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Summary Arguably, symbiotic arbuscular mycorrhizal (AM) fungi have the broadest host range of all fungi, being able to intracellularly colonise root cells in the vast majority of all land plants. This raises the question how AM fungi effectively deal with the immune systems of such a widely diverse range of plants.Here, we studied the role of a nuclear‐localisation signal‐containing effector from Rhizophagus irregularis, called Nuclear Localised Effector1 (RiNLE1), that is highly and specifically expressed in arbuscules.We showed that RiNLE1 is able to translocate to the host nucleus where it interacts with the plant core nucleosome protein histone 2B (H2B). RiNLE1 is able to impair the mono‐ubiquitination of H2B, which results in the suppression of defence‐related gene expression and enhanced colonisation levels.This study highlights a novel mechanism by which AM fungi can effectively control plant epigenetic modifications through direct interaction with a core nucleosome component. Homologues of RiNLE1 are found in a range of fungi that establish intimate interactions with plants, suggesting that this type of effector may be more widely recruited to manipulate host defence responses.

To acquire sufficient mineral nutrients such as phosphate (Pi) from the soil, most plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. Attracted by plant-secreted strigolactones (SLs), the fungi colonize the roots and form highly branched hyphal structures called arbuscules inside inner cortex cells. The host plant must control the different steps of this interaction to maintain its symbiotic nature. However, how plants sense the amount of Pi obtained from the fungus, and how this determines the arbuscule lifespan, are far from understood. Here, we show that Medicago truncatula SPX-domain containing proteins SPX1 and SPX3 regulate root Pi starvation responses, in part by interacting with PHOSPHATE RESPONSE REGULATOR2, as well as fungal colonization and arbuscule degradation. SPX1 and SPX3 are induced upon Pi starvation but become more restricted to arbuscule-containing cells upon the establishment of symbiosis. This induction in arbuscule-containing cells is associated with the presence of cis-regulatory AW-boxes and transcriptional regulation by the WRINKLED1-like transcription factor WRI5a. Under Pi-limiting conditions, SPX1 and SPX3 facilitate the expression of the SL biosynthesis gene DWARF27, which could help explain the increased fungal branching in response to root exudates. Later, in arbuscule-containing cells, SPX1 and SPX3 redundantly control arbuscule degradation. Thus, SPX proteins play important roles as phosphate sensors to maintain a beneficial AM symbiosis., In Medicago, two phosphate-sensing SPX proteins control phosphate homeostasis and the degradation of arbuscules to ensure a beneficial mycorrhizal 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.

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.

Summary NIN (NODULE INCEPTION) is a transcription factor that plays a key role during root nodule initiation. However, its role in later nodule developmental stages is unclear.Both NIN mRNA and protein accumulated at the highest level in the proximal part of the infection zone in Medicago truncatula nodules. Two nin weak allele mutants, nin‐13/16, form a rather normal nodule infection zone, whereas a fixation zone is not formed. Instead, a zone with defence responses and premature senescence occurred and symbiosome development gets arrested.Mutations in nin‐13/16 resulted in a truncated NIN lacking the conserved PB1 domain. However, this did not cause the nodule phenotype as nin mutants expressing NINΔPB1 formed wild‐type‐like nodule. The phenotype is likely to be caused by reduced NIN mRNA levels in the cytoplasm. Transcriptome analyses of nin‐16 nodules showed that expression levels of defence/senescence‐related genes are markedly increased, whereas the levels of defence suppressing genes are reduced. Although defence/senescence seems well suppressed in the infection zone, the transcriptome is already markedly changed in the proximal part of infection zone.In addition to its function in infection and nodule organogenesis, NIN also plays a major role at the transition from infection to fixation zone in establishing a functional symbiosis.

