Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).     

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.     

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.     

Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and α-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy α-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.     

Contribution of transgenic Casuarinaceae to our knowledge of the actinorhizal symbioses

The Casuarinaceae family is a group of 96 species of trees and shrubs that are tolerant to adverse soil and climatic conditions. In the field, Casuarinaceae bears nitrogen-fixing root nodules (so called actinorhizal nodules) resulting from infection by the soil actinomycete Frankia. The association between Casuarina and Frankia is of tremendous ecological importance in tropical and subtropical areas where these trees contribute to land stabilization and soil reclamation. During differentiation of the actinorhizal nodule, a set of genes called actinorhizal nodulins is activated in the developing nodule. Understanding the molecular basis of actinorhizal nodule ontogenesis requires molecular tools such as genomics together with gene transfer technologies for functional analysis of symbiotic genes. Using the biological vectors Agrobacterium rhizogenes and A. tumefaciens, gene transfer into the two species Allocasuarina verticillata and Casuarina glauca has been successful. Transgenic Casuarinaceae plants proved to be valuable tools for exploring the molecular mechanisms resulting from the infection process of actinorhizal plants by Frankia.     

Jasmonate biosynthesis in legume and actinorhizal nodules

Jasmonic acid (JA) is a plant signalling compound that has been implicated in the regulation of mutualistic symbioses. In order to understand the spatial distribution of JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined the localization of allene oxide cyclase (AOC) which catalyses a committed step in JA biosynthesis. In all nodule types analysed, AOC was detected exclusively in uninfected cells. The levels of JA were compared in the roots and nodules of the three plant species. The nodules and noninoculated roots of the two actinorhizal species, and the root systems of M. truncatula, noninoculated or nodulated with wild-type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not show significant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance. To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1-sense or MtAOC1-RNAi construct were inoculated with S. meliloti. Neither an increase nor reduction in AOC levels resulted in altered nodule formation. These data suggest that jasmonates are not involved in the development and function of root nodules.     

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.     

A novel nuclear protein interacts with the symbiotic DMI3 calcium- and calmodulin-dependent protein kinase of Medicago truncatula

Many higher plants establish symbiotic relationships with arbuscular mycorrhizal (AM) fungi that improve their ability to acquire nutrients from the soil. In addition to establishing AM symbiosis, legumes also enter into a nitrogen-fixing symbiosis with bacteria known as rhizobia that results in the formation of root nodules. Several genes involved in the perception and transduction of bacterial symbiotic signals named "Nod factors" have been cloned recently in model legumes through forward genetic approaches. Among them, DMI3 (Doesn't Make Infections 3) is a calcium- and calmodulin-dependent kinase required for the establishment of both nodulation and AM symbiosis. We have identified, by a yeast two-hybrid system, a novel protein interacting with DMI3 named IPD3 (Interacting Protein of DMI3). IPD3 is predicted to interact with DMI3 through a C-terminal coiled-coil domain. Chimeric IPD3::GFP is localized to the nucleus of transformed Medicago truncatula root cells, in which split yellow fluorescent protein assays suggest that IPD3 and DMI3 physically interact in Nicotiana benthamiana. Like DMI3, IPD3 is extremely well conserved among the angiosperms and is absent from Arabidopsis. Despite this high level of conservation, none of the homologous proteins have a demonstrated biological or biochemical function. This work provides the first evidence of the involvement of IPD3 in a nuclear interaction with DMI3.     

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

The promoter of the Vicia faba L. leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and nonlegume plants

The VfLb29 leghemoglobin gene promoter was polymerase chain reaction-amplified from a Vicia faba genomic library and was fused to the gusAint coding region. Expression of the chimeric gene was analyzed in transgenic hairy roots of the legumes V. faba, V. hirsuta, and Medicago truncatula as well as in transgenic Nicotiana tabacum plants. The VfLb29 promoter was found to be specifically active not only in the infected cells of the nitrogen-fixing zone of root nodules but also in arbuscule-containing cells of transgenic V. faba and M. truncatula roots colonized by the endomycorrhizal fungus Glomus intraradices. In addition to these two legumes, specific expression in arbuscule-containing cells was also observed in the nonlegume N. tabacum. All studies were done in comparison to the V. faba leghemoglobin gene promoter VfLb3 that as VfLb29 was expressed in the infected cells of root nodules but showed no activity in endomycorrhiza. An activation of the VfLb29 promoter due to hypoxia in metabolically active tissues was excluded. The conserved activation in arbuscule-containing cells of legumes and the nonlegume N. tabacum suggests a conserved trigger for this promoter in legume and nonlegume endomycorrhiza symbioses.     

Is the Legume Nodule a Modified Root or Stem or an Organ sui generis?

The legume nodule, which houses nitrogen-fixing rhizobia, is a unique plant organ. Its homology with lateral roots has been inferred by a comparison with other nitrogen-fixing nodules, especially those formed on actinorhizal plants in response to Frankia inoculation or on Parasponia roots following inoculation with Bradyrhizobium species. These nodules are clearly modified lateral roots in terms of their structure and development. However, legume nodules differ from lateral roots and these other nodules in their developmental origin, anatomy, and patterns of gene expression, and, consequently, several other evolutionary derivations, including from stems, wound or defense responses, or the more ancient vesicular-arbuscular mycorrhizal symbiosis, have been postulated for the legume nodule. In this review, we first present a broad view of the legume family showing the diversity of nodulation occurrence and types in the different subfamilies and particularly within the subfamily Papilionoideae. We then define the typological and molecular criteria used to discriminate the basic organs — root, stem, leaf— of the plant. Finally, we discuss the possible origins of the legume nodule in terms of these typological and molecular bases.     

Fungal symbiosis in rice requires an ortholog of a legume common symbiosis gene encoding a Ca2+/calmodulin-dependent protein kinase

In natural ecosystems, many plants are able to establish mutually beneficial symbioses with microorganisms. Of critical importance to sustainable agriculture are the symbioses formed between more than 80% of terrestrial plants and arbuscular mycorrhizal (AM) fungi and between legumes and nitrogen-fixing rhizobial bacteria. Interestingly, the two symbioses share overlapping signaling pathways in legumes, suggesting that the evolutionarily recent root nodule symbiosis may have acquired functions from the ancient AM symbiosis. The Medicago truncatula DMI3 (DOESN'T MAKE INFECTIONS3) gene (MtDMI3) and its orthologs in legumes are required for both bacterial and fungal symbioses. MtDMI3 encodes a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for the transduction of the Ca(2+) signal induced by the perception of Nod factors. Putative orthologs of MtDMI3 are also present in non-legumes, but their function in AM symbiosis has not been demonstrated in any non-legume species. Here, we combine reverse genetic approaches and a cross-species complementation test to characterize the function of the rice (Oryza sativa) ortholog of MtDMI3, namely, OsDMI3, in AM symbiosis. We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also is able to complement a M. truncatula dmi3 mutant, indicating an equivalent role of MtDMI3 orthologs in non-legumes.