Purification and partial characterization of the Rhizobium leguminosarum biovar viciae Ca2+-dependent adhesin, which mediates the first step in attachment of cells of the family Rhizobiaceae to plant root hair tips.

The Ca2+-dependent adhesin which mediates the first step in attachment of bacteria of the family Rhizobiaceae to plant root hair tips was isolated from the surface of Rhizobium leguminosarum biovar viciae cells; its ability to inhibit attachment of R. leguminosarum to pea root hair tips was used as a bioassay. Isolated adhesin was found to be able to inhibit attachment of both carbon-limited and manganese-limited R. leguminosarum cells. A multicolumn purification procedure was developed which resulted in pure adhesin, as judged from silver staining of isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electropherograms. The crucial step in purification was the elution of rhizobial proteins by a CaCl2 gradient from a hydroxyapatite matrix. The specific activity increased 1,250 times during purification. The isoelectric point of the adhesin was determined to be 5.1, and the molecular mass was 14 kilodaltons (kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using gel filtration in the presence and absence of Ca2+, the molecular mass of the adhesin was determined to be 15 and 6 kDa, respectively. The adhesin appeared to be a calcium-binding protein. The purified adhesin inhibited attachment of various other rhizobia to pea root hair tips. Also, cell surface preparations of several other rhizobial strains, including Agrobacterium, Bradyrhizobium, and Phyllobacterium spp., showed adhesin activity, suggesting that a common plant receptor is used for attachment of Rhizobiaceae cells and that the adhesin is common among Rhizobiaceae. No attachment-inhibiting activity was detected in cell surface preparations from various other bacterial strains tested. Cell surface preparations from Sym or Ti plasmid-cured Rhizobium and Agrobacterium strains, respectively, also showed adhesin activity, indicating that Sym or Ti plasmid-borne genes are not required for the synthesis and biogenesis of the adhesin. The adhesin was also found to be involved in the attachment of rhizobia to the root hairs of various other legumes and nonlegume plants, including monocotyledonous ones. Since the adhesin appears to be specific for Rhizobiaceae and is Ca2+ dependent, we propose to designate it rhicadhesin. A more detailed model for rhizobial attachment to plant root hairs is discussed.     

Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips.

As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R. leguminosarum initiates nodulation. This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth. Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation. Attachment of R. leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions. Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips. Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment. Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM. R. leguminosarum produced extracellular fibrils. A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.     

Involvement of both cellulose fibrils and a Ca2+-dependent adhesin in the attachment of Rhizobium leguminosarum to pea root hair tips.

We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process. Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986). In the present paper the chemical and functional characterization of the fibrils of R. leguminosarum is described. Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose. Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay. Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process. Tn5-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas Tn5-induced fibril-negative mutants lost this ability. None of these Tn5 insertions appeared to be located on the Sym plasmid. Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used. The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms. However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis. This phenomenon was found for several Rhizobium spp. It was concluded that both cellulose fibrils and a Ca2+ -dependent adhesin(s) are involved in the attachment of R. leguminosarum to pea root hair tips. A model cap formation as a two-step process is discussed.     

A novel polar surface polysaccharide from Rhizobium leguminosarum binds host plant lectin

Rhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin-glycan mechanism under slightly acidic conditions.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis.

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.     

Entry of Rhizobium leguminosarum bv.viciae into root hairs requires minimal Nod factor specificity, but subsequent infection thread growth requires nodO or nodE

Using various mutant strains of Rhizobium leguminosarum bv. viciae, we have investigated the role of nodO in stimulating infection thread development in vetch and pea. Analysis of R. leguminosarum bv. viciae nodE and nodO mutants revealed no significant difference from the wild-type infection phenotype. Conversely, an R. leguminosarum bv. viciae nodE nodO double mutant was severely impaired in its ability to form normal infection threads. This strain displayed a number of novel infection-related events, including intracellular accumulations of bacteria at the base of root hairs, distended and enlarged infection threads, and reversed threads growing up root hairs. Since normal infection was seen in a nodE mutant, nodO must suppress these abnormal infection phenomena A deletion mutant, retaining only the nodD and nodABCIJ genes, also formed intracellular accumulations at the base of root hairs. Addition of R. leguminosarum bv. viciae nodO could alleviate this phenotype and restore some infection thread formation, although these threads appeared to be abnormal. Exogenous application of R. leguminosarum bv. viciae Nod factors could not alleviate the aberrant infection phenotype. Our results show that the most basic Nod factor structure can allow bacterial entry into the root hair, and that nodO can promote subsequent infection thread development.     

