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The unbearable naivety of legumes in symbiosis
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The unbearable naivety of legumes in symbiosis Griet Den Herder and Martin Parniske The mechanisms by which legumes choose their rhizobial partners operate independently from their ability to fix nitrogen. As a result of this naivety, symbiotic nitrogen fixation is often suboptimal. The initial recognition of the bacterial partner and the subsequent signal transduction in the host root utilises components that are functionally conserved between legumes and probably actinorhiza host plants. However, the later steps, which largely determine symbiotic performance, are subject to ongoing evolutionary diversification of molecular mechanisms. For example, the impact of bacterial effector proteins, the occurrence of terminal bacteroid differentiation and the expression of bacterial hydrogenase, all depend on the plant genotype. Strategies towards increased nitrogen fixation of legumes in agriculture need to encompass this diversification of mechanisms. Address Genetics, Faculty of Biology, University of Munich (LMU), Grosshaderner Strasse 2-4, 82152 Martinsried-Mu¨nchen, Germany Corresponding author: Parniske, Martin ([email protected])
Current Opinion in Plant Biology 2009, 12:491–499 This review comes from a themed issue on Biotic Interactions Edited by Xinnian Dong and Regine Kahmann Available online 23rd July 2009 1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2009.05.010
Introduction All legume crops are able to establish nitrogen fixing root nodule symbiosis with rhizobia [1]. However, nitrogen fixation efficiency within a single legume species can vary more than 10-fold, providing a huge potential for optimisation. A major challenge is that the plant has to choose a bacterial partner before this bacterium starts to deliver nitrogen. The plant’s decision is governed by a display of bacterial signals and effectors that have little or nothing to do with their ability to fix nitrogen. This ‘naı¨ve’, imperfect selection process provides ample opportunities for inefficient, parasitic bacteria that induce root nodules and infect them, but fix nitrogen only poorly or not at all. Such bacterial freeloaders are omnipresent and are a practical problem in agriculturally important legumes such as bean and soybean [2]. An extreme case is a Bradyrhizobium japonicum mutant defective for a nitrogenase-encoding gene and hence www.sciencedirect.com
completely unable to fix nitrogen, which induces fully developed root nodules on soybean and competes successfully with the wild type for infection [3]. Multiple loss-of-nodulation events apparent in the phylogeny of legumes [4] are perhaps an indication that giving up nodulation altogether was advantageous over coping with such cheaters. The diversified recognition specificity among legumes towards bacterial Nod factor (NF) may be another reflection of the inefficiency of this selection mechanism. However, there are indications that selection by legumes exists [2] and that it shaped the genomes of rhizobia. For example, nodules with persistent meristems have the potential to stop nodule growth if the rhizobia do not perform within the nitrogen-fixing zone. Moreover, rhizobial nod-genes are typically found in close proximity to the nif-genes linked on mobile genetic elements such as plasmids (pSymA of Sinorhizobium meliloti) or symbiosis islands (Bradyrhizobium japonicum; Mesorhizobium loti) promoting co-transmission of both abilities to potential recipients. A second challenge for optimisation strategies is the finely tuned functional compatibility between adapted host and symbiont pairs at the later stages of symbiosis that can be observed between legumes in their native habitats and endogenous rhizobia [5]. Since allelic variation in multiple unknown host genes determines this functional compatibility, optimal allele combinations need to be re-established after hybridisation steps in plant breeding programs, which will only be possible once the key determinants are identified. In this opinion, we briefly summarise recent progress in unravelling the molecular events underlying the nodule symbiosis, highlighting the diversified processes that operate during the establishment and function in different legume bacterial genotype combinations.
Rhizobia signal with multiple molecules A key bacterial determinant of host specificity is the structure of the secreted lipochitin oligosaccharide NFs, which is recognised by plant LysM containing NF receptor kinases, in which single amino acid changes lead to altered recognition specificities [6]. Biotechnological alteration of the NF recognition specificity of crop legumes to exclusively allow nodulation by a rhizobial high performance inoculum strain while at the same time reduce nodule occupancy by parasitic bacteria has now become a tempting possibility. This recognition event initiates subsequent signal transduction leading to symbiotic responses of the host root (Box 1). The identification of orthologues for genes Current Opinion in Plant Biology 2009, 12:491–499
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encoding early signal transduction components from different legume and actinorhiza host species suggests that the pathway for the inception of nodulation is ancient and conserved among nodulating plants [7,8,9]. In addition, the precise polysaccharide composition of the rhizobial cell wall (EPS, LPS and cyclic glucans) is important for successful infection. The panel and impact of polysaccharides varies between rhizobia, with the EPS of S. meliloti preventing defence responses [10,11]. The cellulase CelC2 of R. leguminosarum was found to be important for infection and shown to lyse the host plant cell wall at the point of entry [12]. Bacterial effector proteins injected into the host cell via type 3 or type 4 secretion systems can also influence symbiotic performance. Some of the rhizobial effectors share sequence similarity with the effectors of human or plant pathogens [13]. In the broad host range Rhizobium NGR234, an acetyltransferase of the Yersinia outer protein L (YopL) family was shown to acetylate MAPkinases in vitro and to affect symbiosome differentiation [14]. NopT is a protease that improved the symbiosis with Phaseolus but was detrimental for the interaction with Crotalaria juncea [15]. The quantitative and specific effects of rhizobial effectors make them likely determinants of symbiotic performance. It is therefore important to identify the molecular targets of rhizobial effectors in the legume host cell as well as the stage at which they act during development [16].