Legume and rhizobium species can establish a nitrogen-fixing nodule symbiosis. Previous studies have shown that several transcription factors that play a role in (lateral) root development are also involved in nodule development. Chromatin remodeling factors, like transcription factors, are key players in regulating gene expression. However, studies have not investigated whether chromatin remodeling genes that are essential for root development are also involved in nodule development. Here, we studied the role of Medicago (Medicago truncatula) histone deacetylases (MtHDTs) in nodule development. Arabidopsis (Arabidopsis thaliana) orthologs of HDTs have been shown to play a role in root development. MtHDT expression is induced in nodule primordia and is maintained in the nodule meristem and infection zone. Conditional, nodule-specific knockdown of MtHDT expression by RNAi blocks nodule primordium development. A few nodules may still form, but their nodule meristems are smaller, and rhizobial colonization of the cells derived from the meristem is markedly reduced. Although the HDTs are expressed during nodule and root development, transcriptome analyses indicate that HDTs control the development of each organ in a different manner. During nodule development, the MtHDTs positively regulate 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 (MtHMGR1). Decreased expression of MtHMGR1 is sufficient to explain the inhibition of primordium formation.

Legume and rhizobium can establish a nitrogen-fixing nodule symbiosis. Previous studies have shown that several transcription factors that play a role in (lateral) root development are also involved in nodule development. Chromatin remodelling factors, like transcription factors, are key players in regulating gene expression. However, it has not been studied whether chromatin remodelling genes that are essential for root development get involved in nodule development. Here we studied the role of Medicago histone deacetylases (MtHDTs) in nodule development. Their Arabidopsis orthologs have been shown to play a role in root development. The expression ofMtHDTsis induced in nodule primordia and is maintained in nodule meristem and infection zone. Conditional knock-down of their expression in a nodule-specific way by RNAi blocks nodule primordium development. A few nodules still can be formed but their nodule meristems are smaller and rhizobial colonization of the cells derived from the meristem is markedly reduced. Although the HDTs are expressed during nodule and root development, transcriptome analyses indicate that HDTs control the development of these organs in a different manner. During nodule development the MtHDTs positively regulate3-hydroxy-3-methylglutaryl coenzyme a reductase 1(MtHMGR1). The decreased expression ofMtHMGR1is sufficient to explain the block of primordium formation.ONE SENTENCE SUMMARYPlant-specific histone deacetylases regulate the expression of3-hydroxy-3-methylglutaryl-coenzyme A reductasesto control root nodule development.

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.

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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.

Interactions between plants and the soil’s microbial & fungal flora are crucial for the health of soil ecosystems and food production. Microbe-plant interactions are difficult to investigate in situ due to their intertwined relationship involving morphology and metabolism. Here, we describe an approach to overcome this challenge by elucidating morphology and the metabolic profile of Medicago truncatula root nodules using Magnetic Resonance (MR) Microscopy, at the highest magnetic field strength (22.3 T) currently available for imaging. A home-built solenoid RF coil with an inner diameter of 1.5 mm was used to study individual root nodules. A 3D imaging sequence with an isotropic resolution of (7 μm)3 was able to resolve individual cells, and distinguish between cells infected with rhizobia and uninfected cells. Furthermore, we studied the metabolic profile of cells in different sections of the root nodule using localised MR spectroscopy and showed that several metabolites, including betaine, asparagine/aspartate and choline, have different concentrations across nodule zones. The metabolite spatial distribution was visualised using chemical shift imaging. Finally, we describe the technical challenges and outlook towards future in vivo MR microscopy of nodules and the plant root system.

Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume‐rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF‐Y) complex. It is hypothesized that nodulation has a single evolutionary origin ~ 110 million years ago, followed by many independent losses. Despite a significant body of knowledge of the legume‐rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF‐YA genes in rhizobium nodulation in a non‐legume system. Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed in nodules. By analyzing single, double and higher‐order CRISPR‐Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF‐YA1 are PanNIN‐dependent and that PanNF‐YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF‐YA1 commit conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF‐YA1 represent core transcriptional regulators in this symbiosis.