Temperature impact on localization of "free" phenolic compounds in the root tissues and deformation of root hairs in pea seedlings inoculated by Rhizobium

The study was focused on localization of "free" phenolic compounds in pea Pisum sativum L. seedling roots grown at 22 and 8 degrees C 24 h after their inoculation with Rhizobium leguminosarum bv. viceae bacteria. A comparison of phenolic compound distribution along the root in root tissues, and results of observation of root hair development on the root surface, response of root hairs to inoculation, manifesting itself in various deformation degree (bends, twists, ect.) enabled us to reveal differences between roots grown at different temperatures. These differences are basically referred to a sector localized 0-5 mm away from the root tip containing meristematic and extending cells. A distribution of phenolic compounds in sectors with root hairs responding to inoculation by various degrees of contortion practically did not depend on the temperature of plant growth. The evidence provided in the course of this work enabled us to suggest that inhibition of pea root infection at low temperature is caused by decelerated growth processes characteristic of both the root itself and root hairs, as well by a slow increase in the root hair zone.     

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.     

Role of Lectins in the Specific Recognition of Rhizobium by Lotononis bainesii

Fluorescein isothiocyanate (FITC)-labeled lectin purified from the root of Lotononis bainesii Baker was bound by cells of five out of seven L. bainesii-nodulating strains of Rhizobium under culture conditions. With the exception of a strain of Rhizobium leguminosarum, strains of noninfective rhizobia failed to bind the root lectin under these conditions. The two nonlectin binding L. bainesii-specific strains did not bind root lectin on the L. bainesii rhizoplane although this was observed with three other L. bainesii-nodulating strains. A single Rhizobium japonicum strain bound root lectin on the L. bainesii rhizoplane. There was no evidence of an interaction between the L. bainesii seed lectin and the Rhizobium strains tested.

Root lectin-specific FITC-labeled antibodies were bound to the tips of developing root hairs and lateral growth points of more mature root hairs of L. bainesii seedlings. The damaged edges of severed root hairs always bound FITC-labeled root lectin antibody. Seed lectin-specific FITC-labeled antibodies were not bound to the roots of L. bainesii. The preemergent root hair region of L. bainesii was most susceptible to infection by rhizobia but nodules also emerged in the developing and mature root hair regions. Lectin exposed at growth points on L. bainesii root hairs may provide a favorable site for host plant recognition of infective strains of Rhizobium.

     

Bioengineering of symbiotic systems: creation of new associative symbiosis with the use of lectins on the example of tobacco and colza

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.     

Modulation of development, growth dynamics, wall crystallinity, and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii.

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC- mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC- mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.     

Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment or lectin binding.

Rhizobium japonicum USDA 31 demonstrated marked polarity by binding homologous fluorescent antibody (FA) heavily on one end of the cell. FA prepared against R. japonicum strains 110 and 138, and against R. trifolii TA1 cross-reacted with strain 31 only in the polar tip region. No polar immunofluorescing tips could be seen with FA against two other strains of R. japonicum or with those against several unrelated microorganisms. Common antigens localized only in a polar region were seen in many rhizobia stained with R. japonicum 31 FA: 22 of 23 strains of R. japonicum, 10 of 17 strains of R. trifolii, 3 of 7 strains of R. melitolii, 3 of 6 strains of R. phaseoli, and 3 of 9 strains of R. leguminosarum had some cells with detectable polar tips. The proportion of R. japonicum 31 cells with polar tips was high throughout the growth cycle. Polar tip staining was not affected by drastic cell treatments. A function was proposed for the polar tip region as a site for attachment. R. japonicum 31 cells attached to each other in a tip-to-tip fashion and endwise to fungal hyphae with the polar tip in contact with the hyphal wall. Binding of fluorescein isothiocyanate-labeled soybean lectin to certain strains of R. japonicum gave additional evidence of polarity. Polar binding of both antibody and lectin may provide insights into relationships between rhizobia and roots of host legumes.     

