Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement

Intracellular symbiosis is widespread in the insect world where it plays an important role in evolution and adaptation. The weevil family Dryophthoridae (Curculionoidea) is of particular interest in intracellular symbiosis evolution with regard to the great economical and ecological features of these invasive insects, and the potential for comparative studies across a wide range of host plants and environments. Here, we have analyzed the intracellular symbiotic bacteria of 19 Dryophthoridae species collected worldwide, representing a wide range of plant species and tissues. All except one (Sitophilus linearis) harbor symbiotic bacteria within specialized cells (the bacteriocytes) assembled as an organ, the bacteriome. Phylogenetic analysis of the 16S rDNA gene sequence of the Dryophthoridae endosymbionts revealed three endosymbiotic clades belonging to gamma3-Proteobacteria and characterized by different GC contents and evolutionary rate. The genus name Candidatus Nardonella was proposed for the ancestral clade infesting Dryophthoridae 100 MYA and represented by five of nine bacterial genera studied. For this clade showing low GC content (40.5% GC) and high evolutionary rate (0.128 substitutions/site per 100 Myr), a single infection and subsequent cospeciation of the host and the endosymbionts was observed. In the two other insect lineage endosymbionts, with relatively high GC content (53.4% and 53.8% GC), competition with ancestral pathogenic bacteria might have occurred, leading to endosymbiont replacement in present-day last insects.     

Assessment of bacterial endosymbiont diversity in Otiorhynchusspp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

Background

Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species.

Results

High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of “Candidatus Blochmannia” and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and “Candidatus Blochmannia”, we identified a new phylogenetic clade of Rickettsia as well as “Candidatus Nardonella” endosymbionts in Otiorhynchus spp. which are closely related to “Candidatus Blochmannia” bacteria.

Conclusions

Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and “Candidatus Nardonella” endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.

     

Assessment of bacterial endosymbiont diversity in <Emphasis Type="Italic">Otiorhynchus</Emphasis>spp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

BACKGROUND: Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species. RESULTS: High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of "Candidatus Blochmannia" and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and "Candidatus Blochmannia", we identified a new phylogenetic clade of Rickettsia as well as "Candidatus Nardonella" endosymbionts in Otiorhynchus spp. which are closely related to "Candidatus Blochmannia" bacteria. CONCLUSIONS: Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and "Candidatus Nardonella" endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.     

Wolbachia在我国广赤眼蜂种群内的感染

Wolbachia是广泛分布于节肢动物生殖组织内的一类细胞内共生菌,它属于原细菌的α亚类,能够通过调控寄主的生殖活动而促进其在寄主种群中的扩散。通过对wsp 基因的克隆及PCR-RFLP分析确定了Wolbachia在我国广赤眼蜂种群内的存在,并发现有2种Wolbachia 菌系的感染,命名为wEvaA和wEvaB。经过克隆分离得到了这2种Wolbachia的wsp基因序列,在GenBank的登录号为AY390279和 AY390280 ,并由基于wsp基因的聚类树中发现,这两种Wolbachia菌系均属于A组。     

Bakterien,die in Acanthamöben leben: Dasein im Verborgenen

One fourth of Acanthamoebaisolates studied contain obligate bacterial endosymbionts. These intracellular bacteria have recently been assigned to four different, previously unknown phylogenetic lineages within the Proteobacteriaand the Chlamydiales. The symbiotic association of these amoebae and their bacterial endosymbionts might be a valuable model system for the analysis of bacterial adaptations and mechanisms for intracellular survival. In addition, Chlamydia‐related amoebal endosymbionts have been implicated as causative agents for respiratory disease suggesting that these protozoa may be sources of new emerging pathogens.     

Host and symbiont intraspecific variability: The case of Paramecium calkinsi and “Candidatus Trichorickettsia mobilis”

Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species “Candidatus Trichorickettsia mobilis” (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split “Candidatus Trichorickettsia” into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed “Candidatus Trichorickettsia” hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture.     