Root nodules differ in structure and molecular mechanisms Nodule structure, bacteroid differentiation and molecular mechanisms underlying development and function vary strongly between legumes, are host-determined and may, in part, reflect adaptations to different climatic conditions [4] (Table 1). A major differentiating trait is the presence of a persistent meristem. Indeterminate nodules have a persistent apical meristem and, in the elongated mature nodule, a spatial differentiation resulting in different developmental zones (Figure 1). Infection threads (ITs) arrive in the differentiating and expanding cells of the infection zone and release rhizobia into unwalled, membrane-bound infection droplets. In the fixation zone, the bacteria differentiate to become nitrogen-fixing bacteroids. Leghemoglobin oxygen carriers in the plant cytoplasm protect nitrogenase from molecular oxygen while facilitating bacteroid respiration [17]. Yshaped terminally differentiated bacteroids are aligned around the vacuole, perpendicular to the cell wall, a structural feature that is correlated with the spatial organisation of the cytoskeleton [18]. A senescence zone in the basal region contains degrading bacteroids and collapsing plant cells. Similar differentiation steps featuring several important differences (Table 1) occur during the development of typically round-shaped determinate nodules, but in a temporal succession. The importance of tight spatial control of cellular differentiation is illustrated by the complexity of post-tran-
Box 1 Signal transduction from Nod factor (NF) perception to nuclear events assuming Ca2+-spiking continues during pre-infection thread (PIT) and infection thread (IT) formation Recognition of rhizobial NF is mediated by LysM receptor-like kinases (RLKs) (Lotus japonicus NF receptors NFR1 and NFR5, Medicago truncatula NFP), which are associated with remorins (REM) (B Lefe`bvre, T Ott, unpublished data). NFs induce early downstream responses such as ion fluxes, ROS concentration changes, Ca2+spiking and early nodulin gene expression (for recent reviews see [47,48]). Reorganisation of the cytoskeleton [18] contributes to root hair deformation and curling. The microtubule (MT) network and actin filaments (AF) extend into the curled root hair, and are remodelled during IT formation and growth [18,49]. At least eight of the genes encoding signal transduction components downstream of the NF receptors are also required for arbuscular mycorrhiza (AM) establishment and are referred to as common symbiosis (SYM) genes. Upstream of Ca2+-spiking, the LRR-RLK SYMRK is represented by three distinct gene structures in different angiosperms. SYMRK may have contributed to the predisposition of the eurosid clade to evolve nodulation, because its nodulation competent longest version was exclusively found in this clade [8]. CASTOR and POLLUX are believed to function as counter ion channels during calcium efflux or as trigger for opening calcium channels and induction of Ca2+-spiking [50]. The nucleoporins NUP85 [51], NUP133 [52] and NENA (M Groth, M Parniske, unpublished data), are perhaps required for the transport of a component essential for calcium spiking into the nucleus. Downstream of Ca2+-spiking, the nuclear, preassembled CCaMK–CYCLOPS complex is positioned at a bifurcation of the pathways leading to infection and organogenesis, with CYCLOPS functioning predominantly in the infection process [53].
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The transcriptional regulators NSP1 and NSP2 heterodimerise, perhaps only after NSP2 migration into the nucleus [54]. Through NSP1 and dependent on CCaMK, the heterodimer directly binds the ENOD11, NIN and ERN1 promoters [55]. MtERN1 and MtERN2, potentially in the same complex with NSP1/NSP2, have a role in ENOD11 induction, whereas MtERN3 is a repressor [56,57]. The ENOD11 promoter-binding site of NSP1/NSP2 is located in close proximity to the ERN binding site [55], and NIN negatively regulates the spatial pattern of ENOD11 expression in the epidermis [58]. Additionally, these TFs are also required in the cortex for spontaneous nodulation [36,59,60]. MtRPG, a nuclear-localised long coiled-coil RRP-protein is required for polar infection thread (IT) growth in the epidermis [61]. The IT grows inward as an apoplastic tube, driven by bacterial division and discontinuous sliding movements of bacterial files, and by cell wall matrix disposition and ROS-mediated crosslinking (#) of (hydroxy)proline-rich (glyco) protein (GP) and extensins (EXT) resulting in transition of a fluid-to-solid extracellular matrix (ECM), which limits bacterial division. A confined 60 mm region of the growing IT tip contains the bacteria dividing with a four-hour doubling time [62]. Golgi-derived vesicles accumulate at the tip and fuse to deliver membrane and cell wall compounds, and perhaps nutrients to the dividing bacteria [62]. The requirement for nodulation (nod) gene induction not only in the epidermis but also for the invasion of cortical cells was shown in G. max, M. truncatula and S. rostrata [29,63,64]. b, bacteria; CP, cytoplasm; ER, endoplasmatic reticulum; v, vacuole; PIT, pre-infection thread.
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Box 1 (Continued)
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Figure 1
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Table 1 Two main types of root nodules found in agriculturally important legumes Determinate
Indeterminate
Species (examples) Persistent nodule meristem N-transport Phenotype of nitrogenase mutant
Soybean, bean, Lotus No Ureides Normal bacteroid development
Local auxin transport AON
No change required for ccd Abortion of initiated NP
Pea, Medicago (Galegoids) Trifolium, Vetch Yes Amides Normal bacteroid development, but longer senescence zone and ceased meristematic activity. Flavonol-mediated inhibition required for ccd Inhibition of NP initiation
Bacteroids Poly-b-hydroxy-butyrate accumulation DNA content Number per symbiosome Symbiosome distribution Size, shape Membranes Terminal differentiation Senescence
++ 1C/2C Multiple Random Same as free living (1–2 mm), rod-shaped Not permeable to PI No Survival
– Polynucleoid, mean 24C Single Perpendicular to CW Longer than free living (5–10 mm), Y-shaped Higher permeability to PI Yes Digested by plant
There is considerably larger variation in the legumes, and the bacteroid features listed in this table are based on very few tested legume species [44]. It is therefore possible that the listed features are not consistently distinct between all determinate and indeterminate nodulators. ccd, cortical cell division; AON, autoregulation of nodulation; NP, nodule primordium; CW, cell wall; PI, propidium iodide.
scriptional regulatory mechanisms active during nodule development. The transcription factor (TF) gene MtHAP2-1, for instance, is expressed in the apical nodule meristem, and expression is spatially restricted at the edge of the infection zone by miR169-targeted mRNA degradation and by a trans-acting regulatory peptide uORF1p, a result of alternative splicing in its 50 -leader intron sequence [19,20] (Figure 1).