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.     

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.     

Attachment of Rhizobium to Legume Roots as the Basis for Specific Interactions

Experiments were conducted to elucidate the basis of the observation that different strains of Rhizobium infect particular legumes. Rhizobia specific for a variety of legumes were grown with ¹³PO^2?₄ and exposed to pea roots (Pisum sativum L.), R. leguminosarum 128C53, which nodulates pea, did not attach to the roots in greater numbers than those strains of rhizobia incapable of infecting pea roots. A complex of R. leguminosarum 128C53 conjugated to a fluorochrome-labeled antibody exhibited a striking attachment to the tips of pea root hairs, where infection normally occurs, but this fluorescent complex also bound to the root hairs of Canavalia en siformis DC., Lupinus polyphyllus Lindl., Trifolium pratense L., and Medicago sativa L., which are not infected by this bacterium. A reproducible, quantitative technique developed for studying interactions between fluorochrome-labeled lectins and rhizobia revealed no relationship between lectin-Rhizobium interactions and the capacity to infect a plant. The data are interpreted as suggesting that simple attachment of Rhizobium to a legume root is not the basis of host-symbiont specificity in this system.     

Artificial colonization of non-symbiotic plants roots with the use of lectins

Rhizobia have the ability to increase growth of non-legume plants due to the production of phytohormones and protection of plant from diseases and pathogens. However, the practical use of these beneficial bacteria sometimes fails because of their inability to effectively colonize rhizoplane and rhizosphere of inoculated plants. We chose the legume lectins as a factor that allows plants to form associative symbiosis with rhizobia. To test the fact that transgenic tobacco, tomato and rape roots with pea lectin gene may affect specific interaction with rhizobia, transgenic roots have been artificially inoculated by fluorescently-labeled pea rhizobia R. leguminosarum and east galega rhizobia Rhizobium galega. Microscopic and microbiological tests have shown that the number of adhered R. leguminosarum onto tobacco, rape and tomato roots which transformed with pea lectin gene is higher in comparison with the control, but no such effect through inoculation of these plants with R. galegae has been found. This confirms the interaction of R. leguminosarum with pea lectin at the surface of transformed roots. Undoubtedly, the improvement of recognition and attachment processes by using lectins can lead to the achievement of a stable associative relationship between non-symbiotic plants and rhizobia.     

The effect of inoculation with Rhizobium leguminosarum on the contents of cytoplasmic protein and free amino acids in the roots of pea seedlings

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root region (0-5 mm) and decreased in the root regions susceptible to rhizobia (5-20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.     

Use of potato extract broth for culturing root-nodule bacteria

Liquid media containing potato extract and 1% of glucose or sucrose were used to culture root-nodule bacteria (rhizobia) in shaken Erlenmeyer flasks. For comparison, these bacteria were also cultured in yeast extract-mannitol broth (YEMB) as a standard medium. Proliferation of rhizobia was monitored by measuring optical densities (OD550) of the cultures and by plate counting of the viable cells (c.f.u) of the bacteria. In general, multiplication of the rhizobia in potato extract-glucose broth (PEGB) and potato extract-sucrose broth (PESB) was markedly faster, as indicated by higher values of OD550, than in YEMB. The numbers of R. leguminosarum by. vicae GGL and S. meliloti 330 in PEGB and PEGB were high and ranged from 1.2 x 10(10) to 4.9 x 10(10) mL(-1) after 48 h of incubation at 28 degrees C. B. japonicum B3S culture in PEGB contained 6.4 x 10(9) c.f.u. ml(-1) after 72 h of incubation. PEGB and YEMB cultures of the rhizobia were similar with respect to their beneficial effects on nodulation of the host-plants of these bacteria.