International audience; Arbuscular mycorrhizal (AM) fungi greatly improve mineral uptake by host plants in nutrient-depleted soil and can intracellularly colonize root cortex cells in the vast majority of higher plants. However, AM fungi possess common fungal cell wall components such as chitin that can be recognized by plant chitin receptors to trigger immune responses, raising the question as to how AM fungi effectively evade chitin-triggered immune responses during symbiosis. In this study, we characterize a secreted lysin motif (LysM) effector identified from the model AM fungal species Rhizophagus irregularis, called RiSLM. RiSLM is one of the highest expressed effector proteins in intraradical mycelium during the symbiosis. In vitro binding assays show that RiSLM binds chitin-oligosaccharides and can protect fungal cell walls from chitinases. Moreover, RiSLM efficiently interferes with chitin-triggered immune responses, such as defence gene induction and reactive oxygen species production in Medicago truncatula. Although RiSLM also binds to symbiotic (lipo)chitooligosaccharides it does not interfere significantly with symbiotic signalling in Medicago. Host-induced gene silencing of RiSLM greatly reduces fungal colonization levels. Taken together, our results reveal a key role for AM fungal LysM effectors to subvert chitin-triggered immunity in symbiosis, pointing to a common role for LysM effectors in both symbiotic and pathogenic fungi.

How cells control the proper delivery of vesicles and their associated cargo to specific plasma membrane (PM) domains upon internal or external cues is a major question in plant cell biology. A widely held hypothesis is that expansion of plant exocytotic machinery components, such as SNARE proteins, has led to a diversification of exocytotic membrane trafficking pathways to function in specific biological processes. A key biological process that involves the creation of a specialized PM domain is the formation of a host–microbe interface (the peri-arbuscular membrane) in the symbiosis with arbuscular mycorrhizal fungi. We have previously shown that the ability to intracellularly host AM fungi correlates with the evolutionary expansion of both v- (VAMP721d/e) and t-SNARE (SYP132α) proteins, that are essential for arbuscule formation in Medicago truncatula. Here we studied to what extent the symbiotic SNAREs are different from their non-symbiotic family members and whether symbiotic SNAREs define a distinct symbiotic membrane trafficking pathway. We show that all tested SYP1 family proteins, and most of the non-symbiotic VAMP72 members, are able to complement the defect in arbuscule formation upon knock-down/-out of their symbiotic counterparts when expressed at sufficient levels. This functional redundancy is in line with the ability of all tested v- and t-SNARE combinations to form SNARE complexes. Interestingly, the symbiotic t-SNARE SYP132α appeared to occur less in complex with v-SNAREs compared to the non-symbiotic syntaxins in arbuscule-containing cells. This correlated with a preferential localization of SYP132α to functional branches of partially collapsing arbuscules, while non-symbiotic syntaxins accumulate at the degrading parts. Overexpression of VAMP721e caused a shift in SYP132α localization toward the degrading parts, suggesting an influence on its endocytic turn-over. These data indicate that the symbiotic SNAREs do not selectively interact to define a symbiotic vesicle trafficking pathway, but that symbiotic SNARE complexes are more rapidly disassembled resulting in a preferential localization of SYP132α at functional arbuscule branches.

Nitrogen-fixing root nodule symbiosis can sustain the development of the host plants under nitrogen-limiting conditions. Such symbiosis occurs only in a clade of angiosperms known as the nitrogen-fixing clade (NFC). It has long been proposed that root nodule symbiosis evolved several times (in parallel) in the NFC. Two recent phylogenomic studies compared the genomes of nodulating and related non-nodulating species across the four orders of the NFC and found that genes essential for nodule formation are lost or pseudogenized in the non-nodulating species. As these symbiosis genes are specifically involved in the symbiotic interaction, it means that the presence of pseudogenes and the loss of symbiosis genes strongly suggest that their ancestor, which still had functional genes, most likely had a symbiosis with nitrogen-fixing bacteria. These findings agree with the hypothesis that nodulation evolved once at the common ancestor of the NFC, and challenge the hypothesis of parallel evolution. In this chapter, we will cover the current understandings on actinorhizal-type and legume nodule development, and discuss the evolution of the legume nodule type.

Legumes and actinorhizal plants are capable of forming root nodules symbiosis with rhizobia and Frankia bacteria. All these nodulating species belong to the nitrogen fixation clade. Most likely, nodulation evolved once in the last common ancestor of this clade. NIN (NODULE INCEPTION) is a transcription factor that is essential for nodulation in all studied species. Therefore, it seems probable that it was recruited at the start when nodulation evolved. NIN is the founding member of the NIN-like protein (NLP) family. It arose by duplication, and this occurred before nodulation evolved. Therefore, several plant species outside the nitrogen fixation clade have NLP(s), which is orthologous to NIN. In this review, we discuss how NIN has diverged from the ancestral NLP, what minimal changes would have been essential for it to become a key transcription controlling nodulation, and which adaptations might have evolved later.

