Role of Lectins in Plant - Microorganism Interactions

The influence of rhizosphere/rhizoplane culture conditions on the ability of various rhizobia to bind soybean seed lectin (SBL) was examined. Eleven strains of the soybean symbiont, Rhizobium japonicun4 and six strains of various heterologous Rhizobium species were cultured in root exudate of soybean ( Glycine max IL.I Merr.) and in association with roots of soybean seedlings which were growing either hydroponically or in montmorillonite clay soil amendment (Turface). All II of the R.japonicum strains developed biochemically specific receptors for the lectin when cultured under these conditions, whereas six of the II did not develop such receptors when cultured in synthetic salts medium. Two cowpea strains also developed receptors for SBL. The other four heterologous strains of rhizobia gave no evidence of biochemically specific SBL binding in either synthetic salts media or rhizosphere/rhizoplane cultures. These results demonstrate that the environment provided by plant roots is an important factor in the development of specific lectin receptors on the cell surface of R. japonicum lation between SBL binding and host-specific infectivity could be discerned. In a previous study (2) we provided evidence in confirmation of Bohlool and Schmidt's original observations (3) on the SBLbinding properties of more than a dozen homologous and heterologous Rhizobium strains. Furthermore, the ability of strains of R. japonicum to bind SBL in a biochemically specific manner was shown to depend greatly on the growth phase of the bacteria in artificial culture media (2). It appeared that the SBL receptors on R. japonicum were transient, rather than constitutive, components of the cell surface, and that the appearance of these receptors might be critically dependent on the growth environment of the bacteria. We suggested that those strains of R. japonicum which gave no evidence of lectin binding at any stage of growth in artificial media might develop SBL receptors under in vivo culture conditions. A study of the influence of root exudate and rhizoplane culture conditions on the development of SBL receptors by various strains of rhizobia is reported here. MATERIALS AND METHODS The ability of plants to respond to the presence of pathogenic or symbiotic microorganisms is an important aspect of their physiology. In order to respond appropriately, a plant must recognize a particular microorganism as a potential pathogen or symbiont. It has been suggested that the carbohydrate-binding plant proteins known as lectins might function in the recognition of microbial pathogens and symbionts by binding to characteristic carbohydrate receptors on the microbial cell surfaces (1, 3). Several recent studies have indicated that microorganism recognition mechanisms based on lectin (or agglutinin) binding may indeed be operative in a variety of plant species (3, 7, 8, 11, 12, 15-17, 20). The results of studies on the role of soybean lectin as a determinant of host specificity in the soybean/Rhizobium japonicum symbiosis, however, have been variously supportive, contradictory, and enigmatic (2-5, 10, 13, 20). The initial study by Bohlool and Schmidt (3) indicated that SBL' bound to 22 of 25 strains of R. japonicum, the soybean symbiont. These authors reported that SBL did not bind to three of the R. japonicum strains or to any of 23 strains representative of various heterologous Rhizobium species. In three subsequent studies (4, 5, 10), however, it was reported that SBL bound to several heterologous strains and failed to bind to several homologous strains, with the result that no clear corre' Supported in part by National Science Foundation Grant BMS 7517710. Contribution No. 605 from the Charles F. Kettering Research Laboratory. 2 whom reprint reque..1s should be sent. 3Abbreviations: FITC: fluorescein isothiocyanate; SBL: soybean lectin; PBS: phosphate-buffered saline. Chemicals and Plant Materials. Eriochrome black was obtained from ICN, Cleveland, Ohio, methyl green from Bio-Rad Laboratories, FITC and D-galactose from Sigma Chemical Company, and N-acetyl-D-galactosamine from Aldrich Chemical Company. Montmorillonite clay soil amendment ('Turface,' regular size) was purchased from Wyandotte Chemical Co. Seeds of soybean (Glycine max [L.] Merr. var. Beeson) were obtained from Dewine and Hanna Seed Co., Yellow Springs, Ohio, and seeds of pea (Pisum sativum var. Wando) from a local garden store. SBL was purified from defatted seed flour (Soya Fluff 200W, Central Soya Chemungy, Chicago, Ill.) by affinity chromatography, labeled with FITC, and repurified by affinity chromatography as described previously (2). Rhizobium Cultures. The sources, maintenance, and culture of the R. japonicum strains have been described (2). Strains of Rhizobium spp. (cowpea group) 641 le and 3G4b4 were obtained froip Dr. J. D. Paxton, University of Illinois, Urbana. The heterologous Rhizobium strains were cultured in the synthetic salts medium (2) supplemented with biotin (0.5 ,tg/l). Preparation of Inoculum. Cultures of Rhizobium strains grown in the synthetic salts medium for 72 hr were used as inoculum. The cultures were harvested by centrifugation at 20,000g for 5 min in sterile centrifuge tubes, washed twice with 30 ml of autoclaved phosphate-buffered saline (PBS, see ref. 2) and suspended in sterile, filtered, Jensen's medium (19). Bacteria in these suspensions were counted with a Petroff-Hauser bacterial counter and diluted with filtered Jensen's medium so that the suspensions contained 2 x 109 cells/ml. Aliquots of such suspensions were used to inoculate the synthetic salts medium, the root exudate media, the seedlings grown under hydroponic conditions, and the seedlings grown in Turface. 