Host Recognition in the Rhizobium -Soybean Symbiosis

Abstract

Polar binding of Rhizobiumjaponicum to roots and root hairs of Glycine soja (L.) Sieb. and Zucc. is specifically inhibited by D-galactose and N-acetyl-D-galactosamine, haptens of Glycine max seed lectin. A protein, immunologically cross-reactive with the C. max seed lectin, is present in G. soja seed extracts. Peptide mapping of the purified G. max and G. soja lectins indicates that the two are similar in structure. Soybean lectin can be localized on the surface of both G. max and G. soja roots by indirect immunolatex techniques. These observations indicate that the Rhizobium-binding lectin, previously isolated from seeds, also is present on the root surface-the site of the initial steps in the infection. This lectin is capable of binding Rhizobiumjaponicum to the root. Symbiotic nitrogen fixation is a complex process involving physiological and biochemical properties of both the bacterium and the host plant. The first step in this process involves the selective attachment and penetration of the plant by the bacterium. Lectins, proteins that bind carbohydrates, have been implicated as important determinants in the specificity of this first step (2, 3, 10, 15, 32). In the soybean system, a 120,000 dalton glycoprotein with specificity for D-galactose and N-acetyl-D-galactosamine binds specifically to infective strains of Rhizobium japonicum (2, 3, 19-21). This lectin is found in seeds, leaves, stems, and roots of several varieties of Glycine max (23, 26), although it appears to be absent or in very low quantity in a few varieties (26, 29). Even with all of the evidence indicating that the lectin specifically binds to R. japonicum, there has been little direct evidence showing that this lectin actually binds the bacteria to the root. In only one legume system, that of white clover, has direct evidence been presented that a lectin binds the bacterium to the root (9, 10, 12). In that case, the lectin was localized at the site of infection. A hapten of the clover lectin, 2-deoxyglucose, specifically prevents binding of Rhizobium tr#folii to clover roots (9, 1 1). Similar studies to those performed with clover have not been done with soybeans chiefly because of the difficulty of adapting light microscopic studies to a large plant. Here, we overcame the difficulties of using a large plant such as G. max by using Glycine soja, a small-seeded soybean. The nod-ulation specificities of G. max and G. soja are very similar (6). Hymowitz (18) suggested, on cytogenetic evidence, that the two be considered the same species. Therefore, we propose the use of G. soja as a model system to study infection by R. japonicum ' This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and National Science Foundation Grants PCM-7624271 and PFR-7700879 to W. J. B., and SPI-7914901 to G. S. microscopically. The binding of capsular polysaccharides isolated from R. japonicum to the root hairs of G. soja has already been demonstrated (17). We extend these studies by showing that the binding of R. japonicum cells to G. soja roots can be followed easily by light microscopy and that D-galactose and N-acetyl-D-galactosamine will specifically prevent this binding. Furthermore, the G. soja and G. max lectin can be localized on the root surface. The lectin isolated from seeds of G. soja is very similar to that isolated from G. max. This explains the binding similarities of the two plants. MATERIALS AND METHODS Organisms and Plant Material. R. japonicum 31 lb 110, Rhizo-bium meliloti 102F5 1, Rhizobium lupini 96A6 and 96B8, and R. "lotus" 95E8 were generous gifts of J. C. Burton, The Nitragin Co., Milwaukee, Wis. G. max var. Corsoy and G. soja were used for infection and binding studies. Initial seed supplies of G. soja were kindly supplied by Paul Bishop, North Carolina State University. G. max seeds were obtained from the Agronomy Department , University of Wisconsin. The Rhizobium inoculum was grown in yeast extract-mannitol broth (30) at 30 C. The plant nutrient solution used (W. Leps, unpublished results) was as follows: 2 mm CaSO4.2H20; 5 mM K2SO4; 5 mm NaH2PO4. H20; 40 AM ethylene diaminetetraacetic acid ferric salt; 0.5 /IM CoCl2-6H20; 0.5 iM MgSO4.7H20; and I ml/l micronutrient solution (30). The pH of the PNS was adjusted to 7.0 with NaOH after autoclaving. Plants were grown at light intensity of 300 tiE/M2.* swith an 18-h photoperiod at 23-25C. Assay for Binding of Bacteria to Roots. G. soja seeds were sterilized by swirling them for 10 min in Clorox (The Clorox Co., Oakland, Calif.). The seeds were rinsed with sterile distilled H20 and scarified by soaking them in concentrated H2SO4 for 5 min. The seeds were rinsed with sterile H20 and soaked for an additional 10 min in 0.01 N HCI. After the HCI treatment, seeds were rinsed repeatedly with sterile H20 and germinated on sterile moist filter paper for 2 days. R. japonicum binding to G. soja roots was examined by using the slide-culture method of Fahraeus (13). The slide assemblies were modified slightly by raising the coverslip approximately 1-2 mm on a cushion of silicon cement (Dow Corning Co., Midland, Mich.). This provides ample room for root growth and is within the working distance of microscopic observation. Initial binding studies were performed by inoculating each plant-slide assembly with approximately 5 x 108 cells of R. japonicum 311 bl 0. Plants were then incubated for 4 days in sterile, capped beakers containing 35 ml of PNS2. At the end of incubation, the plant-slide assemblies were removed and the plants rinsed with 20 ml of PNS. The plants were then examined using phase contrast or dark field optics at magnifications of 200 and 400. 2 Abbreviations: PNS: plant nutrient solution; PBS: phosphate-buffered saline; SBA: soybean hemagglutinin. 609