A Fluorescent Antibody Assay for Hyphae and Glomalin From Arbuscular

Plant and Soil 226: 171–177, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 171 A fluorescent antibody assay for hyphae and glomalin from arbuscular mycorrhizal fungi S. F. Wright∗ Soil Microbial Systems Laboratory, United States Department of Agriculture–Agricultural Research Service, Beltsville, MD 20705, USA Received 31 March 1999. Accepted in revised form 22 November 1999 Key words: glycoprotein, immunofluorescence, monoclonal antibody, soil biology Abstract Studies o
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  Plant and Soil 226: 171–177, 2000.© 2000 Kluwer Academic Publishers. Printed in the Netherlands. 171 A fluorescent antibody assay for hyphae and glomalin from arbuscularmycorrhizal fungi S. F. Wright ∗ Soil Microbial Systems Laboratory, United States Department of Agriculture–Agricultural Research Service, Beltsville, MD 20705, USA Received 31 March 1999. Accepted in revised form 22 November 1999 Key words: glycoprotein, immunofluorescence,monoclonal antibody, soil biology Abstract Studies on the role of arbuscular mycorrhizal (AM) fungi in soil have been aided by the use of a monoclonalantibody that detects a molecule common to all isolates of these fungi studied to date. The molecule, glomalin, isa glycoprotein that forms on hyphae, but apparently sloughs off and adheres to soil particles or imbedded plasticmesh. An indirect immunofluorescence (IF) assay is described for detection of glomalin on hyphae attached toroots, in roots, on hyphae traps and on the surface of soil aggregates. Small sieves are used to process hyphaeattached to roots and soil aggregates. Glomalin on hyphae and glomalin attached to plastic or nylon are assayed ona 1 cm 2 section of meshes. Examples of IF assay results are shown and discussed. Introduction Antibodies have been used to study arbuscular my-corrhizal (AM) fungi at the level of host-symbiontinteractions (Bonfante-Fasolo et al., 1991; Perotto etal., 1994), as taxonomy aids (Aldwell et al., 1985;Hahn et al., 1993; Sanders et al., 1992; Wright etal., 1987)and in ecologicalinvestigationsof hyphaeinsoil (Aldwell and Hall, 1986; Fries and Allen, 1991;Kough and Linderman, 1986; Wilson et al., 1983).Three of these studies employed monoclonal antibod-ies (Hahn et al., 1993; Perotto et al., 1994; Wright etal., 1987).Monoclonalantibodies are highlyspecific antibod-ies generated by hybridoma technology (Köhler andMilstein, 1975). A monoclonal antibody (MAb) is asingle antibody type that detects a few amino acids,nucleic acids or monosaccharides. As a taxonomy aid,a hybridoma may be chosen because the MAb is re-active with one genus and species (Hahn et al., 1993;Wright et al., 1987) or with a broad range of genera ∗ FAX No: (301) 504-8370. TEL No: (301) 504-8156.E-mail: 1 The U.S. Government’s right to retain a non-exclusive royalty freelicence in and to any copyright is acknowledged. within a taxonomic group. Because hybridomas canbe preserved and regrown, they provide a consistentand plentiful supply of the antibody.During an attempt to produce a MAb that wouldreact specifically with Glomus intraradices Schenck and Smith as a taxonomy aid, MAb32B11 was pro-duced. This MAb was generated using freshly collec-ted spores as the immunogen. The reactive moleculewas detected by indirect immunofluorescence on thesurface of fresh, young spores of  G. intraradices ,on hyphae of this species and on hyphae of isolatesfrom all other culturable AM fungal genera (Wright etal., 1996). Low cross-reactivity with the pathogenicfungus Leptosphaeria korrea J C Walker and A MSmith was not considered to be a significant imped-iment in the use of MAb32B11 to study the targetmolecule on AM fungi.MAb32B11 is an IgM antibody. This class of anti-body has five pairs of heavy and light chains giving atotal of 10 potentialreactive sites to attach to the targetmolecule. This is in contrast to the two pairs of heavyand light chains of an IgG antibody, the predominantantibody class in polyclonal antisera. Generally, onlyone or two of the reactive sites of an IgM antibodyattach to an immobilized target molecule. However,  172the 10 heavy chains providenumeroussites for attach-ment of the FITC-tagged secondary antibody used toreveal the MAb resulting in a strong fluorescent signal(Goding, 1986).The first use of the immunofluorescenceassay wasto assess the build up and decline of a sheath-likecoating on hyphae from early colonization to plantsenescence (Wright et al., 1996). After initially be-ing revealed by MAb32B11 in immunofluorescenceassay, the antibody-reactive molecule was extractedfrom