RPG interacts with E3-ligase CERBERUS to mediate rhizobial infection in Lotus japonicus.

Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

CERBERUS is critical for stabilization of VAPYRIN during rhizobial infection in Lotus japonicus.

CERBERUS (also known as LIN) and VAPYRIN (VPY) are essential for infection of legumes by rhizobia and arbuscular mycorrhizal fungi (AMF). Medicago truncatula LIN (MtLIN) was reported to interact with MtVPY, but the significance of this interaction is unclear and the function of VPY in Lotus japonicus has not been studied. We demonstrate that CERBERUS has auto-ubiquitination activity in vitro and is localized within distinct motile puncta in L. japonicus root hairs and in Nicotiana benthamiana leaves. CERBERUS colocalized with the trans-Golgi network/early endosome markers. In L. japonicus, two VPY orthologs (LjVPY1 and LjVPY2) were identified. CERBERUS interacted with and colocalized with both LjVPY1 and LjVPY2. Co-expression of CERBERUS with LjVPY1 or LjVPY2 in N. benthamiana led to increased protein levels of LjVPY1 and LjVPY2, which accumulated as mobile punctate bodies in the cytoplasm. Conversely, LjVPY2 protein levels decreased in cerberus roots after rhizobial inoculation. Mutant analysis indicates that LjVPY1 and LjVPY2 are required for rhizobial infection and colonization by AMF. Our data suggest that CERBERUS stabilizes LjVPY1 and LjVPY2 within the trans-Golgi network/early endosome, where they might function to regulate endocytic trafficking and/or the formation or recycling of signaling complexes during rhizobial and AMF symbiosis.

Atypical Receptor Kinase RINRK1 Required for Rhizobial Infection But Not Nodule Development in Lotus japonicus.

During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 (Rhizobial Infection Receptor-like Kinase1), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception (NIN), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection.

Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides.

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

The rules of engagement in the legume-rhizobial symbiosis.

Rhizobial bacteria enter a symbiotic association with leguminous plants, resulting in differentiated bacteria enclosed in intracellular compartments called symbiosomes within nodules on the root. The nodules and associated symbiosomes are structured for efficient nitrogen fixation. Although the interaction is beneficial to both partners, it comes with rigid rules that are strictly enforced by the plant. Entry into root cells requires appropriate recognition of the rhizobial Nod factor signaling molecule, and this recognition activates a series of events, including polarized root-hair tip growth, invagination associated with bacterial infection, and the promotion of cell division in the cortex leading to the nodule meristem. The plant's command of the infection process has been highlighted by its enforcement of terminal differentiation upon the bacteria within nodules of some legumes, and this can result in a loss of bacterial viability while permitting effective nitrogen fixation. Here, we review the mechanisms by which the plant allows bacterial infection and promotes the formation of the nodule, as well as the details of how this intimate association plays out inside the cells of the nodule where a complex interchange of metabolites and regulatory peptides force the bacteria into a nitrogen-fixing organelle-like state.

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium, but not nitrogen-fixing rhizobial symbiosis.

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad-spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen-associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae-specific PRR ELONGATION FACTOR-THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF-Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF-Tu-derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR-Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.

Bacterial Biosensors for in Vivo Spatiotemporal Mapping of Root Secretion.

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea (Pisum sativum) rhizosphere, a suite of bioreporters has been developed in Rhizobium leguminosarum bv viciae strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial lux fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo. Using different bacterial mutants (nodC and nifH), the process of colonization and symbiosis has been analyzed, revealing compounds important in the different steps of the rhizobium-legume association. Dicarboxylates and sucrose are the main carbon sources within the nodules; in ineffective (nifH) nodules, particularly low levels of sucrose were observed, suggesting that plant sanctions affect carbon supply to nodules. In contrast, high myo-inositol levels were observed prior to nodule formation and also in nifH senescent nodules. Amino acid biosensors showed different patterns: a γ-aminobutyrate biosensor was active only inside nodules, whereas the phenylalanine bioreporter showed a high signal also in the rhizosphere. The bioreporters were further validated in vetch (Vicia hirsuta), producing similar results. In addition, vetch exhibited a local increase of nod gene-inducing flavonoids at sites where nodules developed subsequently. These bioreporters will be particularly helpful in understanding the dynamics of root exudation and the role of different molecules secreted into the rhizosphere.

