Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots

Common components of the infection thread matrix and the intercellular space identified by... The EMBO Journal vol.8 no.2 pp.335 - 342, 1989 Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots cell wall (Mort and Grover, 1988), and are released into Kathryn A.VandenBosch1"3, Desmond an 'infection droplet' structure contained by an invagination J1.Bradley" J.Paul Knox', Silvia Perottol 5, of the plant cell membrane (Robertson et al., 1985). Geoffrey W.Butcher2 and Nicholas J.Brewin' Individual bacterial cells are then internalized into the plant and AFRC Institute of Plant Science Research, 'John Innes Institute where they normally develop into nitrogen- cell cytoplasm, Colney Lane, Norwich, NR4 7UH and 2Monoclonal Antibody Centre. fixing endosymbiotic bacteroids enclosed by a plant-derived AFRC Institute of Animal Physiology and Genetics. Babraham. peribacteroid membrane (Brewin et al., 1985). However, CB2 4AT. UK Cambridge, not all legumes are invaded by rhizobia through the 3Biology Department. Texas A and M University. Present addresses: of infection threads (Rolfe and Gresshoff, 1988; development 4Plant Biology Laboratory. Salk College Station. TX 77843-3258. The alternative route involves 'crack entry' Torrey, 1988). Institute for Biological Studies. PO Box 85800, San Diego, CA 92138, through the root epidermis and/or the root cortex by way USA. 5Centro di Studio Sulla Micologia del Terreno. Dipartimento di Biologia Vegetale dell'UniversitA. Viale P.A. Mattioli 25. Torino 10125, of intercellular spaces or the middle lamella as, for example, Italy in Arachis (Chandler, 1978) and (Chandler et Sy'losanthes D. A. Hopwood 1982). Our present data reconcile these two apparently Conmmunicated by al., of entry by suggesting that the infection different modes lines have been isolated which as a specialized anatomical Three rat hybridoma cell thread should be considered produce monoclonal antibodies identifying a nodule- modification of the normal intercellular space, rather than soluble component of Pisum sativum root as a unique symbiosis-specific structure. enhanced, These antibodies each recognized a protease- We have used monoclonal antibodies to identify a 95K nodules. on SDS-polyacrylamide gels. which was considerably more abundant in sensitive band (Mr 95K) plant component by isoelectric focusing into than in uninfected root tissue. The 95K antigen was resolved developing pea nodules the cytological distribution of this acidic and neutral components which were separately Information concerning immunogold labelling, which detected by AFRC MAC 236 and MAC 265 respectively. antigen was obtained by infection thread The third antibody (MAC 204) reacted with both acidic showed it to be a major component of the components through an epitope that was matrix. Additionally, silver enhancement of immunogold and neutral to oxidation. These monoclonal labelling permitted investigation by light microscopy, and sensitive periodate were for localizations at light revealed an extracellular location for the 95K antigen antibodies used immunogold this in Three antibodies reacting against and electron microscopic levels. In each case, the antigen intercellular spaces. antigens, and a fourth that reacts with was shown to be present in the matrix that surrounds infection thread matrix the relationship rhizobia in infection threads and infection cell wall pectin, were used to re-examine the invading as in the intercellular spaces between between the infection thread and the intercellular spaces droplets, as well and also of uninfected roots. formed at the between plant cell walls during the plant cell walls of nodules junctions monoclonal antibody, AFRC JIM normal process of cell division (Jeffree et al., 1986). By contrast, a fourth in the walls of infection 5, labelled a component pectic and 5 was also found to label the middle threads, JIM Results at three-way of cell walls, especially lamella plant of antibodies by Western blot analysis between cells. The composition and structure Screening junctions were examined after one- of the infection thread lumen is thus comparable to that Root and nodule proteins and immuno- electroblotting of an intercellular space. dimensional SDS-PAGE, MAC MAC sativiu with the monoclonal antibodies 204, words: infection thread/nodules/Pisui staining Key The blots are MAC 265 I). resulting pictured 236 and (Table in 1. All three antibodies recognized prominent, Figure -95 kd in mol. wt. MAC band in nodule diffuse tissue, Introduction a similar band in MAC 265 also young 204 and recognized reaction with root tissue was rather faint is intracellular tunnel which root tissue, although infection thread an through The was used as a Incubation of are conducted across the when MAC 236 probe. rhizobia legunme epidermal invading MAC 83 irrelevant blots with antibody) across the cortical cells of the nitrocellulose (an root hair cell and also of in no root nodule and This resulted non-specific labelling plant components. (Turgeon Bauer, 1985). developing of the nodule is cell membrane and plant cell wall After two-dimensional electrophoresis tunnel sheathed by plant the 95K band was resolved into a and Torrey, 1981). Rhizobia do not supernatant fraction, materials (Callaham form and a neutral acidic form (or slightly alkaline) the to fix within the strongly usually develop capacity nitrogen and these two forms showed of the (Figure 2), thread Faria et ait.. 1986). which represents antigen infection (de MAC 204 both of internalization of bacteria different specificities. recognized first in the antibody only the stage process whereas MAC 236 detected the rhizobia the forms of the macromolecule, cells. penetrate plant by plant Subsequently IRL Press : K.A.VandenBosch et al. Table I. Monoclonal antibodies Name Antigens recognized AFRC MAC 204 Matrix of infection glycoprotein threads AFRC MAC 236 Matrix of infection glycoprotein threads AFRC MAC 265 Matrix of infection glycoprotein threads JIM 5 Polygalacturonic acid only the acidic form and MAC 265 detected the only neutral ..... i1)4 I, t < I; form. An immunoaffinity column was constructed by coupling MAC 236 to sepharose, and this was used to isolate _ ji ee a, MAC 236 from antigen the nodule supernatant fraction. After elution of the material retained on the column, this was subjected to gel electrophoresis and immunoblotting. As expected, a 95K band could be detected by immuno- staining with MAC but 236, this band was also found to cross-react with MAC that both 204, suggesting epitopes are carried on the same macromolecule. Biochemical analysis of the antigens Pre-treatment of the nodule supernatant fraction with proteinase K prior to electrophoresis and electroblotting resulted in the complete removal of all three antigens from immunoblots stained with MAC 236 and 204, 265, indicating a protein for component these antigens. Similarly, pretreat- ment of immunoblots with 200 mM sodium metaperiodate under 1. Immunochemical acidic conditions resulted in identification of infection loss of the Fig. thread complete ability glycoproteins monoclonal antibodies MAC MAC 236 to bind MAC using 204, and MAC 265, 204 a antibody, indicating carbohydrate epitope after one-dimensional gel electrophoresis, to electroblotting for both acidic and neutral components. The epitopes nitrocellulose sheets and treatment with anti-rat IgG antibody recognized by MAC 236 and MAC 265 were resistant even to alkaline Lanes conjugated 3 and 5 contain root phosphatase. 