Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

Hanging by a thread: invasion of legume plants by rhizobia

Nitrogen-fixing nodules on plants such as alfalfa, pea and vetch arise from the root inner cortex and grow via a persistent meristem. Thus, these nodules are defined as indeterminate. The formation of functional indeterminate nodules requires that symbiotic bacteria, collectively called rhizobia, gain access to the interior of roots and root nodules via infection threads. Recent work has begun to elucidate the important functions of the root cell cytoskeleton in infection thread formation. It has also recently become apparent that rhizobial Nod factors and rhizobial exopolysaccharides play key roles in the initiation and elongation of infection threads.     

Hormone modulation of legume‐rhizobial symbiosis

Leguminous plants can establish symbiotic associations with diazotropic rhizobia to form nitrogenfixating nodules, which are classified as determinate or indeterminate based on the persistence of nodule meristem. The formation of nitrogen-fixing nodules requires coordinating rhizobial infection and root nodule organogenesis. The formation of an infection thread and the extent of nodule formation are largely under plant control, but vary with environmental conditions and the physiological state of the host plants. Many achievements in these two areas have been made in recent decades.Phytohormone signaling pathways have gradually emerged as important regulators of root nodule symbiosis. Cytokinin, strigolactones(SLs) and local accumulation of auxin can promote nodule development. Ethylene,jasmonic acid(JA), abscisic acid(ABA) and gibberellic acid(GA) all negatively regulate infection thread formation and nodule development. However, salicylic acid(SA) and brassinosteroids(BRs) have different effects on the formation of these two nodule types. Some peptide hormones are also involved in nodulation. This review summarizes recent findings on the roles of these plant hormones in legume-rhizobial symbiosis, and we propose that DELLA proteins may function as a node to integrate plant hormones to regulate nodulation.     

Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.     

Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.     

Effects of Boron on Rhizobium-Legume Cell-Surface Interactions and Nodule Development

Boron (B) is an essential micronutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum). By using monoclonal antibodies that recognize specific glycoconjugate components implicated in legume root-nodule development, we investigated the effects of low B on the formation of infection threads and the colonization of pea nodules by Rhizobium leguminosarum bv viciae. In B-deficient nodules the proportion of infected host cells was much lower than in nodules from plants supplied with normal quantities of B. Moreover, the host cells often developed enlarged and abnormally shaped infection threads that frequently burst, releasing bacteria into damaged host cells. There was also an over-production of plant matrix material in which the rhizobial cells were embedded during their progression through the infection thread. Furthermore, in a series of in vitro binding studies, we demonstrated that the presence of B can change the affinity with which the bacterial cell surface interacts with the peribacteroid membrane glycocalyx relative to its interaction with intercellular plant matrix glycoprotein. From these observations we suggest that B plays an important role in mediating cell-surface interactions that lead to endocytosis of rhizobia by host cells and hence to the correct establishment of the symbiosis between pea and Rhizobium.     

Rhizobial secreted proteins as determinants of host specificity in the rhizobium–legume symbiosis

Rhizobia are Gram-negative bacteria than can elicit the formation of specialized organs, called root nodules, on leguminous host plants. Upon infection of the nodules, they differentiate into nitrogen-fixing bacteroids. An elaborate signal exchange precedes the symbiotic interaction. In general, both rhizobia and host plants exhibit narrow specificity. Rhizobial factors contributing to this specificity include Nod factors and surface polysaccharides. It is becoming increasingly clear that protein secretion is important in determining the outcome of the interaction as well. This paper discusses our current understanding of the symbiotic role played by rhizobial secreted proteins, transported both by secretion systems that are of general use, such as the type I secretion system, and by specialized, host-targeting secretion systems, such as the type III, type IV and type VI secretion systems.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.     

