105 256 _THS_ L, mm 3‘: A It“ ‘.. Hewsrnt—uufi Stags-irrev- chm—b Universit This is to certify that the thesis entitled Quantitation and Localization of Extensin by Immunochemical Methods presented by Theresa Ann Conrad has been accepted towards fulfillment of the requirements for MS degree in Plant Pathology him ' 0 Major professor Date \3 MMJK V154: 0-7639 MS U is an Afimau‘w Action/Equal Opportunity Institution IV1£SI_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from n your record. FINES will be charged if book is returned after the date stamped below. WITATION AND LOCALIZATION OF EXTENSIN BY WCAL MEIT'DDS TheresaAnnCom'ai A THEIS ahnitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIM Department of Botany and Plant Pathology 1986 PBSTRACT QUANTITATION AND LOCALIZATION OF EXTENSIN BY IMMUNOCHEMICAL ms 3‘! TheresaAnnCcnral A canpetitive indirect enzyme-l inked immunosorbent assay (ELISA) was developed for the rapid quantitation of the glycosylated and deglycosylated forms of the monomeric soluble extensin precursor submits P1 and P2. Linear response rave for each kind of precursor in the competition curve was between 1 ug/ml and 10 pg/ml. 'lhe cellular distribution of insoluble extensin was investigated in hypocotyls of cucumber (Cucumis sativus L.) seedlings subjected to a disease resistance indacing heat shock followed by inoculation with the fmgal pathogen Clmnun mrimm 211 and Arth., using mtibodies to the soltble precursor forms of extensin. Fluorescence microscopy revealed that anti-precursor antibodies bound only to the upper and lower epidermal cell walls of sections from inoculated and non—inoculated heat-shocked plants. Tomyyarents ii TABIE OF (DNTENTS LISTOFTABLES.. LISTOFFIGIRB I. Extensin . . . . . . . . . . . II. Invovement of extensin.in disease resistance . . . . III. Methods to quantitate aid localize soluble extensin . A.. Quantitation by enzyme-linked immuncacrbent assay. B. Localizationbyinmumflmresence........ LiteratureCited. PARTI DETEIYI‘ION 0F ammo AND DMWTED EXTENSIN PRECIRSORSBYINDIRHZTMITIVEEISA INTRODUCTION........... MAMIAISANDME'HDDSHH RESULTSAIDDISCUSSION............ PART II ImALIZATION OF ECI'ENSIN BY momma MICW DISCUSSION LITERATURE CITED . HP . nOmmOH-‘H .17 .18 20 U 24 O 33 .35 O 36 38 41 .49 .52 APPENDICES Appendix A; Flow charts of indirect ELISA . Appendix B:Flow chart for indirect fluorescent antibody pnxxfihre . iv 9393 O 55 59 LIST OF TABLES Table PART I 1 . Sensitivity of various extensin precursor antibodies to extensin precursor antigens in indirect cometitive ELISA(CEIA).................. 32 LIST OF FIGRES Figure PARTI ELISAtitration of rabbit anti-P1 antibody. phoorbanoe of preinmune serum at same dilutions were subtracted from absortenceusingimnsemm.... ..... Conpetitive indirect ELISA standard curve to 0P1. Each point represents triplicate determinations in a single microtiter plate. In this stanrhrd curve, aborbarce obtained with 0.1ug/ml of free precursor was signifi— cantly different (P=0.05 by Student's t-test) than absor- mnce obtained using precursor diluent only. Absortance values represent the mean of four replications. Starrhrd deviation was always 0.02 absorbnce units or less. The absorba'ice valuewhen preinlmme serumwas usedwas 0.00. . . PART II View of cross section of cucumber hypocotyl indirectly stained with P1 antibody followed by mti-rabbit-FI'IC 72hafterheatshock (40seccndsat500). . . . . . . View of cross section of cucumber hgpocotyl indirectly stained with P1 antibody followed by mti-rabbit-FI‘I'C 72 h after heat sock and 48 h after inoculation with spores of Cladospgrium cuamerinum (3 X 106 spores per ml). . View of cross section of cucumber hypocotyl indirectly stained with rabbit normal serum followed by anti-rabbit- FI'l‘C?2hafterheatshock........ .. ..... View of cross section of tmshocloed, unincculated cucmber hypocotyls indirectly stained with P1 antibody followed byanti-rabbit-FITC. . . . . . . . . . . . ......... View of cross section of unshocked cucumber hypccotyls indirectly stained with P1 antibody followed by anti— rabbi t-FIm. o e e e eeeeeeeeeee e e e o e o e 0 vi .28 .30 42 42 44 LITERATLRE REVIEW Lbrtensin The first evidence for a protein which is an integral part of the primary cell wall, and which contains virtually all of the hydroocyproline of the cell was presented by Import and Northcote and independently by Dougal and Shimbayashi in 1960 (28.9). In 1965 (29), Lamport hypothesized that this hydraxyproline—rich protein must play a structural role in the cell wall and, therefore, must inevitably be involved in cell extension. This idea was referred to as the extaisin hypothesis and led to the hydroxyproline-rich protein being given the name extensin (by analogy with the structural proteins collagen and elastin). In 1967 (30) Lamport reported the isolation of arabinosyl- hydroxyproline. a new type of carbohydrate-amino acid link. out of partial alkaline hydrolysates of tomato cell walls. Extenein was described as a polypeptide backbone with hydroxyproline residues involved in 0—glyccsidic links to short arabinosides. It was pointed out that these arebinose oligosaccharides might serve as attachments for other wall polysaccharides. enabling a small amount of extensin to cross-link a large amount of wall polysaccharide. Law concentrations of hydroxyproline residues could thus play an important part in determining the properties of the primary cell wall. Analysis of enzymatic degradation products of cell walls from 1 2 cultured tomato cells confirmed the presence of hydroxyproline— arabinosides and demonstrated that galactose was an additional sugar capcnent of the glycoprotein (31). In 1970 Lamport (32) sugested that the short arabincse side—chains might represent the beginning of much larger polysaccharide chains which were alkali sensitive and therefore not recovered from earlier alkaline treatments used to obtain the hydrmyproline—arabincsides. Lamport and Miller (33) later demtrated that the arabincsyl-hydrcncyproline linkage is widely distribited in the plant kingiom. A second amino acid-sugar link, 0-galactosyl serine, was subsequently identified in 1973 by Lamport et al. (35). Galactoeyl- serine was identified in glycopeptides prepared from cell walls of cultured tomato cells by acid hydrolysis (to remove arabinosides) fol lowed by trypsin digestion. Lamport, et a1. (35) and Lamport (34) developed a technique for estimating the degree of serine glycosylation in the wall. This technique was based on the finding that glycosylated serine residues were degraded when treated with hydrazine, whereas serine residues free of sugars were stable. They reported results of preliminary experiment: whichshmed that therewasadecrewe insugar—free serine residiss in the cell wall with increasing age of the suspensicn culture. This was consistent with the extensin hypothesis which called for increased cross-linking as cel 1 extension decreased (29). Esquerre-Tugaye and Lamport (14) and Esquerre—Tugaye and Mazau (15), after a study of the glycosylation patterns of the hydroxyproline-rich glycoprotein which accumulates in the cell wall of Col letotrichum my! infected melon plants, suggested more specific functions for the carbohydrate side- chains. Subsequent work confirmed and expanded that the extent of glycosylation of hydroxyproline was higher in infected plants (14). In cartrast, the extent of serine glycosylation did not vary significmtly upon infection, but decreased with age in healthy as wel 1 as infected plants. This led them to suggest that sure of the galactosylated serine residues could provide temporary links for orienting other wall polymers. while the arabinosides attached to hydroxyproline could have a different role. This role may, perhaps, include an involvement in the disease response mice the arabincee residues were known to protect the wall glycoprotein against proteolysis. In particular, proteolytic enzymes which may be involved in pathogenisis (by Q. lggemrium) had been sham to be ineffective on cell walls without prior deglycosylation of the hydrmcyprolire residues (14). Mort and Lamport (42) applied a new technique to the problem of studying extensin. They used anhydrous hydrogen fluoride which specifically hydrolyzes the polysaccharides of the cell wall and leaves peptide bonds intact. They found that the cell wall remained as an insoluble resiche rather than canpletely