Systemic and local (hormone) regulation of nodulation Plants control the costs of nitrogen fixation via mechanisms that limit the nodule number. Negative autoregulation of nodulation (AON) occurs via systemic feedback from the shoot to the root (for a recent review see [21]). In Lotus, the shoot-control is mediated by the leucine-rich repeat (LRR) containing CLV1-like receptor HAR1 (orthologues from Medicago: SUNN, or soybean: NARK), mutation of which leads to hypernodulation. The candidates for root-to-shoot communication comprise CLE-RS1/2 peptides [22], perhaps functioning analogous to CLV3 as the ligand of the CLV1-like receptors [23]. The shoot-to-root inhibitory
signal involves hormone transport changes, maybe in combination with other mobile signals [24]. A Rhizobiuminduced NARK-dependent jasmonic acid (JA) synthesis in soybean leaves, for instance, suggests a negative regulatory role for JA in AON [24]. The HAR1-induced inhibitory signal, JA or a mobile signal induced downstream, limits the nodule number, perhaps via Too-Much-Love (TML), which is a negative regulator of nodulation in the inoculated root [25]. In M. truncatula, systemic AON signalling is associated with a reduced auxin transport rate from the shoot to the root [26]. The shoot-controlled sickle (SKL) hypernodulating mutant with a mutation in the EIN2 ethylene signalling gene [27] for instance, also lacks this longdistance auxin transport inhibition. Together with flavonoid-mediated local inhibition of auxin transport in the root cortex, the resulting auxin gradients control meristem induction during indeterminate nodulation in the root cortex [28]. Interestingly, flavonoid-mediated auxin transport inhibition appears not to be involved in determinate nodulation [29].
( Figure 1 Legend ) Nodule differentiation and function. Indeterminate nodule with differentiation zones: the apical meristem (zone I), the infection zone II with invading ITs, the fixation zone III with infected cells (IC) and uninfected cells (UC) and the senescent zone IV. HAP2-1 functions in nodule meristem maintenance, spatially restricted by miR169 and uORF1p. MtERF For Differentiation (EFD) TF mediates differentiation probably via CK signalling inhibition [65]. MtSINA E3 ligases are involved in symbiosome formation [66]. SNARE MtSYP132 [67] and Rab [68] are attached to the membrane at the symbiotic interface, probably mediating vesicle-docking to physically mediate bacterial uptake and symbiosome differentiation. The Astragalus sinicus NODF32 cysteine protease is a determinant of the senescence zone, as delayed senescence was observed after RNAi silencing, resulting in substantially longer indeterminate nodules [69]. A bacteroid within an infected cell is shown enlarged in zone III. Carbon sources are delivered via the DctA transporter into the bacteroid to feed the TCA cycle. Nitrogenase activity results in the production of ammonium, which is delivered to the infected cell via the ammonium transporter AMT1 and is assimilated through GS-GOGAT. A by-product from the nitrogenase reaction is hydrogen, from which valuable reduction equivalents can be rescued through hydrogenase. The induction of transcription of bacterial hydrogenase and its maturation rely on plant-derived factor(s) (PF) perhaps including Nickel (Ni) [45]. Legumes differ with respect to the nitrogen transport form from root to shoot. The amides glutamine and asparagine are transported apoplastically in many legumes, especially those with indeterminate nodules. Tropical legumes with determinate nodules tend to transport ureides (purine derivatives) symplastically. The conversion of ammonium to ureides takes place in uninfected nodule cortical cells. ETC, electron transport chain; Lb, leghemoglobin; PBS, peribacteroid space; PBM, peribacteroid membrane. www.sciencedirect.com
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Initiation of infection in the epidermis is also limited to a specific zone of the developing root. Cell-to-cell communication in the epidermis perhaps via small secreted peptides, such as CLE, RALF1 or DVL1 [30], limits the number of responsive root hairs and restricts root hair curling and IT formation. Local as well as shoot-derived signals potentially contribute, because har1, nin and common sym mutants show supernumerous responsive root hairs or an extension of the responsive root zone. Local concentrations of hormones fine-tune symbiotic responsiveness of the root at several levels. The negative regulators of nodulation — ethylene, JA and abscisic acid (ABA) — similarly affect maintenance and NF-concentration responsiveness of Ca2+-spiking but have opposing effects (ABA and JA versus ethylene) on spiking frequency [31,32]. Furthermore, L. japonicus nodulation signalling is negatively influenced by gibberellins (GA) downstream of cytokinin (CK) [33]. The central roles of Lotus Histidine Kinase 1 (LHK1) and M. truncatula MtCRE1 CK receptor genes in nodule organogenesis [34–36] point towards CK as key player in coordinating the infection in the epidermis with nodule meristem induction and maintenance in the cortex (recently reviewed by [37]). Expression of the transcriptional regulator Nodule Inception (NIN) is induced by both, NF and CK, and depends on MtCRE1 in the cortical cells [34], indicating a dual role for NIN in epidermis (Box 1) and cortical NF-induced signalling. Purine derivatives were isolated from nod gene-lacking photosynthetic bradyrhizobia capable to nodulate some Aeschynomene species [38], suggesting that bacterially derived CK compounds may replace NF in de novo organ formation in this symbiosis. Nodule number and nitrogen fixation are subject to an exquisite quantitative regulation at multiple levels. This regulation has evolved to maximise fitness under natural conditions. However, in agricultural systems different criteria apply and slightly altered regulation that allows fixation also in the presence of environmental nitrogen sources may be favourable for sustainability and could perhaps be achieved by introduction of weak alleles of har1 into cultivars.
Symbiosome development Symbiosomes consist of bacteroids surrounded by a plantderived peribacteroid membrane (PBM) (Figure 1). The peribacteroid or symbiosome space is located between the PBM and the bacteroid outer membrane and contains plant proteins that are targeted to this compartment with specific N-terminal signal peptide motifs [39]. The differentiation of bacteria into bacteroids is associated with drastic changes in bacterial lifestyle [40,41]. The metabolism and respiration are adapted to provide sufficient energy for nitrogen fixation at low oxygen concentrations, by the expression of Current Opinion in Plant Biology 2009, 12:491–499
an alternative electron transport chain including a high affinity terminal oxidase, cbb3. Transcriptional regulation of nitrogen fixation genes is complex and differs between rhizobia [42]. The LPS composition changes [10]. There are substantial differences between the symbiosomes in determinate and indeterminate nodules (Table 1). Bacteroid differentiation is, in extreme cases, irreversible, cell division stops, and in Sinorhizobium DNA endoreduplication occurs. In Medicago truncatula, specific cysteine-rich secreted defensin-like peptides (NCRs) [43] from the nodule appear to mediate bacteroid differentiation (W. Van de Velde, P Mergaert and E. Kondorosi, unpublished data). S. meliloti BacA is involved in the initiation of bacteroid differentiation possibly via the perception or uptake of NCRs or other peptides [10,44].