A comparative transcriptome analysis of Poncirus trifoliata and four herbaceous model plants identifies a core set of arbuscular mycorrhiza-induced genes that are likely to play key roles in 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.

Some plant species establish mutualistic cooperation with nitrogen-fixing bacteria to overcome nitrogen shortage. The interaction between legume plants and rhizobial bacteria is the canonical example of such mutualism. It leads to the formation of root nodules, which provide the environment for the bacteria to convert atmospheric dinitrogen into ammonia. Nodule formation is initiated by rhizobium-secreted compounds called nodulation (Nod) factors. Perception of Nod factors by receptors in the plant root cells initiates nodule organogenesis and is essential for bacterial infection (1). Several legumes evolved a narrow host range such that only one or a few rhizobium species can initiate nodulation (2). This specificity is largely determined by the Nod factor receptors. On page 663 of this issue, Bozsoki et al. (3) provide structural characterization of the binding site of a Nod factor receptor in legumes. The structural basis of rhizobial Nod factor recognition is a key to understanding the evolution of specificity in symbioses.

BackgroundThe Chinese chestnut (Castanea mollissima) is widely cultivated in China for nut production. This plant also plays an important ecological role in afforestation and ecosystem services. To facilitate and expand the use of C. mollissima for breeding and its genetic improvement, we report here the whole-genome sequence of C. mollissima.FindingsWe produced a high-quality assembly of the C. mollissima genome using Pacific Biosciences single-molecule sequencing. The final draft genome is ∼785.53 Mb long, with a contig N50 size of 944 kb, and we further annotated 36,479 protein-coding genes in the genome. Phylogenetic analysis showed that C. mollissima diverged from Quercus robur, a member of the Fagaceae family, ∼13.62 million years ago.ConclusionsThe high-quality whole-genome assembly of C. mollissima will be a valuable resource for further genetic improvement and breeding for disease resistance and nut quality.

Background Legumes can utilize atmospheric nitrogen by hosting nitrogen-fixing bacteria in special lateral root organs, called nodules. Legume nodules have a unique ontology, despite similarities in the gene networks controlling nodule and lateral root development. It has been shown that Medicago truncatula NODULE ROOT1 (MtNOOT1) is required for the maintenance of nodule identity, preventing the conversion to lateral root development. MtNOOT1 and its orthologs in other plant species -collectively called the NOOT-BOP-COCH-LIKE (NBCL) family- specify boundary formation in various aerial organs. However, MtNOOT1 is not only expressed in nodules and aerial organs, but also in developing roots, where its function remains elusive. Results We show that Mtnoot1 mutant seedlings display accelerated root elongation due to an enlarged root apical meristem. Also, Mtnoot1 mutant roots are thinner than wild-type and are delayed in xylem cell differentiation. We provide molecular evidence that the affected spatial development of Mtnoot1 mutant roots correlates with delayed induction of genes involved in xylem cell differentiation. This coincides with a basipetal shift of the root zone that is susceptible to rhizobium-secreted symbiotic signal molecules. Conclusions Our data show that MtNOOT1 regulates the size of the root apical meristem and vascular differentiation. Our data demonstrate that MtNOOT1 not only functions as a homeotic gene in nodule development but also coordinates the spatial development of the root.

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. Further, also the endodermis can be reprogrammed into stem cells in some species.

Plants form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which facilitates the acquisition of scarce minerals from the soil. In return, the host plants provide sugars and lipids to its fungal partner. However, the mechanism by which the AM fungi obtain sugars from the plant has remained elusive. In this study we investigated the role of potential SWEET family sugar exporters in AM symbiosis in Medicago truncatula. We show that M. truncatula SWEET1b transporter is strongly upregulated in arbuscule-containing cells compared to roots and localizes to the peri-arbuscular membrane, across which nutrient exchange takes place. Heterologous expression of MtSWEET1b in a yeast hexose transport mutant showed that it mainly transports glucose. Overexpression of MtSWEET1b in M. truncatula roots promoted the growth of intraradical mycelium during AM symbiosis. Surprisingly, two independent Mtsweet1b mutants, which are predicted to produce truncated protein variants impaired in glucose transport, exhibited no significant defects in AM symbiosis. However, arbuscule-specific overexpression of MtSWEET1bY57A/G58D , which are considered to act in a dominant-negative manner, resulted in enhanced collapse of arbuscules. Taken together, our results reveal a (redundant) role for MtSWEET1b in the transport of glucose across the peri-arbuscular membrane to maintain arbuscules for a healthy mutually beneficial symbiosis.

