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Pods and Nods: a new look at symbiotic nitrogen fixing Nicholas J Brewin John Innes Centre, Norwich, UK How can growing a crop plant make fields more fertile? With legumes, this is precisely what happens. Working in partnership with symbiotic bacteria that create root nodules on their chosen host, legumes can fix atmospheric nitrogen and enhance the nitrogen status of soils. How does this symbiosis develop? And how did it evolve? Bacterial and plant genomics are beginning to provide the answers. It has been known since ancient times that legumes contribute significantly to soil fertility. Pliny the Elder (circa 70 AD) wrote: ‘It is universally agreed that no manure is more beneficial than a crop of lupins turned in by the plough or with forks before the plants form pods.’ The important connection between legumes, root nodules and nitrogen fixation was established in 1888 by two German soil chemists, Hellriegel and Wilfarth. They demonstrated how the unique capacity of leguminous plants to ‘fix’ atmospheric nitrogen for plant growth was related to the development of root nodules following infection by soil microorganisms (reviewed by Quispels, 1988). In the same year, Beyerinck (following in the footsteps of Koch and Pasteur) successfully cultured the microorganisms from lupin and pea nodules. It was demonstrated that the strain of ‘Bacillus radicicola’ isolated from pea nodules (title image) was capable of forming nodules on peas but not on lupin, whereas the converse was true for the lupin-derived strain. However, another German microbiologist, named Frank, was not convinced by Beyerinck’s identification. Because he suspected that fungi or myxomycetes might be the causal Title image. Pea nodules, the site of biological nitrogen fixation. The physiology of a legume root nodule is adapted to promote the activity of the oxygen-sensitive nitrogenase system of rhizobium bacteria by reducing oxygen concentrations in the central tissues. Biologist (2002) 49 (3) agents of nodulation, Frank proposed the neutral name Rhizobium leguminosarum, and this name has endured from 1889 to the present day. Although there were some minor refinements in nomenclature, not much changed over the next 100 years. Rhizobium genera and species were defined in the context of cross-inoculation groups. A rhizobial species was observed to be co-adapted to a particular range of host plants. Some strains showed a high degree of host specificity, while others had very broad host ranges (particularly with tropical legumes). This anecdotal information was used as the basis for an inoculum industry that underpinned the globalisation of agriculture. As legume crops were introduced from one region of the world to another (soybeans and lucerne to North America, clovers to Australia), it was recognised that they needed to be inoculated with an appropriate rhizobium strain in order to reap the benefits of biological nitrogen fixation. However, there was no real understanding of the underlying scientific issues. Why is a particular strain of rhizobium capable of nodulating one legume host but not another? What do rhizobial strains have in common and how do they differ? Finally, what unique feature of legumes allows them to develop this unique symbiosis with rhizobia? Over the past two decades, some fascinating answers have begun to emerge. 1 P o d s a n d N o d s Figure 1. Colonisation of host cells by rhizobia (courtesy of Simon Walker and Allan Downie). Rhizobium-derived Nod-factor acts as a signal for root hair deformation (left-hand micrograph). The infection thread orginates as an intrusion of the host cell wall (arrows) and propagates from cell to cell as a transcellular tunnel in the root cortex (right-hand micrograph). nod genes and Nod-factors With the onset of bacterial genetics, it has been possible to define a number of key genes (nod genes) that are critical for the initiation of nodulation on legumes, but are not necessary for survival as free-living soil bacteria. Analysis of the functions of nod genes showed that they arecomponents of a molecular dialogue that results in the initiation of nodule development on the appropriate host legume. Apparently, all legumes exude flavonoid compounds from their roots. Therefore, each legume species has a distinctive ‘odour’ that is sensed specifically by the nodD gene product(s) of an infective rhizobium strain. Following this recognition