Nitrogen fixation and root nodule metabolism Malate and succinate are the main carbon sources used by the bacteroid [40], and are transported by plant (unknown) and bacterial (dctA) dicarboxylic acid transporters across the PBM and the bacteroid plasma membrane, respectively. Nitrogenase reduces atmospheric nitrogen to ammonium. However, as a side reaction it reduces protons to molecular hydrogen, which constitutes a substantial loss of energy (Figure 1). Rhizobia expressing hydrogenase activity can rescue some of the reduction power. However, rhizobial isolates differ substantially in their ability for hydrogen oxidation. Importantly, expression of hydrogenase in R. leguminosarum is dependent on the host genotype and grafting experiments in pea implicated shoot-derived diffusible factors [45], providing a huge potential for optimisation on the plant side. Ammonium is believed to be the main transport form out of the symbiosome and a PBM located ammonium transporter has been cloned [46]. However, the bacterial amino acid transporter mutants aap and bra result in nitrogen fixing nodules, but the nitrogen is not made available to the plant [41] (Figure 1).
Conclusion It appears that the interaction between legumes and rhizobia is still in an actively diversifying evolutionary phase. In contrast to the early stages at which different legumes appear to share nearly identical signalling mechanisms (Box 1), there are substantial differences among legumes not only in nodule structure (determinate versus indeterminate), but also in metabolism as well as bacteroid development (Table 1). Considering how comparatively little we know about the functional stages of the symbiosis, these examples probably constitute only the tip of the iceberg. Strategies towards optimising nitrogen fixation in legumes should embrace this intrinsic variation. Forward genetic approaches in model legumes will continue to be fruitful for the identification of genes involved in nodule develwww.sciencedirect.com
The unbearable naivety of legumes Den Herder and Parniske 497
opment and function. However, the genes responsible for fine-tuning the symbiosis may not necessarily be the ones that were identified as essential for symbiosis through genetic screens. A direct route towards key genes that determine the efficiency of nitrogen fixation and the agricultural performance of legumes is the exploitation of natural variation. In the near future, routine (re-)sequencing of entire plant genomes through third-generation sequencing technologies will become an economic reality. Sequence comparisons between entire genomes of ecotypes or cultivars will provide us with unprecedented tools for identifying genes that are under positive selection and hence likely to contribute to adaptation to different environmental conditions. In combination with functional genetic approaches, this will ultimately lead to a catalogue of functionally defined SNPs and other genetic variation bringing a genome wide dimension to the concept of molecular breeding for improved nitrogen fixation. This fine-tuning of matching genotypes for full compatibility at the nitrogen fixing stages will be especially fruitful when infection of the inoculated elite rhizobial strain can be promoted and the infection of endogenous competitive but under-performing rhizobia can be suppressed. Therefore, modification of host NF receptors for specific recognition of the NF structure of the elite rhizobium strain may be a promising strategy, although introduction of foreign rhizobia in New Zealand has shown that symbiosis genes can travel fast within the microbial population of the soil thereby eroding the selective advantage of the initially introduced strain [70]. Durable strategies need to be developed that deal with the intrinsic naivety of legumes in choosing their partners.
Acknowledgements The authors would like to thank A Schu¨ßler and A Brachmann for critical reading of the manuscript.
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38. Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre JC, Jaubert M, Simon D, Cartieaux F, Prin Y et al.: Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Science 2007, 316:1307-1312. Bacterial signals other than NF can trigger nodulation, since bacterial nodulation genes are not required for nodulation in certain species. 39. Hohnjec N, Lenz F, Fehlberg V, Vieweg MF, Baier MC, Hause B, Kuster H: The signal peptide of the Medicago truncatula modular nodulin MtNOD25 operates as an address label for the specific targeting of proteins to nitrogen-fixing symbiosomes. Mol Plant Microbe Interact 2009, 22:63-72. 40. Prell J, Poole P: Metabolic changes of rhizobia in legume nodules. Trends Microbiol 2006, 14:161-168. 41. White J, Prell J, James EK, Poole P: Nutrient sharing between symbionts. Plant Physiol 2007, 144:604-614. 42. Becker A, Berges H, Krol E, Bruand C, Ruberg S, Capela D, Lauber E, Meilhoc E, Ampe F, de Bruijn FJ et al.: Global changes in gene expression in Sinorhizobium meliloti 1021 under microoxic and symbiotic conditions. Mol Plant Microbe Interact 2004, 17:292-303. 43. Mergaert P, Nikovics K, Kelemen Z, Maunoury N, Vaubert D, Kondorosi A, Kondorosi E: A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol 2003, 132:161-173. 44. Mergaert P, Uchiumi T, Alunni B, Evanno G, Cheron A, Catrice O, Mausset AE, Barloy-Hubler F, Galibert F, Kondorosi A et al.: Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium–legume symbiosis. Proc Natl Acad Sci U S A 2006, 103:5230-5235. Demonstration that bacteroid differentiation in indeterminate nodulation differs substantially from determinate nodulation, a difference that might be due to the presence of NCRs. 45. Brito B, Toffanin A, Prieto RI, Imperial J, Ruiz-Argueso T, Palacios JM: Host-dependent expression of Rhizobium leguminosarum bv. viciae hydrogenase is controlled at transcriptional and post-transcriptional levels in legume nodules. Mol Plant Microbe Interact 2008, 21:597-604. 46. Kaiser BN, Finnegan PM, Tyerman SD, Whitehead LF, Bergersen FJ, Day DA, Udvardi MK: Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules. Science 1998, 281:1202-1206. 47. Crespi M, Frugier F: De novo organ formation from differentiated cells: root nodule organogenesis. Sci Signal 2008, 1:re11. 48. Oldroyd GE, Downie JA: Coordinating nodule morphogenesis with rhizobial infection in legumes. Annu Rev Plant Biol 2008, 59:519-546. 49. Yokota K, Fukai E, Madsen LH, Jurkiewicz A, Rueda P, Radutoiu S, Held M, Hossain MS, Szczyglowski K, Morieri G et al.: Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti. Plant Cell 2009, 21:267-284. 50. Charpentier M, Bredemeier R, Wanner G, Takeda N, Schleiff E, Parniske M: Lotus japonicus CASTOR and POLLUX are ion channels essential for perinuclear calcium spiking in legume root endosymbiosis. Plant Cell 2008, 20:3467-3479. 51. Saito K, Yoshikawa M, Yano K, Miwa H, Uchida H, Asamizu E, Sato S, Tabata S, Imaizumi-Anraku H, Umehara Y et al.: NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus. Plant Cell 2007, 19:610-624. 52. Kanamori N, Madsen LH, Radutoiu S, Frantescu M, Quistgaard EM, Miwa H, Downie JA, James EK, Felle HH, Haaning LL et al.: A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc Natl Acad Sci U S A 2006, 103:359-364. 53. Yano K, Yoshida S, Muller J, Singh S, Banba M, Vickers K, Markmann K, White C, Schuller B, Sato S et al.: CYCLOPS, a www.sciencedirect.com
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62. Gage DJ: Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev 2004, 68:280-300. 63. Den Herder J, Vanhee C, De Rycke R, Corich V, Holsters M, Goormachtig S: Nod factor perception during infection thread growth fine-tunes nodulation. Mol Plant Microbe Interact 2007, 20:129-137. 64. Zhang J, Subramanian S, Stacey G, Yu O: Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti. Plant J 2009, 57:171-183.
