Multiple domains in MtENOD8 protein including the signal peptide target it to the symbiosome

Symbiotic nitrogen fixation occurs in nodules, specialized organs on the roots of legumes. Within nodules, host plant cells are infected with rhizobia that are encapsulated by a plant-derived membrane forming a novel organelle, the symbiosome. In Medicago truncatula, the symbiosome consists of the symbiosome membrane, a single rhizobium, and the soluble space between them, called the symbiosome space. The symbiosome space is enriched with plant-derived proteins, including the M. truncatula EARLY NODULIN8 (MtENOD8) protein. Here, we present evidence from green fluorescent protein (GFP) fusion experiments that the MtENOD8 protein contains at least three symbiosome targeting domains, including its N-terminal signal peptide (SP). When ectopically expressed in nonnodulated root tissue, the MtENOD8 SP delivers GFP to the vacuole. During the course of nodulation, there is a nodule-specific redirection of MtENOD8-SP-GFP from the vacuole to punctate intermediates and subsequently to symbiosomes, with redirection of MtENOD8-SP-GFP from the vacuole to punctate intermediates preceding intracellular rhizobial infection. Experiments with M. truncatula mutants having defects in rhizobial infection and symbiosome development demonstrated that the MtNIP/LATD gene is required for redirection of the MtENOD8-SP-GFP from the vacuoles to punctate intermediates in nodules. Our evidence shows that MtENOD8 has evolved redundant targeting sequences for symbiosome targeting and that intracellular localization of ectopically expressed MtENOD8-SP-GFP is useful as a marker for monitoring the extent of development in mutant nodules.     

Localization of H+-ATPases in soybean root nodules

The localization of H⁺-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H⁺-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H⁺-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids. Received: 25 November 1998 / Accepted: 6 January 1999     

Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.     

Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.

The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.     

<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 .     

Medicago truncatula ENOD40-1 and ENOD40-2 are both involved in nodule initiation and bacteroid development

The establishment of a nitrogen-fixing root nodule on legumes requires the induction of mitotic activity of cortical cells leading to the formation of the nodule primordium and the infection process by which the bacteria enter this primordium. Several genes are up-regulated during these processes, among them ENOD40. Here it is shown, by using gene-specific knock-down of the two Medicago truncatula ENOD40 genes, that both genes are involved in nodule initiation. Further, during nodule development, both genes are essential for bacteroid development.     

A Peanut Nodule Lectin in Infected Cells and in Vacuoles and the Extracellular Matrix of Nodule Parenchyma

Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. The function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most abundant in the nodule parenchyma, where it accumulated in vacuoles, suggesting a possible role as a vegetative storage protein. Lectin was also detected in the extracellular matrix in the nodule parenchyma, a location that corresponds to the tissue layer forming a barrier to oxygen diffusion. The potential for interactions between PNL and other cell wall components, including a previously described high-molecular weight glycoprotein that co-localizes with PNL, is discussed. Within infected cells, lectin was not detectable by immunogold labeling within the cytoplasm, but light labeling was suggestive of lectin localization within the symbiosome lumen. Analysis of fractionated symbiosomes by the more sensitive ELISA technique confirmed that lectin was present within the symbiosome, but was not bound to bacteroids. Our results indicate that PNL probably plays several roles in this nitrogen-fixing symbiosis.     

A novel ankyrin-repeat membrane protein, IGN1, is required for persistence of nitrogen-fixing symbiosis in root nodules of Lotus japonicus

Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix(-) mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8-9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis.     

ENOD12, an early nodulin gene, is not required for nodule formation and efficient nitrogen fixation in alfalfa.

To demonstrate the importance of an extensively studied early nodulin gene ENOD12 in symbiotic nodule development, plants of different Medicago sativa subspecies were tested for the presence or absence of ENOD12 alleles. In M. s. ssp coerulea w2 (Mcw2), two ENOD12 genes were detected, whereas in M. s. ssp quasifalcata k93 (Mqk93) only one gene was present. In both plants, the ENOD12 genes were expressed in nodules induced by Rhizobium meliloti. The nucleotide sequence of the ENOD12 genes showed that the two Mcw2-specific genes were similar to the ENOD12A and ENOD12B genes of the tetraploid M. s. ssp sativa. ENOD12 from Mqk93 was similar to the corresponding gene found in M. truncatula. From the aligned ENOD12 sequences, an evolutionary tree was constructed. Genetic analysis of the progenies of a cross between Mqk93 and Mcw2 showed that several offspring in F1 carried a null allele originating from Mcw2, and among the F2 progenies, plants with the null allele only lacking the ENOD12 gene appeared. Surprisingly, the ENOD12-deficient plants were similar to their wild-type parents in viability, nodule development, nodule structure, and nitrogen fixation efficiency. Therefore, we concluded that in Medicago the ENOD12 gene is not required for symbiotic nitrogen fixation. Furthermore, we proposed that the heterozygous nature of these legumes can be exploited for the identification of mutated alleles of other known nodulin genes; this will permit the construction of plant mutants deficient in these genes.     