Arbuscular mycorrhizal (AM) fungi and rhizobium bacteria are accommodated in specialized membrane compartments that form a host-microbe interface. To better understand how these interfaces are made, we studied the regulation of exocytosis during interface formation. We used a phylogenetic approach to identify target soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) that are dedicated to symbiosis and used cell-specific expression analysis together with protein localization to identify t-SNAREs that are present on the host-microbe interface in Medicago truncatula. We investigated the role of these t-SNAREs during the formation of a host-microbe interface. We showed that multiple syntaxins are present on the peri-arbuscular membrane. From these, we identified SYNTAXIN OF PLANTS 13II (SYP13II) as a t-SNARE that is essential for the formation of a stable symbiotic interface in both AM and rhizobium symbiosis. In most dicot plants, the SYP13II transcript is alternatively spliced, resulting in two isoforms, SYP13IIα and SYP13IIβ. These splice-forms differentially mark functional and degrading arbuscule branches. Our results show that vesicle traffic to the symbiotic interface is specialized and required for its maintenance. Alternative splicing of SYP13II allows plants to replace a t-SNARE involved in traffic to the plasma membrane with a t-SNARE that is more stringent in its localization to functional arbuscules.

In root nodules rhizobia enter host cells via infection threads. The release of bacteria to a host cell is possible from cell wall-free regions of the infection thread. We hypothesized that the VAMP721d and VAMP721e exocytotic pathway, identified before in Medicago truncatula, has a role in the local modification of cell wall during the release of rhizobia. To clarify the role of VAMP721d and VAMP721e we used Glycine max, a plant with a determinate type of nodule. The localization of the main polysaccharide compounds of primary cell walls was analysed in control vs nodules with partially silenced GmVAMP721d. The silencing of GmVAMP721d blocked the release of rhizobia. Instead of rhizobia-containing membrane compartments - symbiosomes - the infected cells contained big clusters of bacteria embedded in a matrix of methyl-esterified and de-methyl-esterified pectin. These clusters were surrounded by a membrane. We found that GmVAMP721d-positive vesicles were not transporting methyl-esterified pectin. We hypothesized that they may deliver the enzymes involved in pectin turnover. Subsequently, we found that GmVAMP721d is partly co-localized with pectate lyase. Therefore, the biological role of VAMP721d may be explained by its action in delivering pectin-modifying enzymes to the site of release.

Significance Fixed nitrogen is essential for plant growth. Some plants, such as legumes, can host nitrogen-fixing bacteria within cells in root organs called nodules. Nodules are considered to have evolved in parallel in different lineages, but the genetic changes underlying this evolution remain unknown. Based on gene expression in the nitrogen-fixing nonlegume Parasponia andersonii and the legume Medicago truncatula, we find that nodules in these different lineages may share a single origin. Comparison of the genomes of Parasponia with those of related nonnodulating plants reveals evidence of parallel loss of genes that, in legumes, are essential for nodulation. Taken together, this raises the possibility that nodulation originated only once and was subsequently lost in many descendant lineages., 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.