1, to high concentrations of and periodate but lanes 4 and 6 contain (200 mM), because proteins 2, nodule supernatant proteins. Positions of the mol. wt markers these are epitopes are carried on the same (Pharmacia, UK) indicated at the macromolecules that right. the MAC carry 204 it is epitope, concluded that all three antibodies related recognize plant glycoproteins. Further evidence that the antibodies recognized carbohydrate groups was sought by monitoring antigen -antibody interactions in ELISA under assays conditions where the addition of monosaccharides or complex glycoproteins might interfere with the reaction binding (Anderson et al., 1987). It was shown that Gum arabic (50 a complex extracellular Atg/ml), glycoprotein from Acacia et senegal (Fincher al., 1983), gave > 90% inhibition of antigen for binding MAC 204 and 50% inhibition for MAC 236 and MAC 265. However, none of the following sugars (100 mM) were inhibitory for antigen -antibody in an binding ELISA competition assay: L-arabinose, D-galactose, D-glucuronic acid, D-galacturonic acid, L-rhamnose, D-arabinose, D-glucose, D-mannose, D-xylose, L-fucose and N-acetyl D-glucosamine. These negative data might be consistent with epitopes that were complex carbohydrates, rather than simple repeating sugar homopolymers, but a more detailed characterization of the epitopes recognized by MAC 204, 236 and 265 must await purification of the antigens in larger quantities than have been obtained so far. H 7: 4 $ pH Immunocytochemical labelling of infection thread pH matrix and wall components i.lg. lmmunochemical staining of infection thread glycoproteins Following immunogold labelling and silver enhancement from nodules pea after two-dimensional gel electrophoresis, (Figure 3), the and distribution of antigens electroblotting immunostaining with detected by MAC MAC 236, MAC 204 and MAC 265. mol. 204, 236 High wt regions of each and 265 was gel are shown and found to be identical. the Heavy labelling position of the 95K antigen is indicated by arrows. The pH was values observed over infection threads and unwalled infection were deduced by slicing a tube gel into 0.5 cm segments and droplets. Additionally, label occurred over some intercellular equilibrating each slice with 0.5 ml 'Analar' water for 30 min. 336 Components of infection thread matrix D,"- T I I T -Sis is a-,' Fig. 3. Localization of the MAC 236 antigen following immunogold labelling and silver enhancement. Views A, B and C depict labelling after incubation with MAC 236. Omission of the primary antibody results in no labelling of comparable adjacent sections (D, E, F). 20 800 x. Bars, 'Um. (A) Labelling of young, infected tissue in a pea nodule. Dense silver deposits indicate the presence of the MAC 236 antigen in infection threads (IT). (B) Some intercellular spaces (IS) in the uninfected nodule cortex clearly contain the MAC 236 antigen. (C) MAC 236 identifies a component of intercellular spaces in cortical tissue from uninfected pea roots. spaces, notably in the inner cortex of the nodule, two to three plant cytoplasm from the infection droplet. Similarly, the cell layers outside the zone of infected cells (Figure 3B). membrane was never observed to be labelled peribacteroid Some intercellular spaces inside the central infected zone also with any of the antibodies. In addition, label was also became labelled. of uninfected Similarly intercellular spaces localized over the matrix present in some intercellular spaces, roots were clearly labelled (Figure 3C), but root cap slime as distinct from the cell wall itself. Labelling of Golgi bodies and root cells were not cap labelled by any of these antibodies was only observed with MAC 265, which sparsely labelled not (data shown). peripheral vesicles associated with the Golgi stack, probably of Electron microscopy confirmed the distribution antigens at the secretory face (Figure 5). 4A-C). The matrix As of observed by light microscopy (Figure a contrast to the labelling the infection thread matrix bacteria in the infection threads and with MAC 236 and the monoclonal JIM material surrounding 204, 265, antibody was and labelled with 5 was used to label sections of pea nodule tissue (Figure infection droplets specifically heavily an earlier 4D-F). JIM 5 recognizes polygalacturonic acid, a major gold particles, confirming preliminary study using MAC 204 the of molecules. The cell walls (Bradley et al., 1988). Moreover, plasma component pectin primary in divided cells near was also in of showed a high pectin content recently membrane labelled, particularly regions material. in accumulation of infection thread matrix the nodule meristem. However, older, non-dividing cells, By contrast, was discernible in the label was restricted to the middle lamella. At material and no largely no matrix antigen in immature cells where the between the distribution of the JIM even three-way junctions cells, peribacteroid spaces, taken into the 5 did not with that of MAC 236 would have been antigen (pectin) overlap bacteria only recently up 337 et al. K.A.VandenBosch M A 2. ':) w " ./ IT MAC 236 I Ti I T ID MAC 2-36 A4 -9OI with monoclonal antibodies with infection thread glycoprotein (MAC 236 4. Thin sections of nodules after immunogold labelling reacting Fig. pea Bars = 0.5 of the infection thread matrix with MAC 265. Note the and MAC or acid (JIM 5). ym. (A) Labelling 265) polygalacturonic the membrane to the infection thread (IT) 22 000 x. (B) After release from an accumulations of matrix material (arrowheads) along plasma adjacent rhizobia (R) enclosed by peribacteroid membrane are no longer surrounded by the infection thread infection thread and infection droplet (ID), as identified MAC 236. 16 500 x. (C) Presence of the MAC 236 antigen in an intercellular space (arrow). The antigen is not glycoprotein, by x. of acid in an infection detectable in the cell wall or middle lamella (double arrowhead). 23 000 (D) Demonstration polygalacturonic (arrowhead) 5 of into the infection droplet, the JIM 5 antigen cannot be detected thread wall using JIM antibody. 22 000 x. (E) Following release bacteria with JIM 5, but the intercellular space (arrow) and around the bacteria. 24 000 x. (F) The middle lamella (double arrowhead) is heavily labelled cell wall (arrowhead) are free of label. 27 000 x. whereas the 95K matrix component): pectin, MAC 265 were present as a matrix, filling the triangular antigen (i.e. the outer zone of the cell as detected JIM marked wall, intercellular space. Similarly, JIM 5 caused heavy labelling by 5, identified MAC MAC 204 and thread but matrix material of infection the 95K components by 236, of infection walls, 338 Components of infection thread matrix MAC 236 j .-- C D MAC 236 A400, PM Four views of Golgi bodies in pea root nodules following Fig. 5. immunogold labelling with MAC 265. 50 000 x. Bar = 0.25 ym. The label tends to occur at the periphery of the Golgi stack, or on associated vesicles. (A, B, C) Cross-sectional views of Golgi bodies. Fig. 7. Immunogold labelling of the MAC 236 antigen on thin sections (D) Face view. of soybean nodules. 28 000 x. Bars = 0.5 ym. (A) The infection thread matrix (arrow), although less abundant in soybean than in peas, MAC 236 is shown to contain the MAC 236 antigen. (B) Abundant labelling by MAC 236 of the matrix (arrow) of an intercellular space. Note the labelling associated with the plasma membrane (PM) in the cells slight the space. bordering Occurrence of the MAC 236 epitope in other plant species Protein preparations from Phaseolus bean and soybean nodules were fractionated by SDS-PAGE, transferred to nitrocellulose sheets and examined by immunostaining with MAC 236. The bands detected were similar to those obtained from nodules, but of slightly different mobility (Figure pea MAC 236 was used as an immunocytochemical 6). When the matrix of infection threads and intercellular spaces probe, in soybean nodules (Figure 7), as observed for was labelled of the peribacteroid space were not labelled pea. The contents in of the preparations from soybean, by MAC 236 any Phaseolus or pea. isolated from the medium of carrot Glycoproteins MAC 236 The also carried the epitope. suspension cultures 1 2 3 4 5 one near and a diffuse two bands, lOOK, antibody recognized This observation indicates at 50K 6. Detection of MAC 236 antigen in legumes and in carrot. band roughly (Figure 6). Fig. the as revealed one-dimensional electrophoresis and immunoblotting. not restricted to but by that the MAC 236 epitope is legumes, Lanes 1, 2 and 3 represent the antigen in pea, bean and soybean in unrelated species. occurs on extracellular glycoproteins nodules, respectively. Lane 4, secreted proteins from carrot suspension known whether either band it is not protein However, culture Lane 5, Gum arabic from Acaicia. supernatant. carrot was related to the band recognized in the preparation in nodules. because Gum arabic detected legume Lastly, shown to react with all three was not labelled with JIM 5, had been threads and infection droplets (from Acacia senegal) and this was also a lack of this in the matrix. At MAC 236 antigen indicating