Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma.

Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis.

Guidelines for genetic nomenclature and community governance for the model legume Medicago truncatula.

At the 2nd Medicago meeting (a satellite of the 1999 IS-MPMI meeting in Amsterdam), investigators perceived a need for standardization of genetic nomenclature in Medicago truncatula, due to the rapid growth of research on this species in the past few years. Establishment of such standards grew out of discussions begun at this meeting and continued electronically throughout the M. truncatula community. The proposed standards presented here are the consensus results of those discussions. In addition to standards for gene nomenclature, a method for community governance and a website for cataloging gene names and submitting new ones are presented. The purpose of implementing these guidelines is to help maintain consistency in the literature, to avoid redundancy, to contribute to the accuracy of databases, and, in general, to aid the international collaborations that have made M. truncatula a model system for legume biology.

Gene structure and differential regulation of the Rhizobium-induced peroxidase gene rip1.

Primary expression of the Rhizobium meliloti-induced peroxidase gene rip1 occurs prior to nodule morphogenesis, specifically at the site of impending rhizobial infection (D. Cook, D. Dreyer, D. Bonnet, M. Howell, E. Nony, K. VandenBosch [1995] Plant Cell 7: 43-55). We examined the distribution and structure of rip1 transcript throughout nodule development. We determined that expression of rip1 in root tips is correlated with the competence of this zone for symbiotic association, whereas after rhizobial infection rip1 transcript is specifically associated with the zone of nodule development, including nascent nodule primordia. rip1 transcripts are characterized by multiple polyadenylation sites distributed within 200 to 400 bp of the translation stop site, and a single major transcription initiation site in close proximity to the rip1 open reading frame. Thus, rip1 expression is likely to be mediated through effects on a single transcription unit. Immediately 5' of the rip1 transcription unit DNA sequence analysis identified a 377-bp DNA element containing extensive repeat structure that is widely distributed in the Medicago truncatula genome.

Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus.

The tobacco etch potyvirus (TEV) capsid protein (CP) is necessary for cell-to-cell and long distance transport of the virus in plants. In this study, the transport phenotypes of TEV mutants containing CPs with a substitution of the highly conserved Ser122 (termed S122W) within the core domain, or with a deletion of sequences encoding 17 amino acid residues comprising most of the variable C-terminal domain (delta C), were analyzed. The S122W and delta C mutant genomes were amplified to levels comparable to parental virus in protoplasts. The S122W mutant was encapsidation-defective, although in transgenic plants expressing wild-type CP a small number of virions were observed after prolonged incubation. Cells infected by the delta C mutant produced virions, indicating that the C-terminal domain is not necessary for encapsidation. The mutants exhibited unique defects in cell-to-cell and long distance movement in plants. The S122W mutant was confined to single, primarily inoculated epidermal cells in nontransgenic plants, but the cell-to-cell movement defect was rescued efficiently by transgenic CP. Long distance movement of this mutant was also rescued in transgenic plants, but accumulation in systemically infected tissue was low compared to parental virus. The delta C mutant exhibited a slow cell-to-cell movement phenotype in inoculated leaves and a complete inability to move systemically in nontransgenic plants. Transgenic CP was able to rescue partially the slow cell-to-cell movement defect of the delta C mutant, but not the long distance transport defect. Taken together with previous results, these data suggest that the core domain of TEV CP provides a function essential during cell-to-cell movement and that the variable N- and C-terminal regions exposed on the virion surface are necessary for long distance transport. In addition, trans-inhibition models are presented to account for the widely differing transgenic complementation efficiencies of the various movement-defective mutants.

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.

Differential accumulation of four phaseolin glycoforms in transgenic tobacco.

An intron-less phaseolin gene was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical beta-phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (beta wti-) and mutant phaseolin glycoforms (beta dgly1, beta dgly2 and beta dgly1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the beta dgly1 and beta dgly2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin beta dgly1,2 gene. Additionally, the profile of 25-29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.

Developmental regulation of a Rhizobium cell surface antigen during growth of pea root nodules.

A monoclonal antibody, AFRC MAC 203, was used to examine the expression of a nodule-induced cell surface antigen associated with lipopolysaccharide in Rhizobium leguminosarum bv. viciae 3841. Silver-enhanced immunogold-labeled tissue sections revealed that, in very young tissues of pea root nodules, the nodule-induced form of lipopolysaccharide antigen was not expressed either by rhizobia in the infection thread or by bacteria recently released into the plant cell cytoplasm. In the more mature regions of the nodule, the antigen was expressed by membrane-enclosed bacteroids, including immature forms that had not yet expressed the enzyme nitrogenase and were not yet Y shaped. Immunogold labeling of thin sections revealed that the MAC 203 antigen, but not the nitrogenase, was also expressed by bacteria in infection threads situated in and between bacteroid-containing plant cells in mature nodule tissue.