dissolving as expected. This insoluble fractim. about 1096 of the wall, carsisted of empal wants of wall protein and an unknown, possibly phenolic, component (37). They suggested that this insoluble residue must contain some other, as yet unidentified, cross-links. In addition, it may be cross-linked to itself independently of any possible links to cell wall polysaccharides. However, Lamport (3?) pointed out that proof of this idea required the isolation of cross—linked peptides and the identification of caponents involved at the cross-link region. Based on adiitional results, Import presented (38) the concept of two semi-independent cell wall mtworks: protein an! carbohydrate. He suggested that the tyrosine derivatives, which he had earlier been unable to identify (36) could be possible candidates for the cross-links, but the isolation of cross-linked peptides was still needed for proof. More recent evidence which srpports the concept of an independent glycoprotein network was presented by Fry (19). He isolated a new public anino acid frcn cell wall hydrolysates. The new amino acid was shown to be an oxidatively coupled dimer of tyrosine with the two tyrosine units linked by a dighenyl ether bridge. He proposed the name isodityrosine for this compound. Fry reported that the amount of isodityrosine in cell wall hydrolysate was proporticmal to the anount of hydroxyproline and suggested it was a component of extensin. He also suggested that isodityrosine might be the uncharacterized tyrosine derivative reported by Ianport (36) and that the glycoprotein is held in the cell wall by interchain isodityrosine cross-links. Cooper and Varner (8) demonstrated thatmuch of the extensin in waunded carrot tissue arrives at the wall in soluble form md gradal ly becomes insoluble. They were successful in isolating soluble extensin and speculated that an increase in the extractability of carrot extensin could be facilitated by inhibition of peroxidase nediated crosslinkim of extensin. This finding is in agresnent with Fry (19) who previously speculated that the crosslinking of eoctensin by isodityrosine could be control led by peroxichse. The ability to isolate soluble extensin frail minded carrot root tissue made that system ideal for studies on the biosynthesis of We (3). Soluble extensin was found to accumulate as a salt-extractable hydroxyproline-rich glycoprotein (HRGP) in the cell wall (49,51). The glycoprotein was found to have a molecular weight of approximately 86,000 D and consisted of approxinately two-thirds carbohydrate and one- third protein. The amino acid canpcsition resanbled the canpcsiticn of the insoluble extensin peptides described by Lamport (36) as well as that of soluble extensin eluted from intact tanato cell suspensions (47) and a hydroxyproline-rich bacterial agglutinin (39,41). Smith et a1. (41) further found that the soluble extensin eluted from the cell wall of intact tomato cell suspensions yielded two components (P1 and P2) that displayed kinetic and chemical properties which indicated their role as precursors of insoluble extensin. P1 md P2 were characterized by tryptic degradation of the m-deglyccsylated polypeptides. DP1 and DP2 (47). Cooper and Varner (8) suggest that the lack of acidic amino acids and the ahmdaice of lysine and histidine in soluble extensin give this macranolecule a high isoelectric point (in the range of 10 to 12). Such an isoelectric point explains its activity as a non-specific acterial mlutinin (39) (by birding to acidic bacterial cell wall comments), and males it a likely aididate for interaction with acidic pectins. The idea of a glycoprotein network in the plant cell wall has been extended to a possible glycoprotein-phenolic couplex cartaining lignin. “there (55) smgested that a: early stage of notification may involve cross-linking of the protein during polymerization of lignin manners. In experiments with lignin precursors in combination with various proteins and polysaccharides, be observed peroxidase—mediated bonding of polyphenolic substances to proteins, especially those containing hyclrcncyproline. In further experiments, using cell walls frat: tissue culture which were incubated with coniferyl alcohol and hydrogen peroxide, thinners (56) demonstrated the formation of lignin which was bound to carbohydrate and hydrcnqproline—ccntaining proteins. thitmore (57) attended his previous argument for lignin-protein structural buds by conparing the nine acid distributions of proteins associated with lignin and those of the whole cell wall. He suggested that, when polymerizing, lignin links covalmtly with cell wall glycoprotein, and that the bonds may be formed preferentially with hydroxyproline. However, no evidence for this has stbsequantly been presented. In conclusion, the concept of a glycoprotein, or perhaps glycoprotein-lignin network in some cell walls seens to be a credible explanation for the attachment of extensin in the cell wall. The evidence of a hydrogen fluoride-insoluble residue, the finding of a potential protein-protein link in isodityrosine, and the high isoelectric point supports this idea. The notion that an extensin- pectin network inercalated with xyloglucan coated cellulose microfibrils is the most ml working nodel of cell wall architecture (7). II. Involvanent 91 extensin in gi_sease resistance Begierre-Tmaye and New (15) suggested the involvsnent of extensin in disease resistance. The levels of HRG’Ps have been shown to increase greatly in melon seedlings infected with the fungus galletotrichum lgerarium, the causal agent of anthracnose (14). This increase in extensin was correlated with resistance to fungal infection (16). Esquerre-Tugaye et al. (13) concluded that the accumulation of this glycoprotein acts as a defense mechanism which becomes efficient if started early in the host. Toppan et a1. (50) found indirect evidence that ethylene regulates this defense mechanism. They aimed that both ethylene and “c-hydroutyproiim deposition was significuitly lowered in the cell wall of infected tissue. F'urther. treabnent of healthy tissues with natural precursor of ethylene stimulated both the production of ethylene and incorporation of I‘D—Wine into cell vall protein. The increase in cell wall hydra-cyproline content observed in med carrot discs has also been suggested to occur as part of a wound response in excised tissue (2). Chrispeels et al. (3) suggested that the increase in extensin bicsynthesis my be part of the plant's defense mechanism against invading pathogens. Fukuda and Kagimoto (20) similarly observed an increase in the cell wal l and hydroxyproline levels on aging sections of sweet pepper fruits. They thought the increase in hydrdoxyproline during aging derived fran the bicsynthesis of the HRGPs and its precursor in response to wounding of the tissues. It is possible that production of HRGPs in each of these stress situations may be controlled by ethylene, hearse wounding, qing, and infections are al 1 known to cause plants to release large amounts of ethylene (50). Stermer and Hammerschmidt (68) describe how a brief disease resistance inducing heat shock stimulates the synthesis of ethylene in cucumber seedlings, enhances peroxidase activity, and increases the accumulation of bound extensin in their cell val ls. They suggested that perhaps a heat-shock-induced increase in ethylene production could stimulate the peroxidase mediated accumrlation of bound extensin in the cell wall, and this increase in extensin may confer resistance to attack by a pathogen. In via: of the possible existence of a glycoprotein—phenolic cell wall network containing lignin, it is significant that phenolic compounds have also been observed to accumulate during infection. Glazener (21) dencnstrated that lignin-like materials are synthesized by young tomato fruits after infection by Botrytis cineria, and has suggested that the formation of a polyphenolic layer around the infection helps limit the spread of the fungus. Grand and Rossignol (22) and I-hrmerscl'midt and Kuc (24) described changes in ligrificration involved in systemic protection of melons and cucumber, to Col letotrichum W and gadgritm cuc_tgegi_m_r_m_, respectively. An initial inoculation with the fungus appears to stimulate the enzymes required for lignin synthesis so that in a later exposure to the pathogen rapid lignificaticn occurs and restricts the infection. Hannerschnidt