Background Diazotrophic Bradyrhizobium spp. are well known for their ability to trigger nodule formation on a variety of legume species. In nodules, Bradyrhizobium utilizes plant-derived carbohydrates in exchange for fixed nitrogen. The genes essential for the nodulation and nitrogen-fixation trait are clustered in a genomic region, which is known as the ‘symbiotic island’. Recently, novel non-diazotrophic Bradyrhizobium spp. have been found to be highly abundant in soils, suggesting that these species can also have a ‘free-living’ life history. However, whether non-diazotrophic Bradyrhizobium spp. can live in association with plants remains elusive. Results In this study, we show that Bradyrhizobium spp. are common root endophytes of non-legume plant species – including Arabidopsis thaliana (Arabidopsis) – grown in an ecological setting. From a single Arabidopsis root, four Bradyrhizobium sp. strains (designated MOS001 to MOS004) were isolated. Comparative genome analysis revealed that these strains were genetically and functionally highly diverse, but did not harbour the nodulation and the nitrogen fixation gene clusters. Comparative colonization experiments, with MOS strains and nitrogen-fixing symbiotic strains, revealed that all tested Bradyrhizobium spp. can colonize the root endophytic compartment of Arabidopsis. Conclusion This study provides evidence that both diazotrophic and non-diazotrophic Bradyrhizobium spp. colonize the root endophytic compartment of a wide variety of plant species, including the model species Arabidopsis. This demonstrates that plant roots form a major ecological niche for Bradyrhizobium spp., which might be ancestral to the evolution of the nodulation and nitrogen-fixation trait in this genus. Electronic supplementary material The online version of this article (10.1186/s12870-018-1272-y) contains supplementary material, which is available to authorized users.

Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.

Arbuscular mycorrhizal fungi form the most wide-spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host-dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host- and stage-dependent manner to contribute to their extremely wide host range. We investigated the expression of SP-encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA-seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host-dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.

Ochrobactrum intermedium strain SA148 is a plant growth-promoting bacterium isolated from sandy soil in the Jizan area of Saudi Arabia. Here, we report the 4.9-Mb draft genome sequence of this strain, highlighting different pathways characteristic of plant growth promotion activity and environmental adaptation of SA148.

Symbiotic bacteria (rhizobia) are maintained and conditioned to fix atmospheric nitrogen in infected cells of legume root nodules. Rhizobia are confined to the asymmetrical protrusions of plasma membrane (PM): infection threads, cell wall-free unwalled droplets and symbiosomes. These compartments rapidly increase in surface and volume due to the microsymbiont expansion, and, remarkably, the membrane resources of the host cells are targeted to interface membrane quite precisely. We hypothesized that the change in the membrane tension around the expanding microsymbionts creates a vector for membrane traffic toward the symbiotic interface. To test this hypothesis, we selected calcium sensors from the group of synaptotagmins: MtSyt1, Medicago truncatula homolog of AtSYT1 from Arabidopsis thaliana known to be involved in membrane repair, and two other homologs expressed in root nodules: MtSyt2 and MtSyt3. Here we show that MtSyt1, MtSyt2 and MtSyt3 are expressed in the expanding cells of the meristem, zone of infection and proximal cell layers of zone of nitrogen fixation (MtSyt1, MtSyt3). All three GFP-tagged proteins delineate the interface membrane of infection threads and unwalled droplets and create a subcompartments of PM surrounding these structures. The localization of MtSyt1 by EM immunogold labelling has shown the signal on symbiosome membrane and endoplasmic reticulum (ER). To specify the role of synaptotagmins in interface membrane formation, we compared the localization of MtSyt1, MtSyt3 and exocyst subunit EXO70i, involved in the tethering of post-Golgi secretory vesicles and operational in tip growth. The localization of EXO70i in root nodules and arbusculated roots was strictly associated with the tips of infection threads and the tips of arbuscular fine branches, but the distribution of synaptotagmins on membrane subcompartments was broader and includes lateral parts of infection threads, the membrane of unwalled droplets as well as the symbiosomes. The double silencing of synaptotagmins caused a delay in rhizobia release and blocks symbiosome maturation confirming the functional role of synaptotagmins. In conclusion: synaptotagmin-dependent membrane fusion along with tip-targeted exocytosis is operational in the formation of symbiotic interface. Keywords: symbiosis, synaptotagmi1, membrane repair, interface membrane, root nodule, arbuscular mycorrhiza, Medicago truncatula

Enterobacter sp. Sa187 is a plant endophytic bacterium, isolated from root nodules of the desert plant Indigofera argentea , collected from the Jizan region of Saudi Arabia. Here, we report the genome sequence of Sa187, highlighting several genes involved in plant growth–promoting activity and environmental adaption.