pectic component antibodies 204, 265, and to see when bacteria were released from SDS-PAGE immunoblotting, a later developmental stage examined by wt could be but the threads into infection the intensity of cell a low mol. antigen identified, infection droplets, whether to be of mol. wt and did not JIM 5 was dramatically reduced, although was shown high wall labelling by antigen very labelled MAC 236. the material was still by enter separating gel. the matrix strongly 339 K.A.VandenBosch et al. Discussion the intercellular in the space root cortex sub-epidermal and As the (Turgeon infection thread Bauer, 1985). spreads The three monoclonal antibodies MAC 204, MAC 236 and from cell to cell an creating intracellular tunnel the through MAC 265 appear to recognize different epitopes on the 95K nodule a similar of cell wall developing cortex, process infection thread macromolecules (Figure 1). The two and/or must each disruption degradation accompany cycle epitopes recognized by MAC 236 and MAC 265 were of and exit from the cortical cells. It is entry into, thus mutually exclusive, as seen by isoelectric focusing (Figure to interesting that a common speculate component of the 2), whereas MAC 204 recognized a periodate-sensitive infection thread matrix and the intercellular space might epitope common to both the acidic and neutral forms of 95K to be a cell wall prove or degrading loosening enzyme (Fry, macromolecule. (In older root and leaf tissue from peas, Cassab and 1988; Varner, 1988). MAC 204 also a 40K data recognized glycoprotein: not In the Ulmaceous plant Parasponia (the only non-legume shown.) in order to However, establish whether the known to form a with the rhizobia symbiosis Rhizobium), differences between neutral and acidic forms of the 95K enter the root a break in the at through epidermis the site antigen reside in the or polypeptide carbohydrate com- of Rhizobium-induced cortical cell proliferation and (Lancelle ponents, it will first be to these necessary purify macro- in the more Torrey, 1984). Similarly, primitive legume molecules in substantial from quantities nodule tissue. the invasion of associations, primary rhizobia into the plant The extracellular distribution of the 95K as antigens roots is an intercellular infection by (Torrey, For 1988). visualized by immunogold with labelling MAC MAC 236, in example, peanut (Arachis), infection occurs at the point 204 or MAC 265 antibodies 4 (Figures and 7) was unique of of a lateral root emergence In both (Chandler, 1978). in comparison to other extracellular localized components and Arachis the Parasponia bacteria enter and proliferate with gold probes. The matrix glycoprotein was found in in the intercellular of spaces the root surrounded cortex, by intercellular spaces outside the wall and middle lamella. It an extracellular matrix. Subsequently, infection threads did not overlap in distribution with the pectic component of within develop these cortical and this in cells, turn leads to the wall identified by JIM and it is not an 5, clearly integral the of rhizobia from cell to spread cell, and also in most wall component (Fry, 1988). By JIM 5 contrast, recognized to the release of bacteria as legumes endosymbiotic nitrogen- an epitope with a similar distribution to that very reported bacteroids In fixing (Torrey, such as 1988). legumes pea for rhamnogalacturonan I (Moore and Staehelin, both 1988): and the root hair infection soybean, thread may represent antigens were localized in the middle lamella and were a more specialized proliferation of intercellular which space at especially pronounced three-way junctions between cells. the conveys invading rhizobia through the root epidermis On the other the extracellular hand, extensin glycoprotein an by creating apoplastic (extracellular) tunnel into the root was detected in the cell wall but not in the middle lamella hair cell. The pre-existing mechanism for creating passages or intercellular spaces (Stafstrom and and Staehelin, 1988), between cells thus may have been the exploited by symbiosis a similar distribution was shown also for the hemicellulose for the establishment of an infection thread. xyloglucan (Moore and Staehelin, 1988). It will be interesting to examine whether and how the The infection thread matrix component identified MAC by of the synthesis macromolecules recognized by MAC 204, 204, MAC 236 and MAC 265 is a nodule-enhanced protein, 236 and 265 is stimulated close contact by between plant but it is not a nodulin in the sense defined by Legocki and cells and and how infecting rhizobia, material originating Verma (1980), because it also occurs in uninfected roots from Golgi bodies is (Figure 5) specifically targetted into (Figure where it 1), is restricted to intercellular spaces the infection thread matrix the (or intercellular As space). (Figure 3). Such intercellular spaces are created where a cell regards the function of these macromolecules, it will be plate fuses with the parent cell wall et (Jeffree al., 1986). to how important investigate they interact biochemically with This space between three or more cells is lined with pectic other of cell components plant walls the (Fry, 1988), components (Moore and Staehelin, 1988; this study), and bacterial cell surface and other, as yet unidentified, it is that likely its formation involves the localized action components of the infection thread matrix. of cell wall degrading or loosening enzymes (Jeffree et al., 1986). There are obvious Materials and similarities between the methods structure and formation of these intercellular spaces occurring at three- Biological materials way junctions between cells and the structure and forma- Peas sativum L. cv. (Pisum Wisconsin Perfection) were grown as previously tion of the infection thread which is also frequently initiated described et (Brewin al., 1983), and inoculated with Rhizobium at a three-way cellular junction leguminosarum bv. viceae strain between, for example, a 3841 (Brewin et al., 1985). Soybeans (Glycine max Merr. cv. were curled root hair cell (L.) Pride) inoculated with and two adjacent Bradyrhizobium epidermal cells strain japonicum, USDA 110, and beans (Phaseolus vulgaris L. cv. (Turgeon and Bauer, 1985). Both structures are bounded by Tendergreen) were inoculated with Rhizobium leguminosarum bv. phaseoli, cell pectic wall components and contain the same soluble strain 8002. Both soybeans and beans were grown as previously described matrix material. A very localized role for cell wall disrup- for beans et (Borthakur al., 1986). Nodules were isolated from plants 3 -4 tion and/or weeks after germination and used for degrading enzymes has been postulated immunochemical to explain analysis. Young nodules were harvested 3-4 weeks after germination how, during plant cell and division, the maturing cell plate fuses fractionated using differential centrifugation on sucrose cushions (Brewin with the mother cell wall (Jeffree et al., 1986). Similarly, et al., The 1985). nodule cytoplasmic supernatant fraction was obtained cell wall disruption and/or degradation has been postulated after homogenization of nodules in buffer (50 mM Tris-HCI, pH 7.5; to explain the inception of infection 10 mM thread formation in the dithiothreitol; 0.5 M sucrose; 5 mM p-aminobenzamidine; 5% (w/v) insoluble curled root hair polyvinylpyrrolidone) and centrifugation in an cell (Callaham and Eppendorf Torrey, 1981), and also microfuge at 10 000 for min. g 1 Cytoplasmic proteins from uninoculated roots the mechanism were by which the infection thread exits through similarly obtained by homogenization and centrifugation from the apical the opposite wall of the root hair cell, releasing rhizobia into portions of primary and lateral roots harvested 10 days after germination. 