et a1. (23) showed hydroxyproline and lignin enhancenent in cucumber cell walls is associated with resistance to W cucmerimm. These results also suggested 81 association between ligrin deposition aid hydroxyproline enhancement. Further evidence for the possible role of extensin in the defense mechanism of plants can be found in the structural similarity of extensin to potato lectin (46) which is prtatively involved in disease resistance. Potato lectin was reported to strongly agglutinate avirulent strains of the bacterial pathogen Pseudomcnas solanacearum. The virulent isolates, however, produce a: artracel lular pclysaccharide which apparently protected the cells fran binding with the lectin (46). Seqzeira et al. (45) demonstrated a similar aglutinaticn of avirulent cells of g. sclanaceargrg in tobacco cells, and showed tint the bacteria were specifically attached to the cell walls. These workers postulate that these glycoproteins may function in binding of bacteria to the plant cell wall. However, related work done more recently by Leach et 'al. (39) demonstrated that potato lectin was not responsible for the agglutination activity, but rather an HRGP was involved in qglutination. III. Methods 39 guantitate and localize luble extensin _—*_‘ 9 A. Quantitation by enzyme-l inked immcscrbent assay. Immunoassays have replaced many other methods used to detect or quantitate substances with important biologic properties. The high levels of sensitivity ard specificity achieved with immcaesays result from the specific, high affinity, reversible binding of antigens to antibodies, and from the existence of methods for attachment of sensitively detected labels such as isotopes, fluorescent ccnpcunds, ard emymes to antigens or antibodies. Among the first applications of enzymes as labels was the use of enzyme-antibody conjugates to detect and localize antigenic cellular components by light and electron microscopy ( 1). Later, the use of enzyme-antigen and enzyme-antibody conjugates in immunoassays was reported by Engvall ad Perlmann (12), ad irdepently by Van Weenem and Schuurs (52). The manna-linked immmorbent assay (ELISA) has been the subject of several reviews (10,11,54,55). The ELISA is tased on the principle that the ancunt of an enzyme-label led antigen bound by a fixed level of mtibcdy is inversely proportional to the amount of unlabelled antigen present. The unlabelled antigen canpetes with the label led antigen for mtibcdy birding. This, a dcse-respctse curve for serial dilutions of samples containing unknown levels of a particular antigen can be ccpared to a stardard curve derived from dilutions of a known qrantity of the prrified antigen. ‘lhe key to the sensitivity, specificity, ard precision of the assay is the antibody. The radioinmuncassay (RIA) in which a radioactive tracer label led antigen along with the ELISA are knam as ligard-birding assays. The sensitivity of both these assays is determined by the specificity of the binding reagent and its affinity for the ligrd. The extreme specificity ard high affinity of antibodies 10 for artigenic determinants ideally suits them for this role. The development of an ELISA for a particular antigen involves several aspects, all of which contribute to the precisian, sensitivity. specificity, ad reproducibility of the assay (25). Time include the purification of the antigen to hcrncgeneity urder conditions which do not alter its immunlcgical properties relevant to the samples to be tested. Seccrdly, a specific antiserum must be chosen which ideally will have higher titres of high avidity antibodies which at the same time are immunologically specific. Thirdly, reaction conditions must be established to provide precision and sensitivity for the assay. Finally, the data must be salyzed both with regard to the qrantitative and qualitative patterns of inhibition of binding. An example of the use of these assays to quantify a plant protein was demonstrated by Saunders et al. (44). The assay was used to (quantify phytochrcme, a plant protein, frcm extracts of plant tissue. 8. Localizatian by imrncflmrescence. Imnflucrescence technology provides a very important carriecticn between biochemical or immunological approaches ard cytological methods in the study of many biological problem. Cellular products which can be isolated ard characterized can also be localized an or in cells using inunflucrescence techniques. The method, first outlined by (loan in 1941 (4) and further refined in 1942 (5) and in 1950 (6), has been used actensively in the bicmdical field. The technique Ins the dvantge of being capable of detecting canponents on living cells. Immunofluorescence analysis also has the advantages of innuncchaical methodology such as extrene specificity ard sensitivity. This technique has many of the same potential problems as any 11 immunological method such as non-specific artifacts and impure antibodies with which to work. Therefore, all experiments using the technique require strict cantrols for non—specific staining, ard the use of the purest fluorescent tracer regents available. Much mre detailed information on the methodology involved in immmcflmrescence, ard its limitations, can be obtained fran many excel lent reviews an the stbject (18,25,43). Immcflucrescence techniqres have been used previously to localize glycoproteins in plant cells. For sample, Kilpatrick et al. (27) demonstrated that hydroxyproline-rich glycoproteins from m stmim were localized in the cytoplasm ard presented immunological evidence for structural similarly within the HRGP lectins from the Solanaceae (26). In addition, Etzler et al. (17) demonstrated that a molecule which cross-reacts with arom the Solanaceae (26). In addition, Etzler et al. (17) demonstrated that a molecule which cross-reacts with anti—Dolichos biflorus lectin is located in the cell walls of stuns, leaves, and cotyledons. Leach et al. (40) demonstrated that proteins similar to the HRGP-bacterial agglutinin extracted from potatoes are located on or in many varying species of mcnccct ard dicct cell wal ls. 10. 11. 12. LIW CITE) Avramas, S. (1976). Immunoenzymic techniques for biomedical analysis. Methods Enzymcl., 44, 709. . Chrispeels, M.J. (1969). 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A hydroxyproline-rich bacterial agglutinin from potato: its localization by immunofluorescence. Physiol. Pl. Pathol. 21, 319-25. Melon, J. E. and Helgeson, J. P. (1982). Interaction of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogers. Pl. Physiol., 70, 401-405. Mort, A. M. and Lamport, D. T. A. (1975). Evidence for pclysaccharide attachnent to extensin in cell walls obtained from tomato cell suspension cultures. Pl. Physiol., 56, 80. Ploem, J. S., Tanke, H. J., Al, I. and Deelder, A. M. (1978). Recent developments in immunofluorescence microscopy in Immunofluorescence and related staining techniques. Eds. W. Knapp. K. Holuber, and G. Wick. Saurnders, J. J., Cordcnnier, M.-—M., Palevitz, B. A. and Pratt, L. H. (1983). Immunofluorescance visualization of phytochrome in Pisum sativum L.epicctyls using monoclonal antibodies. Planta, 159, 545-53. Sequeira, L., Gaard, G. and De Zoeten, G. A. (1977). Interaction of bacteria and host cel l walls: its relation to mechanisms of induced resistance. Physiol. Pl. Pathol., 10, 43—50. Sequeira, L. ad Grahmn, T. L. (1977). Agglutinaticn of avirulent strains of Pseudomonas _s:olanacearum by potato lectin. Physiol. Pl. Pathol., 11, 43—54. Smith, J. J., Muldoon, E. P. and Lamport, D. T. A. (1984). Isolation of extensin precursors by direct elution of intact tunato cell suspension cultures. Phytochem. 23. 1233-1239. Sterner, B. A. and Hammerschmidt, R. (1985). The induction of disease resistance by heat shock _:_l_r_n Cellular arnd Molecular Biology of Plant Stress. Ed. J. L. Key and T. Kosuge. pp. 291- 302. Stuart, D. A. and Varner, J. E. (1980). Purification and characterization of a salt-extractable hydroxyproline-rich glycoprotein fraa aerated carrot dises. Pl. Physiol. 66, 787- 792. Tcppan, A., Roby. D. and Esquerre-Tugaye, M. T. (1982). Cell surfaces in plant-microorganism interactions. Pl. Physiol.. 70. 82—6. 51. 52. 53. 54. 55. 56. 57. 