Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

The tomato cytochrome P450 CYP712G1 catalyses the double oxidation of orobanchol en route to the rhizosphere signalling strigolactone, solanacol.

Strigolactones (SLs) are rhizosphere signalling molecules and phytohormones. The biosynthetic pathway of SLs in tomato has been partially elucidated, but the structural diversity in tomato SLs predicts that additional biosynthetic steps are required. Here, root RNA-seq data and co-expression analysis were used for SL biosynthetic gene discovery. This strategy resulted in a candidate gene list containing several cytochrome P450s. Heterologous expression in Nicotiana benthamiana and yeast showed that one of these, CYP712G1, can catalyse the double oxidation of orobanchol, resulting in the formation of three didehydro-orobanchol (DDH) isomers. Virus-induced gene silencing and heterologous expression in yeast showed that one of these DDH isomers is converted to solanacol, one of the most abundant SLs in tomato root exudate. Protein modelling and substrate docking analysis suggest that hydroxy-orbanchol is the likely intermediate in the conversion from orobanchol to the DDH isomers. Phylogenetic analysis demonstrated the occurrence of CYP712G1 homologues in the Eudicots only, which fits with the reports on DDH isomers in that clade. Protein modelling and orobanchol docking of the putative tobacco CYP712G1 homologue suggest that it can convert orobanchol to similar DDH isomers as tomato.

Synthetic bacterial community derived from a desert rhizosphere confers salt stress resilience to tomato in the presence of a soil microbiome.

The root bacterial microbiome is important for the general health of the plant. Additionally, it can enhance tolerance to abiotic stresses, exemplified by plant species found in extreme ecological niches like deserts. These complex microbe-plant interactions can be simplified by constructing synthetic bacterial communities or SynComs from the root microbiome. Furthermore, SynComs can be applied as biocontrol agents to protect crops against abiotic stresses such as high salinity. However, there is little knowledge on the design of a SynCom that offers a consistent protection against salt stress for plants growing in a natural and, therefore, non-sterile soil which is more realistic to an agricultural setting. Here we show that a SynCom of five bacterial strains, originating from the root of the desert plant Indigofera argentea, protected tomato plants growing in a non-sterile substrate against a high salt stress. This phenotype correlated with the differential expression of salt stress related genes and ion accumulation in tomato. Quantification of the SynCom strains indicated a low penetrance into the natural soil used as the non-sterile substrate. Our results demonstrate how a desert microbiome could be engineered into a simplified SynCom that protected tomato plants growing in a natural soil against an abiotic stress.

Cannabis labelling is associated with genetic variation in terpene synthase genes.

Analysis of over 100 Cannabis samples quantified for terpene and cannabinoid content and genotyped for over 100,000 single nucleotide polymorphisms indicated that Sativa- and Indica-labelled samples were genetically indistinct on a genome-wide scale. Instead, we found that Cannabis labelling was associated with variation in a small number of terpenes whose concentrations are controlled by genetic variation at tandem arrays of terpene synthase genes.

Apple Ripening Is Controlled by a NAC Transcription Factor.

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of pre-harvest ripening and associated textural changes following harvest are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from ∼800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders (ACS1, ACO1, and PG1), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after 3 months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein that is orthogolous to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). We introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program.

Origin and Evolution of the Cannabinoid Oxidocyclase Gene Family.

Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects, we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species, we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic analyses, we propose a comprehensive classification of three main clades and seven subclades that are intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid phenotype is primarily determined by the presence/absence of single-copy genes. Although wild populations of Cannabis are still unknown, increased sampling of landraces and wild/feral populations across its native geographic range is likely to uncover additional cannabinoid oxidocyclase sequence variants.

A new P450 involved in the furanocoumarin pathway underlies a recent case of convergent evolution.