Identification,origin, and evolution of leaf nodulating symbionts of <Emphasis Type="Italic">Sericanthe</Emphasis> (Rubiaceae)

Bacterial leaf symbiosis is an intimate association between bacteria and plants in which endosymbionts are housed within leaf nodules. This phenomenon has been reported in three genera of Rubiaceae (Pavetta, Psychotria, and Sericanthe), but the bacterial partner has only been identified in Psychotria and Pavetta. Here we report the identification of symbiotic bacteria in two leaf nodulating Sericanthe species. Using 16S rRNA data and common housekeeping genetic markers (recA and gyrB) we studied the phylogenetic relationships of bacterial endosymbionts in Rubiaceae. Endosymbionts of leaf nodulating Rubiaceae were found to be closely related and were placed as a monophyletic group within the genus Burkholderia (β-Proteobacteria). The phylogenetic analyses revealed a pattern of strict host specificity and placed the two investigated endosymbionts at two distinct positions in the topology of the tree, suggesting at least two different evolutionary origins. The degree of sequence divergence between the Sericanthe endosymbionts and their relatives was large enough to propose the Sericanthe endosymbionts as new species (‘Candidatus Burkholderia andongensis’ and ‘Candidatus Burkholderia petitii’). In a second part of this study, the pylogenetic relationships among nodulating and non-nodulating Sericanthe species were investigated using sequence data from six chloroplast regions (rps16, trnG, trnL-trnF, petD, petA-psbJ, and atpI-atpH). Overall, genetic variation among the plastid markers was insufficient to enable phylogenetic estimation. However, our results could not rule out the possibility that bacterial leaf symbiosis originated once in a common ancestor of the Sericanthe species.     

Symbiosis in the green leafhopper,Cicadella viridis (Hemiptera,Cicadellidae). Association in statu nascendi?

The green leafhopper, Cicadella viridis lives in symbiotic association with microorganisms. The ultrastructural and molecular analyses have shown that in the body of the C. viridis two types of bacteriocyte endosymbionts are present. An amplification and sequencing of 16S rRNA genes revealed that large, pleomorphic bacteria display a high similarity (94–100%) to the endosymbiont ‘Candidatus Sulcia muelleri’ (phylum Bacteroidetes), whereas long, rod-shaped microorganisms are closely related to the γ-proteobacterial symbiont Sodalis (97–99% similarity). Both endosymbionts may be harbored in their own bacteriocytes as well as may co-reside in the same bacteriocytes. The ultrastructural observations have revealed that the Sodalis-like bacteria harboring the same bacteriocytes as bacterium Sulcia may invade the cells of the latter. Bacteria Sulcia and Sodalis-like endosymbionts are transovarially transmitted from one generation to the next. However, Sodalis-like endosymbionts do not invade the ovaries individually, but only inside Sulcia cells. Apart from bacteriocyte endosymbionts, in the body of C. viridis small, rod-shaped bacteria have been detected, and have been identified as being closely related to γ-proteobacterial microorganism Pectobacterium (98–99% similarity). The latter are present in the sheath cells of the bacteriomes containing bacterium Sulcia as well as in fat body cells.     

Experimental evolution of nodule intracellular infection in legume symbionts

Soil bacteria known as rhizobia are able to establish an endosymbiosis with legumes that takes place in neoformed nodules in which intracellularly hosted bacteria fix nitrogen. Intracellular accommodation that facilitates nutrient exchange between the two partners and protects bacteria from plant defense reactions has been a major evolutionary step towards mutualism. Yet the forces that drove the selection of the late event of intracellular infection during rhizobium evolution are unknown. To address this question, we took advantage of the previous conversion of the plant pathogen Ralstonia solanacearum into a legume-nodulating bacterium that infected nodules only extracellularly. We experimentally evolved this draft rhizobium into intracellular endosymbionts using serial cycles of legume-bacterium cocultures. The three derived lineages rapidly gained intracellular infection capacity, revealing that the legume is a highly selective environment for the evolution of this trait. From genome resequencing, we identified in each lineage a mutation responsible for the extracellular–intracellular transition. All three mutations target virulence regulators, strongly suggesting that several virulence-associated functions interfere with intracellular infection. We provide evidence that the adaptive mutations were selected for their positive effect on nodulation. Moreover, we showed that inactivation of the type three secretion system of R. solanacearum that initially allowed the ancestral draft rhizobium to nodulate, was also required to permit intracellular infection, suggesting a similar checkpoint for bacterial invasion at the early nodulation/root infection and late nodule cell entry levels. We discuss our findings with respect to the spread and maintenance of intracellular infection in rhizobial lineages during evolutionary times.     