16 Van Holst, G. J. ard Varner. J. E. (1984). minforced pclyproline II conformation in an hydroxyproline-rich cell wall glycoprotein fran carrot root. Pl. Physiol. Van Weeman, B. K. and Schurs, A. H. W. M. (1971). Immunoassay using antigen-enzyme canjugates. FEBS Lett., 15, 232. Voller, A., Bartlett, A., and Bidwell, D. E. (1978). Enzyme immunoassays with special reference to ELISA techniques. J. Clin. Pathol., 31, 507. Watson, D. (1976). ELISA, a replacement for radioimmunoassay. Lancet, 2. 570. Whitmore, F. W. ( 1978). Lignin—protein complex catalyzed by peroxidase. P1. Sci. Lett., 13, 241-5. Whitmore, F. W. (1978). Lignin-carbohydrate complex formed in isolated cell walls of callus. Phytcchem, 17, 421-5. Rnitmcre, F. W. (1982). Lignin—protein canplex in cell walls of Pinus elliottii: amino acid canstituents. Phytochen., 21, 315. PARTI DETECTION OF GLYCOSYLA'I'ED AND BMW-MED MENSIN W BY INDIRECT WITIVE ELISA ABSTRACT A competitive indirect enzyme-l inked inmuncsorbent assay (ELISA) was developed for the rapid quantitation of the glycosylated and deglyccsylated forms of the monomeric soluble extensin precursor subunits P1 and P2. Immunization of rabbits with each extensin precursor resulted in anti-precursor antibody titers of 40 to 1600 in 18 weeks. A competitive indirect ELISA was conducted by simultaneously incutating precursor with anti-precursor antiserum over precursor solid fiase and then determining bound rabbit inununcglohrlin with goat anti- rabbit peroxidase conjugate. Linear response range fcr each kind of precursor in the canpetiticn curve was between 1 ug/ml and 10 m/ml. 17 WIN Extensin is a plant cell wall-bound hydroxyproline—rich glycoprotein (HEP). Recently, Snith et a1. (11) were able to isolate two different soluble forms of extensin out of salt eluates fran tomato suspension cel l walls. These HRGPs were shown to vary in amino acid canpcsiticn. These two actansins, called P1 and P2, represent manneric precursors to insoluble extensin. Although the evidence is correlative, extensin nay function as part of the resistance mechaniann against potentially pathogenic microorganisms (2,3,8,9,12). Extensin accumulation has been demonstrated to be correlated with wound and pathogen induced responses (2,3,8,13). These responses include the accumulation of extansin in the cell walls of wourded carrots (13), the lccalizatian of an HRGP as a bacterial aglutinin in potato cell walls (8), and the accnnmnlaticn of HRGPs in the cell walls of diseased plants in response to fungal infection (2,3). Recent work by Stermer and Hanunerschnidt has sham that extansin accumlates in the cell walls of cucuuber in response to a disease resistance indicing heat shock (12). The accumulation of extensin in cell walls may make the wall more resistant to degradation by pathogen induced cell wall degrading enzymes (3). Leach, et al. suggest that the HRGP may act in resistance to bacteria as an aglutinin which acts by inlncbilizing inccnpatible bacteria (8,9). The precise functim and role of extensin in dime resistance have, however, not been unequivocally proven. A principal question in ascertaining the role of extensin in disease resistance is 18 19 whetl'rer it accumulates to sufficient concentrations at infection sites at or before the time that pathogen growth stops. Satisfactory evidence has not been provided on this point, partly becaise of the irability to quantitate soluble extensin accurately in plant cell wal ls. Currant techniques for quantitation of the accumulation of HRGPs in cell walls involve spectrophotometric measurement of hydroxyproline released after acid hydrolysis (7). Such methods are, however, cumbersome and require two days to obtain the final results. We report here the development of a highly sensitive and rapid enzyme- linked immunosorbent assay (ELISA) for the precise quantitation of extensin precursors. MATERIAISANDME'IHCDS Materials. All inorganic chemicals were reagent graie or better. Bovine serum albumin (BSA) (fatty acid free and fraction V), polyoxyethylenesorbitan monolaurate (Tween 20), and 2,2'-azino-di(3— ethylbenzthiozaline) sulfonic acid (ABTS) were obtained from Sigma Chemical Co., St. Louis, MO; Freund's complete and incomplete adjuvmts, and goat mtirabbit IgG conjugated to horseraiish peroxidase (antirabbit-peroxidase) were from Cooper Biomedical, Malvem, PA; and immunoassay microtiter plates (immunoplates) were frcm Nunc Intenned, Roskilde, Dermark. W9: W; The extensin precursors, P1 ani P2, were isolated by direct elution of the cell surface of intact tomato cel 1 suspension cultures and further purified by column chromatography according to the mthcd of anith, et al. (11). Deglycosylation of P1 and P2 involved hydrolysis of the carbohydrate moieties fran the glycoprotein with HP—MeOI-l according to the method of Smith, et al. (11). m gimmimtion. P1, P2, and deglyccsylated P1 and P2 (DP1 an DP2) were used as immogens. Initial injectim of all four inmmogens enployed a modification of the multiple site method of Vaitukaitis, et al. (14). For the two glycosylated immunogens, P1 and P2, 0.5 mg precursor in 1.0 ml of 0.9% saline was emulsified with 2.0 ml Freund's couplets adjuvmt. For the two deglyccsylated immogens DP1 and DP2. 0.5mg precursor in 0.5ml of 0.9% saline was emulsified with 1.5ml 20 21 Freund's complete adjuvant. With all four precursors, the pregaration was injected intradermally into 4 sites on the back of a New Zealand white doe rabbit. Subsequent injections were maie at six week intervals using Freund's incomplete adjuvant emulsified in the same ratios and concentrations as described above, but at one-half the volume. Rabbits were bled through the marginal ear vein and sera purified by three amnonium sulfate (35% saturated) precipitations (4) followed by dialysis overnight at 4°C against 0.1M ptnsphate biffer in 0.15M saline (PBS, pH 7.5). Antibody titration g _i__n_direct ELISA. Appendix A srmmarins the indirect ELISA protocol used for attibcdy titratim in a flaw diagram Wells in polystyrene immunoplates were coated with 0.2 ml of 50 mM mutate-bicarbonate buffer, [H 9.6, camtaining precursor diluted to 10 ug/ml. The plates were incubated overnightat 4°C. They were then washed with tap water for 1 minute using an immunoplate washer. The plate washer consisted of a plexiglass box in which holes (1 mm in diureter) hai been made corresponding to wells of a 96 well microtiter plate. Tap water was forced into the box and through the holes over which an immunoplate was inverted and washed. After the washing procedure, the plate was shaken dry and 200 ul of PBS containing 1% (wt/vol) BSA was adied to each well to block urbcumi solid phase sites 8):! minimize nonspecific binding. The plates were then imbated for 30 min at 37°C, and washed as described above. Fifty ul of antiserum hanologous to the coating precursor was diluted in PBS containing 1.0% BSA and 0.1:; Tween 20, pH 7.5, were added to each well, and the plate was incubated at 37°C for 60 min. In all titration procedures and in the indirect carpetitive procedures (to be described), the mtibody used 22 was always Innologous to the precursor tlat was used to coat the plate. The plate was washed as described above and 50 ul of goat anti-rabbit peroxidase ccmjtmte (diluted 1:2000 (v/v) in PBS cmtaining 0.1% BSA- Tween) was added to each well. After incubation at 37°C for one hour, the plate was washed for 2 min and 0.1 ml of peroxidase substrate cmtaining 1.2 mM hydrogen peroxide aid 0.4mm of ABTS was aided. Thirty minutes after incumtion at 37°C, the reaction was terminated by aiding 0.1 ml of stopping reagent (hydrofluoric acid-ethylenediamine tetra- acetic acid (10). The absorbaice at 410nm was determined in a Dynatech Minireader (Dynatech Instrunnts, Alexardria, Va.) after imbation at roan tenperature. There were three replicatims of each treatment. Determinatim 9; war mantrations g indirect cgtitive M Appendix A summarizes the indirect ELISA protocols in flow diagrams. ‘me indirect competitive ELISA was identical to the irrlirect titration procedure described above except for an aided preimbaticn before aiding the antibody to the polystym wells. Twenty-five ul of 1* BSA in PBS-Tween was first adied to each well to medics unspecific antibody binding. To obtain reproducible competition data, it was necessary to preincutate 25 ul of antisera along with 25 ul of varying dilutions of free glycosylated precursor in glass test tubes at room temperature for 2 hours. After 2 hours, 50 ul of the preincubated antibody/antigen mixture was added to microtiter wells and the assay proceeded as in the antibody titration procedure described