event, transcription of a set of nod genes takes place, which culminates in the synthesis and secretion of specific signal molecules, termed Nod-factors. Structurally, Nod-factors are decorated oligomers of N-acetyl glucosamine (the constituent of chitin) and, characteristically, they each carry an unsaturated acyl chain N-linked onto the terminal non-reducing sugar. Nod-factors specifically activate host cells of the appropriate legume (Stougaard, 2001). By a process that is still not understood at the molecular level, Nod-factors initiate the twin processes of nodule initiation (by inducing cortical cell divisions) and cell colonisation (by provoking a re-organisation of cell wall growth in epidermal cells and root hairs). Rhizobial diversity From the above analysis, an operational definition of rhizobia emerges. They are soil bacteria carrying a set of nod genes that allow them to nodulate one or more legume hosts. There is a presumption that rhizobia also carry nitrogenase (or nif) genes that allow them to develop the capacity for biological nitrogen fixation as part of the root nodule symbiosis. Generally, it is found that the nod and nif genes are closely linked, but this is where the taxonomy begins to get interesting. When rhizobial strains are analysed by standard taxonomic criteria based on gene sequence comparisons, it is clear that these bacteria do not form part of a homogeneous evolutionary clade. The Rhizobiaceae as a distinct taxonomic group does not exist (Spaink et al., 1998). The clear implication is that nod, nif and associated symbiosis genes have been transferred ‘horizontally’ between various groups of soil bacteria to create new genetic combinations that are optimally adapted, both for survival in the soil and for the nodulation of some particular legume host. 2 Until recently, most of the characterised rhizobial strains had been derived from the standard but limited range of cultivated legume species. All these bacterial isolates belong to three distinct branches within the alpha-2 subgroup of Proteobacteria. In each case, rhizobia are phylogenetically intertwined with non-symbiotic bacteria (Moulin et al., 2001). The largest branch includes the genus Rhizobium, which nodulates peas and clovers, and Sinorhizobium, which nodulates alfalfa (lucerne); however, Sinorhizobium is much more closely related to the plant pathogen Agrobacterium and to Brucella (an intracellular animal pathogen) than it is to Rhizobium. A second branch includes the genus Bradyrhizobium, with species that nodulate soybean, lupin and many tropical legumes; however, closely related species include Rhodopseudomonas (a photosynthetic free-living bacterium). The third group includes Azorhizobium, which is closely related to the chemiautotroph Xanthobacter. This diversity and heterogeneity may represent only the tip of the iceberg for rhizobial strains. There are about 18 000 species in the family Leguminosae and, so far, symbionts from only 50 of the 750 genera have been sampled. In the last year alone, two completely new rhizobial groupings have been identified. Methylobacterium nodulans, isolated from Crotalaria nodules, represents a fourth class of alpha-2 subgroup Proteobacteria (the first rhizobium strain reported to grow on methanol), while bacteria isolated from Aspalathus nodules were found to belong to the genus Burkholderia, a member of the phylogenetically distant beta-subclass of Proteobacteria (Moulin et al., 2001). Although nitrogen-fixing bacteria exist in other proteobacterial subclasses, e.g., Herbaspirillum (which colonises sugar cane tissues) and Azoarcus (which colonises rice roots), none has previously been found to harbour the nod genes essential for establishing legume symbiosis. Horizontal gene transfer How does horizontal transfer of nod and nif genes occur? The clues are to be found in the genomic structures of those rhizobia that have been examined so far. In the case of Rhizobium leguminosarum, the cassette of symbiosis genes is carried on a self-transmissible plasmid (c. 250 kilobases): biovar viciae (which infects peas and vetches) and biovar trifolii (which nodulates clover) harbour plasmids that confer different host specificities. Depending on the ecological niche (e.g., a sward of vetch or clover), it is easy to see how the symbiotic plasmid with a selective advantage is likely to become established as the dominant biovar in the soil population. In the case of Sinorhizobium strain