Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses

Most land plants can form a root symbiosis with arbuscular mycorrhizal (AM) fungi for assimilation of inorganic phosphate from the soil. In contrast, the nitrogen-fixing root nodule symbiosis is almost completely restricted to the legumes. The finding that the two symbioses share common signaling components in legumes suggests that the evolutionarily younger nitrogen-fixing symbiosis has recruited functions from the more ancient AM symbiosis. The recent advances in cloning of the genes required for nodulation and AM symbioses from the two model legumes, Medicago truncatula and Lotus japonicus, provide a unique opportunity to address biological questions pertaining to the evolution of root symbioses in plants. Here, we report that nearly all cloned legume genes required for nodulation and AM symbioses have their putative orthologs in nonlegumes. The orthologous relationship can be clearly defined on the basis of both sequence similarity and microsyntenic relationship. The results presented here serve as a prelude to the comparative analysis of orthologous gene function between legumes and nonlegumes and facilitate our understanding of how gene functions and signaling pathways have evolved to generate species- or family-specific phenotypes.     

Divergence of evolutionary ways among common sym genes: CASTOR and CCaMK show functional conservation between two symbiosis systems and constitute the root of a common signaling pathway

In recent years a number of legume genes involved in root nodule (RN) symbiosis have been identified in the model legumes, Lotus japonicus (Lotus) and Medicago truncatula. Among them, a distinct set of genes has been categorized as a common symbiosis pathway (CSP), because they are also essential for another mutual interaction, the arbuscular mycorrhiza (AM) symbiosis, which is evolutionarily older than the RN symbiosis and is widely distributed in the plant kingdom. Based on the concept that the legume RN symbiosis has evolved from the ancient AM symbiosis, one issue is whether the CSP is functionally conserved between non-nodulating plants, such as rice, and nodulating legumes. We identified three rice CSP gene orthologs, OsCASTOR, OsPOLLUX and OsCCaMK, and demonstrated the indispensable roles of OsPOLLUX and OsCCaMK in rice AM symbiosis. Interestingly, molecular transfection of either OsCASTOR or OsCCaMK could fully complement symbiosis defects in the corresponding Lotus mutant lines for both the AM and RN symbioses. Our results not only provide a conserved genetic basis for the AM symbiosis between rice and Lotus, but also indicate that the core of the CSP has been well conserved during the evolution of RN symbiosis. Through evolution, CASTOR and CCaMK have remained as the molecular basis for the maintenance of CSP functions in the two symbiosis systems.     

OsIPD3, an ortholog of the Medicago truncatula DMI3 interacting protein IPD3, is required for mycorrhizal symbiosis in rice

Medicago truncatula IPD3 (MtIPD3) is an interacting protein of DMI3 (does not make infections 3), a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for both arbuscular mycorrhizal (AM) and rhizobial symbioses. However, the function of MtIPD3 in root symbioses has not been demonstrated in M. truncatula, because of a lack of knockout mutants for functional analysis. In this study, the availability of IPD3 knockout mutants in rice (Oryza sativa) was exploited to test the function of OsIPD3 in AM symbiosis. Three independent retrotransposon Tos17 insertion lines of OsIPD3 were selected and the phenotypes characterized upon inoculation with the AM fungus Glomus intraradices. Phenotypic and genetic analyses revealed that the Osipd3 mutants were unable to establish a symbiotic association with G. intraradices. In conclusion, IPD3 represents a novel gene required for root symbiosis with AM fungi in plants.     

Are common symbiosis genes required for endophytic rice-rhizobial interactions?

Legume plants are able to establish root nodule symbioses with nitrogen-fixing bacteria, called rhizobia. Recent studies revealed that the root nodule symbiosis has co-opted the signaling pathway that mediates the ancestral mycorrhizal symbiosis that occurs in most land plants. Despite being unable to induce nodulation, rhizobia have been shown to be able to infect and colonize the roots of non-legumes such as rice. One fascinating question is whether establishment of such associations requires the common symbiosis (Sym) genes that are essential for infection of plant cells by mycorrhizal fungi and rhizobia in legumes. Here, we demonstrated that the common Sym genes are not required for endophytic colonization of rice roots by nitrogen-fixing rhizobia.     

A novel type of thioredoxin dedicated to symbiosis in legumes

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.     

Antiquity and function of CASTOR and POLLUX, the twin ion channel-encoding genes key to the evolution of root symbioses in plants

Root symbioses with arbuscular mycorrhizal fungi and rhizobial bacteria share a common signaling pathway in legumes. Among the common symbiosis genes are CASTOR and POLLUX, the twin homologous genes in Lotus japonicus that encode putative ion channel proteins. Here, we show that the orthologs of CASTOR and POLLUX are ubiquitously present and highly conserved in both legumes and nonlegumes. Using rice (Oryza sativa) as a study system, we employ reverse genetic tools (knockout mutants and RNA interference) to demonstrate that Os-CASTOR and Os-POLLUX are indispensable for mycorrhizal symbiosis in rice. Furthermore, a cross-species complementation test indicates that Os-POLLUX can restore nodulation, but not rhizobial infection, to a Medicago truncatula dmi1 mutant.     