above. When antisera to deglyccsylated precursor was used, the preincubation step described above was not used; rather, 25 ul of free precursor was adied to the wells followed by imediate addition of 25 ul of diluted antisera. Antisera dilutions used were 1:1500 (v/v). Cross-reactivity of the various precursors in the indirect ELISA was determined as above 23 except that heterologous imtai of homologous mtigen was adled alaag with homologous antibody. Antibodies against glycosylated and deglyccsylated forms of each precursor were tested. RESULTS AND DISCUSSICm Production 9_i_‘_ antibody against extensin precursors. Two glycosylated (P1 and P2) and two deglyccsylated (DP1 and 1P2) extensin precursors were used to elicit anti-extensin antibody responses in rabbits. Production of antibodies was similar to a previously described method (5). An indirect ELISA was developed to mmitor titers of anti- extensin antiserum. In this assay, rabbit antiserum was incutated over a microtiter plate solid phase coated with 10 ug/ml precursor and total bound antibodies were subsequently detected with goat-antirabbit peroxichse conjugate. The serum dilution showing absorbance distinct from that of a preimmune serum control at the same dilution was arbitrarily designated as tie titer. Figure 1 arms the titration curve for rabbit P-1 serum (18 wks after initial inoculation). Preimmune serum controls shcwed negligible absorbance at each dilution whereas this antiserum was diluted 6400-fold and still had significant absortance. Antisera to the other three precursors could be diluted at least 6400—fold and produce a significant response. Conggtitive indirect ELISA. A competitive indirect ELISA was carried out by simultaneously incutsting precursor with an appropriate diluticrn (1:1000-l:1600) of homlogous rabbit anti-precursor antiserum over a precursor sol id phase and then determining bound rabbit antibody with a goat anti-rabbit peroxidase conjugate. A typical competition curve for deglyccsylated antigen is shown in Figure 2. The response range for this curve was between 0.01 and 1000 ng/ml. Each precursor 24 25 produced a similar competition curve with minimum sensitivity of 10 pg/ml. For each curve a level of 1.0 ng/ml could be determined reaiily without the aid of a spectrophotaneter. An essential canpanent in our indirect ELISA was the incutntion of glycosylated precursor with diluted anti-precursor antiserum for 2 hours in glass test tubes at roan teaperature prior to determination of bound rabbit antibody. Reproducible canpetition curves could not be obtained for glycosylated precursor without preincubation. However, this preincubation step was not necessary when assaying deglyccsylated precursor. Spgificigy 9; anti—Erecursor antibody. In order to ascertain the specificity of the rabbit anti-precursor antiserum, the ability of each precursor to compete with each bound precursor was evaluated. Concentratians of the four precursors which resulted in 50% inhibition of anti-precursor antibody binding to the solid phase are presented in Table 1. The 50% inhibition value is a standard means of comparing cross—reactivities among similar antigens to an antiserum (1). These values were calculated as the amount (by weight) of free precursor required to reduce antibody binding by 50% (Table 1). Concentrations which resulted in 50% inhibition of anti-glycosylated P1 antibody binding to the solid phase were 0.03, 0.07, 0.03, and 0.16 ug/ml for glycosylated precursors 1 and 2, and deglyccsylated precursors 1 and 2 respectively (Table 1). This, Pl antibody appears to react similarly with all four precursors. Concentrations which resulted in 50% inhibition of solid phase-bouni anti-P2 antibody were 0.41, 0.17, 14.0, ard 21.0 ug/ml for P1, P2, DP1, and DP2 respectively (Table 1). Anti-P2 antibody thus appears to react more strongly with the glycosylated 26 precursors than with the deglyccsylated precursors. Therefore, it appears that this antiserum binds primarily to the carbohydrate moiety of the native glycosylated protein. Both the anti-DP1 and anti—DP2 antisera bound strongly and equivalently with both deglyccsylated precursors but not at al 1 (at 10 ug/ml) with the glycosylated forms (Table 1). These results suggest that the carbohydrate moiety on the mtive glycosylated precursors do not al low the molecule to react with the antibodies uade to the deglyccsylated precursors presunnbly because the protein portion of the glycosylated molecule is unavailable for mtibcdy binding. Smith, et al. (11) determined that the molar ratio of each glycosylated precursor form consisted of approximately two-thirds carbohydrate and one-third protein. Therefore, it would take three times as many moles of DP1 to inhibit antibody binding than it does P1 even though, by weight, it takes a similar amount of DP1 as P1 to inhibit binding of P1 mtibcdy. Extensin is a naturally occurring glycoprotein in plant cell val ls (6). Soluble extensin, or HRGPs, isolated from carrots (13) and sycamore ('1), as well as bacterial agglutinin (8), have similar amino acid composition. It is conceivable, therefore, that antibodies prepared against tomato extensin precursors will recognize similar sequences of precursors present in cucunber. The differential precursor cross-reactivities of these four mtisera can be used for determinations of the different precursor forum present in a plant extract. If an extract is mixed with each antiserum and then used in the competitive indirect ELISA, the amount of cross- reactivity (inhibition) with each antiserum should reveal the precursor profile of that sauple. For example, cross-reaction with P1 antiserun 27 and not DP1 antiserum establishes the presence of glycosylated precursors in the sample (Table 1). This result can be confirmed by similar results with P2 mtiserum. Cross-reaction with P1 antiserum and DP1 antiserum but minimally with P2 antiserum would be expected with a sample containing only deglyccsylated forms of the precursor (Table 1). Other precursor profiles should give reactions as indicated in Table 1. The antibodies prepared in this study have cross-reactivities similar to precursor antibodies previously characterized (5). As explained above, these differim antibody specificitiss could be useful in assayirg for precursors present in biologin systans. In conclmion, n have described procedures for the production of anti-extensin precursor antibody in rabbits and for the use of these antibodies in the competitive indirect ELISA for extensin. Indirect competitive ELISA would have significant advantages over existing methods (7) for quantifying low levels of soluble extensin in plant extracts. 28 Figure 1. ELISA titration of rabbit anti—P1 antibody. Absorbance of preinnmne serum at same dilutions were subtracted fran absorbance using immune serum. 29 QBSORBQNCE-410nm #0004 4300” ...N00: #000: 0.000.. 0.000.. 0300.. 0.M00.. 980.. a a n.» um us As ...m m.» as we a UHFCfiHOZ X HOS 30 Figure 2. Coupetitive indirect ELISA standard curve to DP1. Each data point represents triplicate determinations in a single microtiter plate. In this standard curve, absorbance obtained with 0.1ug/ml of free precursor was significantly different (P=0.05 by Student's t-test) than absorbance obtained using precursor di luent only. Absortance values represent the mean of four replicaticns. Standard deviation vas always 0.02 absorbance units or less. The absorbance value when preimmune serum was used was 0.00. WNIQD-HDSBDOBUd Ida 901 31 PER CENT INHIBITION d . oomflmthNd 59999939999930 I a: i .5 Ii “1... I5 n l d Q d O ABSORBQNCE-klflnm 9... 32 Table 1. Sensitivity of various extensin precursor antibodies to extensian precursor antigens in indirect competitive ELISA (CEIA). bound to immo— Free extensin precursor plate (sol id phase) added in cm 5095 inhibitionb P1 P1 0.03 P1 P2 0.07 P1 DP1 0.03 P1 0P2 0.16 P2 P1 0.41 P2 P2 0.1; P2 DP1 14 .0 P2 DP2 21 .0d DP1 P1 *‘3 an P2 * DP1 DP1 0.01 DP1 DP2 0.07 0P2 P1 . DP2 P2 - DP2 DP1 0.06 DP2 DP2 0.32 8All values are in ug/ml of extensin precursor in 0.1M PBS, pH 7.5. Antisera homologous to the bound precursor was added with free precursor, incubated 1 hour, then vashed. Mount of bound antibody Pas htermined with anti-rabbit peroxidase fol loved by peroxifle substrate. There were four replications per imrnoplate for each treatment. hch experiment was performed at least two tires with comarable results. hLag/ml extensin precursor required to inhibit binding of antibody by 5096 to the precursor solid phase. Inhibition vas determined by regression analysis. The correlation coefficient (r) for all curves except the P2— DP1 combination were greater than 9.93. The r value for the P2-DP1 curve was 0.77. °*=-no competition danonstrated at 10 ug/ml free extensin precursor. c’values were extrapolated tased on experimental values lower than the value indicated. LITERATLRE CITED 1. Bosch, A. M. 0., van Hell, 11., Brands, J., and Schuurs, A. H. W. M. (1978). Specificity, sensitivity and reproducibility of enzyme- immunoassays _i_._n_ S. B. Pal, ed. Enzyme Labelled Immunoassay of Hormones ard Drugs. Falter de Gruyter, Berlin, pp. 175-187. 