NGR234, there is a very large symbiotic plasmid (Freiberg et al., 1997). The extremely broad host range of this strain is apparently the consequence of the long-term accumulation of a diversity of nodD variants and a corresponding range of nod genes, which synthesise a diversity of Nod-factors. Similarly in the case of Mesorhizobium loti, a ‘symbiosis island’ allows non-symbiotic bacteria to evolve into symbionts in a single quantum leap (Sullivan and Ronson, 1998). The 500 kb cassette of symbiosis genes has become inserted by integration into the chromosome at the phe-tRNA locus, in a process mediated by a P4 integrase encoded within the island. Apparently, the symbiosis island is one of a number of fitness islands that can be acquired by related bacteria. These mechanisms of horizontal gene transfer contribute to the ecological adaptation of soil bacteria. In the last year, the complete genomes of Sinorhizobium meliloti and Biologist (2002) 49 (3) P o d s a n d N o d s Figure 2. Electron micrograph showing transverse section of a tubular infection thread (IT) bounded by a primary cell wall containing pectin and cellulose. Immunogold staining with monoclonal antibody MAC265 highlights root nodule extensin, a legume-derived glycoprotein that is secreted through the plasma membrane (arrowheads) and surrounds the invading rhizobia in the infection thread lumen. Scale bar = 0.5 microns Mesorhizobium loti have been determined. Thus, comparative analysis of genome structure and function will help to define the minimum set of functions associated with a root-nodulating (rhizobium) strain. Adaptations for tissue and cell colonisation It is clear that the nod and nif genes do not represent the whole of the story as far as nodulation is concerned. For example, when these genes are introduced into E. coli nothing happens. What other genes are required for symbiosis and what attributes of non-symbiotic soil bacteria predispose them to become rhizobia? Once again, the clues are to be found in comparative genome analysis among the non-symbiotic relatives of rhizobia. The answers may be related to the ability of rhizobia and potential rhizobial strains to colonise host tissues (Figures 1 and 2) and to survive under the special conditions found inside the tissues and cells of the host plant (Figure 3). For example, the ability of rhizobia to survive in a hypo- or hyper-osmotic environment may be an adaptive characteristic. The nature of bacterial cell surfaces (e.g., lipopolysaccharide and exopolysaccharide) has also been shown to affect the ability of rhizobia to colonise host tissues and cells (Pellock et al., 2000). Intracellular symbionts and pathogens Fitness for host cell colonisation is not a trait that is unique to rhizobia. It is interesting to note that Sinorhizobium meliloti (a symbiont of alfalfa) is phylogenetically related to Brucella abortus (a mammalian pathogen). Both strains establish chronic intracellular infections in their respective hosts, without the induction of host defence responses. In these two very different relationships, a similar gene encoding a putative membrane transport protein (bacA) was found to be of critical importance for the maintenance of bacteria within the host cell (LeVier et al., 2000). An even more curious coincidence has recently emerged, with the discovery that endophytic arbuscular mycorrhizae Biologist (2002) 49 (3) Figure 3. Endosymbiotic rhizobia are morphologically differentiated as bacteroids. In central tissues of the root nodule, host cells harbour thousands of nitrogen-fixing bacteroids. Host cells have large central nuclei and the vacuole is reduced or absent (upper micrograph). The electron micrograph (lower micrograph) shows that each bacteroid is bounded by a plant-derived peribacteroid membrane, forming an organelle-like structure termed a symbiosome. (which colonise 90% of all land plants, not just legumes) sometimes harbour intracellular bacteria that are related to Burkholderia (Ruiz-Lozano and Bonfante, 2000). Because Burkholderia has recently been characterised as a rhizobial genus, this raises some interesting questions about the direction of evolutionary change. Have intracellular symbiotic microorganisms from mycorrhizae subsequently acquired the ability to colonise legume cells or have Burkolderia-type rhizobia developed the capacity to survive as endosymbionts of mycorrhizal hyphae? The joint occurrence of Burkolderia spp as endosymbionts of