Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum

Abstract

Plant and bacterial antigens contributing to nodule development and symbiosis in pea (Pisum sativum L.) roots were identified after isolation of a set of monoclonal antibody (McAb)-producing hybridoma lines. Rats were immunised with the peribacteriod material released by mild osmotic shock treatment from membrane-enclosed bacteroids of Rhizobium leguminosarum bv. viceae. In order to diversify the range of McAb specificities, this material was either used as immunogen directly (method 1), or after immunodepletion of a set of glycoprotein and lipopolysaccharide antigens (method 2), or after deglycosylation (method 3). After fusion and screening of cloned hybridoma lines, these three immunisation methods gave respectively 4, 2 and 1 classes of McAb with unique antigen specificities. Ultrastructural immunogold localisation studies showed four different antigens to be present on peribacteriod and plasma membranes (identified by MAC 64, 202, 206 or 209); in addition, a glycoprotein of plant origin but present in the infection-thread matrix was identified by MAC 204. Although none of the epitopes recognised by these McAb was nodule-specific, several were found to be more abundant in extracts of nodule tissue than in uninfected roots (MAC 64, 202, 204, 206). Two McAb reacted with new bacterial antigens: MAC 203 identified a bacterial antigen expressed upon infection but not in free-living cultures of Rhizobium, and MAC 115 identified a bacterial polypeptide (55 kdaltons) that was present in both free-living and bacteroid forms. There were also some McAb of broader specificity that react with antigens present in both plant and bacterial cytoplasms.

Sequential deposition of plant glycoproteins and polysaccharides at the host-parasite interface ofUromyces vignae andVigna sinensis

“…This contrasts with single layered host-parasite interfaces of mycorrhizal cells from GIomus versiforme, where deposition of plant cell wall polysaccharides, such as un-esterified pectin and hydroxyproline-rich glycoproteins at the host-parasite interface, has also been detected (Bonfante-Fasolo et al 1992). Furthermore, Bradley et al (1988) observed labelling of peribacteroid membranes of Rhizobium leguminosarum and plant plasma membrane of Pisum sativum with arabinogalactan antibodies (MAC 209). Their labelling pattern is similar to the result we obtained with the AG/AGP-antibody (CCRC-M-8), the ema was labelled close to the ehm (Fig.…”

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

“…Wild-type, cesa5-1, and sos5-2 seeds were labeled with JIM13 [epitope: Knox et al, 1991;Yates and Knox, 1994;Yates et al, 1996;Majewska-Sawka and Nothnagel, 2000], MAC207 [epitope: a-GlcA-(1,3)-a-GalA-(1,2)-a-Rha; Bradley et al, 1988;Pennell et al, 1989;Yates and Knox, 1994;Yates et al, 1996;Pattathil et al, 2010], JIM8 (epitope: arabinogalactan; Pennell et al, 1991;Majewska-Sawka and Nothnagel, 2000), and CCRC-M7 (epitope: recognizes 6-linked b-D-Gal oligomers that contain arabinose; Puhlmann et al, 1994;Steffan et al, 1995;Pattathil et al, 2010) antibodies (Supplemental Figs. S7 and S8).…”

“…The specificities of the primary antibodies JIM5 and JIM7 (CarboSource) have been extensively described (Liners et al, 1989;Knox et al, 1990;Knox, 1997;Willats et al, 2001aWillats et al, , 2001bWillats et al, , 2001cMacquet et al, 2007a;Pattathil et al, 2010;Xu et al, 2011). JIM13, MAC207, JIM8, and CCRC-M7 (CarboSource) have also been previously described (Bradley et al, 1988;Pennell et al, 1989Pennell et al, , 1991Knox et al, 1991;Puhlmann et al, 1994;Yates and Knox, 1994;Steffan et al, 1995;Yates et al, 1996;Majewska-Sawka and Nothnagel, 2000;Pattathil et al, 2010). CBM3a, specific to more crystalline cellulose, and CBM28, specific to more amorphous cellulose regions (Blake et al, 2006), were treated as primary antibodies in identical solutions before treatment with mouse anti-histidine (Qiagen).…”

Oxidative cross‐linking of plasma membrane arabinogalactan proteins

“…The anti-AGP mAbs were from immunizations with different kinds of plant cell preparations and extracts (Bradley et aL, 1988;Dewey et aL, 1997;Knox et al, 1989;Norman etaL, 1986;Pennell etal., 1989). Sugar beet (Beta vulgaris) cv.…”