Closely coupled evolutionary history of ecto‐ and endosymbionts from two distantly related animal phyla

The level of integration between associated partners can range from ectosymbioses to extracellular and intracellular endosymbioses, and this range has been assumed to reflect a continuum from less intimate to evolutionarily highly stable associations. In this study, we examined the specificity and evolutionary history of marine symbioses in a group of closely related sulphur‐oxidizing bacteria, called Candidatus Thiosymbion, that have established ecto‐ and endosymbioses with two distantly related animal phyla, Nematoda and Annelida. Intriguingly, in the ectosymbiotic associations of stilbonematine nematodes, we observed a high degree of congruence between symbiont and host phylogenies, based on their ribosomal RNA (rRNA) genes. In contrast, for the endosymbioses of gutless phallodriline annelids (oligochaetes), we found only a weak congruence between symbiont and host phylogenies, based on analyses of symbiont 16S rRNA genes and six host genetic markers. The much higher degree of congruence between nematodes and their ectosymbionts compared to those of annelids and their endosymbionts was confirmed by cophylogenetic analyses. These revealed 15 significant codivergence events between stilbonematine nematodes and their ectosymbionts, but only one event between gutless phallodrilines and their endosymbionts. Phylogenetic analyses of 16S rRNA gene sequences from 50 Cand. Thiosymbion species revealed seven well‐supported clades that contained both stilbonematine ectosymbionts and phallodriline endosymbionts. This closely coupled evolutionary history of marine ecto‐ and endosymbionts suggests that switches between symbiotic lifestyles and between the two host phyla occurred multiple times during the evolution of the Cand. Thiosymbion clade, and highlights the remarkable flexibility of these symbiotic bacteria.     

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts

Acanthamoebae are ubiquitous free-living amoebae and important predators of microbial communities. They frequently contain obligate intracellular bacterial symbionts, which show a worldwide distribution. All Acanthamoeba spp. described so far harboured no or only a single specific endosymbiont phylotype, and in some cases evidence for coevolution between the symbiotic bacteria and the amoeba host has been reported. In this study we have isolated and characterized an Acanthamoeba sp. (strain OEW1) showing a stable symbiotic relationship with two morphologically different endosymbionts. 16S rRNA sequence analysis assigned these symbionts to the candidate genus Procabacter (Betaproteobacteria) and the genus Parachlamydia (Chlamydiae) respectively. Fluorescence in situ hybridization and transmission electron microscopy confirmed the affiliation of the endosymbionts and showed their co-occurrence in the amoeba host cells and their intracellular location within separate compartments enclosed by host-derived membranes. Further analysis of this stable relationship should provide novel insights into the complex interactions of intracellular multiple-partner associations.     

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the α-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.     

The Inductive Role of Bacterial Symbionts in the Morphogenesis of a Squid Light Organ

SYNOPSIS. The association of the sepiolid squid Euprymna scolopeswith its marine luminous bacterial symbiont Vibrio fischeriis an emerging model system to study the initiation and developmentof bacterial symbioses in higher animals, in particular theinfluence of bacteria on the ontogenic development of symbiotic-specifichost tissues. Experiments comparing the development of juvenilesquid infected with symbiotic V. fischeri with that of uninfectedjuveniles suggest postembryonic development of the light organrequires cell-cell interactions with the bacterial symbionts.The presence of symbiotic bacteria induces specific morphologicalchanges by affecting such fundamental processes as cell deathand cell differentiation. The surface of the juvenile organis largely composed of ciliated cells that appear to facilitateinfection of the light organ. These cells begin to undergo celldeath within hours of infection with symbiotic V. fischeri.Within three days the epithelial cells that form the bacteriacontainingcrypts of the light organ increase in size; these cells do notappear mitotically active, and may represent a terminally differentiatedstate. The light organs of uninfected juvenile E. scolopes,however, do not exhibit any of these early postembryonic developmentalevents but remain in a state of arrested morphogenesis.     