71 www.plantphysiol.org on January 28, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. BHUVANESWARI AND BAUER Growth of Plants for Collection of Root Exudate and for Hydroponic Culture. Undamaged soybean seeds were surfacesterilized for 5 min in 0.1% HgCl2, then rinsed and soaked in sterile distilled H20 for 3 hr. The seeds were germinated on yeast extract mannitol agar (19) at 28 ± I C in the dark. Yeast extract mannitol agar was used to detect the growth of any seed-borne microorganisms, including rhizobia. After 72 hr, the seedlings that were free of any detectable microbial contamination were affixed to a sterile piece of Teflon by means of a rubber band and transferred to a test tube (30 x 300 mm) containing 15 ml of filtered Jensen's (N-free) solution. The rootlets of these seedlings remained immersed in the solution. The seedlings were maintained hydroponically in a growth chamber at 28 ± I C and 7,500 lux from fluorescent lamps (16-hr photoperiod). After 7 days, the Jensen's medium containing the root exudate from several plants was pooled, centrifuged at 20,000g for 15 min, and filtered through a Millipore membrane (0. I-,im pore size) to remove small particles of plant debris. The seedlings were then supplied with 15 ml of fresh Jensen's medium containing 0.5 ml of the inoculum suspension, prepared as indicated above. At least four plants were maintained for each strain of Rhizobium tested. Several uninoculated plants were also maintained as controls. Plants and Cultures of Nodulation and Rhizoplane Studies. Aseptically germinated seedlings were planted in test tubes (30 x 200 mm) containing sterilized Turface to a depth of 5 cm. Before planting, 7 ml of Jensen's nutrient solution was added to each tube. These seedlings were inoculated at the time of planting with I ml of the inoculum suspension of a Rhizobium strain. The plants were maintained in the growth chamnber as described earlier. Seven to 10 plants were maintained for each strain of Rhizobium tested. Of these, five to six plants were used for observations of rhizoplane cultures over a 7-day period. The remaining plants were grown for an additional 14 days and then checked for the presence of nodules. Uninoculated plants were used as controls for both studies. RhizoblumCultures in Root Exudate or Synthetic Salts Media. Aliquots (100 Al) of the Rhizobium inoculum suspensions were added to test tubes (15 x 100 mm) containing either 2 ml of the synthetic salts medium or the filter-sterilized root exudates, or to 1.8 ml of the root exudate supplemented with 0.2 ml of 10-foldconcentrated synthetic salts medium. These cultures were incubated at 25 ± I C without shaking. Binding Studies with. FITC-SBL. Rhizobia growing in the hydroponic seedling cultures, root exudate media, and synthetic medium were collected by centrifugation at 20,000g for 5 min, washed once with PBS, and suspended in 500 ,ul of PBS. FITCSBL (25 ,ul of a 3.0 mg/ml solution in PBS) was added to these cell suspensions. After 10-min incubation at room temperature, the cells were sedimented, washed once with PBS, and suspended in a small volume (200 ,ul) of PBS. A portion of this suspension was placed in a Petroff-Hauser counter and the binding of FITCSBL to the bacteria was determined. The lectin-treated bacteria were examined with both fluorescence and phase contrast optics in order to assure that any observed fluorescence was associated with individual bacteria or clumps of bacteria, and in order to estimate the proportion of cells in a sample that bound FITC-SBL. Three replicate determinations were generally made. In addition, the biochemical specificity of FITC-SBL binding to the bacteria was determined by hapten inhibition for each sample. In the hapten inhibition tests, a portion of the cell suspension labeled with FITC-SBL was washed twice with 100 mm galactose in PBS, resuspended in the galactose-PBS solution, and observed again to check the hapten reversibility of binding. In those instances where 100 mm galactose did not remove the bound FITC-SBL, the cells were washed again with 5 mM N-acetyl-D-galactosamine. N-Acetyl-D-galactosamine is also an effective hapten inhibitor of SBL binding (2). The binding of FITC-SBL to the bacteria was normally determined at 36, 60, 84, and 108 hr after inoculation. The sensitivity of the FITC-SBLbinding assay was estimated by treating 250-,ul aliquots of suspensions of 84-hr-old cultures of R. japonicum 311b 138 in synthetic salts medium containing 1.4 x 109 cells/ml with 10-,l aliquots of FITC-SBL containing either 7.2, 0.72, or 0.072 ,ug of lectin. The number of fluorescing cells and the number of nonfluorescing cells were then determined for each sample. A Leitz ortholux II instrument, equipped with a Ploemopak 2.2 incident fluorescence illuminator, FITC-filter module H (2 x KP490/TK5 10/K515), and with 40 X/N.A. 1.3 and 100 X/N.A. 1.25 fluorescence objectives, was used for all microscopy. Binding of FITC-SBL to Rhizobia Growing on Soybean Root Surfaces. Seedlings grown in Turface were collected 2 or 3 days after inoculation and washed with distilled H20 to remove the adhering clay particles. The washed roots were dipped in a dilute solution of FITC-SBL (6 ,ug/ml) in PBS for 10 min, washed with PBS, and then counterstained with eriochrome black and methyl green according to the procedure of Schenk and Churukian (14) to reduce autofluorescence. Older portions of the root system were observed without this counterstaining as these tissues did not have strong autofluorescence.