2. Esquerre-Tugaye, M. T., Lafitte, C., Mazau, D.. Toppan, A. and Touze, A. (1979). Cell surfaces in plant—microorganism interactions. II. Evidence for the accumulation of hydroxyprolire—rich glycoproteins in the cell wall of diseased plants as a defense mechanism. Plant Physiol. 64, 320-326. 3. Hammerschmidt, R., Lamport, D. T. A. and Muldoon, E. P. (1984). Cell wall hydroxyproline enhaicemt ad lignin deposition as an early event in the resistance of cucumber to _C__l_adosporium cuarnerinum. Physiol. Pl. Pathol. 24, 43-47. 4. Hebert, G. A., Pelham, P. L. and Pittman, B. (1973). Determinination of the optimal ammonium sulfate concentration for the fractionation of rabbit, sheep, horse, ard goat antisera. Appl. Microbiol. 25, 26-36. 5. Kieliszanski, M. ad Lamport, D. T. A. (1985). Cross-reactivities of polyclcnal antibodies mainst extensin precursors determined via ELISA techniques. Phytochem. in press 6. Lamport, D. T. A. (1980). Structure and function of plant glycoproteins in P. K. Stumpf, E. E. 0am, eds. The Biochemistry of Plants, Vol. 3. Acadenic Press, New York, pp. 501-541. 7. Lamport, D. T. A. and Miller, D. A. (1971). Hydroxyproline arabincsides in the plat kingdan. Plant Physiol. 48, 454-456. 8. Leach, J. E., Cantrell, M. A. and Sequeira, L. (1982). A hydroxyproline-rich bacterial agglutinin from potato: extraction, purification, and characterization. Plant Physiol. 70. 1353—1358. 9. Leach, J. E., Cantrell, M. A. and Sequeira, L. (1982). A hydroxyproline-rich bacterial agglutinin from potato: its localization by imnmofluorescence. Physiol. Plant Pathol. 21, 319-325. 10. Pestka, J. J., Gaur, P.K. and Chu, F. S. (1980). Quantitation of aflatoxin Bi and aflatoxin Bl antibody by an enzyme-linked inlmmcsorbent microassay. Ippl. Environ. Microbiiol. 40, 1027- 1031. 33 11. 12 13. 14. 34 Smith, J. J., Muldoon, E. P. and Lamport, D. T. A. (1984). Isolation of extensin precursors by direct elution of intact tanato cell suspersicn cultures. Phytoctmistry 23, 1233-1239. . Stermer, B. A. and Hamerschmidt, R. (1985). Heat shock increases the synthesis of ethylene and enhances the accumulation of hydrmryprwoline—rich glycoprotein in cucumber seedlings _._1_r_1 J. L. Key, T. Kosuge, eds. Cellular and Molecular Biology of Plant Stress, Alan R. Liss, New York, pp. 291-302. Stuart, D. A. and Varner, J. E. (1980). Purification and characterization of a salt-extractable hydroxyproline—rich glycoprotein fran aerated carrot dises. Plant Physiol. 66, 787- 792 . Vaitukaitis, J., Robbins, J. B., Neischlag, E. and Ross. 6. T. (1971). A method for producing specific antisera with small dosesof imrncgen. J. Clin. Bdocr. 33, 988-991. PART II WTIM 0? mm BY WE MICIDSCOPY ABSTRACT We have investigated the cellular distribution of insoluble extensin in hypocotyls of cucumber (Cucumis sativus L.) seedlings subjected to a disease resistance inducing heat shock followed by inoculation with the frugal pathogen Qlfiadcsporium cucumerinum Ell and Arth., using antibodies to the soluble precursor forms of extensin. Antisera to glycosylated precursor (P1 and P2) and deglyccsylated precursor (DP1 and DP2) forms of extensin were prochced. Plant tissue cross—sections were treated with anti-precursor antibodies follaoed by fluorescein isothiocyanate-conjugated anti—rabbit immunoglobulin. Fluorescence microscopy revealed that anti-precursor antibodies bound only to the upper and lower epidermal cell wal ls of sections from inoculated and non-inoculated heat-shocked plants. Anti-precursor antibodies did not bind to sections from non heat-shocked plants. Antibodies to all four forms of precursor bound to the epidermis and sub-epidermal cell val ls, irdicating that proteins ttet cross—react with these antibodies are present in heat shocked, inoculated seedlings. The significance of these findings in relation to disease resistance is discmsed. 35 WION The role that hydroxyproline-rich glycoproteins (HRGPs), such as extensin, play in disease resistance is not known. Hammerschmidt, et al. (11) have shown that extensin increases in the cell walls, of cucumber seedlings infected with the fungus Cladospcriun cucmginum £11. and Arth. the cause of cucumber scab. This increase in extensin correlates with resistance to g. acumerimrm. Further evidence for the possible role of extensin in the defense mechanism of plants was demonstrated by Leach et al. (14,15) who showed that the bacterial aglutinin from potato 0B8 similar to soluble extensin. Recent mark by Stermer and Hamerschmidt ( 19) has shown that a brief heat shock enhances the accumulation of extensin in the cell walls of cucumber seedlings. Inoculaticns of heat shocked seedlings with Q. wwimm 24h after the shock resulted in further enhancement of extensin. The increase in cell wall hydroxyproline cartent observed in aged carrot discs has also been postulated to occur as part of a response to wourding in excised tissue (2). In each instance described above soluble extensin becomes insolubilized in plant cell walls with time. Other vorluers have mggested that the increase in extensin biosynthesis may be part of the plant's defense mechanisn gainst invading pathogens (3,5). The solrble extensins produced in rounded carrot root tissue and from tomato cell suspension cultures have been well characterized biochemically (1.18.22). A common amino acid sequence is present in both carrot and tomato extensin (1,18). Because a similar amino acid 36 37 composition is present in both these kinds of tissue we believed that crusher extensin may also curtain a similar amino acid sequer'ne. The purpose of this study was to determine the distribution of extensin in cucmber seedlings subjected to heat shock ard infected with the fungus (_3_,_ cucumerinum. Recently developed antibodies to glycosylated (P1 and P2) and deglyccsylated (DP1 and DP2) forms of tomato extensin (4) were used to visualize immocytochemically these glycoproteins in micunber seedlings. MAMA") METHODS Materials. All inorganic chemicals were reagent graie or better. Bovine serum albumin (BSA) (fatty acid free and fraction V), polyoxyethylenesorbitan monolaurate (Tween 20), and 2,2-azino-di (3- ethylbenzthiozaline) sulfonic acid (ABTS) were obtained from Sigma Genital Co., St. Louis, Mo; Freund's mlete ard implete aijuvants, sheep normal IgGs, and fluorescein conjugated sheep anti-rabbit IgG (PITC—IgG) fran Cooper Biomedical, Malvern, PA; immmoassay microtiter plates (implates) fran Nuns Intermed, Rcskilde, Denmark; and Nalgae Sterilization filter units, from Helge Company, Rochester, New York. Purified glycosylated and deglyccsylated form of extensin precursors were gereraisly provided by J.J. Snith (18). Antigen production. Glycosylated precusors, (P1 and P2), and deglyccsylated precursors, (DP1 and DP2), were prepared by direct eluticn of the cell surface of intact tanato cell suspension cultures according to the method of anith, et al. (18). Immizatim protocol. This protocol is described in previous work (4). Pour rabbits were injected repeatedly for more than one year. They were bled weekly and the titer was checked by the indirect ELISA method described in previous work (4). Plant and fungl material. Cucumber (Cucumis sativus L., cv. Marketer; Burpee Seed Co., Warminster, PA) susceptible to g. Writ-um were grown in rol led germination paper and placed in darkness for 5 days at 22 C (10). _C_._ floweerm cultures were grown on potato 38 39 dextrose agar at 18 C (21). that shock g inoculatim pf film”. Seedlirgs were treated by dipping the cotylecns ad hypocotyls in a 50 C water mth for 40 seca'ds (19). The shocked seedlings were then rolled on moistened germination paper and incubated in the