mycorrhizal fungi and as endosymbionts of legume nodule cells is particularly intriguing. Genetic analysis of symbiotically defective legumes has revealed that about half of the plant mutants that fail to establish the early stages of nodule symbiosis are also impaired in the establishment of the mycorrhizal symbiosis (Bonfante et al., 2000). Yet, the mycorrhizal fungi evolved the capacity to colonise host plant cells over 400 million years ago, while the legume symbiosis is only about 60 million years old. Apparently, rhizobia have exploited this more ancient host-symbiont signalling system as part of the signal transduction cascade for Nod-factor. As an added twist to this curious paradox, it should be pointed out that rhizobium Nod-factors are simply decorated fragments of oligochitin, and chitins are, of course, the key component of fungal cell walls. A fresh look at legume taxonomy Let us now examine the rhizobium-legume symbiosis from the viewpoint of the host plant. Can we identify what unique features of legumes enable them to establish an interaction with rhizobia? Once again, a taxonomic and evolutionary perspective may be helpful. Among the 18 000 3 P o d s a n d 1 N o d s Pisium Medicago Lotus Glycine Vigna Papilionoideae Parasponia has been shown to be dependent on the presence of functional nod genes (Marvel et al., 1987). These observations suggest two important conclusions. First, the predisposition to form nodules is not restricted to the Leguminosae, but rather to the Rosid clade I. Second, at least one non-leguminous member of the clade is capable of recognising the products of rhizobial nod genes. Swartzia New excitement for a new century 2 Leucaena Acacia Mimoisoideeae Ceratonia 3 Chaemaecrista Caesalpiniodeae Bauhinia Dialium Figure 4. Simplified evolutionary tree for the Leguminosae (Fabaceae), showing the nodulating sectors in red and non-nodulating groups in blue. The three putative origins for nodulation capability are indicated. (Adapted from Doyle, 1998.) legume species, there are a number of groups that do not nodulate. In some cases, the symbiotic character may have been lost during evolution. However, in the Caesalpinioid sub-family, only 23% of all species are nodulated. Therefore, it seems probable that nodulation has never developed in the more primitive legume groups (Doyle, 1998). Plant taxonomy is now based on comparative sequence analysis for several well-defined genes (of nuclear, chloroplast and mitochondrial origin). On this basis, and applying the principle of parsimony, it seems that nodulation must have evolved at least three times during the evolution of legumes (Figure 4). This implies some form of predisposition to establish a nodulating symbiosis. To put it another way, rhizobia may originally have been colonisers of host cells and intercellular spaces in legume roots in much the same way that mycorrhizal fungi colonise root cortical cells. Only subsequently would the rhizobium-legume symbiosis have evolved the capacity to form a nodule-like structure as a physiological adaptation that serves to reduce oxygen damage to the oxygen-sensitive nitrogenase enzyme system. The legume-nodulation paradigm is further complicated when we step outside the family Leguminosae (Fabaceae). Following a reconfiguration of the taxonomy of higher plants, the Leguminosae now belong to a grouping termed Rosid clade I (Doyle, 1998). Unlike all other groups of higher plants, this clade contains probably seven separate instances of the evolution of non-legume root nodule symbioses (although nodule anatomy is more reminiscent of a modified lateral root than is the case with legume nodules). In six of these symbioses, the nodule endophyte is not a rhizobial strain (as currently defined) but is instead an actinomycete, Frankia, a filamentous Gram-positive bacterium (Table 1). Examples of actinorhizal plants include Alnus, Caeanothus and Casuarina. The seventh example of non-legume root nodule symbiosis involves the ulmaceous shrub Parasponia, but in this case the endophyte is unquestionably a rhizobium strain. Moreover, nodulation of 4 With so much exciting new information, how should the experiments of Hellriegel, Beyerinck and Frank be interpreted today? First, in answer to Hellriegel and Wilfarth, nitrogen fixation is a property of the bacterial symbiont and host specificity is related to different chemical structures for rhizobium-derived Nod-factors. These are chemically decorated