340 of infection thread matrix Components The supernatant fractions of the nodule and in root preparations were microtitre as used described et plates previously (Bradley Protease al., 1988). for the screening antibodies by Western treatment of blotting. to was modified from a antigens prior gel electrophoresis The extracellular glycoprotein derived from the conditioned medium of et previous (Brewin al., Protein procedure 1985). preparations (2 a carrot mg/mil) (Daucus cell carota) suspension culture was prepared by were incubated precipitation at 28°C with Triton X-100 overnight 0.5% and (v/v) with 50% (v/v) ethanol. Gum arabic and other chemical K reagents were As a (0.1 K was proteinase control, omitted. Protease mg/nil). proteinase obtained from Sigma (Poole, unless UK) otherwise was stated. arrested for 3 min in digestion Laemmli solubiliza- by boiling samples tion buffer (Laemmli, 1970). Monoclonal antibodies The rat monoclonal antibodies used in this study are listed in Table I. The and Microscopy immunocytochemistry derivation of MAC 204 has been previously described Pea and (Bradley et al., 1988). nodules and root were fixed in soybean pea tips It involved glutaraldehyde cell line myeloma Y3 AgI.2.3 (Galfre et w/v in al., 1979) and spleen 0.2 M sodium (2.5% in cacodylate buffer, pH 7.0), dehydrated cells from a rat LOU/iap that had been immunized with ethanol and material embedded in LR released White resin Aids, acrylic (Agar Stansted, UK) osmotic shock by treatment of at low peribacteroid-membrane-enclosed and bacteroids. immunolabelled as temperature, described previously is that (Bradley (It probable infection droplet structures et co-purified with For electron membrane- al., thin 1988). sections mounted on microscopy, gold enclosed bacteroids grids the during nodule fractionation were incubated in procedure, and this would diluted culture followed hybridoma supernatant, by account for the goat of presence MAC 204 in antigen the anti-rat nm 'peribacteroid' obtained from fraction.) (15 Janssen Life gold Sciences particles Products, MAC 236 is a similar cell line obtained from the same fusion, and and MAC with Wantage, UK), acetate and lead citrate. finally post-stained uranyl 265 was obtained from a similar fusion The performed for of subsequently with a sections procedure semi-thin mounted on slides labelling glass LOU/C rat. was modified from that of VandenBosch IntenSE II (1986), using silver JIM 5 was raised following immunization of a LOU/C rat with Life protoplasts Sciences to (Janssen enhance the developer Products) gold from labelling carrot prepared cells. The for suspension myeloma line was IR 983 F an black Labelled light microscopy by creating opaque semi- precipitate. (Bazin, 1982). Using apple pectin (Sigma P2157) as on thin sections were antigen microtitre counterstained with azure and blue in II sodium methylene in plates (500 Ag/ml phosphate-buffered incubated and were saline, overnight at with 4°C), metaborate, Ilford Pan F film. photographed Controls for a 200-fold dilution of JIM 5 cell hybridoma culture supernatant included omission of gave 90% the non-specific labelling and substitu- primary antibody maximal was binding. 50% inhibited Binding by 0.8 tion of the polygalacturonic irrelevant MAC 83 for the ltg/ml antibody primary or 6 antibody. mM acid, or D-galacturonic acid, 100 mM D-glucuronic acid. L- rhamnose, L-arabinose, D-glucose, D-xylose, or L-fucose, D-galactose gave no inhibition at 100 mM. Acknowledgements MAC 83 is a rat from the Monoclonal IgM Centre Antibody (AFRC Institute of Animal and K.VdB and Physiology Genetics, J.P.K. were Babraham, Cambridge, the and UK). Food Research supported by Agriculture It the CD2 on recognizes antigen porcine as and S.P. a sectoral T-lymphocytes and, it does Council from the Commission. by training grant European not cross-react with root nodule it was We thank tissue, used as a K.A.Hadfield for negative control assistance with 2-D gels, L.Richardson, J.Cooke, in Western blot and immunocytochemical and for assays. with cell J.King C.Cooper culture and help hybridoma J.A.Downie for comments on the manuscript. Affinity of MAC 236 purification antigen The MAC 236 antibody (30 mg was to protein) coupled 5 ml of cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, References Sweden) according to the manufacturer's instructions, following purification of antibody by from ascitic fluid precipitation made 40% with saturating ammonium Anderson,M.A., Harris,P.J., and Bonig,I. Clarke,A.E. (1987) Planta, 171, This was sulphate. precipitate in 438-442. resuspended phosphate-buffered saline. The column was loaded with affinity a high speed 000 Bazin,H. Prot. Biol. (80 g) supernatant 615-618. (1982) Fluids, 29, fraction derived from nodule homogenates (10 which had been and ml), Bittner,M., Anal. partially Kupferer,P. Norris,C.F. (1980) Biochem., 102, fractionation with purified by ammonium 459-471. sulphate (30-40% saturating), and was with premixed (1: 1) phosphate-buffered saline I % Borthakur,D., Barber,C.E., containing (v/v) Lamb,J.W., and Daniels,M.J., Downie,J.A. The NP40. column was washed under with PBS 0.5% v/v Mol. gravity Johnson,A.W.B. Gen. NP40. (1986) Genet., 320-323. 203, After 10 buffer was column-volumes, as eluting applied recommended by Bradley,D.J., Wood,E.A., Larkins,A.P., Galfre,G., Butcher,G.W. and the manufacturer M 0.5 M (0.2 2.8 + glycine, NaCl, pH 10% Brewin,N.J. 149-160. (v/v) (1988) Planta, dioxane) 173, and fractions were collected. (1 ml) Brewin,N.J., and Wood,E.A. J. Gen. Young,J.P.W. (1983) Microbiol., 2973-2977. 129, SDS - PAGE and Western blots gels Brewin,N.J., Robertson,J.G., Wood,E.A., Wells,B., Larkins,A.P., Proteins were SDS-PAGE and separated by 12% Galfre,G. (Laemmli, Butcher,G.W. EMBO 605-611. 1970) using (1985) J., 4, with of acrylamide 5 loaded mini-gels, lane. The and ug protein per Callaham,D.A. Can. J. proteins Torrey,J.G. (1981) Bot., 1647-1664. 59, were then transferred et and to nitro- Cassab,G.I. Annu. electrophoretically (Towbin al., Varner,J.E. Rev. Plant 1979) (1988) Physiol. Plant Mol. cellulose sheets and 321 (Schleicher Schuell, for 16 h at 10 V Biol., 39, -353. Dassell, FRG) in 25 mM sodium buffer 6.8 et a Chandler,M.R. J. phosphate pH (Bittner al., (1978) Bot., 29, 749-755. 1980) Exp. using Biorad 'Transblot' or apparatus with a blotter Date,R.A. and (Watford, UK), Chandler,M.R., J. semi-dry Roughley,R.J. Bot., (1982) Exp. 33, (Sartorius, the methods 47-57. and buffers of Gottingen, FRG) using Kyhse- Anderson After the nitrocellulose sheets de (1984). were incubated Faria,S.M., and blotting, Can. J. McInroy,S.G. Sprent,J.I. (1986) Bot., 65, with a 553 -558. sequentially solution, anti-rat alkaline blocking primary antibody, goat and as phosphatase conjugate detailed Fincher,G.B., Stone,B.A. and chromogenic substrates, Clarke,A.E. Annu. Rev. (1983) Plant previously et al., 47 - 70. (Bradley 1988). Phvsiol., 34, Two-dimensional were run as described The O'Farrell Plant Cell Wall: gels Fry,S.C. (1988) Growing Chemical and Metabolic by (1975). mix for the first dimension contained urea Acrylamide gel Analysis. Scientific and Longman Technical, Harlow, UK. (9.5 M), Essex, 4% acrylamide NP40 Milton Galfre,G., and (w/v), (2% v/v), (Pharmacia, Milstein,C. Wright,B. Nature, 227, ampholines (1979) 131-133. 3.5-10 and 5-7 Keynes, UK) pH (3% Jeffree,C.E., Dale,J.E. and v/v) (2% 132, ampholines pH v/v): Fry,S.C. (1986) Protoplasma, 90-98. this was in 10.5-cm tubes. After the were (1984) J. Biochem. polymerized Khyse-Anderson,J. Methods, 10, 203 electrophoresis, Biophys. -209. gels electroblotted to nitrocellulose in the normal Laemrnli,U.K. (1970) Nature, 277, 680-685. way. Lancelle,S.A. and Torrey,J.G. (1984) Protoplasma, 123, 26-37. of Analysis and Verma,D.P.S. epitopes Legocki,R.P. (1980) Cell, 20, 153-163. To ascertain the nature of the and Moore,P.J. Staehelin,L.A. MAC (1988) Planta, 174, 433-445. carbohydrate epitopes recognized by MAC 236 and MAC transferred to Mort,A.J. and 204, 265, nitrocellulose were Grover,P.B. Plant (1988) 86, 638-641. proteins Physiol., oxidized treatment to to O'Farrell,B. J. (1975) Biol. Chem., 250, by periodate the 4007-4017. prior immunostaining, according of et al. Woodward Nitrocellulose sheets were procedure incubated Robertson,J.G., Wells,B., Brewin,N.J., (1985). Wood,E.A., and Knight,C.D. Downie,J.A. for I h in 20 or 200 (1985) J. Cell Sci. mM sodium metaperiodate in Suppl., 2, 50 mM sodium 317-331. acetate buffer (pH 4.5), or in buffer alone, followed and Annu. Rev. Plant by 30 min in 50 mM sodium Rolfe,B.G. Gresshoff,P.M. (1988) 39, Physiol., borohydride in Tris-buffered saline 297 -3 19. (pH 7.5). ELISA assays were performed http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots

Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/common-components-of-the-infection-thread-matrix-and-the-intercellular-IsL9dolhKt