hyphae and studied further (Wright et al., 1996).Harshness of conditions necessary to extract the mo-lecule suggested that it was very insoluble and stable.This led to use of the extraction procedure on soil andassessment of the role of the molecule in soil stability(Wright and Upadhyaya, 1996, 1998). Also, becauseof the uniqueness and abundance of the molecule insoil andapparentproductionbymembersofthe fungalorder Glomales, the name glomalin was ascribedto it (Wright and Upadhyaya, 1996). The presenceof glomalin on hyphae and glomalin sloughed fromhyphae onto plastic was detected by immunofluor-escence which led to use of hyphae traps to estim-ate activity of hyphae and glomalin production overtime periods of 12–14 weeks (Wright and Upadhyaya,1999). Extracted glomalin was also subjected to ca-pillary electrophoresis analysis which demonstratedthat the antibody-reactive compound is a glycoprotein(Wright et al., 1998).This paper describes several variations of the im-munofluorescence assay developed to assess glomalinproduction on hyphae in the rhizosphere, monitor hy-phal growth and production of glomalin during plantgrowth, and to reveal glomalin on the surface of waterstable soil aggregates. Materials and methods  Monoclonal antibody Monoclonal antibody (MAb) 32B11 was producedagainst fresh spores of  Glomus intraradices FL208(Dazzo and Wright, 1996). Briefly, a BALB/c mousewas immunized with four intraperitoneal injections of 5000 spores crushed in 0.5 mL physiological solu-tion at 3-week intervals. Hybridomas were producedand hybridoma 32B11 was selected based on pos-itive enzyme-linked immunosorbent assay reactionswith five spores of the targeted isolate, other mor-phologically similar AM fungi and isolates of othergenera of AM fungi. The antibody was produced inbulk by overgrowth of the hybridoma in tissue cul-ture medium. Antibodies in cell-free supernatant werestored at 4 ◦ C as a sterile solution. Batches of MAb32B11 were produced as needed and tested for re-activity against known amounts of extracted glomalin(Wright et al., 1996). Staining sieves Small sieves to hold root and soil samples during in-cubations with antibodies and in washing steps weremade from 10 mm ID polyvinyl chloride tubing orother plastic plumbing tubing. Tubing was cut into 7mm sections. A fine nylon mesh (40 µ m openings)was glued to one end of each section of tubing withepoxy glue, and after the glue dried, the mesh wascut to fit the outer diameter. Sieves fit into wells of a 12-well tissue culture plate. Samples Root pieces with attached hyphae AM fungi were cultured in sand or a sand-coal mix-ture on sudangrass ( Sorghum sudanese (Piper) Staph).Glomalin, naturally present in small amounts on sand,was extracted before the sand was used. This was ac-complished by flooding large batches of sand with 50mM citrate and autoclaving for 1 h. The sand wasrinsed thoroughlyto removethe citrate and then dried.Dried sand and coal were sterilized by autoclaving for1 h. Inoculated plants were grown for 3–4 months.At harvest, several pieces of root (ca. 3 cm lengths)were placedin a sieve. Root pieces were examinedun-der a microscope to aid in choosing sections to assay,but they were not allowed to dry before processing.Immunoreactive material remained on hyphae for aminimum of several weeks if root pieces were kept inwater.  Intraradical colonization Roots to be examined for internal colonization weredried before processing. To clear roots, boiling hotKOH (10% w/v) was poured over root pieces of ca. 1cm length and incubated at room temperature (RT) for15 min. The material was transferred to a small sieve,KOH was neutralized with 3N HCl for 5 min followedby 3 × 5 min washes in PBS.  173  Hyphae traps Hyphae traps made of plastic horticultural mesh(WrightandUpadhyaya,1999)were placedin pot cul-tures to monitor growth and productionof extraradicalhyphae in a root-free zone. Nylon mesh (40 µ m) isusedinpotculturestoconfineroots.Trapswereplacedagainst the inner wall of the pot at the time seeds wereplanted and inoculated. At biweekly or monthly in-tervals, or at the termination of the experiment, stripswere carefully removed using a spatula to push sandawayfromthe trap andpreventthe sand fromabradingmaterial on the trap as it was pulled out of the pot. Thetop2cm ofa stripwas cutawayanddiscardedbecausethis area is often contaminated with algae. Several 1cm 2 sections were cut out of the remaining materialand placed directly into a well of a 12-well plate. Soil aggregates Aggregates measuring 1–2 mm or 0.25–1 mm weresieved from air-dry soils and placed in the stainingsieves in amounts that coveredthe bottom of the sieve.Water stability of the batch from which aggregateswere sampled for the immunoassay was determinedon other samples using the method of Kemper andRosenau (1986). Stability ≥ 70% is required for ag-gregates to maintain integrity during processing.  