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.     

Arbuscular Mycorrhiza–Specific Signaling in Rice Transcends the Common Symbiosis Signaling Pathway

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.     

Development of the arbuscular mycorrhizal symbiosis

The arbuscular mycorrhizal (AM) symbiosis formed between plant roots and fungi is one of the most widespread symbiotic associations found in plants, yet our understanding of events underlying its development are limited. The recent integration of biochemical, molecular and genetic approaches into analyses of the symbiosis is providing new insights into various aspects of its development. In the past year there have been advances in our understanding of the signals required for the formation of appressoria, the molecular changes in the root in response to colonisation, and components of the signal transduction pathways common to both the AM and Rhizobium symbioses.     

Functional adaptation of a plant receptor-kinase paved the way for the evolution of intracellular root symbioses with bacteria

Nitrogen-fixing root nodule symbioses (RNS) occur in two major forms—Actinorhiza and legume-rhizobium symbiosis—which differ in bacterial partner, intracellular infection pattern, and morphogenesis. The phylogenetic restriction of nodulation to eurosid angiosperms indicates a common and recent evolutionary invention, but the molecular steps involved are still obscure. In legumes, at least seven genes—including the symbiosis receptor-kinase gene SYMRK—are essential for the interaction with rhizobia bacteria and for the Arbuscular Mycorrhiza (AM) symbiosis with phosphate-acquiring fungi, which is widespread in occurrence and believed to date back to the earliest land plants. We show that SYMRK is also required for Actinorhiza symbiosis of the cucurbit Datisca glomerata with actinobacteria of the genus Frankia, revealing a common genetic basis for both forms of RNS. We found that SYMRK exists in at least three different structural versions, of which the shorter forms from rice and tomato are sufficient for AM, but not for functional endosymbiosis with bacteria in the legume Lotus japonicus. Our data support the idea that SYMRK sequence evolution was involved in the recruitment of a pre-existing signalling network from AM, paving the way for the evolution of intracellular root symbioses with nitrogen-fixing bacteria.     

Molecular genetic mechanisms used by legumes to control early stages of mutually beneficial (mutualistic) symbiosis

Recent data on the plant control of early stages of mutually beneficial (mutualistic) symbioses of legumes, the mechanisms of perception and transmission of the microsymbiont’s molecular signals in the macrosymbiont’s cells, and induction of the genetic programs of the development of symbiotic compartments and organs of the plant are summarized. It is demonstrated that the genetic system of the plant controlling the development of nitrogen-fixing symbiosis of legumes (symbiotic root nodules), which emerged 70–80 Ma ago, has undoubtedly evolved on the basis of the genetic system controlling the development of the symbiosis with arbuscular mycorrhizal fungi (which emerged 400–500 Ma ago). Interactions between genes and between gene products, as well as exchange of molecular signals, form the basis of mutually beneficial (mutualistic) plant-bacterium interactions. Even in the case of a highly specific nitrogen-fixing symbiosis of legumes (symbiotic nodules), the receptors perceiving the signal from root-nodule bacteria may function in different ways. The development of arbuscular mycorrhiza and nitrogen-fixing symbiosis in legumes is a multistep process involving hundreds of genes of both the macro- and microsymbionts. For the symbioses to develop successfully, these genes should act in a coordinated way in the newly formed superorganismal system. Further studies are necessary to shed light onto the complexity of the plant genetic control of the development of mutualistic symbioses in legumes and provide information required for improving their functions in adaptive plant-breeding systems.     

Endomycorrhizal and rhizobial symbiosis: How much do they share?

Abstract

Legume plants enter two important endosymbioses – with soil fungi, forming phosphorus acquiring arbuscular mycorrhiza (AM), and with nitrogen-fixing bacteria, leading to the formation of nitrogen-fixing root nodules. Both symbioses have been studied extensively because these symbioses have great potential for agricultural applications. Although 80% of all living land plants form AM, the nitrogen-fixing root nodule symbiosis with rhizobia is almost exclusively restricted to legumes. Despite varying degree of differences in the morphological responses induced by both endosymbionts in the host plants, significant similarities in the development of both fungal and bacterial symbioses have been reported. The signal perception and signal transduction cascades that initiate nodulation and mycorrhization in legumes partially overlap. Legume genes have been identified that are required for the establishment of both AM and root nodule symbiosis and are referred to as the common SYM genes. Genetic dissection of the common SYM signal transduction pathway required for bacterial and fungal root endosymbiosis has not only unraveled the players involved but also provided a first glimpse at conservation and specialization of signaling cascades essential for nodulation and mycorrhiza development. Based on the observation of common signaling cascades, it is tempting to speculate that the root nodule symbiosis, where fossil records date back to the late Cretaceaous, adopted and subsequently modified more ancient signal transduction pathways leading to AM formation, having already been in place 400 million years ago. This review discusses the common aspects of recognition of mycorrhizal fungi and Rhizobium by the host, and further signal transduction that leads to an effective symbiosis.