Acinetobacter radioresistens strain SA188 is a plant endophytic bacterium, isolated from root nodules of the desert plants Indigofera spp., collected in Jizan, Saudi Arabia. Here, we report the 3.2-Mb draft genome sequence of strain SA188, highlighting characteristic pathways for plant growth–promoting activity and environmental adaptation.

The legume–rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant–bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix−) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts’ differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.

Background Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level. Results Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase. Conclusions By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1261-8) contains supplementary material, which is available to authorized users.

In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria.

Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.

Pseudomonas argentinensis strain SA190 is a plant endophytic-inhabiting bacterium that was isolated from root nodules of the desert plant Indigofera argentea collected from the Jizan region of Saudi Arabia. Here, we report the genome sequence of SA190, highlighting several functional genes related to plant growth–promoting activity, environment adaption, and antifungal activity.

International audience; Life in arid regions and, in particular, hot deserts is often limited due to their harsh environmental conditions, such as large temperature fluctuations and low amounts of water. These extreme environments can influence the microbial community present on the surface sands and any rhizosphere members surrounding desert plant roots. The Jizan desert area, located in Saudi Arabia, supports particular vegetation that grows in the large sandy flat terrain. We examined five different samples, four from the rhizosphere of pioneer plants plus a surface sand sample, and used pyrosequencing of PCR-amplified V1-V3 regions of 16S rDNA genes from total extracted DNA to reveal and compare the bacterial population diversity of the samples. The results showed a total of 3,530 OTUs in the five samples, calculated using ≥ 97% sequence similarity levels. The Chao1 estimation of the bacterial diversity fluctuated from 637 to 2,026 OTUs for a given sample. The most abundant members found in the samples belong to the Bacteroidetes, Firmicutes and Proteobacteria phyla. This work shows that the Jizan desert area of Saudi Arabia can contain a diverse bacterial community on the sand and surrounding the roots of pioneer desert plants. It also shows that desert sand microbiomes can vary depending on conditions, with broad implications for sandstone monument bacterial communities

Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the At CHR12/ 23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato ( Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated Sl CHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of Sl CHR1 show reduced growth in all developmental stages of tomato. This confirms that Sl CHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non- GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value.

Parasponia trees are the only non-legume species that form nitrogen-fixing root nodules with rhizobium. Based on its taxonomic position in relation to legumes (Fabaceae), it is most likely that both lineages have gained this symbiotic capacity independently. Therefore, Parasponia forms a bridging species to understand the evolutionary constraints underlying this symbiosis. However, absence of key technologies to genetically modify Parasponia seriously impeded studies on these species. We employed Agrobacterium rhizogenes to create composite Parasponia andersonii plants that harbour transgenic roots. Here, we provide an optimized protocol to infect P. andersonii as well as its non-symbiotic sister species Trema tomentosa with A. rhizogenes. We show that the transformation efficiency is temperature dependent. Whereas the optimal growth temperature for both these species is 28 °C, the transformation is most efficient when co-cultivation with A. rhizogenes occurs at 21 °C. Using this optimized protocol up to 80 % transformation efficiency can be obtained. These robust transformation platforms will provide a strong tool to unravel the Parasponia–rhizobium symbiosis.

Chantier qualité GA; International audience; 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 fixation1. 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 species2. 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.

For over two decades now, it is known that the nodule symbiosis between legume plants and nitrogen fixing rhizobium bacteria is set in motion by the bacterial signal molecule named nodulation (Nod) factor.1 Upon Nod factor perception a signaling cascade is activated that is also essential for endomycorrhizal symbiosis (Fig. 1). This suggests that rhizobium co-opted the evolutionary far more ancient mycorrhizal signaling pathway in order to establish an endosymbiotic interaction with legumes.2 As arbuscular mycorrhizal fungi of the Glomeromycota phylum can establish a symbiosis with the vast majority of land plants, it is most probable that this signaling cascade is wide spread in the plant kingdom.3 However, Nod factor perception generally is considered to be unique to legumes. Two recent breakthroughs on the evolutionary origin of rhizobium Nod factor signaling demonstrate that this is not the case.4,5 The purification of Nod factor-like molecules excreted by the mycorrhizal fungus Glomus intraradices and the role of the LysM-type Nod factor receptor PaNFP in the non-legume Parasponia andersonii provide novel understanding on the evolution of rhizobial Nod factor signaling.

Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.

In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homolog FtsZ at mid-cell to form a cell division scaffold (the Z ring), followed by recruitment of the other divisome components. The current view of bacterial cell division control starts from the principle of negative checkpoints that prevent incorrect Z-ring positioning. Here we provide evidence of positive control of cell division during sporulation of Streptomyces, via the direct recruitment of FtsZ by the membrane-associated divisome component SsgB. In vitro studies demonstrated that SsgB promotes the polymerization of FtsZ. The interactions are shown in vivo by time-lapse imaging and Förster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM), and are corroborated independently via two-hybrid studies. As determined by fluorescence recovery after photobleaching (FRAP), the turnover of FtsZ protofilaments increased strongly at the time of Z-ring formation. The surprising positive control of Z-ring formation by SsgB implies the evolution of an entirely new way of Z-ring control, which may be explained by the absence of a mid-cell reference point in the long multinucleoid hyphae. In turn, the localization of SsgB is mediated through the orthologous SsgA, and premature expression of the latter is sufficient to directly activate multiple Z-ring formation and hyperdivision at early stages of the Streptomyces cell cycle.

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

Plants can establish intracellular interactions with symbiotic as well as pathogenic microbes. Such intracellular accommodation of microbes always involves the formation of a host membrane compartment--the interface between the cytoplasm of the host and the microbe. These are the so-called perimicrobial compartments. In this review we will focus on the rhizobial legume symbiosis in which the microbes are hosted in organelle-like compartments, which are named symbiosomes. The signaling events leading to infection and symbiosome formation are discussed. Further the role of the host cell endomembrane system in symbiosome formation is described and compared with the processes involved in arbuscule and haustorium formation during the interaction of plants and biotrophic fungi.

Rhizobium bacteria form N2-fixing organelles, called symbiosomes, inside the cells of legume root nodules. The bacteria are generally thought to enter the cells via an endocytosis-like process. To examine this, we studied the identity of symbiosomes in relation to the endocytic pathway. We show that in Medicago truncatula, the small GTPases Rab5 and Rab7 are endosomal membrane identity markers, marking different (partly overlapping) endosome populations. Although symbiosome formation is considered to be an endocytosis-like process, symbiosomes do not acquire Rab5 at any stage during their development, nor do they accept the trans-Golgi network identity marker SYP4, presumed to mark early endosomes in plants. By contrast, the endosomal marker Rab7 does occur on symbiosomes from an early stage of development when they have stopped dividing up to the senescence stage. However, the symbiosomes do not acquire vacuolar SNAREs (SYP22 and VTI11) until the onset of their senescence. By contrast, symbiosomes acquire the plasma membrane SNARE SYP132 from the start of symbiosome formation throughout their development. Therefore, symbiosomes appear to be locked in a unique SYP132- and Rab7-positive endosome stage and the delay in acquiring (lytic) vacuolar identity (e.g., vacuolar SNAREs) most likely ensures their survival and maintenance as individual units.

SUMMARY A semi-automated method to quantify fluorescence intensity of objects in intact organs and tissues, composed of several cell layers, has been designed. The method has been developed on whole-mount propidium-iodide stained Arabidopsis thaliana (Arabidopsis) root tips, in which the DNA content of individual nuclei could be quantified. A diameter of less than 150 microm makes this organ most appropriate for whole-mount imaging. Further advantages are the lack of chlorophyll and transparent cell walls, with only a little background fluorescence. The method has a great advantage over flow cytometry, as the information regarding the positions of nuclei is maintained, and nuclei with aberrant DNA content can be re-assessed individually, which facilitates the efficient distinction between technical artefact and aberrant DNA content. Our averaging 3D method calculates the average of the summed fluorescence intensities of all sections of a nucleus and interpolates the missing sections, thereby allowing for the correction of detection problems. Furthermore, this method has the advantage of detecting objects in tissues covering multiple cell layers. The results of our method in Arabidopsis root tips showed that the quiescent centre cells, which rarely divide, are diploid, and are arrested in G1 or G0. Most stem cells, with the exception of those of the vascular tissue, are diploid cells, and their rather low division rate is caused by an elongated G1 phase. In contrast, the majority of the vascular stem cells are tetraploid.