MtLAX2, a Functional Homologue of the Arabidopsis Auxin Influx Transporter AUX1, Is Required for Nodule Organogenesis.

Most legume plants can form nodules, specialized lateral organs that form on roots, and house nitrogen-fixing bacteria collectively called rhizobia. The uptake of the phytohormone auxin into cells is known to be crucial for development of lateral roots. To test the role of auxin influx in nodulation we used the auxin influx inhibitors 1-naphthoxyacetic acid (1-NOA) and 2-NOA, which we found reduced nodulation of Medicago truncatula. This suggested the possible involvement of the AUX/LAX family of auxin influx transporters in nodulation. Gene expression studies identified MtLAX2, a paralogue of Arabidopsis (Arabidopsis thaliana) AUX1, as being induced at early stages of nodule development. MtLAX2 is expressed in nodule primordia, the vasculature of developing nodules, and at the apex of mature nodules. The MtLAX2 promoter contains several auxin response elements, and treatment with indole-acetic acid strongly induces MtLAX2 expression in roots. mtlax2 mutants displayed root phenotypes similar to Arabidopsis aux1 mutants, including altered root gravitropism, fewer lateral roots, shorter root hairs, and auxin resistance. In addition, the activity of the synthetic DR5-GUS auxin reporter was strongly reduced in mtlax2 roots. Following inoculation with rhizobia, mtlax2 roots developed fewer nodules, had decreased DR5-GUS activity associated with infection sites, and had decreased expression of the early auxin responsive gene ARF16a Our data indicate that MtLAX2 is a functional analog of Arabidopsis AUX1 and is required for the accumulation of auxin during nodule formation in tissues underlying sites of rhizobial infection.

SCARN a Novel Class of SCAR Protein That Is Required for Root-Hair Infection during Legume Nodulation.

Rhizobial infection of legume root hairs requires a rearrangement of the actin cytoskeleton to enable the establishment of plant-made infection structures called infection threads. In the SCAR/WAVE (Suppressor of cAMP receptor defect/WASP family verpolin homologous protein) actin regulatory complex, the conserved N-terminal domains of SCAR proteins interact with other components of the SCAR/WAVE complex. The conserved C-terminal domains of SCAR proteins bind to and activate the actin-related protein 2/3 (ARP2/3) complex, which can bind to actin filaments catalyzing new actin filament formation by nucleating actin branching. We have identified, SCARN (SCAR-Nodulation), a gene required for root hair infection of Lotus japonicus by Mesorhizobium loti. Although the SCARN protein is related to Arabidopsis thaliana SCAR2 and SCAR4, it belongs to a distinct legume-sub clade. We identified other SCARN-like proteins in legumes and phylogeny analyses suggested that SCARN may have arisen from a gene duplication and acquired specialized functions in root nodule symbiosis. Mutation of SCARN reduced formation of infection-threads and their extension into the root cortex and slightly reduced root-hair length. Surprisingly two of the scarn mutants showed constitutive branching of root hairs in uninoculated plants. However we observed no effect of scarn mutations on trichome development or on the early actin cytoskeletal accumulation that is normally seen in root hair tips shortly after M. loti inoculation, distinguishing them from other symbiosis mutations affecting actin nucleation. The C-terminal domain of SCARN binds to ARPC3 and ectopic expression of the N-terminal SCAR-homology domain (but not the full length protein) inhibited nodulation. In addition, we found that SCARN expression is enhanced by M. loti in epidermal cells and that this is directly regulated by the NODULE INCEPTION (NIN) transcription factor.