References (0)

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 1989
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1002/j.1460-2075.1989.tb03382.x
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal vol.8 no.2 pp.335 - 342, 1989 Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots cell wall (Mort and Grover, 1988), and are released into Kathryn A.VandenBosch1"3, Desmond an 'infection droplet' structure contained by an invagination J1.Bradley" J.Paul Knox', Silvia Perottol 5, of the plant cell membrane (Robertson et al., 1985). Geoffrey W.Butcher2 and Nicholas J.Brewin' Individual bacterial cells are then internalized into the plant and AFRC Institute of Plant Science Research, 'John Innes Institute where they normally develop into nitrogen- cell cytoplasm, Colney Lane, Norwich, NR4 7UH and 2Monoclonal Antibody Centre. fixing endosymbiotic bacteroids enclosed by a plant-derived AFRC Institute of Animal Physiology and Genetics. Babraham. peribacteroid membrane (Brewin et al., 1985). However, CB2 4AT. UK Cambridge, not all legumes are invaded by rhizobia through the 3Biology Department. Texas A and M University. Present addresses: of infection threads (Rolfe and Gresshoff, 1988; development 4Plant Biology Laboratory. Salk College Station. TX 77843-3258. The alternative route involves 'crack entry' Torrey, 1988). Institute for Biological Studies. PO Box 85800, San Diego, CA 92138, through the root epidermis and/or the root cortex by way USA. 5Centro di Studio Sulla Micologia del Terreno. Dipartimento di Biologia Vegetale dell'UniversitA. Viale P.A. Mattioli 25. Torino 10125, of intercellular spaces or the middle lamella as, for example, Italy in Arachis (Chandler, 1978) and (Chandler et Sy'losanthes D. A. Hopwood 1982). Our present data reconcile these two apparently Conmmunicated by al., of entry by suggesting that the infection different modes lines have been isolated which as a specialized anatomical Three rat hybridoma cell thread should be considered produce monoclonal antibodies identifying a nodule- modification of the normal intercellular space, rather than soluble component of Pisum sativum root as a unique symbiosis-specific structure. enhanced, These antibodies each recognized a protease- We have used monoclonal antibodies to identify a 95K nodules. on SDS-polyacrylamide gels. which was considerably more abundant in sensitive band (Mr 95K) plant component by isoelectric focusing into than in uninfected root tissue. The 95K antigen was resolved developing pea nodules the cytological distribution of this acidic and neutral components which were separately Information concerning immunogold labelling, which detected by AFRC MAC 236 and MAC 265 respectively. antigen was obtained by infection thread The third antibody (MAC 204) reacted with both acidic showed it to be a major component of the components through an epitope that was matrix. Additionally, silver enhancement of immunogold and neutral to oxidation. These monoclonal labelling permitted investigation by light microscopy, and sensitive periodate were for localizations at light revealed an extracellular location for the 95K antigen antibodies used immunogold this in Three antibodies reacting against and electron microscopic levels. In each case, the antigen intercellular spaces. antigens, and a fourth that reacts with was shown to be present in the matrix that surrounds infection thread matrix the relationship rhizobia in infection threads and infection cell wall pectin, were used to re-examine the invading as in the intercellular spaces between between the infection thread and the intercellular spaces droplets, as well and also of uninfected roots. formed at the between plant cell walls during the plant cell walls of nodules junctions monoclonal antibody, AFRC JIM normal process of cell division (Jeffree et al., 1986). By contrast, a fourth in the walls of infection 5, labelled a component pectic and 5 was also found to label the middle threads, JIM Results at three-way of cell walls, especially lamella plant of antibodies by Western blot analysis between cells. The composition and structure Screening junctions were examined after one- of the infection thread lumen is thus comparable to that Root and nodule proteins and immuno- electroblotting of an intercellular space. dimensional SDS-PAGE, MAC MAC sativiu with the monoclonal antibodies 204, words: infection thread/nodules/Pisui staining Key The blots are MAC 265 I). resulting pictured 236 and (Table in 1. All three antibodies recognized prominent, Figure -95 kd in mol. wt. MAC band in nodule diffuse tissue, Introduction a similar band in MAC 265 also young 204 and recognized reaction with root tissue was rather faint is intracellular tunnel which root tissue, although infection thread an through The was used as a Incubation of are conducted across the when MAC 236 probe. rhizobia legunme epidermal invading MAC 83 irrelevant blots with antibody) across the cortical cells of the nitrocellulose (an root hair cell and also of in no root nodule and This resulted non-specific labelling plant components. (Turgeon Bauer, 1985). developing of the nodule is cell membrane and plant cell wall After two-dimensional electrophoresis tunnel sheathed by plant the 95K band was resolved into a and Torrey, 1981). Rhizobia do not supernatant fraction, materials (Callaham form and a neutral acidic form (or slightly alkaline) the to fix within the strongly usually develop capacity nitrogen and these two forms showed of the (Figure 2), thread Faria et ait.. 1986). which represents antigen infection (de MAC 204 both of internalization of bacteria different specificities. recognized first in the antibody only the stage process whereas MAC 236 detected the rhizobia the forms of the macromolecule, cells. penetrate plant by plant Subsequently IRL Press : K.A.VandenBosch et al. Table I. Monoclonal antibodies Name Antigens recognized AFRC MAC 204 Matrix of infection glycoprotein threads AFRC MAC 236 Matrix of infection glycoprotein threads AFRC MAC 265 Matrix of infection glycoprotein threads JIM 5 Polygalacturonic acid only the acidic form and MAC 265 detected the only neutral ..... i1)4 I, t < I; form. An immunoaffinity column was constructed by coupling MAC 236 to sepharose, and this was used to isolate _ ji ee a, MAC 236 from antigen the nodule supernatant fraction. After elution of the material retained on the column, this was subjected to gel electrophoresis and immunoblotting. As expected, a 95K band could be detected by immuno- staining with MAC but 236, this band was also found to cross-react with MAC that both 204, suggesting epitopes are carried on the same macromolecule. Biochemical analysis of the antigens Pre-treatment of the nodule supernatant fraction with proteinase K prior to electrophoresis and electroblotting resulted in the complete removal of all three antigens from immunoblots stained with MAC 236 and 204, 265, indicating a protein for component these antigens. Similarly, pretreat- ment of immunoblots with 200 mM sodium metaperiodate under 1. Immunochemical acidic conditions resulted in identification of infection loss of the Fig. thread complete ability glycoproteins monoclonal antibodies MAC MAC 236 to bind MAC using 204, and MAC 265, 204 a antibody, indicating carbohydrate epitope after one-dimensional gel electrophoresis, to electroblotting for both acidic and neutral components. The epitopes nitrocellulose sheets and treatment with anti-rat IgG antibody recognized by MAC 236 and MAC 265 were resistant even to alkaline Lanes conjugated 3 and 5 contain root phosphatase. 