycosides in MJ-treated cells. DNA microarray analysis revealed rapid, massive induction of early (iso)flavonoid pathway gene transcripts in response to yeast elicitor, but not MJ, and differential induction by the two elicitors of sets of genes encoding transcription factors, ABC transporters, and [beta]-glucosidases. In contrast, both elicitors induced genes encoding enzymes for conversion of the isoflavone formononetin to medicarpin. Four MJ-induced [beta]-glucosidases were expressed as recombinant enzymes in yeast, and three were active with isoflavone glucosides. The most highly induced [beta]-glucosidase was nuclear localized and preferred flavones to isoflavones. The results indicate that the genetic and biochemical mechanisms underlying accumulation of medicarpin differ depending on the nature of the stimulus and suggest a role for MJ as a signal for rapid hydrolysis of preformed, conjugated intermediates for antimicrobial biosynthesis during wound responses. glucosidase | methyl jasmonate | phytoanticipin | cell culture | elicitation

Backyard gardens, dump heaps, and kitchen middens are thought to have provided important venues for early crop domestication via generation of hybrids between otherwise isolated plant species. However, this process has rarely been demonstrated empirically. For the majority of polyploid crops, it remains uncertain to what extent hybridization and polyploidization preceded domestication or were precipitated by human activities. Using archaeological, ethnobotanical, geographical, and genetic data, we investigate the extent and significance of predomestication cultivation, backyard sympatry, and spontaneous hybridization for the Mimosoid legume tree Leucaena, which is used as a food crop throughout south-central Mexico. We show that predomestication cultivation was widespread, involved numerous independent transitions from the wild to cultivation, and resulted in extensive artificial sympatry of 2-6 species locally and 13 species in total. Using chloroplast and rapidly evolving nuclear-encoded DNA sequences, we demonstrate that hybridization in Leucaena has been extensive and complex, spawning a diverse set of novel hybrids as a result of juxtaposition of species in cultivation. The scale and complexity of hybridization in Leucaena is significantly greater than that documented for any other Mexican plant domesticates so far. However, there are striking parallels between Leucaena and the other major Mexican perennial domesticates Agave and Opuntia, which show very similar domestication via backyard hybridization pathways. Our results suggest that backyard hybridization has played a central role in Mesoamerican crop domestication and demonstrate that the simple step of bringing species together in cultivation can provide a potent trigger for domestication. domestication | Leguminosae | Leucaena | Mesoamerica

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133, essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the [Ca.sup.2+] spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear-cytoplasmic communication after host-plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger. legume symbiosis | nucleoporin | nuclear pore | plant-microbe interaction

Legumes are simultaneously one of the largest families of crop plants and a cornerstone in the biological nitrogen cycle. We combined molecular and phylogenetic analyses to evaluate genome conservation both within and between the two major clades of crop legumes. Genetic mapping of orthologous genes identifies broad conservation of genome macrostructure, especially within the galegoid legumes, while also highlighting inferred chromosomal rearrangements that may underlie the variation in chromosome number between these species. As a complement to comparative genetic mapping, we compared sequenced regions of the model legume Medicago truncatula with those of the diploid Lotus japonicus and the polyploid Glycine max. High conservation was observed between the genomes of M. truncatula and L. japonicus, whereas lower levels of conservation were evident between M. truncatula and G. max. In all cases, conserved genome microstructure was punctuated by significant structural divergence, including frequent insertion/deletion of individual genes or groups of genes and lineage-specific expansion/contraction of gene families. These results suggest that comparative mapping may have considerable utility for basic and applied research in the legumes, although its predictive value is likely to be tempered by phylogenetic distance and genome duplication.

Within papilionoid legumes, characterized by flowers with strong bilateral symmetry, a derived condition within angiosperms, Cadia (Cadia purpurea) has reverted to radially symmetrical flowers. Here, we investigate the genetic basis of this morphological reversal. Two orthologues of the floral symmetry gene CYCLOIDEA (CYC) demarcate the adaxial (dorsal) region of the flower in typical papilionoid legumes. In the model legume Lotusjaponicus, one of these LegCYC genes has been shown, like CYC, to be required for the establishment of floral bilateral symmetry. This study shows that these genes are expressed in the adaxial region of the typical papilionoid flower of Lupinus, which belongs to the same papilionoid subclade as Cadia. In Cadia, these genes also are expressed, but the expression pattern of one of these has expanded from the adaxial to the lateral and abaxial regions of the corolla. This result suggests that the radial flowers of Cadia are dorsalized and, therefore, are not a true evolutionary reversal but an innovative homeotic transformation, where, in this case, all petals have acquired dorsal identity. This study raises a question over other putative reversals in animals and plants, which also may be cryptic innovations. developmental gene | floral evolution | CYCLOIDEA | Leguminosae (Fabaceae)