56. Andriankaja A, Boisson-Dernier A, Frances L, Sauviac L, Jauneau A, Barker DG, de Carvalho-Niebel F: AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif. Plant Cell 2007, 19:2866-2885.
65. Vernie T, Moreau S, de Billy F, Plet J, Combier JP, Rogers C, Oldroyd G, Frugier F, Niebel A, Gamas P: EFD is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula. Plant Cell 2008, 20:2696-2713.
57. Middleton PH, Jakab J, Penmetsa RV, Starker CG, Doll J, Kalo P, Prabhu R, Marsh JF, Mitra RM, Kereszt A et al.: An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction. Plant Cell 2007, 19:12211234.
66. Den Herder G, De Keyser A, De Rycke R, Rombauts S, Van de Velde W, Clemente MR, Verplancke C, Mergaert P, Kondorosi E, Holsters M et al.: Seven in absentia proteins affect plant growth and nodulation in Medicago truncatula. Plant Physiol 2008, 148:369-382.
58. Marsh JF, Rakocevic A, Mitra RM, Brocard L, Sun J, Eschstruth A, Long SR, Schultze M, Ratet P, Oldroyd GE: Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulindependent protein kinase. Plant Physiol 2007, 144:324-335.
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68. Cheon CI, Lee NG, Siddique AB, Bal AK, Verma DP: Roles of plant homologs of Rab1p and Rab7p in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de novo during root nodule symbiosis. EMBO J 1993, 12:41254135. 69. Li Y, Zhou L, Li Y, Chen D, Tan X, Lei L, Zhou J: A nodule-specific plant cysteine proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus. New Phytol 2008, 180:185-192. Functional evidence for a cysteine protease in regulating or executing nodule senescence. 70. Sullivan JT, Ronson CW: Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene. Proc Natl Acad Sci USA 1998, 95:5145-5149.
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The unbearable naivety of legumes in symbiosis Griet Den Herder and Martin Parniske The mechanisms by which legumes choose their rhizobial partners operate independently from their ability to fix nitrogen. As a result of this naivety, symbiotic nitrogen fixation is often suboptimal. The initial recognition of the bacterial partner and the subsequent signal transduction in the host root utilises components that are functionally conserved between legumes and probably actinorhiza host plants. However, the later steps, which largely determine symbiotic performance, are subject to ongoing evolutionary diversification of molecular mechanisms. For example, the impact of bacterial effector proteins, the occurrence of terminal bacteroid differentiation and the expression of bacterial hydrogenase, all depend on the plant genotype. Strategies towards increased nitrogen fixation of legumes in agriculture need to encompass this diversification of mechanisms. Address Genetics, Faculty of Biology, University of Munich (LMU), Grosshaderner Strasse 2-4, 82152 Martinsried-Mu¨nchen, Germany Corresponding author: Parniske, Martin ([email protected])
Current Opinion in Plant Biology 2009, 12:491–499 This review comes from a themed issue on Biotic Interactions Edited by Xinnian Dong and Regine Kahmann Available online 23rd July 2009 1369-5266/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2009.05.010
Introduction All legume crops are able to establish nitrogen fixing root nodule symbiosis with rhizobia [1]. However, nitrogen fixation efficiency within a single legume species can vary more than 10-fold, providing a huge potential for optimisation. A major challenge is that the plant has to choose a bacterial partner before this bacterium starts to deliver nitrogen. The plant’s decision is governed by a display of bacterial signals and effectors that have little or nothing to do with their ability to fix nitrogen. This ‘naı¨ve’, imperfect selection process provides ample opportunities for inefficient, parasitic bacteria that induce root nodules and infect them, but fix nitrogen only poorly or not at all. Such bacterial freeloaders are omnipresent and are a practical problem in agriculturally important legumes such as bean and soybean [2]. An extreme case is a Bradyrhizobium japonicum mutant defective for a nitrogenase-encoding gene and hence www.sciencedirect.com
completely unable to fix nitrogen, which induces fully developed root nodules on soybean and competes successfully with the wild type for infection [3]. Multiple loss-of-nodulation events apparent in the phylogeny of legumes [4] are perhaps an indication that giving up nodulation altogether was advantageous over coping with such cheaters. The diversified recognition specificity among legumes towards bacterial Nod factor (NF) may be another reflection of the inefficiency of this selection mechanism. However, there are indications that selection by legumes exists [2] and that it shaped the genomes of rhizobia. For example, nodules with persistent meristems have the potential to stop nodule growth if the rhizobia do not perform within the nitrogen-fixing zone. Moreover, rhizobial nod-genes are typically found in close proximity to the nif-genes linked on mobile genetic elements such as plasmids (pSymA of Sinorhizobium meliloti) or symbiosis islands (Bradyrhizobium japonicum; Mesorhizobium loti) promoting co-transmission of both abilities to potential recipients. A second challenge for optimisation strategies is the finely tuned functional compatibility between adapted host and symbiont pairs at the later stages of symbiosis that can be observed between legumes in their native habitats and endogenous rhizobia [5]. Since allelic variation in multiple unknown host genes determines this functional compatibility, optimal allele combinations need to be re-established after hybridisation steps in plant breeding programs, which will only be possible once the key determinants are identified. In this opinion, we briefly summarise recent progress in unravelling the molecular events underlying the nodule symbiosis, highlighting the diversified processes that operate during the establishment and function in different legume bacterial genotype combinations.