1, to high concentrations of and periodate but lanes 4 and 6 contain (200 mM), because proteins 2, nodule supernatant proteins. Positions of the mol. wt markers these are epitopes are carried on the same (Pharmacia, UK) indicated at the macromolecules that right. the MAC carry 204 it is epitope, concluded that all three antibodies related recognize plant glycoproteins. Further evidence that the antibodies recognized carbohydrate groups was sought by monitoring antigen -antibody interactions in ELISA under assays conditions where the addition of monosaccharides or complex glycoproteins might interfere with the reaction binding (Anderson et al., 1987). It was shown that Gum arabic (50 a complex extracellular Atg/ml), glycoprotein from Acacia et senegal (Fincher al., 1983), gave > 90% inhibition of antigen for binding MAC 204 and 50% inhibition for MAC 236 and MAC 265. However, none of the following sugars (100 mM) were inhibitory for antigen -antibody in an binding ELISA competition assay: L-arabinose, D-galactose, D-glucuronic acid, D-galacturonic acid, L-rhamnose, D-arabinose, D-glucose, D-mannose, D-xylose, L-fucose and N-acetyl D-glucosamine. These negative data might be consistent with epitopes that were complex carbohydrates, rather than simple repeating sugar homopolymers, but a more detailed characterization of the epitopes recognized by MAC 204, 236 and 265 must await purification of the antigens in larger quantities than have been obtained so far. H 7: 4 $ pH Immunocytochemical labelling of infection thread pH matrix and wall components i.lg. lmmunochemical staining of infection thread glycoproteins Following immunogold labelling and silver enhancement from nodules pea after two-dimensional gel electrophoresis, (Figure 3), the and distribution of antigens electroblotting immunostaining with detected by MAC MAC 236, MAC 204 and MAC 265. mol. 204, 236 High wt regions of each and 265 was gel are shown and found to be identical. the Heavy labelling position of the 95K antigen is indicated by arrows. The pH was values observed over infection threads and unwalled infection were deduced by slicing a tube gel into 0.5 cm segments and droplets. Additionally, label occurred over some intercellular equilibrating each slice with 0.5 ml 'Analar' water for 30 min. 336 Components of infection thread matrix D,"- T I I T -Sis is a-,' Fig. 3. Localization of the MAC 236 antigen following immunogold labelling and silver enhancement. Views A, B and C depict labelling after incubation with MAC 236. Omission of the primary antibody results in no labelling of comparable adjacent sections (D, E, F). 20 800 x. Bars, 'Um. (A) Labelling of young, infected tissue in a pea nodule. Dense silver deposits indicate the presence of the MAC 236 antigen in infection threads (IT). (B) Some intercellular spaces (IS) in the uninfected nodule cortex clearly contain the MAC 236 antigen. (C) MAC 236 identifies a component of intercellular spaces in cortical tissue from uninfected pea roots. spaces, notably in the inner cortex of the nodule, two to three plant cytoplasm from the infection droplet. Similarly, the cell layers outside the zone of infected cells (Figure 3B). membrane was never observed to be labelled peribacteroid Some intercellular spaces inside the central infected zone also with any of the antibodies. In addition, label was also became labelled. of uninfected Similarly intercellular spaces localized over the matrix present in some intercellular spaces, roots were clearly labelled (Figure 3C), but root cap slime as distinct from the cell wall itself. Labelling of Golgi bodies and root cells were not cap labelled by any of these antibodies was only observed with MAC 265, which sparsely labelled not (data shown). peripheral vesicles associated with the Golgi stack, probably of Electron microscopy confirmed the distribution antigens at the secretory face (Figure 5). 4A-C). The matrix As of observed by light microscopy (Figure a contrast to the labelling the infection thread matrix bacteria in the infection threads and with MAC 236 and the monoclonal JIM material surrounding 204, 265, antibody was and labelled with 5 was used to label sections of pea nodule tissue (Figure infection droplets specifically heavily an earlier 4D-F). JIM 5 recognizes polygalacturonic acid, a major gold particles, confirming preliminary study using MAC 204 the of molecules. The cell walls (Bradley et al., 1988). Moreover, plasma component pectin primary in divided cells near was also in of showed a high pectin content recently membrane labelled, particularly regions material. in accumulation of infection thread matrix the nodule meristem. However, older, non-dividing cells, By contrast, was discernible in the label was restricted to the middle lamella. At material and no largely no matrix antigen in immature cells where the between the distribution of the JIM even three-way junctions cells, peribacteroid spaces, taken into the 5 did not with that of MAC 236 would have been antigen (pectin) overlap bacteria only recently up 337 et al. K.A.VandenBosch M A 2. ':) w " ./ IT MAC 236 I Ti I T ID MAC 2-36 A4 -9OI with monoclonal antibodies with infection thread glycoprotein (MAC 236 4. Thin sections of nodules after immunogold labelling reacting Fig. pea Bars = 0.5 of the infection thread matrix with MAC 265. Note the and MAC or acid (JIM 5). ym. (A) Labelling 265) polygalacturonic the membrane to the infection thread (IT) 22 000 x. (B) After release from an accumulations of matrix material (arrowheads) along plasma adjacent rhizobia (R) enclosed by peribacteroid membrane are no longer surrounded by the infection thread infection thread and infection droplet (ID), as identified MAC 236. 16 500 x. (C) Presence of the MAC 236 antigen in an intercellular space (arrow). The antigen is not glycoprotein, by x. of acid in an infection detectable in the cell wall or middle lamella (double arrowhead). 23 000 (D) Demonstration polygalacturonic (arrowhead) 5 of into the infection droplet, the JIM 5 antigen cannot be detected thread wall using JIM antibody. 22 000 x. (E) Following release bacteria with JIM 5, but the intercellular space (arrow) and around the bacteria. 24 000 x. (F) The middle lamella (double arrowhead) is heavily labelled cell wall (arrowhead) are free of label. 27 000 x. whereas the 95K matrix component): pectin, MAC 265 were present as a matrix, filling the triangular antigen (i.e. the outer zone of the cell as detected JIM marked wall, intercellular space. Similarly, JIM 5 caused heavy labelling by 5, identified MAC MAC 204 and thread but matrix material of infection the 95K components by 236, of infection walls, 338 Components of infection thread matrix MAC 236 j .-- C D MAC 236 A400, PM Four views of Golgi bodies in pea root nodules following Fig. 5. immunogold labelling with MAC 265. 50 000 x. Bar = 0.25 ym. The label tends to occur at the periphery of the Golgi stack, or on associated vesicles. (A, B, C) Cross-sectional views of Golgi bodies. Fig. 7. Immunogold labelling of the MAC 236 antigen on thin sections (D) Face view. of soybean nodules. 28 000 x. Bars = 0.5 ym. (A) The infection thread matrix (arrow), although less abundant in soybean than in peas, MAC 236 is shown to contain the MAC 236 antigen. (B) Abundant labelling by MAC 236 of the matrix (arrow) of an intercellular space. Note the labelling associated with the plasma membrane (PM) in the cells slight the space. bordering Occurrence of the MAC 236 epitope in other plant species Protein preparations from Phaseolus bean and soybean nodules were fractionated by SDS-PAGE, transferred to nitrocellulose sheets and examined by immunostaining with MAC 236. The bands detected were similar to those obtained from nodules, but of slightly different mobility (Figure pea MAC 236 was used as an immunocytochemical 6). When the matrix of infection threads and intercellular spaces probe, in soybean nodules (Figure 7), as observed for was labelled of the peribacteroid space were not labelled pea. The contents in of the preparations from soybean, by MAC 236 any Phaseolus or pea. isolated from the medium of carrot Glycoproteins MAC 236 The also carried the epitope. suspension cultures 1 2 3 4 5 one near and a diffuse two bands, lOOK, antibody recognized This observation indicates at 50K 6. Detection of MAC 236 antigen in legumes and in carrot. band roughly (Figure 6). Fig. the as revealed one-dimensional electrophoresis and immunoblotting. not restricted to but by that the MAC 236 epitope is legumes, Lanes 1, 2 and 3 represent the antigen in pea, bean and soybean in unrelated species. occurs on extracellular glycoproteins nodules, respectively. Lane 4, secreted proteins from carrot suspension known whether either band it is not protein However, culture Lane 5, Gum arabic from Acaicia. supernatant. carrot was related to the band recognized in the preparation in nodules. because Gum arabic detected legume Lastly, shown to react with all three was not labelled with JIM 5, had been threads and infection droplets (from Acacia senegal) and this was also a lack of this in