Furanocoumarins are phytoalexins often cited as an example to illustrate the arms race between plants and herbivorous insects. They are distributed in a limited number of phylogenetically distant plant lineages, but synthesized through a similar pathway, which raised the question of a unique or multiple emergence in higher plants. The furanocoumarin pathway was investigated in the fig tree (Ficus carica, Moraceae). Transcriptomic and metabolomic approaches led to the identification of CYP76F112, a cytochrome P450 catalyzing an original reaction. CYP76F112 emergence was inquired using phylogenetics combined with in silico modeling and site-directed mutagenesis. CYP76F112 was found to convert demethylsuberosin into marmesin with a very high affinity. This atypical cyclization reaction represents a key step within the polyphenol biosynthesis pathway. CYP76F112 evolutionary patterns suggests that the marmesin synthase activity appeared recently in the Moraceae family, through a lineage-specific expansion and diversification. The characterization of CYP76F112 as the first known marmesin synthase opens new prospects for the use of the furanocoumarin pathway. It also supports the multiple acquisition of furanocoumarin in angiosperms by convergent evolution, and opens new perspectives regarding the ability of cytochromes P450 to evolve new functions related to plant adaptation to their environment.

New Insights Into the Evolution of C4 Photosynthesis Offered by the Tarenaya Cluster of Cleomaceae.

Cleomaceae is closely related to Brassicaceae and includes C3, C3-C4, and C4 species. Thus, this family represents an interesting system for studying the evolution of the carbon concentrating mechanism. However, inadequate genetic information on Cleomaceae limits their research applications. Here, we characterized 22 Cleomaceae accessions [3 genera (Cleoserrata, Gynandropsis, and Tarenaya) and 11 species] in terms of genome size; molecular phylogeny; as well as anatomical, biochemical, and photosynthetic traits. We clustered the species into seven groups based on genome size. Interestingly, despite clear differences in genome size (2C, ranging from 0.55 to 1.3 pg) in Tarenaya spp., this variation was not consistent with phylogenetic grouping based on the internal transcribed spacer (ITS) marker, suggesting the occurrence of multiple polyploidy events within this genus. Moreover, only G. gynandra, which possesses a large nuclear genome, exhibited the C4 metabolism. Among the C3-like species, we observed intra- and interspecific variation in nuclear genome size as well as in biochemical, physiological, and anatomical traits. Furthermore, the C3-like species had increased venation density and bundle sheath cell size, compared to C4 species, which likely predisposed the former lineages to C4 photosynthesis. Accordingly, our findings demonstrate the potential of Cleomaceae, mainly members of Tarenaya, in offering novel insights into the evolution of C4 photosynthesis.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Quantitative comparison between the rhizosphere effect of Arabidopsis thaliana and co-occurring plant species with a longer life history.

As a model for genetic studies, Arabidopsis thaliana (Arabidopsis) offers great potential to unravel plant genome-related mechanisms that shape the root microbiome. However, the fugitive life history of this species might have evolved at the expense of investing in capacity to steer an extensive rhizosphere effect. To determine whether the rhizosphere effect of Arabidopsis is different from other plant species that have a less fugitive life history, we compared the root microbiome of Arabidopsis to eight other, later succession plant species from the same habitat. The study included molecular analysis of soil, rhizosphere, and endorhizosphere microbiome both from the field and from a laboratory experiment. Molecular analysis revealed that the rhizosphere effect (as quantified by the number of enriched and depleted bacterial taxa) was ~35% lower than the average of the other eight species. Nevertheless, there are numerous microbial taxa differentially abundant between soil and rhizosphere, and they represent for a large part the rhizosphere effects of the other plants. In the case of fungal taxa, the number of differentially abundant taxa in the Arabidopsis rhizosphere is 10% of the other species' average. In the plant endorhizosphere, which is generally more selective, the rhizosphere effect of Arabidopsis is comparable to other species, both for bacterial and fungal taxa. Taken together, our data imply that the rhizosphere effect of the Arabidopsis is smaller in the rhizosphere, but equal in the endorhizosphere when compared to plant species with a less fugitive life history.