Rhizobia signal with multiple molecules A key bacterial determinant of host specificity is the structure of the secreted lipochitin oligosaccharide NFs, which is recognised by plant LysM containing NF receptor kinases, in which single amino acid changes lead to altered recognition specificities [6]. Biotechnological alteration of the NF recognition specificity of crop legumes to exclusively allow nodulation by a rhizobial high performance inoculum strain while at the same time reduce nodule occupancy by parasitic bacteria has now become a tempting possibility. This recognition event initiates subsequent signal transduction leading to symbiotic responses of the host root (Box 1). The identification of orthologues for genes Current Opinion in Plant Biology 2009, 12:491–499
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encoding early signal transduction components from different legume and actinorhiza host species suggests that the pathway for the inception of nodulation is ancient and conserved among nodulating plants [7,8,9]. In addition, the precise polysaccharide composition of the rhizobial cell wall (EPS, LPS and cyclic glucans) is important for successful infection. The panel and impact of polysaccharides varies between rhizobia, with the EPS of S. meliloti preventing defence responses [10,11]. The cellulase CelC2 of R. leguminosarum was found to be important for infection and shown to lyse the host plant cell wall at the point of entry [12]. Bacterial effector proteins injected into the host cell via type 3 or type 4 secretion systems can also influence symbiotic performance. Some of the rhizobial effectors share sequence similarity with the effectors of human or plant pathogens [13]. In the broad host range Rhizobium NGR234, an acetyltransferase of the Yersinia outer protein L (YopL) family was shown to acetylate MAPkinases in vitro and to affect symbiosome differentiation [14]. NopT is a protease that improved the symbiosis with Phaseolus but was detrimental for the interaction with Crotalaria juncea [15]. The quantitative and specific effects of rhizobial effectors make them likely determinants of symbiotic performance. It is therefore important to identify the molecular targets of rhizobial effectors in the legume host cell as well as the stage at which they act during development [16].
Root nodules differ in structure and molecular mechanisms Nodule structure, bacteroid differentiation and molecular mechanisms underlying development and function vary strongly between legumes, are host-determined and may, in part, reflect adaptations to different climatic conditions [4] (Table 1). A major differentiating trait is the presence of a persistent meristem. Indeterminate nodules have a persistent apical meristem and, in the elongated mature nodule, a spatial differentiation resulting in different developmental zones (Figure 1). Infection threads (ITs) arrive in the differentiating and expanding cells of the infection zone and release rhizobia into unwalled, membrane-bound infection droplets. In the fixation zone, the bacteria differentiate to become nitrogen-fixing bacteroids. Leghemoglobin oxygen carriers in the plant cytoplasm protect nitrogenase from molecular oxygen while facilitating bacteroid respiration [17]. Yshaped terminally differentiated bacteroids are aligned around the vacuole, perpendicular to the cell wall, a structural feature that is correlated with the spatial organisation of the cytoskeleton [18]. A senescence zone in the basal region contains degrading bacteroids and collapsing plant cells. Similar differentiation steps featuring several important differences (Table 1) occur during the development of typically round-shaped determinate nodules, but in a temporal succession. The importance of tight spatial control of cellular differentiation is illustrated by the complexity of post-tran-
Box 1 Signal transduction from Nod factor (NF) perception to nuclear events assuming Ca2+-spiking continues during pre-infection thread (PIT) and infection thread (IT) formation Recognition of rhizobial NF is mediated by LysM receptor-like kinases (RLKs) (Lotus japonicus NF receptors NFR1 and NFR5, Medicago truncatula NFP), which are associated with remorins (REM) (B Lefe`bvre, T Ott, unpublished data). NFs induce early downstream responses such as ion fluxes, ROS concentration changes, Ca2+spiking and early nodulin gene expression (for recent reviews see [47,48]). Reorganisation of the cytoskeleton [18] contributes to root hair deformation and curling. The microtubule (MT) network and actin filaments (AF) extend into the curled root hair, and are remodelled during IT formation and growth [18,49]. At least eight of the genes encoding signal transduction components downstream of the NF receptors are also required for arbuscular mycorrhiza (AM) establishment and are referred to as common symbiosis (SYM) genes. Upstream of Ca2+-spiking, the LRR-RLK SYMRK is represented by three distinct gene structures in different angiosperms. SYMRK may have contributed to the predisposition of the eurosid clade to evolve nodulation, because its nodulation competent longest version was exclusively found in this clade [8]. CASTOR and POLLUX are believed to function as counter ion channels during calcium efflux or as trigger for opening calcium channels and induction of Ca2+-spiking [50]. The nucleoporins NUP85 [51], NUP133 [52] and NENA (M Groth, M Parniske, unpublished data), are perhaps required for the transport of a component essential for calcium spiking into the nucleus. Downstream of Ca2+-spiking, the nuclear, preassembled CCaMK–CYCLOPS complex is positioned at a bifurcation of the pathways leading to infection and organogenesis, with CYCLOPS functioning predominantly in the infection process [53].
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The transcriptional regulators NSP1 and NSP2 heterodimerise, perhaps only after NSP2 migration into the nucleus [54]. Through NSP1 and dependent on CCaMK, the heterodimer directly binds the ENOD11, NIN and ERN1 promoters [55]. MtERN1 and MtERN2, potentially in the same complex with NSP1/NSP2, have a role in ENOD11 induction, whereas MtERN3 is a repressor [56,57]. The ENOD11 promoter-binding site of NSP1/NSP2 is located in close proximity to the ERN binding site [55], and NIN negatively regulates the spatial pattern of ENOD11 expression in the epidermis [58]. Additionally, these TFs are also required in the cortex for spontaneous nodulation [36,59,60]. MtRPG, a nuclear-localised long coiled-coil RRP-protein is required for polar infection thread (IT) growth in the epidermis [61]. The IT grows inward as an apoplastic tube, driven by bacterial division and discontinuous sliding movements of bacterial files, and by cell wall matrix disposition and ROS-mediated crosslinking (#) of (hydroxy)proline-rich (glyco) protein (GP) and extensins (EXT) resulting in transition of a fluid-to-solid extracellular matrix (ECM), which limits bacterial division. A confined 60 mm region of the growing IT tip contains the bacteria dividing with a four-hour doubling time [62]. Golgi-derived vesicles accumulate at the tip and fuse to deliver membrane and cell wall compounds, and perhaps nutrients to the dividing bacteria [62]. The requirement for nodulation (nod) gene induction not only in the epidermis but also for the invasion of cortical cells was shown in G. max, M. truncatula and S. rostrata [29,63,64]. b, bacteria; CP, cytoplasm; ER, endoplasmatic reticulum; v, vacuole; PIT, pre-infection thread.