the matrix. At MAC 236 antigen indicating pectic component antibodies 204, 265, and to see when bacteria were released from SDS-PAGE immunoblotting, a later developmental stage examined by wt could be but the threads into infection the intensity of cell a low mol. antigen identified, infection droplets, whether to be of mol. wt and did not JIM 5 was dramatically reduced, although was shown high wall labelling by antigen very labelled MAC 236. the material was still by enter separating gel. the matrix strongly 339 K.A.VandenBosch et al. Discussion the intercellular in the space root cortex sub-epidermal and As the (Turgeon infection thread Bauer, 1985). spreads The three monoclonal antibodies MAC 204, MAC 236 and from cell to cell an creating intracellular tunnel the through MAC 265 appear to recognize different epitopes on the 95K nodule a similar of cell wall developing cortex, process infection thread macromolecules (Figure 1). The two and/or must each disruption degradation accompany cycle epitopes recognized by MAC 236 and MAC 265 were of and exit from the cortical cells. It is entry into, thus mutually exclusive, as seen by isoelectric focusing (Figure to interesting that a common speculate component of the 2), whereas MAC 204 recognized a periodate-sensitive infection thread matrix and the intercellular space might epitope common to both the acidic and neutral forms of 95K to be a cell wall prove or degrading loosening enzyme (Fry, macromolecule. (In older root and leaf tissue from peas, Cassab and 1988; Varner, 1988). MAC 204 also a 40K data recognized glycoprotein: not In the Ulmaceous plant Parasponia (the only non-legume shown.) in order to However, establish whether the known to form a with the rhizobia symbiosis Rhizobium), differences between neutral and acidic forms of the 95K enter the root a break in the at through epidermis the site antigen reside in the or polypeptide carbohydrate com- of Rhizobium-induced cortical cell proliferation and (Lancelle ponents, it will first be to these necessary purify macro- in the more Torrey, 1984). Similarly, primitive legume molecules in substantial from quantities nodule tissue. the invasion of associations, primary rhizobia into the plant The extracellular distribution of the 95K as antigens roots is an intercellular infection by (Torrey, For 1988). visualized by immunogold with labelling MAC MAC 236, in example, peanut (Arachis), infection occurs at the point 204 or MAC 265 antibodies 4 (Figures and 7) was unique of of a lateral root emergence In both (Chandler, 1978). in comparison to other extracellular localized components and Arachis the Parasponia bacteria enter and proliferate with gold probes. The matrix glycoprotein was found in in the intercellular of spaces the root surrounded cortex, by intercellular spaces outside the wall and middle lamella. It an extracellular matrix. Subsequently, infection threads did not overlap in distribution with the pectic component of within develop these cortical and this in cells, turn leads to the wall identified by JIM and it is not an 5, clearly integral the of rhizobia from cell to spread cell, and also in most wall component (Fry, 1988). By JIM 5 contrast, recognized to the release of bacteria as legumes endosymbiotic nitrogen- an epitope with a similar distribution to that very reported bacteroids In fixing (Torrey, such as 1988). legumes pea for rhamnogalacturonan I (Moore and Staehelin, both 1988): and the root hair infection soybean, thread may represent antigens were localized in the middle lamella and were a more specialized proliferation of intercellular which space at especially pronounced three-way junctions between cells. the conveys invading rhizobia through the root epidermis On the other the extracellular hand, extensin glycoprotein an by creating apoplastic (extracellular) tunnel into the root was detected in the cell wall but not in the middle lamella hair cell. The pre-existing mechanism for creating passages or intercellular spaces (Stafstrom and and Staehelin, 1988), between cells thus may have been the exploited by symbiosis a similar distribution was shown also for the hemicellulose for the establishment of an infection thread. xyloglucan (Moore and Staehelin, 1988). It will be interesting to examine whether and how the The infection thread matrix component identified MAC by of the synthesis macromolecules recognized by MAC 204, 204, MAC 236 and MAC 265 is a nodule-enhanced protein, 236 and 265 is stimulated close contact by between plant but it is not a nodulin in the sense defined by Legocki and cells and and how infecting rhizobia, material originating Verma (1980), because it also occurs in uninfected roots from Golgi bodies is (Figure 5) specifically targetted into (Figure where it 1), is restricted to intercellular spaces the infection thread matrix the (or intercellular As space). (Figure 3). Such intercellular spaces are created where a cell regards the function of these macromolecules, it will be plate fuses with the parent cell wall et (Jeffree al., 1986). to how important investigate they interact biochemically with This space between three or more cells is lined with pectic other of cell components plant walls the (Fry, 1988), components (Moore and Staehelin, 1988; this study), and bacterial cell surface and other, as yet unidentified, it is that likely its formation involves the localized action components of the infection thread matrix. of cell wall degrading or loosening enzymes (Jeffree et al., 1986). There are obvious Materials and similarities between the methods structure and formation of these intercellular spaces occurring at three- Biological materials way junctions between cells and the structure and forma- Peas sativum L. cv. (Pisum Wisconsin Perfection) were grown as previously tion of the infection thread which is also frequently initiated described et (Brewin al., 1983), and inoculated with Rhizobium at a three-way cellular junction leguminosarum bv. viceae strain between, for example, a 3841 (Brewin et al., 1985). Soybeans (Glycine max Merr. cv. were curled root hair cell (L.) Pride) inoculated with and two adjacent Bradyrhizobium epidermal cells strain japonicum, USDA 110, and beans (Phaseolus vulgaris L. cv. (Turgeon and Bauer, 1985). Both structures are bounded by Tendergreen) were inoculated with Rhizobium leguminosarum bv. phaseoli, cell pectic wall components and contain the same soluble strain 8002. Both soybeans and beans were grown as previously described matrix material. A very localized role for cell wall disrup- for beans et (Borthakur al., 1986). Nodules were isolated from plants 3 -4 tion and/or weeks after germination and used for degrading enzymes has been postulated immunochemical to explain analysis. Young nodules were harvested 3-4 weeks after germination how, during plant cell and division, the maturing cell plate fuses fractionated using differential centrifugation on sucrose cushions (Brewin with the mother cell wall (Jeffree et al., 1986). Similarly, et al., The 1985). nodule cytoplasmic supernatant fraction was obtained cell wall disruption and/or degradation has been postulated after homogenization of nodules in buffer (50 mM Tris-HCI, pH 7.5; to explain the inception of infection 10 mM thread formation in the dithiothreitol; 0.5 M sucrose; 5 mM p-aminobenzamidine; 5% (w/v) insoluble curled root hair polyvinylpyrrolidone) and centrifugation in an cell (Callaham and Eppendorf Torrey, 1981), and also microfuge at 10 000 for min. g 1 Cytoplasmic proteins from uninoculated roots the mechanism were by which the infection thread exits through similarly obtained by homogenization and centrifugation from the apical the opposite wall of the root hair cell, releasing rhizobia into portions of primary and lateral roots harvested 10 days after germination. 