Succinoglycan Is Required for Initiation and Elongation of Infection Threads during Nodulation of Alfalfa by Rhizobium meliloti

Rhizobium meliloti Rm1021 must be able to synthesize succinoglycan in order to invade successfully the nodules which it elicits on alfalfa and to establish an effective nitrogen-fixing symbiosis. Using R. meliloti cells that express green fluorescent protein (GFP), we have examined the nature of the symbiotic deficiency of exo mutants that are defective or altered in succinoglycan production. Our observations indicate that an exoY mutant, which does not produce succinoglycan, is symbiotically defective because it cannot initiate the formation of infection threads. An exoZ mutant, which produces succinoglycan without the acetyl modification, forms nitrogen-fixing nodules on plants, but it exhibits a reduced efficiency in the initiation and elongation of infection threads. An exoH mutant, which produces symbiotically nonfunctional high-molecular-weight succinoglycan that lacks the succinyl modification, cannot form extended infection threads. Infection threads initiate at a reduced rate and then abort before they reach the base of the root hairs. Overproduction of succinoglycan by the exoS96::Tn5 mutant does not reduce the efficiency of infection thread initiation and elongation, but it does significantly reduce the ability of this mutant to colonize the curled root hairs, which is the first step of the invasion process. The exoR95::Tn5 mutant, which overproduces succinoglycan to an even greater extent than the exoS96::Tn5 mutant, has completely lost its ability to colonize the curled root hairs. These new observations lead us to propose that succinoglycan is required for both the initiation and elongation of infection threads during nodule invasion and that excess production of succinoglycan interferes with the ability of the rhizobia to colonize curled root hairs.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

The development and structure of root nodules on bambara groundnut [Voandzeia (Vigna)subterranea]

The infection of Vigna subterranea (formerly Voandzeia subterranea) by Bradyrhizobium strain MAO 113 (isolated from V. subterranea) was examined by light and transmission electron microscopy. Bacteria accumulated on the epidermis close to root hairs, and subsequently entered the latter via infection threads. Most of the steps involved in nodule formation were generally characteristic of determinate nodules, such as those which form on the closely related V. radiata. For example, nodule meristems were induced beneath the root epidermis adjacent to infected root hairs, but prior to infection of the meristem by rhizobia. Moreover, after the infection of some of the meristematic cells by the infection threads, and the release of the rhizobia into membrane-bound vesicles, the infection process ceased and dissemination of the rhizobia was by division of already-infected host cells. However, there were some aspects of this process in V. subterranea which have been more commonly described in indeterminate nodules. These include long infection threads entering a number of cells within the meristems simultaneously and a matrix within infection threads which was strongly labelled with immunogold monoclonal antibodies, MAC236 and MAC265, which recognize epitopes on an intercellular glycoprotein. The MAC236 and MAC265 antibodies also recognized material in the unwalled infection droplets surrounding bacteria which were newly-released from the infection threads. The amount of labelling shown was more characteristic of the long infection threads seen in indeterminate nodules such as pea (Pisum sativum) and Neptunia plena. The structure of mature V. subterranea nodules was similar to that described for other determinate nodules such as Glycine max, Vigna unguiculata and V.radiata, i.e. they were spherical and the infected zone consisted of both infected and uninfected cells. Surrounding the infected tissue was an inner cortex of uninfected cell layers containing the putative components of an oxygen diffusion barrier (including glycoprotein-occluded intercellular spaces), and an outer cortex with cells containing calcium oxalate crystals.     

豆科植物根瘤中非共生的内生菌遗传多样性研究进展

在豆科植物与根瘤菌之间结合形成的固氮共生体中,其典型的特征是由特定的微共生体诱导形成的根瘤或茎瘤,除了根瘤菌外,在根瘤中同样也分离出多种与根瘤菌共生固氮无关的内生菌类群,而且根瘤菌与内生菌通常可以共存于同一个根瘤内是普遍存在的客观现象,这些非共生的内生菌生活史的一部分存在于根瘤内且不会引起植物发病,但有关它们的生态学作用还知之甚少,由于其生态上的重要性,近年来对该现象的研究不断深入.就近年来根瘤中隶属于变形菌门,放线菌门、后壁菌门的非共生的内生菌遗传多样性所取得的最新研究结果进行了总结,并介绍了根瘤中相关内生菌多样性研究的新进展.同时,指出了该研究领域存在的问题,并对未来相关研究方向做了展望.     