Indirect immunofluorescence assay All incubation and washing steps were performed atRT on a rotating platform (50–75 rpm). Care wastaken to add sufficient volumes of solutions to coversamples. Samples were blocked to prevent nonspe-cific adsorption of antibodies using 2% (w/v) non-fatdry milk in phosphate buffered saline (PBS) (10 mMNa 2 HPO 4 , 138 mM NaCl, 2.7 mM KCl, pH 7.4) andincubated for 15 min. Sieves or hyphae traps wereremoved from wells with tweezers and placed on apaper towel to blot off excess milk, wells were emp-tied, and sieves and hyphae traps were replaced inwells. A 1:2 mixture of MAb32B11 and PBS was ad-ded to wells, and samples were incubated for 1 h forall except for cleared roots which were incubated for2 h. Samples were washed for 3 × 5 min in PBS with0.05% Tween 20. A commercially available goat anti-mouse IgM antibody ( µ -chain reactive) tagged withfluorescein isothiocyanate (FITC) was diluted as re-commended by the supplier in PBS with 0.1% bovineserumalbumin. TheFITC-labeledantibodywas addedto wells and incubated for 1 h. Samples were washedthree times as described above. PBS without Tween20 was used for the fourth and final wash. For con-trols, an anti-  Rhizobium IgM MAb was used in placeof MAb32B11.Autofluorescence of roots can be quenched byerichrome black (Bolhool, 1987). The staining solu-tion consists of 9 mg of dye dissolved in 1 mLof dimethyl sulfoxide (DMSO) and 5 mL of che-lating solution (DMSO 50 mL; deionized water 20mL; 0.1 M aluminum chloride 10 mL; 1.0 M aceticacid 10 mL). The dye and chelating solution mixtureis adjusted to pH 5.2 and then diluted to 100 mLwith deionizedwater. After antibody-tagging,sampleswere flooded with erichrome black until the root wascolored pink to dark red. Excess stain was removedbywashing in PBS.Aggregates were viewed in a hanging drop slide.A Pasteur pipette with the tip broken off at the neck toprovide a bore larger than the aggregates was used totransfer aggregates. Aggregates in PBS were carefullydrawn into the pipette and transferred to the well of aslide. Excess PBS was removed by blotting, mountingmediumwas addedto coverthe aggregatesanda coverslip was placed over the well.Samples were mounted in a medium formulatedto prevent rapid photobleaching that can be pre-pared (Davidson and Goodwin, 1983) or obtainedcommercially. A coverslip was used. Samples, ex-cept aggregates, mounted in commercially availablemounting medium (Vectashield, Vector Laboratories,Burlingame, CA, USA) can be preserved by freezingat − 20 ◦ C. Aggregates break apart after a few hoursin the mounting medium and thus cannot be preservedthis way.After adding the mounting medium, but beforeplacing the cover slip over roots to be examined forintraradical colonization, the stele was removed andpieces of cortex were torn away so that a single layerof cells could be examined for immunofluorescence.This was done with the aid of a dissecting microscope.An epifluorescence microscope was used with aband pass combination BP450-BP490 exciter filter, adichroic chromatic beam splitter FT-510 filter and alongwave pass LP-520 barrier filter. A digital cameraattached to the microscope and computer with a framegrabber were used to capture and save images. Results Glomalin was detected on hyphae attached to roots,hyphae from the root-free zone of a pot culture, in-traradical structures, on hyphae traps placed in the  174 Figure 1. Results of MAb32Bll immunofluorescence assays for glomalin. (A) Hyphae of an isolate of  Acaulospora mellea on a piece of horticultural plastic. The square-shaped light areas are pores in the plastic mesh (20 × ). (B) A closer view hyphae of  A. mellea on horticulturalmesh shows bright fluorescence and pieces of glomalin attached to the plastic (125 × ). (C) Sudangrass colonized by an isolate of  Glomusmosseae after 4 months of growth (125 × ). (D) Immunofluorescence of an arbuscule in crimson clover ( Trifolium incarnatum L.) root colonizedby Glomus etunicatum BR220 (400 × ). (E) A piece of nylon mesh with glomalin from an isolate of  Glomus viscosum attached (180 × ). (F and G) Aggregates from a silt loam soil coated with glomalin. (F) Shown are 0.25–1 mm size aggregates (63 × ) and (G) is a 1–2 mm size aggregate(63 × ). (H) Root hairs of sudangrass ( Sorghum sudanese (Piper) Staph) (non-colonized) showing auto fluorescence (400 × ).
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