340 of infection thread matrix Components The supernatant fractions of the nodule and in root preparations were microtitre as used described et plates previously (Bradley Protease al., 1988). for the screening antibodies by Western treatment of blotting. to was modified from a antigens prior gel electrophoresis The extracellular glycoprotein derived from the conditioned medium of et previous (Brewin al., Protein procedure 1985). preparations (2 a carrot mg/mil) (Daucus cell carota) suspension culture was prepared by were incubated precipitation at 28°C with Triton X-100 overnight 0.5% and (v/v) with 50% (v/v) ethanol. Gum arabic and other chemical K reagents were As a (0.1 K was proteinase control, omitted. Protease mg/nil). proteinase obtained from Sigma (Poole, unless UK) otherwise was stated. arrested for 3 min in digestion Laemmli solubiliza- by boiling samples tion buffer (Laemmli, 1970). Monoclonal antibodies The rat monoclonal antibodies used in this study are listed in Table I. The and Microscopy immunocytochemistry derivation of MAC 204 has been previously described Pea and (Bradley et al., 1988). nodules and root were fixed in soybean pea tips It involved glutaraldehyde cell line myeloma Y3 AgI.2.3 (Galfre et w/v in al., 1979) and spleen 0.2 M sodium (2.5% in cacodylate buffer, pH 7.0), dehydrated cells from a rat LOU/iap that had been immunized with ethanol and material embedded in LR released White resin Aids, acrylic (Agar Stansted, UK) osmotic shock by treatment of at low peribacteroid-membrane-enclosed and bacteroids. immunolabelled as temperature, described previously is that (Bradley (It probable infection droplet structures et co-purified with For electron membrane- al., thin 1988). sections mounted on microscopy, gold enclosed bacteroids grids the during nodule fractionation were incubated in procedure, and this would diluted culture followed hybridoma supernatant, by account for the goat of presence MAC 204 in antigen the anti-rat nm 'peribacteroid' obtained from fraction.) (15 Janssen Life gold Sciences particles Products, MAC 236 is a similar cell line obtained from the same fusion, and and MAC with Wantage, UK), acetate and lead citrate. finally post-stained uranyl 265 was obtained from a similar fusion The performed for of subsequently with a sections procedure semi-thin mounted on slides labelling glass LOU/C rat. was modified from that of VandenBosch IntenSE II (1986), using silver JIM 5 was raised following immunization of a LOU/C rat with Life protoplasts Sciences to (Janssen enhance the developer Products) gold from labelling carrot prepared cells. The for suspension myeloma line was IR 983 F an black Labelled light microscopy by creating opaque semi- precipitate. (Bazin, 1982). Using apple pectin (Sigma P2157) as on thin sections were antigen microtitre counterstained with azure and blue in II sodium methylene in plates (500 Ag/ml phosphate-buffered incubated and were saline, overnight at with 4°C), metaborate, Ilford Pan F film. photographed Controls for a 200-fold dilution of JIM 5 cell hybridoma culture supernatant included omission of gave 90% the non-specific labelling and substitu- primary antibody maximal was binding. 50% inhibited Binding by 0.8 tion of the polygalacturonic irrelevant MAC 83 for the ltg/ml antibody primary or 6 antibody. mM acid, or D-galacturonic acid, 100 mM D-glucuronic acid. L- rhamnose, L-arabinose, D-glucose, D-xylose, or L-fucose, D-galactose gave no inhibition at 100 mM. Acknowledgements MAC 83 is a rat from the Monoclonal IgM Centre Antibody (AFRC Institute of Animal and K.VdB and Physiology Genetics, J.P.K. were Babraham, Cambridge, the and UK). Food Research supported by Agriculture It the CD2 on recognizes antigen porcine as and S.P. a sectoral T-lymphocytes and, it does Council from the Commission. by training grant European not cross-react with root nodule it was We thank tissue, used as a K.A.Hadfield for negative control assistance with 2-D gels, L.Richardson, J.Cooke, in Western blot and immunocytochemical and for assays. with cell J.King C.Cooper culture and help hybridoma J.A.Downie for comments on the manuscript. Affinity of MAC 236 purification antigen The MAC 236 antibody (30 mg was to protein) coupled 5 ml of cyanogen bromide-activated Sepharose (Pharmacia, Uppsala, References Sweden) according to the manufacturer's instructions, following purification of antibody by from ascitic fluid precipitation made 40% with saturating ammonium Anderson,M.A., Harris,P.J., and Bonig,I. Clarke,A.E. (1987) Planta, 171, This was sulphate. precipitate in 438-442. resuspended phosphate-buffered saline. The column was loaded with affinity a high speed 000 Bazin,H. Prot. Biol. (80 g) supernatant 615-618. (1982) Fluids, 29, fraction derived from nodule homogenates (10 which had been and ml), Bittner,M., Anal. partially Kupferer,P. Norris,C.F. (1980) Biochem., 102, fractionation with purified by ammonium 459-471. sulphate (30-40% saturating), and was with premixed (1: 1) phosphate-buffered saline I % Borthakur,D., Barber,C.E., containing (v/v) Lamb,J.W., and Daniels,M.J., Downie,J.A. The NP40. column was washed under with PBS 0.5% v/v Mol. gravity Johnson,A.W.B. Gen. NP40. (1986) Genet., 320-323. 203, After 10 buffer was column-volumes, as eluting applied recommended by Bradley,D.J., Wood,E.A., Larkins,A.P., Galfre,G., Butcher,G.W. and the manufacturer M 0.5 M (0.2 2.8 + glycine, NaCl, pH 10% Brewin,N.J. 149-160. (v/v) (1988) Planta, dioxane) 173, and fractions were collected. (1 ml) Brewin,N.J., and Wood,E.A. J. Gen. Young,J.P.W. (1983) Microbiol., 2973-2977. 129, SDS - PAGE and Western blots gels Brewin,N.J., Robertson,J.G., Wood,E.A., Wells,B., Larkins,A.P., Proteins were SDS-PAGE and separated by 12% Galfre,G. (Laemmli, Butcher,G.W. EMBO 605-611. 1970) using (1985) J., 4, with of acrylamide 5 loaded mini-gels, lane. The and ug protein per Callaham,D.A. Can. J. proteins Torrey,J.G. (1981) Bot., 1647-1664. 59, were then transferred et and to nitro- Cassab,G.I. Annu. electrophoretically (Towbin al., Varner,J.E. Rev. Plant 1979) (1988) Physiol. Plant Mol. cellulose sheets and 321 (Schleicher Schuell, for 16 h at 10 V Biol., 39, -353. Dassell, FRG) in 25 mM sodium buffer 6.8 et a Chandler,M.R. J. phosphate pH (Bittner al., (1978) Bot., 29, 749-755. 1980) Exp. using Biorad 'Transblot' or apparatus with a blotter Date,R.A. and (Watford, UK), Chandler,M.R., J. semi-dry Roughley,R.J. Bot., (1982) Exp. 33, (Sartorius, the methods 47-57. and buffers of Gottingen, FRG) using Kyhse- Anderson After the nitrocellulose sheets de (1984). were incubated Faria,S.M., and blotting, Can. J. McInroy,S.G. Sprent,J.I. (1986) Bot., 65, with a 553 -558. sequentially solution, anti-rat alkaline blocking primary antibody, goat and as phosphatase conjugate detailed Fincher,G.B., Stone,B.A. and chromogenic substrates, Clarke,A.E. Annu. Rev. (1983) Plant previously et al., 47 - 70. (Bradley 1988). Phvsiol., 34, Two-dimensional were run as described The O'Farrell Plant Cell Wall: gels Fry,S.C. (1988) Growing Chemical and Metabolic by (1975). mix for the first dimension contained urea Acrylamide gel Analysis. Scientific and Longman Technical, Harlow, UK. (9.5 M), Essex, 4% acrylamide NP40 Milton Galfre,G., and (w/v), (2% v/v), (Pharmacia, Milstein,C. Wright,B. Nature, 227, ampholines (1979) 131-133. 3.5-10 and 5-7 Keynes, UK) pH (3% Jeffree,C.E., Dale,J.E. and v/v) (2% 132, ampholines pH v/v): Fry,S.C. (1986) Protoplasma, 90-98. this was in 10.5-cm tubes. After the were (1984) J. Biochem. polymerized Khyse-Anderson,J. Methods, 10, 203 electrophoresis, Biophys. -209. gels electroblotted to nitrocellulose in the normal Laemrnli,U.K. (1970) Nature, 277, 680-685. way. Lancelle,S.A. and Torrey,J.G. (1984) Protoplasma, 123, 26-37. of Analysis and Verma,D.P.S. epitopes Legocki,R.P. (1980) Cell, 20, 153-163. To ascertain the nature of the and Moore,P.J. Staehelin,L.A. MAC (1988) Planta, 174, 433-445. carbohydrate epitopes recognized by MAC 236 and MAC transferred to Mort,A.J. and 204, 265, nitrocellulose were Grover,P.B. Plant (1988) 86, 638-641. proteins Physiol., oxidized treatment to to O'Farrell,B. J. (1975) Biol. Chem., 250, by periodate the 4007-4017. prior immunostaining, according of et al. Woodward Nitrocellulose sheets were procedure incubated Robertson,J.G., Wells,B., Brewin,N.J., (1985). Wood,E.A., and Knight,C.D. Downie,J.A. for I h in 20 or 200 (1985) J. Cell Sci. mM sodium metaperiodate in Suppl., 2, 50 mM sodium 317-331. acetate buffer (pH 4.5), or in buffer alone, followed and Annu. Rev. Plant by 30 min in 50 mM sodium Rolfe,B.G. Gresshoff,P.M. (1988) 39, Physiol., borohydride in Tris-buffered saline 297 -3 19. (pH 7.5). ELISA assays were performed

Journal

The EMBO Journal – Springer Journals

Published: Feb 1, 1989

Keywords: infection thread; nodules; Pisum sativum

There are no references for this article.