Bodo saltans (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple putative toxin-antitoxin systems

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.Subject terms: Microbial ecology, Bacteria     

Endosymbiotic bacteria as a source of carotenoids in whiteflies

Although carotenoids serve important biological functions, animals are generally unable to synthesize these pigments and instead obtain them from food. However, many animals, such as sap-feeding insects, may have limited access to carotenoids in their diet, and it was recently shown that aphids have acquired the ability to produce carotenoids by lateral transfer of fungal genes. Whiteflies also contain carotenoids but show no evidence of the fungus-derived genes found in aphids. Because many sap-feeding insects harbour intracellular bacteria, it has long been hypothesized that these endosymbionts could serve as an alternative source of carotenoid biosynthesis. We sequenced the genome of the obligate bacterial endosymbiont Portiera from the whitefly Bemisia tabaci. The genome exhibits typical signatures of obligate endosymbionts in sap-feeding insects, including extensive size reduction (358.2 kb) and enrichment for genes involved in essential amino acid biosynthesis. Unlike other sequenced insect endosymbionts, however, Portiera has bacterial homologues of the fungal carotenoid biosynthesis genes in aphids. Therefore, related lineages of sap-feeding insects appear to have convergently acquired the same functional trait by distinct evolutionary mechanisms—bacterial endosymbiosis versus fungal lateral gene transfer.     

Use of the Verrucomicrobia-Specific Probe EUB338-III and Fluorescent In Situ Hybridization for Detection of “Candidatus Xiphinematobacter” Cells in Nematode Hosts

Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium “Candidatus Xiphinematobacter” in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the host's molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.     

Evolutionary Relationships of “Candidatus Riesia spp.,” Endosymbiotic Enterobacteriaceae Living within Hematophagous Primate Lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium “Candidatus Arsenophonus insecticola.” We propose that two additional species be added to the genus “Candidatus Riesia.” The new species proposed within “Candidatus Riesia” have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.     

Identification of the Weevil immune genes and their expression in the bacteriome tissue

Background  

Persistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome).     

Structure of the dnaA region of the endosymbiont, Buchnera aphidicola, of aphid Schizaphis graminum

Buchnera aphidicola is an intracellular prokaryote (endosymbiont)that lives in the body cavity of the aphid. Phylogenetic studiesindicated that it is closely related to Escherichia coli andmembers of Enterobacteria. The gene order of the region containingthe dnaA gene is well conserved in many bacteria. Seven genesof the endosymbiont of the aphid Schizaphis graminum, gyrB,dnaN, dnaA, rpmH, rnpA, yidD, and 60K, were found to be homologousin sequence and relative location to those of E. coli. We havefurther sequenced the region downstream of the 60K gene to elucidatethe boundary of the conserved region, and found that one moregene, thdF , is conserved. The comparison of gene organizationsof the dnaA region of the related bacteria supported the closephylogenetic relationship of B. aphidicola to E. coli. In addition,we have identified groES and groEL genesnext to the thdF gene.GroEL protein was reported to be expressed at an elevated levelin the endosymbionts of aphids, and is considered to play animportant role in their association with the aphid host. Comparisonof the structure of the groE operon with that of the endosymbiontof the aphid Acyrthosiphon pisum revealed the conservation ofa sequence resembling the E. coli consensus heat shock promoter,and this sequence may be responsible for the high expressionof the groEL gene in aphid endosymbionts.     

Evolution of signal transduction in intracellular symbiosis

Plant roots form intracellular symbioses with fungi and bacteria resulting in arbuscular mycorrhiza and nitrogen-fixing root nodules, respectively. A novel receptor like-kinase has been discovered that is required for the transduction of both bacterial and fungal symbiotic signals. This kinase defines an ancient signalling pathway that probably evolved in the context of arbuscular mycorrhiza and has been recruited subsequently for endosymbiosis with bacteria. An ancestral symbiotic interaction of roots with intracellular bacteria might have emerged from such a recruitment, in the progenitor of the nodulating clade of plants. Analysis of symbiotic mutants of host plants and bacterial microsymbionts has revealed that present-day endosymbioses require the coordinated induction of more than one signalling pathway for development.