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots.

Three rat hybridoma cell lines have been isolated which produce monoclonal antibodies identifying a noduleenhanced, soluble component of Pisum sativum root nodules. These antibodies each recognized a protease-sensitive band (M(r) 95K) on SDS-polyacrylamide gels. The 95K antigen was resolved by isoelectric focusing into acidic and neutral components which were separately detected by AFRC MAC 236 and MAC 265 respectively. The third antibody (MAC 204) reacted with both acidic and neutral components through an epitope that was sensitive to periodate oxidation. These monoclonal antibodies were used for immunogold localizations at light and electron microscopic levels. In each case, the antigen was shown to be present in the matrix that surrounds the invading rhizobia in infection threads and infection droplets, as well as in the intercellular spaces between plant cell walls of nodules and also of uninfected roots. By contrast, a fourth monoclonal antibody, AFRC JIM 5, labelled a pectic component in the walls of infection threads, and JIM 5 was also found to label the middle lamella of plant cell walls, especially at three-way junctions between cells. The composition and structure of the infection thread lumen is thus comparable to that of an intercellular space.

The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules.

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.

Mutations in Rhizobium phaseoli that lead to arrested development of infection threads.

Two Rhizobium phaseoli mutants, isolated previously by Tn5 mutagenesis, elicited infection threads which ceased development prematurely, usually within root hairs. These infection threads were wide, globular, and otherwise altered in morphology, compared with normal infection threads. Anatomy and division of the root cortical cells during initial stages of nodule morphogenesis appeared normal. However, later nodule differentiation deviated considerably from normal development, and release of bacteria from infection threads was not observed. In tryptone-yeast extract medium the mutants sedimented during growth in shaken cultures and formed rough colonies on agar. Electrophoresis of washed cultures solubilized in dodecyl sulfate revealed that the major carbohydrate band was absent from the mutants. The behavior of this carbohydrate in phenol-water extraction and gel chromatography, its apparent ketodeoxyoctonate content, and its susceptibility to mild acid hydrolysis suggested that it was a lipopolysaccharide. From the results of genetic crosses or reversion analysis, the defect in synthesizing this carbohydrate material and the defect in infection could be attributed to a single mutation in each mutant.

Nodule initiation elicited by noninfective mutants of Rhizobium phaseoli.

Rhizobium phaseoli CE106, CE110, and CE115, originally derived by transposon mutagenesis (Noel et al., J. Bacteriol. 158:149-155, 1984), induced the formation of uninfected root nodule-like swellings on bean (Phaseolus vulgaris). Bacteria densely colonized the root surface, and root hair curling and initiation of root cortical-cell divisions occurred normally in mutant-inoculated seedlings, although no infection threads formed. The nodules were ineffective, lacked leghemoglobin, and were anatomically distinct from normal nodules. Ultrastructural specialization for ureide synthesis, characteristic of legumes that form determinate nodules, was absent. Colony morphology of the mutant strains on agar plates was less mucoid than that of the wild type, and under some cultural conditions, the mutants did not react with Cellufluor, a fluorescent stain for beta-linked polysaccharide. These observations suggest that the genetic lesions in these mutants may be related to extracellular polysaccharide synthesis.

Consequences of Sporangial Development for Nodule Function in Root Nodules of Comptonia peregrina and Myrica gale.

Frankia sp., the actinomycetous endophyte in nitrogen-fixing actinorhizal nodules, may differentiate two forms from its hyphae: vesicles and sporangia. In root nodules of Comptonia peregrina (L.) Coult. and Myrica gale L., sporangia may be either absent or present. Nitrogenase activity and symbiotic efficiency were contrasted in spore(+) and spore(-) nodules of these two host genera. Seedlings of C. peregrina nodulated with the spore(+) inoculum showed only 60% of the nitrogenase activity and 50% of the net size of their spore(-) counterparts after 12 weeks of culture. Measurements of acetylene reduction (i.e., nitrogenase activity) were coordinated with samplings of nodules for structural studies. Significant differences in acetylene reduction rates were discernible between spore(+) and spore(-) nodules commencing 4 weeks after nodulation, concomitant with the maturation of sporangia in the nodule. Spore(+) nodules ultimately reached less than half of the rate of nitrogenase activity of spore(-) nodules. Both types of nodules evolved only small amounts of molecular hydrogen, suggesting that both were equally efficient in recycling electrons lost to the reduction of hydrogen ions by nitrogenase. Respiratory cost of nitrogen fixation, expressed as the quotient of micromole CO(2) to micromole ethylene evolved by excised nodules, was significantly greater in spore(+) than in spore(-) nodules. M. gale spore(-) nodules showed variable effectivity, though all had low CO(2) to ethylene evolution ratios. M. gale spore(+) nodules resembled C. peregrina spore(+), with low effectivity and high respiratory cost for nitrogen fixation.