Developmentally regulated membrane glycoproteins sharing antigenicity with rhamnogalacturonan II are not detected in nodulated boron deficient Pisum sativum

The peribacteroid membrane (PBM) of symbiosomes from pea root nodules developed in the presence of boron (+B) was labelled by anti-rhamnogalacturonan II (RGII) (anti-rhamnogalacturonan II pectin polysaccharide) antiserum. However, in nodules from plants grown at low boron (-B), anti-RGII pectin polysaccharide did not stain PBMs. Given that RGII pectin binds to borate, and that symbiosomes differentiate aberrantly in -B nodules because of abnormal vesicle traffic, anti-RGII pectin polysaccharide antigens were further analysed. Following electrophoresis and electroblotting, anti-RGII pectin polysaccharide immunostained three bands in +B but not in -B nodule-derived PBMs. A similar banding pattern was observed after the immunostaining of membrane fractions from uninfected roots, indicating that anti-RGII pectin polysaccharide antigens are common to both peribacteroid and plasma membranes. Protease treatment of samples led to disappearance of anti-RGII pectin polysaccharide labelling, indicating that the three immunostained bands correspond to proteins or glycoproteins. The immunochemical study of RGII antigen distribution during nodule development showed that it is strongly present on the PBM of dividing (undifferentiated) symbiosomes but progressively disappeared during symbiosome maturation. In B-deficient nodules, PBMs were never decorated with RGII antigens, and there was an abnormal targeting of vesicles containing pectic polysaccharide (homogalacturanan) to cell membranes. Overall, these results indicate that RGII, boron and certain membrane (glyco)-proteins may interact closely and function cooperatively in membrane processes associated with symbiosome division and general cell growth.     

Cells expressing ENOD2 show differential spatial organization during the development of alfalfa root nodules

We have used in situ hybridization to examine the spatial organization of cells expressing the early nodulin gene (ENOD2) during the development of alfalfa root nodules. ENOD2 gene expression was found in the nodule parenchyma, uninfected cells surrounding the symbiotic region of both effective and ineffective nodules. However, in empty nodules, ENOD2 gene expression was found in a mass of parenchyma cells at the base of the nodule. Similar results were also observed in 11-day-old nodules that contained infected cells but that had not yet begun to express leghemoglobin. Although early events of nodulation result in the induction of ENOD2 expression in cells at the nodule base, the pattern of cells expressing ENOD2 during nodule growth appears to be correlated with the development of other peripheral tissues.     

Immunocytological evidence for abnormal symbiosome development in nodules of the pea mutant line Sprint2Fix− (sym31)

Summary Using a series of antibody probes as markers of symbiosome development, we have investigated the impaired development of symbiosomes in nodules formed by the plant mutant line Sprint2Fix⁻ (sym31). In wild-type pea (Pisum sativum L.) nodules, bacteria differentiate into large pleiomorphic, nitrogen-fixing bacteroids and are singly enclosed within a peribacteroid membrane. In thesym31 mutant, several small undifferentiated bacteroids were often enclosed within one peribacteroid membrane, or were found within a vacuole-like compartment. In wild-type nodules, the monoclonal antibody JIM18, which recognizes a plasmalemma glycolipid antigen, bound to the juvenile peribacteroid membrane, and did not recognize the mature peribacteroid membrane. However, in the mutant, the antibody bound to all peribacteroid membranes within the nodule, suggesting that differentiation of the peribacteroid membrane was arrested. Another antibody, MAC266, recognized plant glycoproteins which normally accumulate in symbiosomes at a late stage of nodule development. Binding of this antibody was much reduced within mutant nodules, labelling only a few mature cells. Similarly, MAC301, which normally recognizes a lipopolysaccharide epitope expressed on differentiated bacteroids prior to the induction of nitrogenase, failed to react with rhizobial cell extracts isolated from nodules of thesym31 mutant. On the basis of these developmental markers, the symbiosomes ofsym31 nodules appeared to be blocked at an early stage of development. The distribution of infection structures was also found to be abnormal in the mutant nodules. Models of symbiosome development are presented and discussed in relation to the morphological and developmental lesions observed in thesym31 mutant.     

Adjustment of Host Cells for Accommodation of Symbiotic Bacteria: Vacuole Defunctionalization,HOPS Suppression,and TIP1g Retargeting in Medicago

In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N₂-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria.     

Purification of ENOD8 proteins from Medicago sativa root nodules and their characterization as esterases.

ENOD8 proteins were purified from alfalfa (Medicago sativa) root nodules. After extraction of ENOD8 proteins into an aqueous buffer, they were purified by ammonium sulfate precipitation, concanavalin A Sepharose chromatography, and ion-exchange chromatography. Purification was assessed by comparing silver stained SDS-PAGE gels to Western blots developed with a highly specific ENOD8 antibody. Multiple ENOD8 proteins that co-purified were found. ENOD8 proteins were found to have esterase activity, active on acetyl and butyrl esters but not longer chain aliphatic esters. Thus, ENOD8 proteins are unlikely to be lipases. Kinetic analysis showed that ENOD8 proteins esterase activity exhibited Michaelis-Menten kinetics. Considering ENOD8 protein sequence similarity to an exopolygalacturonase/EP4/iEP4 and lanatoside 15'-O-acetylesterase with the results presented here predicts that ENOD8 substrates could be acetylated oligo- or polysaccharides.