Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium -induced root nodules of pea cross-react with plasma membranes and Golgi bodies

Abstract

Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognise the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold‐stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS‐polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro‐blotting, it was found that MAC 64 bound to a series of protease‐sensitive bands that migrated in the mol. wt. range 50‐85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteriod membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells.

Analysis of Lipopolysaccharide from Root Nodule Bacteroids of Rhizobium leguminosarum Using Monoclonal Antibodies

“…Free-living cultures of R . l~~guminosurum were normally harvested from 2-3-d-old TY slants as previously described (Brewin et al, 1985). Unless otherwise stated, TY medium contained Difco Tryptone, 5 g 1-l; Difco Yeast Extract, 3 g I-' ; CaCI2.…”

“…The fused cell population was initially distributed into separate culture wells in numbers previously ascertained (under local conditions) to permit the outgrowth of hybrid myelomas at or near a frequency of one clone per well. Ten to 15 d after fusion, supernatants from confluent cultures were tested for McAb that reacted with strain B155 bacteroid antigens by dot immunoassay (Brewin et al, 1985). Selected positive cultures were subsequently cloned at 1 cell per well in flatbottomed microtitre plates (the cloning efficiency of our system is generally about 0-4, ranging between 0-1 and 0-6), using 2000 rad-irradiated rat or mouse peritoneal cells as feeders.…”

“…Sections of pea nodules (1 mm3) were fixed overnight at 4 "C in 2.5% (v/v) glutaraldehyde in 0.2 M-cacodylate buffer pH 7.2, post-fixed with 1 % (w/v) osmium tetroxide and saturated uranyl acetate and progressively dehydrated through to 100% (v/v) ethanol. The fixed material was embedded in LR white resin, which was polymerized by incubation for 16 h at 60 "C (Robertson et ul., 1985;Brewin et al, 1985). Thin sections (silver-gold) were treated with saturated sodium metaperiodate/HCl to remove osmium tetroxide before immunogold staining as previously described (Brewin et ul., 1985).…”

“…Compared to rhizobia grown in free-living culture, the bacteroid forms are larger, usually branched or club-shaped, and individually surrounded by a peribacteroid membrane of plant origin (Newcomb et al, 1979;Robertson & Lyttleton, 1984;Brewin et al, 1985). Their cell walls appear thinner (Tu, 1977) and they may lack the capsular polysaccharide present in free-living bacteria.…”

“…Their cell walls appear thinner (Tu, 1977) and they may lack the capsular polysaccharide present in free-living bacteria. As a consequence, bacteroids are more sensitive to osmotic shock and detergents (van Brussel et al, 1977;Sutton et al, 1977). It has been suggested that during the differentiation of R. japonicum bacteroids the outer membrane is entirely sloughed off and replaced by a new outer membrane (Bal et al, 1980;Bal & Wong, 1982).…”