A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny.

Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

Capturing variation in floral shape: a virtual3D based morphospace for Pelargonium.

BACKGROUND: Variation in floral shapes has long fascinated biologists and its modelling enables testing of evolutionary hypotheses. Recent comparative studies that explore floral shape have largely ignored 3D floral shape. We propose quantifying floral shape by using geometric morphometrics on a virtual3D model reconstructed from 2D photographical data and demonstrate its performance in capturing shape variation. METHODS: This approach offers unique benefits to complement established imaging techniques (i) by enabling adequate coverage of the potential morphospace of large and diverse flowering-plant clades; (ii) by circumventing asynchronicity in anthesis of different floral parts; and (iii) by incorporating variation in copy number of floral organs within structures. We demonstrate our approach by analysing 90 florally-diverse species of the Southern African genus Pelargonium (Geraniaceae). We quantify Pelargonium floral shapes using 117 landmarks and show similarities in reconstructed morphospaces for nectar tube, corolla (2D datasets), and a combined virtual3D dataset. RESULTS: Our results indicate that Pelargonium species differ in floral shape, which can also vary extensively within a species. PCA results of the reconstructed virtual3D floral models are highly congruent with the separate 2D morphospaces, indicating it is an accurate, virtual, representation of floral shape. Through our approach, we find that adding the third dimension to the data is crucial to accurately interpret the manner of, as well as levels of, shape variation in flowers.

Lateral root formation involving cell division in both pericycle, cortex and endodermis is a common and ancestral trait in seed plants.

Studies on the model plant Arabidopsis have led to the common view that lateral roots are exclusively formed from pericycle cells and that the latter are unique in their ability to be reprogrammed into stem cells. By analysing lateral root formation in an evolutionary context, we show that lateral root primordium formation in which cortex, endodermis and pericycle are mitotically activated, is a common and ancestral trait in seed plants, whereas the exclusive involvement of pericycle evolved in the Brassicaceae. Furthermore, the endodermis can also be reprogrammed into stem cells in some species.

GeneNoteBook, a collaborative notebook for comparative genomics.

SUMMARY: Analysis and comparison of genomic and transcriptomic datasets have become standard procedures in biological research. However, for non-model organisms no efficient tools exist to visually work with multiple genomes and their metadata, and to annotate such data in a collaborative way. Here we present GeneNoteBook: a web based collaborative notebook for comparative genomics. GeneNoteBook allows experimental and computational researchers to query, browse, visualize and curate bioinformatic analysis results for multiple genomes. GeneNoteBook is particularly suitable for the analysis of non-model organisms, as it allows for comparing newly sequenced genomes to those of model organisms. AVAILABILITY AND IMPLEMENTATION: GeneNoteBook is implemented as a node.js web application and depends on MongoDB and NCBI BLAST. Source code is available at https://github.com/genenotebook/genenotebook. Additionally, GeneNoteBook can be installed through Bioconda and as a Docker image. Full installation instructions and online documentation are available at https://genenotebook.github.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Remote cis-Regulatory Region Is Required for NIN Expression in the Pericycle to Initiate Nodule Primordium Formation in Medicago truncatula.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.

A Resurrected Scenario: Single Gain and Massive Loss of Nitrogen-Fixing Nodulation.

Root nodule endosymbiosis with nitrogen-fixing bacteria provides plants with unlimited access to fixed nitrogen, but at a significant energetic cost. Nodulation is generally considered to have originated in parallel in different lineages, but this hypothesis downplays the genetic complexity of nodulation and requires independent recruitment of many common features across lineages. Recent phylogenomic studies revealed that genes that function in establishing or maintaining nitrogen-fixing nodules are independently lost in non-nodulating relatives of nitrogen-fixing plants. In our opinion, these data are best explained by a scenario of a single gain followed by massively parallel loss of nitrogen-fixing root nodules triggered by events at geological scale.