Manganese transport is essential for N2 -fixation by Rhizobium leguminosarum in bacteroids from galegoid but not phaseoloid nodules.

Rhizobium leguminosarum has two high-affinity Mn2+ transport systems encoded by sitABCD and mntH. In symbiosis, sitABCD and mntH were expressed throughout nodules and also strongly induced in Mn2+ -limited cultures of free-living cells. Growth of a sitA mntH double mutant was severely reduced under Mn2+ limitation and sitA and mntH single mutants were more sensitive to oxidative stress. The double sitA mntH mutant of R. leguminosarum was unable to fix nitrogen (Fix- ) with legumes belonging to the galegoid clade (Pisum sativum, Vicia faba and Vicia hirsuta). The presence of infection thread-like structures and sparsely-packed plant cells in nodules suggest that bacteroid development was blocked, either at a late stage of infection thread progression or during bacteroid-release. In contrast, a double sitA mntH mutant was Fix+ on common bean (Phaseoli vulgaris), a member of the phaseoloid clade of legumes, indicating a host-specific symbiotic requirement for Mn2+ transport.

The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus.

Bacterial accommodation inside living plant cells is restricted to the nitrogen-fixing root nodule symbiosis. In many legumes, bacterial uptake is mediated via tubular structures called infection threads (ITs). To identify plant genes required for successful symbiotic infection, we screened an ethyl methanesulfonate mutagenized population of Lotus japonicus for mutants defective in IT formation and cloned the responsible gene, ERN1, encoding an AP2/ERF transcription factor. We performed phenotypic analysis of two independent L. japonicus mutant alleles and investigated the regulation of ERN1 via transactivation and DNA-protein interaction assays. In ern1 mutant roots, nodule primordia formed, but most remained uninfected and bacterial entry via ITs into the root epidermis was abolished. Infected cortical nodule cells contained bacteroids, but transcellular ITs were rarely observed. A subset exhibited localized cell wall degradation and loss of cell integrity associated with bacteroid spread into neighbouring cells and the apoplast. Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells.

The root hair "infectome" of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for Auxin signaling in rhizobial infection.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula.

Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H+-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H+-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H+-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H+-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.

MgtE From Rhizobium leguminosarum Is a Mg²âº Channel Essential for Growth at Low pH and N2 Fixation on Specific Plants.

MgtE is predicted to be a Rhizobium leguminosarum channel and is essential for growth when both Mg²âº is limiting and the pH is low. N2was only fixed at 8% of the rate of wild type when the crop legume Pisum sativum was inoculated with an mgtE mutant of R. leguminosarum and, although bacteroids were present, they were few in number and not fully developed. R. leguminosarum MgtE was also essential for N2fixation on the native legume Vicia hirsuta but not when in symbiosis with Vicia faba. The importance of MgtE and the relevance of the contrasting phenotypes is discussed.

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots.

Lipopolysaccharide O-chain core region required for cellular cohesion and compaction of in vitro and root biofilms developed by Rhizobium leguminosarum.

The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.

Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.

Legume pectate lyase required for root infection by rhizobia.

To allow rhizobial infection of legume roots, plant cell walls must be locally degraded for plant-made infection threads (ITs) to be formed. Here we identify a Lotus japonicus nodulation pectate lyase gene (LjNPL), which is induced in roots and root hairs by rhizobial nodulation (Nod) factors via activation of the nodulation signaling pathway and the NIN transcription factor. Two Ljnpl mutants produced uninfected nodules and most infections arrested as infection foci in root hairs or roots. The few partially infected nodules that did form contained large abnormal infections. The purified LjNPL protein had pectate lyase activity, demonstrating that this activity is required for rhizobia to penetrate the cell wall and initiate formation of plant-made infection threads. Therefore, we conclude that legume-determined degradation of plant cell walls is required for root infection during initiation of the symbiotic interaction between rhizobia and legumes.