dark at 22 C. Twenty-four hours after heat shock one-half of the seedlings were inoculated by spraying with a spore suspension of 3 X 106 spores per ml. The etiolated seedlings were rolled again in germinaticn paper and incubated at 22 C. Sectioning 93g stainigg procedures. Individual plants were selected for sectioning 6, 12, 18, 24, 48, 72, and 96 h after heat shock. The apical 2 cm. of tissue were excised below the hook region and sectioned imediately. Cross sections were made using a Hooker microtome (Lab-Line Instruments, Inc., Melrose Park, IL). Control sections were prepared from non-heat shocked or non-heat shocked, inoculated plats. Sectims were stained with fluorescent antibody by using the indirect method, modified from Leach, et al. (15) which is described below and sumrized in the form of a flow digram in Apperdix B. Each treatment was performed using at least 3 plants. Label ling of the tissue sections was perform at roan tenperature. The sections were first placed on the upper portions of a Nalgene sterilization filter unit, Type S (115 ml, 0.45 micrm pore) and rimed carefully with 5ml of 0.15M saline (PBS, pH 7.5) in a plastic wash bottle. They were then inunersed in 11111 of sheep normal IgG ”28011!!! =- 1.0) in PBS, incubated for 20 min and rinsed several times with PBS by applying a vacuum below the filter nenbrare for 3-4min to draw the fluid off the sections. The step involving sheep normal 196 was done to prevent nonspecific binding of anti-precursor antibodies. Purified 40 anti-precursor antibodies “280nm a 0.1) in PBS or normal rabbit IgGs (A280nm = 1.0) were added to the sections. After a 20 min. incubation, the sections were rinsed with PBS and auctioned again. PITC-IgG, diluted forty-fold in PBS was added to the sections which were then incumted in the dark for 20 min. Following a final rinse, the sections were carefully washed off the filter unit mto glass slides ard blotted dry. A drop of mounting buffer (9 prts glycerol to 1 part PBS, pi! 7.6) was added to the sections which were then covered with a No. 1 coverslip. Sections were viewed x 250 in a Leitz Laborlux 12 microscope equipped with a high—pressure mercury lamp as an ultraviolet light source ard with epi—illmninaticn. The spectrum of incident ultraviolet light was controlled with exciter filters BP 450-490; a barrier filter (LP 515) was used for protection of the eyes. Photographs were taken with a Wild Heerbrug 35 mm camera, professional Kodak Ektachrome (ASA 100) film ard 15s or 305 exposures. RBUL‘IS Stainigg p_f_ extensin gecursors _i_n cucumber hypocotyls. To localize the precursors, plant tissue sections were first treated with sheep normal immunoglobulins to minimize nonspecific binding of antibodies to the tissues. The staining procedures for inmmofluorescence were done on tissue placed on a Nalgene filter unit which allowed gentle, thorough, rapid rinses. In sections of hypocotyl, antibodies against the glycosylated precursors (P1 ard P2) bound mly to the upper ad lower epidermal cell wal 1s of heat shocked and heat shocked, inoculated seedlings, as indicated by the apple-green color of the fluorescent dye (Fig. 1 and 2). Fluorescence was also visible in the epidermis 12h after heat shock of uninoculated seedlings and immediately after inoculation of heat shocked seedlings. Fluorescence innediately after fungal inoculatim was probably due to the prior heat shock and not the presence of the fungus. No difference in the intensity of fluorescence was visible between heat shocked or heat shocked and inoculated sections treated with antibodies made against P1 or P2. Control sections treated with pre-immme rabbit serum did not bird the FITC-labelled goat anti-rabbit immunoglobul ins (Fig. 3 and 4). Fluorescence (other than autofluorescence) was also seen on the xylem vessels and col lenchyma (Fig 5). Macbraies apparently were not stained. Antibodies specific for the protein portion of the glycosylated precursors were also produced in an attempt to eliminate cross- 41 42 Figure 1. View of cross section of cucumber hypocotyl indirectly stained with P1 antibody followed by arti-rabbit-FITC 72 h after last shock (40 seconds at 50 C). Figure 2. View of cross section of cucumber hypocotyl irdirectly stained with P1 antibody fol lowed by mti-rabbit-FITC 72 h after heat shock ad 48 h after inoculatim with spores of W cucmerinun (3 X 106 spores per ml). 44 Figure 3. View of cross section of cucumber hypocotyl indirectly stained with rabbit normal serun fol lowed by anti-rabbit-FITC 72 h after heat shock. Figure 4. View of cross section of unshocked, uninoculated cucumber hypocotyls irdirectly stained with P1 antibody fol loved by anti-rabbit- FITC. [figure 5. View of cross section of unshocksd cucumber hypocotyls inmrnm 1y Manned with P1 antibody followed by anti—rabbit-FITC. 47 48 reactivity betwear glycosylated and deglyccsylated forms of precursor ard to eliminate cross-reactivity between the carbohydrate moiety of the precursors and polysaccharides present in the cell wall. The carbohydrate component was removed from glycosylated molecules by hydrolysis with hydrogen fluoride (18). Inmunofluorescence of hypocotyl sections treated with antibodies made against both forms of deglyccsylated precursors (DP1 and DP2) indicated that the protein portion of extensin precursors were specifically located in the upper and lower epidermal cell walls of heat shocked and heat shocked, inoculated tisare sections. DISCUSSION The inrmmofluorescence procedures described above indicate that forms of extensin precursor or precursor-like glycoprotein which cross- react with all four types of anti-precursor artibodies accumulate m or in the upper a'd lower epidermal cell walls of cucunbers subjected to a brief heat shock. This demonstration of the accumulation of high extensin precursor levels in response to heat shock is consistent with previous studies dancnstrating the accumrlatim of cell wall HHEPs in cumcumber tissue subjected to heat shock (19). Additionally, the accumlatim of cell wall Me has been demnstrated in plant tissues infected with fungi (6.7.11). Esquerre—Tugaye et al. (8) have ccrncluded that the accumulation of this glycoprotein acts as a defense mechanism which becomes efficient if started early in the host. Stermer and Hammerschmidt (19) have demonstrated that the extensin content of cucumber cell walls increases before the onset of resistance in heat shodmd seedlings. They aygest that the cross-linking of extensin by an increase in peroxidase activity is crucial for disease resistance. The presence of extensin in the epidermal cell wall of heat shocked cucumber tissue is consistent with the theory that the greater cross- linking of extensin could be responsible for the resistance of heat shocked seedling cell walls to digestion 13‘! 91mm W (19). Extensin may function in defense by directly forming a structural barrier to frugal invasion or it nay irdirectly provide sites for lignin deposition. 49 50 It was expected that antibodies prepared aginst soluble extensin frcm tomato bound to the epidermis ard cell val ls of a narsolaxaceous plant. Soluble carrot and tomato extensin have now been well characterized by biochemical and molecular biological approaches (1.18.22). The amino acid composition of the HRGPs is similar for carrot (5) and tomato (18) cell walls. and tobacco (16) and potato agglutinins (14.17). Thus. antibodies prepared against the protein portion of soluble extensin of tomato appear able to bind to common sequences of the glycoprotein found in cucumber. This observation supports the theory that similar forms of extensin are present in all dicotyledonous plants. Results fran enzyme—linked immmosorbent assays discussed in Part I of this thesis show that anti-P1 antibody strongly cross—reacts with all other precursor forms. Anti-P2 antibody appears to bird primarily to the carbohydrate moiety of the native glycosylated protein. whereas. anti-DP1 and anti-DP2 antibodies do not react with anti-P1 or anti-P2 preambly because the protein portim of the glycosylated mlecule is unavailable for antibody binding. Since anti-P1 and anti—P2 glycosylated forms of extensin precursor were able to absorb and fluoresce in the epidermal cell walls of heat shocked tissue. such precursor forms are presumably present in such tissue. More specificel ly. since anti-DP1 ad anti-DP2 forms were able to absorb ard fluoresce. the protein portion of the extensin mlecule is located in cucunber cell walls subjected to heat shock. Since it is unliltely that the deglyccsylated forms of precursor are present in the cell wall (R. Hamerschnidt. personal cammnication). the glycosylated forms must be configured in the cell wall in such a way as to allow antibody birding. 