lipochitin oligosaccharides encoded by nod genes. Second, in answer to Beijerinck and Frank, it is now recognised that root-nodulating bacteria are taxonomically diverse. As a result of horizontal gene transfer, the capacity to nodulate legumes has spread to many bacterial groups in the Proteobacteria. Furthermore, the relationship between the rhizobium-legume symbiosis and the Frankia-Rosid symbiosis is extremely curious. It might be clarified by a genome-sequencing program that establishes whether Frankia spp carry anything equivalent to the nod genes of rhizobia. Legumes are a sub-group of Rosid clade I and there is already one well-documented case of a rhizobium symbiosis with a non-legume. Let us finish on a speculative note. Perhaps the concept of ‘rhizobial strains’ should be extended to include all bacteria capable of inducing nitrogen-fixing root nodules in members of Rosid clade I (including both legumes and non-legumes). After more than a century of research, Frank and his colleagues would probably welcome the re-integration of Frankia into an all-inclusive group of root-nodulating bacteria. Acknowledgements Research on legume nodule development at the John Innes Centre has been sponsored by the Biotechnology and Biological Research Council. Table 1. Families in Rosid Clade I known to include nodulating plants Actinorhizal nodules Rhizobial nodules Rosaceae Ulmaceae Ulmaceae Elaeagnaceae Rhamnaceae Fabaceae Betulaceae Casuarinaceae Myricaceae Coriariaceae Datiscaceaeae Table 1. Rosid clade I is the only group of higher plants in which root nodule symbiosis has sporadically arisen. This table lists the groups that nodulate with Frankia or rhizobia. Biologist (2002) 49 (3) P o d s References Bonfante P, Genre A, Faccio A, Martini I et al. (2000) The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells. Molecular Plant-Microbe Interactions, 13, 1109 – 1120. Doyle J J (1998) Phylogenetic perspectives on nodulation: evolving views of plants and symbiotic bacteria. Trends in Plant Science, 3, 473 – 478. Freiberg C, Fellay R, Bairoch A, Broughton W J et al. (1997) Molecular basis of symbiosis between Rhizobium and legumes. Nature, 387, 394 – 401. LeVier K, Phillips R W, Grippe V K, Roop R M, and Walker G C (2000) Similar requirements of a plant symbiont and a mammalian pathogen for prolonged intracellular survival. Science, 287, 2492 – 2493. Marvel D J, Torrey J G and Ausubel F M (1987) Rhizobium symbiotic genes required for nodulation of legume and nonlegume hosts. Proc Nat Acad Sci USA, 84, 1319 – 1323. Moulin L, Munive A, Dreyfus B and Boivin-Masson C (2001) Nodulation of legumes by members of the beta-subclass of Proteobacteria. Nature 411, 948 – 950. Pellock B J, Cheng H P and Walker G C (2000) Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides. J Bacteriol, 182, 4310 – 4318. Quispel A (1988) Hellreigel and Wilfarth’s discovery of (symbiotic) nitrogen fixation one hundred years ago, p. 3 -12. In Nitrogen Fixation: Hundred Years After. Bothe H et al. (Eds). Gustav Fischer, Stuttgart. Ruiz-Lozano J M and Bonfante P (2000) A Burkholderia strain living inside the arbuscular mycorrhizal fungus Gigaspora margarita possesses the vacB gene, which is involved in host cell colonization by bacteria. Microbial Ecology, 39, 137 – 144. Biologist (2002) 49 (3) a n d N o d s Spaink H P, Kondorosi A and Hooykaas P J J (1998) The Rhizobiaceae. Kluwer, Dordrecht. Stougaard J (2001) Genetics and genomics of root symbiosis. Current Opinion in Plant Biology, 4, 328 – 335. Sullivan J T and Ronson C W (1998) Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene. Proc Nat Acad Sci USA, 95, 5145 – 5149. Websites www.noble.org/medicago/ Describes Medicago truncatula as a model legume for molecular genetic analysis http://wwwifr.toulouse.inra.fr/meliloti.html Describes the complete sequence of Sinorhizobium meliloti, the symbiont of Medicago truncatula http://helios.bto.ed.ac.uk/bto/microbes/microbes.htm A good educational site for plant-microbe interactions www.jic.bbsrc.ac.uk/staff/nick-brewin/index.htm Includes author’s lecture notes on the rhizobium-legume symbiosis Nick Brewin DSc is honorary professor at the University of East Anglia and tutor for graduate studies at the John Innes Centre. He has studied both bacterial and plant aspects of the rhizobium-legume symbiosis, particularly in relation to the mechanisms of tissue and cell invasion. John Innes Centre Norwich, NR4 7UH, UK [email protected] 5