The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling

The symbiotic infection of the model legume Medicago truncatula by Sinorhizobium meliloti involves marked root hair curling, a stage where entrapment of the microsymbiont occurs in a chamber from which infection thread formation is initiated within the root hair. We have genetically dissected these early symbiotic interactions using both plant and rhizobial mutants and have identified a M. truncatula gene, HCL, which controls root hair curling. S. meliloti Nod factors, which are required for the infection process, induced wild-type epidermal nodulin gene expression and root hair deformation in hcl mutants, while Nod factor induction of cortical cell division foci was reduced compared to wild-type plants. Studies of the position of nuclei and of the microtubule cytoskeleton network of hcl mutants revealed that root hair, as well as cortical cells, were activated in response to S. meliloti. However, the asymmetric microtubule network that is typical of curled root hairs, did not form in the mutants, and activated cortical cells did not become polarised and did not exhibit the microtubular cytoplasmic bridges characteristic of the pre-infection threads induced by rhizobia in M. truncatula. These data suggest that hcl mutations alter the formation of signalling centres that normally provide positional information for the reorganisation of the microtubular cytoskeleton in epidermal and cortical cells.     

Rhizoplane colonisation of peas by Rhizobium leguminosarum bv. viceae and a deleterious Pseudomonas putida

Pseudomonas putida strain A313, a deleterious rhizosphere bacterium, reduced pea nitrogen content when inoculated alone or in combination with Rhizobium leguminosarum bv. viceae on plants in the presence of soil under greenhouse conditions. When plants were grown gnotobiotically in liquid media, mixed inocula of A313 and rhizobia gave a higher proportion of small evenly distributed nodules when compared with a single rhizobial inoculation. In addition, the rhizobial root establishment was reduced by A313 irrespective of inoculum density, indicating that A313 has the capacity to interact with the early rhizobial infection process. When pea seedlings were simultaneously inoculated with A313 and rhizobia, A313 colonised the root hairs to the same extent as the rhizobia, according to analysis by immunofluorescence microscopy. This suggests that the root hair colonisation trait of P. putida interferes with the onset of the symbiotic process.     

<Emphasis Type="Italic">Medicago truncatula</Emphasis> syntaxin SYP132 defines the symbiosome membrane and infection droplet membrane in root nodules

Symbiotic association of legume plants with rhizobia bacteria culminates in organogenesis of nitrogen-fixing root nodules. In indeterminate nodules, plant cells accommodate rhizobial infection by enclosing each bacterium in a membrane-bound, organelle-like compartment called the symbiosome. Numerous symbiosomes occupy each nodule cell; therefore an enormous amount of membrane material must be delivered to the symbiosome membrane for its development and maintenance. Protein delivery to the symbiosome is thought to rely on the plant secretory system; however, the targeting mechanisms are not well understood. In this study, we report the first in-depth analysis of a syntaxin localized on symbiosome membranes. Syntaxins help define a biochemical identity to each compartment in the plant secretory system and facilitate vesicle docking and fusion. Here, we present biochemical and cytological evidence that the SNARE MtSYP132, a Medicago truncatula homologue of Arabidopsis thaliana Syntaxin of Plants 132, localizes to the symbiosome membrane. Using a specific anti-MtSYP132 peptide antibody, we also show that MtSYP132 localizes to the plasma membrane surrounding infection threads and is most abundant on the infection droplet membrane. These results indicate that MtSYP132 may function in infection thread development or growth and the early stages of symbiosome formation. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The diversity of actinorhizal symbiosis

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.