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Box 1 (Continued)
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Figure 1
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Table 1 Two main types of root nodules found in agriculturally important legumes Determinate
Indeterminate
Species (examples) Persistent nodule meristem N-transport Phenotype of nitrogenase mutant
Soybean, bean, Lotus No Ureides Normal bacteroid development
Local auxin transport AON
No change required for ccd Abortion of initiated NP
Pea, Medicago (Galegoids) Trifolium, Vetch Yes Amides Normal bacteroid development, but longer senescence zone and ceased meristematic activity. Flavonol-mediated inhibition required for ccd Inhibition of NP initiation
Bacteroids Poly-b-hydroxy-butyrate accumulation DNA content Number per symbiosome Symbiosome distribution Size, shape Membranes Terminal differentiation Senescence
++ 1C/2C Multiple Random Same as free living (1–2 mm), rod-shaped Not permeable to PI No Survival
– Polynucleoid, mean 24C Single Perpendicular to CW Longer than free living (5–10 mm), Y-shaped Higher permeability to PI Yes Digested by plant
There is considerably larger variation in the legumes, and the bacteroid features listed in this table are based on very few tested legume species [44]. It is therefore possible that the listed features are not consistently distinct between all determinate and indeterminate nodulators. ccd, cortical cell division; AON, autoregulation of nodulation; NP, nodule primordium; CW, cell wall; PI, propidium iodide.
scriptional regulatory mechanisms active during nodule development. The transcription factor (TF) gene MtHAP2-1, for instance, is expressed in the apical nodule meristem, and expression is spatially restricted at the edge of the infection zone by miR169-targeted mRNA degradation and by a trans-acting regulatory peptide uORF1p, a result of alternative splicing in its 50 -leader intron sequence [19,20] (Figure 1).
Systemic and local (hormone) regulation of nodulation Plants control the costs of nitrogen fixation via mechanisms that limit the nodule number. Negative autoregulation of nodulation (AON) occurs via systemic feedback from the shoot to the root (for a recent review see [21]). In Lotus, the shoot-control is mediated by the leucine-rich repeat (LRR) containing CLV1-like receptor HAR1 (orthologues from Medicago: SUNN, or soybean: NARK), mutation of which leads to hypernodulation. The candidates for root-to-shoot communication comprise CLE-RS1/2 peptides [22], perhaps functioning analogous to CLV3 as the ligand of the CLV1-like receptors [23]. The shoot-to-root inhibitory
signal involves hormone transport changes, maybe in combination with other mobile signals [24]. A Rhizobiuminduced NARK-dependent jasmonic acid (JA) synthesis in soybean leaves, for instance, suggests a negative regulatory role for JA in AON [24]. The HAR1-induced inhibitory signal, JA or a mobile signal induced downstream, limits the nodule number, perhaps via Too-Much-Love (TML), which is a negative regulator of nodulation in the inoculated root [25]. In M. truncatula, systemic AON signalling is associated with a reduced auxin transport rate from the shoot to the root [26]. The shoot-controlled sickle (SKL) hypernodulating mutant with a mutation in the EIN2 ethylene signalling gene [27] for instance, also lacks this longdistance auxin transport inhibition. Together with flavonoid-mediated local inhibition of auxin transport in the root cortex, the resulting auxin gradients control meristem induction during indeterminate nodulation in the root cortex [28]. Interestingly, flavonoid-mediated auxin transport inhibition appears not to be involved in determinate nodulation [29].
( Figure 1 Legend ) Nodule differentiation and function. Indeterminate nodule with differentiation zones: the apical meristem (zone I), the infection zone II with invading ITs, the fixation zone III with infected cells (IC) and uninfected cells (UC) and the senescent zone IV. HAP2-1 functions in nodule meristem maintenance, spatially restricted by miR169 and uORF1p. MtERF For Differentiation (EFD) TF mediates differentiation probably via CK signalling inhibition [65]. MtSINA E3 ligases are involved in symbiosome formation [66]. SNARE MtSYP132 [67] and Rab [68] are attached to the membrane at the symbiotic interface, probably mediating vesicle-docking to physically mediate bacterial uptake and symbiosome differentiation. The Astragalus sinicus NODF32 cysteine protease is a determinant of the senescence zone, as delayed senescence was observed after RNAi silencing, resulting in substantially longer indeterminate nodules [69]. A bacteroid within an infected cell is shown enlarged in zone III. Carbon sources are delivered via the DctA transporter into the bacteroid to feed the TCA cycle. Nitrogenase activity results in the production of ammonium, which is delivered to the infected cell via the ammonium transporter AMT1 and is assimilated through GS-GOGAT. A by-product from the nitrogenase reaction is hydrogen, from which valuable reduction equivalents can be rescued through hydrogenase. The induction of transcription of bacterial hydrogenase and its maturation rely on plant-derived factor(s) (PF) perhaps including Nickel (Ni) [45]. Legumes differ with respect to the nitrogen transport form from root to shoot. The amides glutamine and asparagine are transported apoplastically in many legumes, especially those with indeterminate nodules. Tropical legumes with determinate nodules tend to transport ureides (purine derivatives) symplastically. The conversion of ammonium to ureides takes place in uninfected nodule cortical cells. ETC, electron transport chain; Lb, leghemoglobin; PBS, peribacteroid space; PBM, peribacteroid membrane. www.sciencedirect.com
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Initiation of infection in the epidermis is also limited to a specific zone of the developing root. Cell-to-cell communication in the epidermis perhaps via small secreted peptides, such as CLE, RALF1 or DVL1 [30], limits the number of responsive root hairs and restricts root hair curling and IT formation. Local as well as shoot-derived signals potentially contribute, because har1, nin and common sym mutants show supernumerous responsive root hairs or an extension of the responsive root zone. Local concentrations of hormones fine-tune symbiotic responsiveness of the root at several levels. The negative regulators of nodulation — ethylene, JA and abscisic acid (ABA) — similarly affect maintenance and NF-concentration responsiveness of Ca2+-spiking but have opposing effects (ABA and JA versus ethylene) on spiking frequency [31,32]. Furthermore, L. japonicus nodulation signalling is negatively influenced by gibberellins (GA) downstream of cytokinin (CK) [33]. The central roles of Lotus Histidine Kinase 1 (LHK1) and M. truncatula MtCRE1 CK receptor genes in nodule organogenesis [34–36] point towards CK as key player in coordinating the infection in the epidermis with nodule meristem induction and maintenance in the cortex (recently reviewed by [37]). Expression of the transcriptional regulator Nodule Inception (NIN) is induced by both, NF and CK, and depends on MtCRE1 in the cortical cells [34], indicating a dual role for NIN in epidermis (Box 1) and cortical NF-induced signalling. Purine derivatives were isolated from nod gene-lacking photosynthetic bradyrhizobia capable to nodulate some Aeschynomene species [38], suggesting that bacterially derived CK compounds may replace NF in de novo organ formation in this symbiosis. Nodule number and nitrogen fixation are subject to an exquisite quantitative regulation at multiple levels. This regulation has evolved to maximise fitness under natural conditions. However, in agricultural systems different criteria apply and slightly altered regulation that allows fixation also in the presence of environmental nitrogen sources may be favourable for sustainability and could perhaps be achieved by introduction of weak alleles of har1 into cultivars.
Symbiosome development Symbiosomes consist of bacteroids surrounded by a plantderived peribacteroid membrane (PBM) (Figure 1). The peribacteroid or symbiosome space is located between the PBM and the bacteroid outer membrane and contains plant proteins that are targeted to this compartment with specific N-terminal signal peptide motifs [39]. The differentiation of bacteria into bacteroids is associated with drastic changes in bacterial lifestyle [40,41]. The metabolism and respiration are adapted to provide sufficient energy for nitrogen fixation at low oxygen concentrations, by the expression of Current Opinion in Plant Biology 2009, 12:491–499
an alternative electron transport chain including a high affinity terminal oxidase, cbb3. Transcriptional regulation of nitrogen fixation genes is complex and differs between rhizobia [42]. The LPS composition changes [10]. There are substantial differences between the symbiosomes in determinate and indeterminate nodules (Table 1). Bacteroid differentiation is, in extreme cases, irreversible, cell division stops, and in Sinorhizobium DNA endoreduplication occurs. In Medicago truncatula, specific cysteine-rich secreted defensin-like peptides (NCRs) [43] from the nodule appear to mediate bacteroid differentiation (W. Van de Velde, P Mergaert and E. Kondorosi, unpublished data). S. meliloti BacA is involved in the initiation of bacteroid differentiation possibly via the perception or uptake of NCRs or other peptides [10,44].
Nitrogen fixation and root nodule metabolism Malate and succinate are the main carbon sources used by the bacteroid [40], and are transported by plant (unknown) and bacterial (dctA) dicarboxylic acid transporters across the PBM and the bacteroid plasma membrane, respectively. Nitrogenase reduces atmospheric nitrogen to ammonium. However, as a side reaction it reduces protons to molecular hydrogen, which constitutes a substantial loss of energy (Figure 1). Rhizobia expressing hydrogenase activity can rescue some of the reduction power. However, rhizobial isolates differ substantially in their ability for hydrogen oxidation. Importantly, expression of hydrogenase in R. leguminosarum is dependent on the host genotype and grafting experiments in pea implicated shoot-derived diffusible factors [45], providing a huge potential for optimisation on the plant side. Ammonium is believed to be the main transport form out of the symbiosome and a PBM located ammonium transporter has been cloned [46]. However, the bacterial amino acid transporter mutants aap and bra result in nitrogen fixing nodules, but the nitrogen is not made available to the plant [41] (Figure 1).
Conclusion It appears that the interaction between legumes and rhizobia is still in an actively diversifying evolutionary phase. In contrast to the early stages at which different legumes appear to share nearly identical signalling mechanisms (Box 1), there are substantial differences among legumes not only in nodule structure (determinate versus indeterminate), but also in metabolism as well as bacteroid development (Table 1). Considering how comparatively little we know about the functional stages of the symbiosis, these examples probably constitute only the tip of the iceberg. Strategies towards optimising nitrogen fixation in legumes should embrace this intrinsic variation. Forward genetic approaches in model legumes will continue to be fruitful for the identification of genes involved in nodule develwww.sciencedirect.com
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opment and function. However, the genes responsible for fine-tuning the symbiosis may not necessarily be the ones that were identified as essential for symbiosis through genetic screens. A direct route towards key genes that determine the efficiency of nitrogen fixation and the agricultural performance of legumes is the exploitation of natural variation. In the near future, routine (re-)sequencing of entire plant genomes through third-generation sequencing technologies will become an economic reality. Sequence comparisons between entire genomes of ecotypes or cultivars will provide us with unprecedented tools for identifying genes that are under positive selection and hence likely to contribute to adaptation to different environmental conditions. In combination with functional genetic approaches, this will ultimately lead to a catalogue of functionally defined SNPs and other genetic variation bringing a genome wide dimension to the concept of molecular breeding for improved nitrogen fixation. This fine-tuning of matching genotypes for full compatibility at the nitrogen fixing stages will be especially fruitful when infection of the inoculated elite rhizobial strain can be promoted and the infection of endogenous competitive but under-performing rhizobia can be suppressed. Therefore, modification of host NF receptors for specific recognition of the NF structure of the elite rhizobium strain may be a promising strategy, although introduction of foreign rhizobia in New Zealand has shown that symbiosis genes can travel fast within the microbial population of the soil thereby eroding the selective advantage of the initially introduced strain [70]. Durable strategies need to be developed that deal with the intrinsic naivety of legumes in choosing their partners.
Acknowledgements The authors would like to thank A Schu¨ßler and A Brachmann for critical reading of the manuscript.
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