51 Previous studies (9,12,13,15) have described the production of highly specific fluorescent antibody preparations to hydroxyproline-rich glycoproteins (HRGPs) in plants. We have described methods for the prediction of anti-extanin precursor antibody in rabbits and for the use of these antibodies for the localization of artensin precursor in heat shocked plant tissue. Our results emport the views of Sterner ard Emersctmidt (11.19) who cmsider the mhancenent of extensin to be a factor in disease resistance. This system could provide a useful method to study the interactions of extensin in lignin synthesis ad cell wall bourd phenols in normal ad abnormal cell wall metabolism (11.23.24). fl 0! 10. LITERATLRE CITE) . Chen. J. and Varner. J. E. (1985). Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDA protein. Proc. Natl. Acad. Sci. 82. 4399-4403. Chrispeels, M.J. (1969). Synthesis and secretion of hydrcntyproline-cmtaining macranolecules in carrots. 1. Kinetic analysis. Pl. Physiol.. 44. 1187-95. Chrispeels, M.J.. Sadava. D. and Cho. Y.P. (1974). Enhancement of extensin biosynthesis in ageing discs of carrot storage tissue. J. Exp. Bot.. 25. 1157-66. Conrad. T. A., Lamport. D.T.A. and Hammerschmidt. R. (1985). Detection of glycosylated ard deglyccsylated extensin precursors with irdirect conpetitive ELISA. ungublished. COOper. J.B.. Chen. J.A. and Varner. J.E. (1984). The glycoprotein component of plant cell walls. I_r_; Structure. function. and biosynthesis of plant cell walls. ed. by WM. Dugger ad S. Dartinicki-Garcia. Rockvile. Marylard. 75-88. . Esquerre-Tugaye. M.T. and Toppan. A. (1976). hydroxyproline-rich glycoproteins of the plant cell wall as an inhibitory envircnnent for the growth of a pathogen in its host. In Cell wall biochemistry related to specificity in host plant pathogen interacticms. ed. 8. Solheim ard J. Rm. Columbia thiv. Press. Esquerre-Tugaye. M.T. ad Import. D.T.A. (1979). Cell surfaces in plant-microorganism interactions. I. A structural investigation of cell wall hydroxyproline-rich glycoproteins which accumulate in fungus-infected plants. Pl. Physiol.. 64. 314-9. Esquerre-Tugaye. M.T.. Lafitte. C., Mazau, D.. Toppan. A. and Touze, A. (1979). Cell surfaces in plant-micro-organism interactions. II. Evidence for the accumulation of hydrcmyproline-rich glycoproteim in the cell wall of diseased plants as a defense mechanism Pl. Physiol.. 64. 320-6. Etzler. M., Gibson. D. and Scates, S. (1979). localization of inactive form of lectin in primary cell walls of leaves and stems of Dolichcs biflorus. Pl. Physiol. 63.34 (Abstr.) Hammerschmidt. R. and Kuc. J. (1982). Lignification as a mechanism for induced systemic resistance of cucumber. Physiol. 52 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 53 Pl. Pathol.. 24. 43-47. Hammerschmidt. R.. Lamport. D.T.A. and Muldoon, E.P. (1984). Cell wall hydroxyproline enhaicemait ad lignin deposition as a: early event in the resistance of cucumber to Cladogrium cuaxmerimn. Physiol. Pl. Pathol.. 24. 43-47. Kilpatrick. D.C.. Yeoman. M.M. and Gould. A.R. (1979). Tissue ad subcel lular distribution of the lectin fran Qatura stranmium Thorn Apple. Biochemistry Journal 184. 215-219. Kilpatrick. D.C.. Jeffree. C.E.. Lockhart. 0.14. and Yeoman. MM. (1980). Immlogical evidence for structural similarity among lectins from species of the Solanaceae. FEBS Letters 113. 129- 133. Leach. J.E.. Cantrell. M.A. and Sequeira, L. (1982). A hydroxyproline-rich bacterial agglutinin from potato: extraction. purificatim. ad characterizaticn. Pl. Physiol. 70: 1353-1358. Leach. J.E.. Cantrell. M.A. and Sequeira, L. (1982). A hydroxyproline-rich bacterial agglutinin from potato: its localization by inlmmofluorescence. Physiol. Pl. Pathol.. 21. 319-325. Mellon. J.E. and Helgeson. J.P. (1982). Interactions of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogaxs. Pl. Physiol. 70. 401-405. Sequeira, L. ad Graham. T.L. (1977). Agglutination of avirulent strains of Pseudomonas solanacearum by potato lectin. Physiol. P1. “trials: 11' ‘8-540 Smith. J. J., Muldoon, E. P. and Lamport. D. T. A. (1984). Isolation of extensin precursors by direct elution of intact taaato cell suspension cultures. Phytochanistry 23. 1233-1239. Sterner. B.A. ad Hamerschmidt. R. (1985). Heat shock increases the synthesis of ethylene and enhances the accumulation of hydrrmyproline-rich glycoprotein in cummer aeedlirm. __Ir_1 J.L. Key. T. Kosuge. eds. Cellular and molecular biology of plant stress. Alan R. Liss. New York. 291-302. Stuart. D.A. and Varner. J.E. (1980). Purification and characterization of a salt-extractable hydroxyproline-rich glycoprotein from aerated carrot discs. Pl. Physiol. 66. 787- 792. Tuite. J. (1969). P1. Pathol. Methods. Burgess Publishing. Minneapolis. Van Holst. G.J. ad Varner. J.E. (1984). Reinforwd polyproline— II cmformation in an hydrcnqproline—rich cell wall glycoprotein fran carrot root. Pl. Physiol. 54 23. Walker. J.C. (1950). Ehvircnnent ad host resistance in relaticn to Mr scab. Phytopathol. 40. 1094-1102. 24. Whitmore, P.W. (1978). Lignin-protein complex catalyzed by peroxidase. Plant Science Letters 13. 241-245. APPBWDIXA MMFORINDIRECTELISA 55 56 All giantities are per well of microtiter plate. Dilutions for reagents vary frcm batch to hatch (e.g. antisera. anyme cantata) will not be givax . Irdirect ELISA— see Part I for reagent sources 200ul lazy/ml precursor in 5011M carbonate-bicarbonate. pH 9.6 incubate 4°C . overnight; wash 1 min. in H20 using microtiter plate washer: air dry 200111 1.0% BSA in PBS incubate 37°C 30 min; wash 1 min. 251.11 1.0% BSA in PBS/O.le Tween-2O + 25ul purified mtisera. diluted in PBS incubate 37°C 1h; wash 1 min. soul goat-mtirabbit peroxidase diluted in 18 BSA/PBS/Twaen imbate 31°C 30 min; wash 2 min. 100 ul ABE/H202 solutim incubate 37°C 30 min.. rooom tanperature 100ul HF/EDI‘A stopping solution Read A410 57 _gdrirect ELISA for giantitation gt deglyccsylated farms gf_ precursor-see Part I for reagent sources. 200111 of DP1 or 1P2 in 50:14 carbonate-bicarbcnate. :8 9.6 incubate 4°C overnigxt; wash 1min with H20 using immunoplate washer 200u1 1.0% BSA in PBS +incutate 37°C 3min; wash 1 min 25ul 1.08 BSA/PBS/0.01% hear 20 + 25ul diluted DP1 or DP2 + 25ul diluted anti-DP1 or anti-DP2 incutate 37% 1h: wash 1min 75ul goat anti-rabbit peroxidase conjugate diluted in 1.0% BSA/PBS/Neai imbate 37°C 3min: wash 2min 100ul AME/11202 solutim incutate 3min. room temperature 100ul HF/EDTA stopping solution 58 _IrLdirect ELISA _fg Quantitation g: glposylated forms _o_f_ precursor-see Part I for reagait sources. 200ul P1 or P2 in 501M mutate-bicarbonate. I“! 9.6 imbate 4°C overnight; wash 1min with H20 using immmoplate washer 200ul 1.0% BSA/PBS incubate 37°C 30 min; wash 1min 25ul'1.0% BSA/PBS/0.01x Tween-20 + 50ul mixture cmtaining diluted P1 or P2 ard aiti-Pi or anti-P2 (me-incubated in glass test tubes for 2h. room tanperature) incubate 37°C 1h; wash 1min 100ul goat anti-rabbit peroxidase conjugate diluted in 1.096 BSA/PBS/Twaen incubate 37°C 30 min: wash 2 min 100ul ARTS/8202 solution incutate 30 min: room temperature 100ul HF/EDTA stoppirg soluticn APPENDIXB MGMFORINDIRMWANIIHJDYW 59 60 Dilutions for reagents that vary from batch to batch (e.g. antisera. FITC-label led enzyme ccnjugate) will not be given. Irdirect fluorescent antibody procedure f9; localizatim pf Eewrair 1;} plant tissue sections-see Part II for reagent sources Tissue sectioned with Hooker microtane sections collected on Nalgene filter unit (0.45um pore); rinsed 3x PBS. pi! 7.5 sections flooded with steep normal IgG in PBS ireubate 2Qnin room temperature; rinse 3x PBS. pH 7.5 Purified alti-precursor antibodies in PBS or normal rabbit 196s in PBS adied to sections incubate 20min rccm temerature; rinse 3x PBS. pH 7.5 PITC-ccajugted sleep anti-rabbit IgGs adied to sections incubate 20min in dark. room teuperature; rinse 3x PBS. pH 7.5 secticre washed off filters mto glass slides + blot dry drop of mounting buffer aided to sections sections covered with No. 1 ccverslip; view sections under epifluorescence 1293 03046 6886 3