Comparative Analysis of the Nodule Transcriptomes of Ceanothus thyrsiflorus (Rhamnaceae, Rosales) and Datisca glomerata (Datiscaceae, Cucurbitales).

Two types of nitrogen-fixing root nodule symbioses are known, rhizobial and actinorhizal symbioses. The latter involve plants of three orders, Fagales, Rosales, and Cucurbitales. To understand the diversity of plant symbiotic adaptation, we compared the nodule transcriptomes of Datisca glomerata (Datiscaceae, Cucurbitales) and Ceanothus thyrsiflorus (Rhamnaceae, Rosales); both species are nodulated by members of the uncultured Frankia clade, cluster II. The analysis focused on various features. In both species, the expression of orthologs of legume Nod factor receptor genes was elevated in nodules compared to roots. Since arginine has been postulated as export form of fixed nitrogen from symbiotic Frankia in nodules of D. glomerata, the question was whether the nitrogen metabolism was similar in nodules of C. thyrsiflorus. Analysis of the expression levels of key genes encoding enzymes involved in arginine metabolism revealed up-regulation of arginine catabolism, but no up-regulation of arginine biosynthesis, in nodules compared to roots of D. glomerata, while arginine degradation was not upregulated in nodules of C. thyrsiflorus. This new information corroborated an arginine-based metabolic exchange between host and microsymbiont for D. glomerata, but not for C. thyrsiflorus. Oxygen protection systems for nitrogenase differ dramatically between both species. Analysis of the antioxidant system suggested that the system in the nodules of D. glomerata leads to greater oxidative stress than the one in the nodules of C. thyrsiflorus, while no differences were found for the defense against nitrosative stress. However, induction of nitrite reductase in nodules of C. thyrsiflorus indicated that here, nitrite produced from nitric oxide had to be detoxified. Additional shared features were identified: genes encoding enzymes involved in thiamine biosynthesis were found to be upregulated in the nodules of both species. Orthologous nodule-specific subtilisin-like proteases that have been linked to the infection process in actinorhizal Fagales, were also upregulated in the nodules of D. glomerata and C. thyrsiflorus. Nodule-specific defensin genes known from actinorhizal Fagales and Cucurbitales, were also found in C. thyrsiflorus. In summary, the results underline the variability of nodule metabolism in different groups of symbiotic plants while pointing at conserved features involved in the infection process.

Comparative transcriptome analysis of Poncirus trifoliata identifies a core set of genes involved in arbuscular mycorrhizal symbiosis.

The perennial woody plants of citrus are one of the most important fruit crops in the world and largely depends on arbuscular mycorrhizal symbiosis (AMS) to obtain essential nutrients from soil. However, the molecular aspects of AMS in citrus and perennial woody plants in general have largely been understudied. We used RNA-sequencing to identify differentially expressed genes in roots of Poncirus trifoliata upon mycorrhization by the AM fungus Glomus versiforme and evaluated their conservation by comparative transcriptome analyses with four herbaceous model plants. We identified 282 differentially expressed genes in P. trifoliata, including orthologs of 21 genes with characterized roles in AMS and 83 genes that are considered to be conserved in AM-host plants. Comparative transcriptome analysis revealed a 'core set' of 156 genes from P. trifoliata whose orthologous genes from at least three of the five species also exhibited similar transcriptional changes during AMS. Functional analysis of one of these conserved AM-induced genes, a 3-keto-acyl-ACP reductase (FatG) involved in fatty acid biosynthesis, confirmed its involvement in AMS in Medicago truncatula. Our results identify a core transcriptional program for AMS that is largely conserved between P. trifoliata and other plants. The comparative transcriptomics approach adds to previous phylogenomics studies to identify conserved genes required for AMS.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

Draft Genome Sequence of the Nitrogen-Fixing Rhizobium sullae Type Strain IS123T Focusing on the Key Genes for Symbiosis with its Host Hedysarum coronarium L.

The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments.