The intercellular biotrophic leaf pathogen Cymadothea trifolii locally degrades pectins, but not cellulose or xyloglucan in cell walls of Trifolium repens

Authors

  • Uwe K. Simon,

    Corresponding author
    1. Universität Tübingen, Lehrstuhl Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, D-72076 Tübingen, Germany;
      Author for correspondence: Uwe K. Simon Tel: +49 7071 2976689 Fax: +49 7071 295344 Email: uwe.simon@uni-tuebingen.de
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  • Robert Bauer,

    1. Universität Tübingen, Lehrstuhl Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, D-72076 Tübingen, Germany;
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  • Danny Rioux,

    1. Natural Resources Canada, Canadian Forest Service – Quebec Region, 1055 du P.E.P.S., PO Box 3800, Sainte-Foy, Quebec, Canada G1V 4C7
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  • Marie Simard,

    1. Natural Resources Canada, Canadian Forest Service – Quebec Region, 1055 du P.E.P.S., PO Box 3800, Sainte-Foy, Quebec, Canada G1V 4C7
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  • Franz Oberwinkler

    1. Universität Tübingen, Lehrstuhl Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, D-72076 Tübingen, Germany;
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Author for correspondence: Uwe K. Simon Tel: +49 7071 2976689 Fax: +49 7071 295344 Email: uwe.simon@uni-tuebingen.de

Summary

  • • The intercellular ascomycetous pathogen Cymadothea trifolii, causing sooty blotch of clover, proliferates within leaves of Trifolium spp. and produces a complex structure called interaction apparatus (IA) in its own hyphae. Opposite the IA the plant plasmalemma invaginates to form a bubble. Both structures are connected by a tube with an electron-dense sheath.
  • • Using immunocytochemistry on high-pressure frozen and freeze-substituted samples, we examined several plant and fungal cell wall components, including those in new host wall appositions at the interaction site, as well as a fungal polygalacturonase.
  • • Within the tube linking IA and host bubble, labelling was obtained for cellulose and xyloglucan but not for rhamnogalacturonan-I and homogalacturonans. The IA labelled for chitin and β-1,3-glucans, and for a fungal polygalacturonase. Plant wall appositions reacted with antibodies against callose, xyloglucans and rhamnogalacturonan-I.
  • • Cymadothea trifolii partly degrades the host cell wall. Structural elements remain intact, but the pectin matrix is dissolved. A fungal polygalacturonase detected in the IA is probably a key factor in this process. Owing to the presence of chitin and β-1,3-glucans, the IA itself is considered an apoplastic compartment.

Introduction

The application of immunocytochemistry in plant pathology made it possible to locate and examine the role of specific enzymes and wall constituents of either host or pathogen as well as newly generated defence products of the invaded organisms. This technique has been widely used to gain further insight into viral (van Lent & Verduin, 1991), bacterial (Brown & Mansfield, 1991; Boher et al., 1996; Vian et al., 1996; Murdoch et al., 1999) and fungal (VandenBosch et al., 1989; Bonfante-Fasolo & Perotto, 1991; Bonfante-Fasolo et al., 1991; Nicole & Benhamou, 1993; Chamberland, 1994; Hippe-Sanwald et al., 1994; Golotte et al., 1995; Benhamou, 1996) infections of plants. However, in relation to host cell wall alteration during such infections, most publications have focused on only a few possibly important molecules. Among these are hydroxyproline-rich and other glycoproteins (VandenBosch et al., 1989; Bonfante-Fasolo et al., 1991; Brown & Mansfield, 1991; Hippe-Sanwald et al., 1994; Golotte et al., 1995; Boher et al., 1996), cellulose (Golotte et al., 1993; Nicole & Benhamou, 1993; Nicole et al., 1994; Rioux & Biggs, 1994; Ouellette et al., 1995; Cole et al., 1998), callose (Golotte et al., 1993; Rodriguez-Galvez & Mendgen, 1995; Cordier et al., 1998), PR-1 protein (Cordier et al., 1998), phenols (Grandmaison et al., 1993; Boher et al., 1996), cell wall degrading or stiffening enzymes (Benhamou et al., 1991; Nicole et al., 1992; Nicole et al., 1994; Tenberge et al., 1999; Hilaire et al., 2001) and pectins (Golotte et al., 1993; Nicole & Benhamou, 1993; Rodriguez-Galvez & Mendgen, 1995; Vian et al., 1996; Murdoch et al., 1999). In rust-infected plants, some work has been done to gain a more precise insight into changes in cell wall chemistry during the interaction between host and fungus, particularly along intracellular haustoria (Xu & Mendgen, 1997; Baka & Lösel, 1998). However, an extensive study of which cell wall components are degraded and which remain intact during the interaction of a host plant and a specific intercellular biotrophic pathogen is lacking.

To fill this gap, we have chosen the biotrophic fungus Cymadothea trifolii, which invades leaves of Trifolium species, because this special relationship displays a strictly extracellular pathogen forming an intricate structure, called interaction apparatus (IA), within its own hyphae. Opposite that probably nutrition-related structure, the plasmalemma of the host cell invaginates to form a bubble. Both compartments are linked by a tube with an electron-dense sheath crossing the cell walls of either organism without disrupting them (Simon et al., 2004). Using immunocytochemistry we were able to document the fate of several plant cell wall components within the tube as well as the chemical nature of the IA and the wall appositions which finally encapsulate the host bubble and probably stop the cellular interaction.

Materials and Methods

Cryofixation and freeze substitution

Discs 1 mm wide and c. 0.5 mm thick were punched out of infected clover leaves. They were infiltrated with 8% (2.5 m) aqueous methanol for c. 5 min at room temperature (RT) to remove intercellular air. Single discs were placed in the 0.3 mm hollow of one half of an aluminium holder, which was covered by another holder with a 0.1 mm hollow. Each sandwich was frozen in a high-pressure freezer (HPM 010; Balzers Union, Liechtenstein), as described in detail by Mendgen et al. (1991), either at the Max-Planck-Institute for Developmental Biology, Tübingen, or at the University of Ulm, Germany, and stored in liquid nitrogen until use.

For substitution the sandwiches were opened in liquid nitrogen and the holders containing the samples were transferred into Eppendorf tubes filled with 2 ml of either 0.5% uranyl acetate in ethanol for substitution with Lowicryl HM 20 and LR White, or 2% OsO4 in acetone for substitution with Epon. Each tube was placed in a copper block in a freeze substitution unit (FSU 010, Balzers Union, Liechtenstein). The temperature was raised from −90°C to −40°C in the course of 3 d before removing the aluminium holders. Samples were rinsed three times with pure acetone or ethanol. The embedding procedure was different for the three resins used. In the case of Epon, the protocol was as follows: 20% in acetone for 1 h and 33% for 9 h at −40°C, then 50% for 2 h on ice and pure resin for 1 d at RT. Polymerization took place at 70°C for 1 d. For HM20, the proportion of the resin, starting with 25% in ethanol, was increased every c. 8–10 h until the samples were placed in pure resin after 3 d, still at −40°C. They were then polymerized by ultraviolet light with indirect radiation for the first 72 h. After another 24 h of direct radiation at −40°C, the prepolymerized tubes were taken out of the substitution apparatus and placed under a UV-lamp at RT. For LR White, the infiltration took place at RT in steps similar to those for HM20. Polymerization was carried out at 60°C.

Cutting and poststaining of samples

Semithin sections (0.5–0.7 µm) of all treatments were made with a glass knife using an ultramicrotome (Ultracut; Reichert-Jung, Leica, Bensheim, Germany) and analysed for thoroughly infected and well-preserved leaf tissue with a light microscope. Ultrathin sections (60–90 nm) were cut from selected samples with a diamond knife (Diatome, Leica, Bensheim, Germany), mounted onto carbon-coated copper or nickel grids covered with formvar film. After immunocytochemical labelling, sections were stained with 1% aqueous uranyl acetate for 15–30 min and Reynold's lead citrate for 10 min; in between and after poststaining, sections were rinsed with distilled water. Some sections were left unstained to better detect 5 nm gold particles. Finally, they were examined with a Zeiss EM 109 transmission electron microscope at 80 kV.

Immunocytochemical labelling

For all labelling protocols, grids with ultrathin sections were placed onto droplets with the respective buffer, blocking and labelling solutions. Successful labelling was achieved for cellulose, chitin, β-1,3-glucans and homogalacturonans with Epon embedded samples, while HM20 and LR White embedded samples were used for labelling of chitin with biotinylated wheat germ agglutinin (WGA), and for detection of polygalacturonase, rhamnogalacturonan-I and xyloglucan epitopes, since the latter were not well preserved in Epon. For better comparison, the labelling protocols are listed in Table 1. To give a representative picture, 20–40 interactions in 200–500 sections were examined for each antibody. In some cases, silver enhancement according to Danscher (1981) and Stierhof et al. (1991) was performed using citric acid (Merck, Darmstadt, Germany), gum arabicum (Merck, Germany), hydrochinon (Merck, Germany), silver lactate (Sigma, Germany) and sodium citrate (Merck). Controls included omission of the first antibody/lectin and, when available, preincubation of the primary antibody/lectin/enzyme with the relevant antigens/chemicals.

Table 1.  Labelling protocols for the enzymes/antibodies/lectins tested
Target moleculeHydratingBlockingIncubation with 1. antibody/ enzyme/lectin1Washing/ blocking1Incubation with 2. antibody1Washing/ blocking1Incubation with 3. antibody1Washing/blocking1
  • BSA, bovine serum albumin; CCRC-M, Complex Carbohydrate Research Centre monoclonal antibody; JIM, John-Innes-Institute monoclonal antibody; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PGA, polygalacturonic acid; RG, rhamnogalacturonan; WGA, wheat germ agglutinin.

  • 1

    Buffers whose pH value is no further specified have the same as those used in the previous steps. All experiments were performed at room temperature, apart from those where a specific temperature is mentioned.

  • 2

    2 Procedures which resulted in cross-labelling of plant and fungal cell walls.

β-1,4-Glucans (cellulose)PBS (pH 6.8), 5 minPBS–PEG (0.02%), 2 × 5 minGold-complexed exoglucanase (diluted 1 : 80 in PBS–PEG), 30 minPBS 3 × 5 min, water 3 × 5 min    
Xyloglucan/ Rhamnogalacturonan I with a terminal α-fucosyl residuePBS (pH 7.6), 10 minPBS–BSA (1%), 3 × 10 minCCRC-M1 (1 : 2 in PBS–BSA), 1 hPBS–BSA (1%), 3 × 5 min; Tris–BSA (1%, pH 8.2), 3 × 5 minDonkey antimouse (1 : 10 in Tris–BSA), 1 hPBS–BSA (1%), 3 × 5 min; water, 3 × 5 min  
Rhamnogalacturonan IPBS (pH 7.6), 10 minPBS–BSA (1%), 3 × 10 minCCRC-M2 (1 : 2 in PBS–BSA), 1 hPBS–BSA (1%), 3 × 5 min; Tris–BSA (1%, pH 8.2), 3 × 5 min Donkey antimouse (1 : 10 in Tris–BSA), 1 hPBS–BSA (1%), 3 × 5 min; water, 3 × 5 min  
Arabinogalactan proteins/ Rhamnogalacturonan IPBS (pH 7.6), 10 minPBS–BSA (1%), 3 × 10 minCCRC-M7 (1 : 2 in PBS–BSA), 1 h PBS–BSA (1%), 3 × 5 min; Tris–BSA (1%, pH 8.2), 3 × 5 min Donkey antimouse (1 : 10 in Tris–BSA), 1 hPBS–BSA (1%), 3 × 5 min; water, 3 × 5 min   
Homogalacturonans with a low to medium degree of esterification (0–50%)0.05 m Tris (pH 7.6), 5 minTris–BSA (0.2%), 3 × 5 minJIM 5 (1 : 20 in Tris–BSA),  ≥ 15 h at 4°CTris–BSA, 3 × 5 min; 0.02 m Tris–BSA (1%, pH 8.2), 3 × 5 minGoat antirat (1 : 20 in Tris–BSA), 1 h Tris–BSA, 3 × 5 min; water, 3 × 5 min  
Homogalacturonans with a medium to high degree of esterification (35–90%)0.05 m Tris (pH 7.6), 5 minTris–BSA (0.2%), 3 × 5 minJIM 7 (1 : 20 in Tris–BSA), ≥ 15 h at 4°CTris–BSA, 3 × 5 min; 0.02 m Tris–BSA (1%, pH 8.2), 3 × 5 min Goat antirat (1 : 20 in Tris–BSA), 1 hTris–BSA, 3 × 5 min; water, 3 × 5 min  
PolygalacturonasePBS (pH 7.4), 5 minPBS–nonfat milkpowder (5%), 3 × 5 minAnti-polygalacturonase (1 : 500 in PBS), 2 hPBS, 5 × 5 minGoat antirabbit (1 : 100 in PBS–nonfat milkpowder), 1 h, or ProteinA–gold (1 : 300 in PBS–BSA–nonfat milkpowder), 1 hPBS, 3 × 5 min; water, 3 × 5 min  
β-1,3-GlucansPBS (pH 7.4), 5 minPBS–BSA (1%), 3 × 5 minAnti-β-1,3-glucans (1 : 30 in PBS–BSA), 1 h at 37°C PBS 3 × 5 min; 0.02 m Tris–BSA (1%)–Tween-20 (0.05%) (pH 8.2), 3 × 5 min Goat antimouse (1 : 20 in Tris–BSA–Tween-20), 30 min Tris 3 × 5 min; water 3 × 5 min  
N-acetylglucosamin (chitin) test 1PBS (pH 7.4), 5 minPBS–BSA (1%), 3 × 5 minWGA (30 µg ml−1 in PBS), 30 minPBS, 3 × 5 min; PBS–PEG (0.02%, pH 6.0), 3 × 5 minOvomucoid–gold complex (1 : 30 in PBS–PEG), 30 minPBS (pH 7.4), 3 × 5 min; water, 3 × 5 min  
N-acetylglucosamin (chitin) test 2PBS (pH 6.8), 5 minPBS–BSA (1%), 3 × 5 minBiotinylated WGA (25 µg ml−1 in PBS), 1 hPBS, 3 × 5 min; 0.02 m Tris–BSA (1%)–Tween-20 (0.05%) (pH 8.2), 3 × 5 min Goat antibiotin (1 : 20 in Tris–BSA–Tween-20), 1 hTris, 3 × 5 min; water, 3 × 5 min  
N-acetylglucosamin (chitin) test 3PBS (pH 7.2), 10 minPBS–BSA (1%)–nonfat milkpowder (0.5%), 3 × 10 minBiotinylated WGA (5 µg ml−1 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA, 6 × 5 minRabbit antibiotin (1 : 300 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA, 6 × 5 minProteinA–gold (1 : 300 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA, 3 × 5 min water, 3 × 5 min
Xyloglucan2PBS (pH 7.4), 10 minPBS–BSA (1%)–nonfat milkpowder (0.5%), 4 × 5 minAnti-xyloglucan (1 : 10 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA (1%)–nonfat milkpowder (0.5%), 6 × 5 minProteinA–gold (1 : 300 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA (1%)–nonfat milkpowder (0.5%), 6 × 5 min; water, 3 × 5 min   
Rhamnogalacturonan-I/ polygalacturonic acids2PBS (pH 7.4), 10 minPBS–BSA (1%)–nonfat milkpowder (0.5%), 4 × 5 minAnti-RG-I/PGA (1 : 10 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA (1%)–nonfat milkpowder (0.5%), 6 × 5 minProteinA–gold (1 : 300 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA (1%)–nonfat milkpowder (0.5%), 6 × 5 min; water, 3 × 5 min   
Extensin2PBS (pH 7.4), 10 minPBS–BSA (1%)–nonfat milkpowder (0.5%), 4 × 5 minAnti-extensin (1 : 10 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA (1%)–nonfat milkpowder (0.5%), 6 × 5 min;ProteinA–gold (1 : 300 in PBS–BSA–nonfat milkpowder), 1 hPBS–BSA (1%)–nonfat milkpowder (0.5%), 6 × 5 min water, 3 × 5 min   

β-1,4-Glucans (cellulose)  Sections were treated with phosphate-buffered saline (PBS), pH 6.8, for 5 min and PBS with 0.02% polyethylene glycol (PEG), pH 6.8, for 2 × 5 min. They were then incubated with a gold-complexed exoglucanase, diluted 1 : 80 in PBS–PEG for 30 min and washed with PBS and water, both for 3 × 5 min. Controls were performed by preincubation of the exoglucanase with β-glucans (0.01 m in PBS) overnight at 4°C. The exoglucanase (EC 3.2.1.91), kindly provided from Dr C. Breuil (University of British Columbia, Vancouver, Canada). The enzyme-gold complex (c. 15 nm in diameter) was prepared according to Frens (1973) and Benhamou et al. (1987). The β-glucans were purchased from Sigma-Aldrich (Oakville, Ontario, Canada).

Xyloglucan/rhamnogalacturonan I with a terminal α-fucosyl residue (1,2)-linked to a galactosyl residue  Sections were treated with PBS buffer (pH 7.6) for 10 min and three subsequent blocking steps with the same buffer containing 1% bovine serum albumin (BSA), each for 10 min. Incubation with the monoclonal antibody CCRC-M1 (kindly provided from Dr M. Hahn, University of Georgia, Athens, GA, USA) diluted 1 : 2 in PBS–BSA lasted for 1 h. Subsequently, washing was done with PBS–BSA for 3 × 5 min, and a Tris buffer (pH 8.2), containing 1% BSA for 3 × 5 min. Sections were then mounted onto a droplet of donkey antimouse antibodies complexed to colloidal gold (6 nm) (Aurion, Australia), diluted 1 : 10 in Tris-BSA for 1 h. Finally, they were washed with Tris-BSA and water, each for 3 × 5 min. For controls, the first antibody was replaced by buffer solution.

Rhamnogalacturonan I  The procedure was the same as for xyloglucan/rhamnogalacturonan I, except that the monoclonal antibody CCRC-M2 was used (kindly provided by Dr M. Hahn).

Arabinogalactan-proteins/rhamnogalacturonan I  The procedure was the same as for xyloglucan/rhamnogalacturonan I, except that the monoclonal antibody CCRC-M7 was used (kindly provided by Dr M. Hahn).

Pectin epitopes with a low to medium degree of esterification (0–50%, Knox et al., 1990)  Sections were treated with 0.05 m Tris buffer (pH 7.6) for 5 min and with the same buffer containing 0.2% BSA for 3 × 5 min. Incubation with the monoclonal antibody JIM 5 (kindly provided from Dr K. Roberts, John-Innes-Institute, Norwich, UK) lasted at least 15 h at 4°C at a dilution of 1 : 20 in Tris–BSA. Sections were then washed with the same buffer for 3 × 5 min and with a 0.02 m Tris-BSA buffer containing 1% BSA (pH 8.2) for 3 × 5 min. Incubation with a goat antirat antibody (IgG) complexed to colloidal gold (6 nm or 15 nm) (Wageningen, the Netherlands) took 1 h at a dilution of 1 : 20 in the latter buffer. Afterwards, sections were washed with the same buffer and with water, both for 3 × 5 min. Controls were performed by either omission of the first antibody or incubation of the first antibody with 1 mg ml−1 citric pectin or polygalacturonic acid (Sigma-Aldrich) for 12 h before the beginning of the experiment.

Pectin epitopes with a medium to high degree of esterification (35–90%, Knox et al., 1990)  The procedure was the same as described in the previous section, except that the monoclonal antibody JIM 7 was used (kindly provided by Dr K. Roberts, John-Innes-Institute, Norwich, UK).

Xyloglucans, rhamnogalacturonan-I/polygalacturonic acids, extensin  Sections were treated with PBS buffer (pH 7.4) for 10 min and blocked with PBS containing 1% BSA and 0.5% nonfat milk powder (pH 7.4) for 4 × 5 min. Grids were incubated with the respective polyclonal antibodies (kindly provided from Dr A. Staehelin, University of Colorado, Boulder, CO, USA), diluted 1 : 10 in PBS–BSA–nonfat milk, for 1 h and washed with the same buffer 6 × 5 min. They were then placed onto a 5 nm proteinA–gold complex (kindly provided from Dr Y. Stierhof, University of Tübingen, Tübingen, Germany), diluted 1 : 300, for 1 h and washed with PBS–BSA–nonfat milk and water, each for 3 × 5 min. Controls were performed by omission of the primary antibody.

Polygalacturonase  Sections were treated with PBS buffer (pH 7.4) for 5 min and blocked with PBS containing 5% nonfat milk powder (pH 7.4) for 3 × 5 min. Incubation took place onto polyclonal anti-polygalacturonase serum (kindly provided by Dr P.-M. Charest, Université Laval, Quebec, Quebec, Canada), diluted 1 : 500 in PBS for 2 h. After washing with PBS for 5 × 5 min, grids were mounted on goat antirabbit antibodies complexed to colloidal gold (15 nm) (Aurion), diluted 1 : 100, or ProteinA-gold (5 nm), diluted 1 : 300 (kindly provided by Dr Y. Stierhof) in PBS containing 5% nonfat milk powder (pH 7.4) for 1 h. Washing was done with PBS and water, each 3 × 5 min. As a control, the primary antibody was omitted.

β-1,3-Glucans  Sections were treated with PBS buffer (pH 7.4) for 5 min and blocked with PBS containing 1% BSA (pH 7.4) for 3 × 5 min. They were then incubated with an anti-β-1,3-glucan antibody (purchased from Biosupplies, Parkville, Australia), diluted 1 : 30 in PBS–BSA for 1 h at 37°C. Sections were washed 3 × 5 min with PBS and blocked with 0.02 m Tris buffer (pH 8.2) with 1% BSA and 0.05% Tween-20. Thereafter, grids were incubated with goat antimouse antibodies complexed to colloidal gold (15 nm) (British BioCell International, Cardiff, UK), diluted 1 : 20 in Tris–BSA–Tween20 for 30 min at RT. Finally, they were washed with Tris buffer and water, both for 3 × 5 min. Controls included omission of the primary antibody and preincubation of the primary antibody with laminarin at 0.5 mg ml−1 (Sigma-Aldrich) overnight at 4°C.

N-acetylglucosamin (chitin)  Three procedures were applied, one with WGA later coupled to ovomucoid-gold and two with biotinylated WGA later bound to secondary antibodies.

  • 1WGA–ovomucoid: sections were treated with PBS buffer (pH 7.4) for 5 min and blocked with PBS containing 1% BSA (pH 7.4) for 3 × 5 min. They were then incubated with 30 µg ml−1 WGA (Sigma-Aldrich) in PBS for 30 min, washed 3 × 5 min with PBS buffer and 3 × 5 min with PBS containing 0.02% PEG (pH 6.0) and then incubated with an ovomucoid-gold complex, diluted 1 : 30 in PBS-PEG for 30 min. This was followed by washing with 3 × 5 min PBS (pH 7.4) and water. Controls were performed leaving out the WGA or preincubation of WGA with chitotriose at 1 mg ml−1 (Sigma-Aldrich, Canada) overnight at 4°C. The ovomucoid-gold complex was prepared according to Benhamou (1988).
  • 2Biotinylated WGA (Sigma-Aldrich): sections were treated with PBS buffer (pH 6.8) for 5 min and blocked with PBS containing 1% BSA (pH 6.8) for 3 × 5 min. They were then incubated with 25 µg ml−1 biotinylated WGA in PBS for 1 h, washed 3 × 5 min with PBS buffer and blocked with 0.02 m Tris buffer (pH 8.2) with 1% BSA and 0.05% Tween-20. They were then incubated with a 20 nm gold goat antibiotin antibody (British Biocell) diluted 1 : 20 in Tris-BSA-Tween20 for 1 h and afterwards washed with Tris buffer and water, each 3 × 5 min. Controls included omission of WGA-biotin and preincubation of the WGA with chitotriose at 1 mg ml−1 overnight at 4°C.
  • 3Biotinylated WGA (Vector Laboratories, Burlingame, CA, USA): sections were first hydrated in PBS pH 7.2 for 10 min and blocked in the same buffer containing 1% BSA and 0.5% nonfat milk powder for 3 × 10 min (also used in every following washing and incubation step). Incubation with WGA lasted 1 h at a concentration of 5 µg ml−1. Subsequent washing for 6 × 5 min was followed by an incubation with a polyclonal rabbit antibiotin antibody (Enzo Diagnostics, Farmingdale, NY, USA) diluted 1 : 300 for 1 h. Sections were washed 6 × 5 min and then incubated with ProteinA gold (5 nm, kindly provided by Dr Y. Stierhof) for 1 h. Finally, they were washed for 3 × 5 min with both buffer and water.

Results

Ultrastructural observations of the interaction between C. trifolii and clover

A thorough description of the ultrastructural details in the interaction between C. trifolii and clover has been presented recently (Simon et al., 2004). Briefly, in its full functioning state the interaction apparatus (IA), which we regard as the structure for nutrient acquisition, is surrounded by a membrane which is continuous with the fungal plasma membrane. The IA consists of a trunk and fine protrusions, which reach deep into the cytoplasm of the hypha in which the IA is made. Opposite the IA the host plasmalemma invaginates to form a bubble. Both compartments are linked by a tube with an electron-dense sheath leading right through the cell wall of either organism (Fig. 1a). Within the tube the plant cell wall seems to remain structurally intact, but is much less electron opaque then adjacent cell wall areas (Fig. 1b). During the course of the interaction, the host bubble becomes encased by increasing amounts of wall appositional material (Fig. 1c). It is finally separated from the host cytoplasm and collapses. At this stage, the delicate protrusions of the IA have disappeared and only its trunk is left (Fig. 1d).

Figure 1.

The interaction between the pathogen Cymadothea trifolii (P) and its clover host (h). (a) The fungus forms an interaction apparatus (IA) within an intercellular hypha opposite to which the host cell invaginates its plasma membrane to form a bubble (bl). Bubble and IA are linked by a tube (arrow) with an electron-dense sheath. Bar, 1 µm. (b) Detail of the interaction zone. Within the tube the plant cell wall is much more electron transparent than at adjacent cell wall sections. The plant cell responds to the interaction by papillae formation (arrowhead). Arrows point to the continuity of the plant and the fungus plasma membrane with the membranes surrounding IA and bubble, respectively. Vesicles (star) possibly add membrane material to the bubble or deposits to the papilla. Bar, 0.2 µm. (c) Beginning encapsulation of the bubble (bl) by host wall appositional material (wa). In the fungal cell, only a fine protrusion of the IA is seen (arrows), because the section is not median. Bar, 0.5 µm. (d) The collapsed bubble (star) has been completely encased by wall appositional material. The IA appears degenerated. Bar, 1 µm. In this and all further figures, plant organelles are indicated by lowercase letters, fungal organelles by uppercase letters. Abbreviations: bl, host bubble; er, endoplasmic reticulum; h, host; hw, host cell wall; IA, interaction apparatus; M, m, mitochondrion; nu, nucleus; P, Pathogen; pl, chloroplast; PW, pathogen cell wall; s, starch grain; wa, wall appositional material.

Immunocytochemical labelling

Cellulose  Labelling for cellulose resulted in numerous gold particles over plant cell walls (Fig. 2a,b). The labelling pattern was not changed within the tube (Fig. 2b–d). Appositional material was free of labelling (Fig. 2d), as were controls (Fig. 2e).

Figure 2.

Labelling for cellulose with a gold-complexed exoglucanase. (a) The plant cell wall is densely labelled, while the wall of Cymadothea trifolii is not. Bar, 1 µm (b) Median section through interaction apparatus (IA), bubble (bl) and tube. Labelling density of the plant cell wall is constant, even within the tube. Bar, 0.2 µm (c) Cross-section through the tube. Bar, 0.1 µm. (d) Wall appositional material (wa) is free of labelling. Bar, 0.2 µm. (e) Control. Bar, 0.5 µm. Abbreviations: bl, host bubble; h, host; IA, interaction apparatus; P, Pathogen; wa, wall appositional material.

Xyloglucan/rhamnogalacturonan-I with a terminal a-fucosyl residue (1,2)-linked to a galactosyl residue  Incubation with the CCRC-M1 antibody clearly labelled the plant cell wall, including the part within the tube (Fig. 3a–d). Plant wall appositional material heavily reacted with the antibody, in particular at late stages of the interaction (Fig. 3d). No labelling was obtained with controls (Fig. 3e).

Figure 3.

Labelling for xyloglucan (rhamnogalacturonan I) with CCRC-M1. (a) The unaltered plant cell wall is labelled as is the area within the tube (arrows). Bar, 0.1 µm. (b) Old interaction. Some labelling is present within the tube (arrow). Bar, 0.1 µm. (c) Cross-section through tube. Labelling is consistent along the plant cell wall, even within the tube. Bar, 0.2 µm. (d) Very old interaction; silver enhancement. The collapsed bubble (star) is encased by enormous amounts of wall appositional material (wa) which heavily reacts with the antibody. Bar, 0.5 µm. (e) Control. Bar, 0.5 µm. Abbreviations: bl, host bubble; IA, interaction apparatus; M, mitochondrion; wa, wall appositional material.

Rhamnogalacturonan I  No labelling was found with the antibody CCRC-M2 (not shown).

Arabinogalactan-proteins/rhamnogalacturonan-I  CCRC-M7, generated against rhamnogalacturonan-I but also recognizing epitopes in plant membrane glycoproteins (Steffan et al., 1995), strongly labelled the inner layer of plant cell walls (Fig. 4a–f). Labelling was absent within the tube (Fig. 4b–e). Wall appositions (Fig. 4b,c,f) and nuclei of either organism (Fig. 4a,b) were densely labelled. Controls were the same as for xyloglucan/rhamnogalacturonan I and were free of labelling (not shown).

Figure 4.

Labelling for arabinogalactan proteins/rhamnogalacturonan I with CCRC-M7. (a–c) Silver enhancement. (a) The plant cell wall and the nuclei of both organisms are densely labelled. Bar, 2 µm. (b) Very old interaction. The tube (arrow) is free of labelling, but the wall appositional material having encased the host bubble (star) is densely labelled. Bar, 1 µm. (c) Dense labelling of the plant cell wall and of wall appositional material encasing the host bubble (star). Bar, 0.5 µm. (d) Labelling with 15 nm gold. No particles are located within the tube whereas a few occur over wall appositional material. Bar, 0.2 µm. (e) Labelling with 5 nm gold is dense over the inner area of the plant wall and appositional material but absent within the tube. Bar, 0.2 µm. (f) Heavy labelling of the wall appositional material using 5 nm gold. Bar, 0.2 µm. Abbreviations: bl, host bubble; h, host; hw, host cell wall; IA, interaction apparatus; Nu, nu, nucleus; P, Pathogen; pl, chloroplast; PW, pathogen cell wall; wa, wall appositional material.

Homogalacturonans with a low to medium degree of esterification (0–50%)  Little to no labelling occurred with the antibody JIM 5 (not shown).

Homogalacturonans with a medium to high degree of esterification (35–90%)  Incubation with the JIM 7 antibody resulted in strong labelling of the plant cell wall. However, no labelling was observed within the tube (Fig. 5a–c). Controls were free of labelling (Fig. 5d).

Figure 5.

Labelling for medium to highly esterified homogalacturonans with JIM 7. (a,b) Labelling with JIM 7 and 6 nm gold (b, enlargement of a). Although the plant cell wall is heavily labelled, no gold particles can be detected within the tube. Bar, 1 µm. (a), 0.1 µm (b). (c) Cross-section through tube, labelling with 15 nm gold. The tube (arrow) is free of labelling. Bar, 0.5 µm. (d) Control (preincubation of JIM 7 with citric pectin). Bar, 0.5 µm. Abbreviations: d, dictyosome; er, endoplasmic reticulum; h, host; hw, host cell wall; IA, interaction apparatus; M, m, mitochondrion; nu, nucleus; P, Pathogen; PW, pathogen cell wall.

Xyloglucans, rhamnogalacturonan-I/polygalacturonic acids, extensin  These antibodies showed dense cross-labelling against unknown epitopes in the fungal cell wall and were therefore not used further (not shown).

Polygalacturonase  Incubation with an antibody against a fungal polygalacturonase showed strong labelling of the IA and a few gold particles along the cell wall of the hyphae (Fig. 6a–c). Old IAs were scarcely labelled (Fig. 6d). No labelling was found in host bubbles (Fig. 6b,c), or in controls (Fig. 6e).

Figure 6.

Labelling for polygalacturonase with an antipolygalacturonase antibody. (a) One hypha produces two IAs to attack two different host cells. Only the IAs and the fungal cell wall (arrows) show labelling for the enzyme. Bar, 1 µm. (b) Specific labelling of the IA. Gold particle size, 15 nm. Bar, 0.5 µm. (c) The labelling becomes more intense at the IA but not along the hyphal wall when 5 nm gold is used. Bar, 0.5 µm. (d) At the end of the interaction, the remnants of the IA show only scattered labelling (arrows). Bar, 0.5 µm. (e) Control. Bar, 0.5 µm. Plant organelles are indicated by lowercase letters, fungal organelles by uppercase letters. Abbreviations: h, host; IA, interaction apparatus; M, m, mitochondrion; Mb, microbody; Nu, nucleus; P, Pathogen; pl, chloroplast.

β-1,3-Glucans  Labelling for these sugars resulted in evenly distributed gold particles all along the cell walls of the hyphae (Fig. 7a–c). Furthermore, all stages of the IA of C. trifolii were consistently labelled (Fig. 7a–d). On the plant side, cell wall appositions, which finally encapsulate the host bubble, were also labelled, indicating the presence of callose (Fig. 7a–d). Within the deposited material, labelling was restricted to bright areas (Fig. 7b,c). Unaltered plant cell walls and controls (Fig. 7e) showed no labelling.

Figure 7.

Labelling for β-1,3-glucans with an anti-β-1,3-glucan antibody. (a–d) Development from a well-functioning to a nonfunctioning interaction. Increasing amounts of callose are added onto the host bubble (bl) which finally collapses. Only the electron-translucent areas of the deposits are labelled (arrows) while the host wall, in general, remains free of labelling. In the fungus, the cell wall and interaction apparatus (IA) are heavily labelled. (e) Control (preincubation of the antibody with laminarin). Bars, 0.5 µm. Abbreviations: bl, host bubble; er, endoplasmic reticulum; h, host; IA, interaction apparatus; M, m, mitochondrion; Nu, nucleus; P, Pathogen; wa, wall appositional material.

Chitin  For chitin, the different detection methods gave more or less congruent results. However, labelling with WGA–ovomucoid was possible with Epon-embedded samples but not with HM 20- or with LR White-embedded samples (not shown). Labelling was not neatly distributed, but nevertheless strong along the cell wall of the fungus (Fig. 8a–c). The IA and its protrusions were also labelled: the older the stage of the IA, the stronger the labelling (Fig. 8a–c). Biotinylated WGA gave a much finer and more regular labelling (Fig. 8d–f), furthermore differentiating between septa and fungal cell walls which seemingly contained less and more chitin, respectively (Fig. 8d). The WGA from Sigma-Aldrich did not mark the IA (Fig. 8e), while the WGA from Vector densely labelled this structure (Fig. 8f). Controls had no labelling (Fig. 8g).

Figure 8.

Labelling for chitin. (a–c) WGA–ovomucoid; (d–f) biotinylated WGA. (a) The fungal cell wall and the IA are labelled, albeit not regularly. Bar, 0.5 µm. (b) Section through upper part of IA. The protrusions are also labelled. Bar, 1 µm. (c) Dense labelling of an old IA. Bar, 0.2 µm. (d) Labelling with biotinylated WGA is much more regular. Arrows point at weaker labelling along a septum, while arrowheads show parallel labelling of two neighbouring hyphal walls. A microbody (MB) gives birth to a Woronin body. Bar, 1 µm. (e) WGA from Sigma yields good labelling along the fungal cell wall, but almost none over the IA. Bar, 0.5 µm. (f) The labelling over the IA becomes much more intense when WGA from Vector is used. Bar, 0.2 µm. (g) Control (preincubation of WGA with chitotriose). Bar, 0.5 µm. Abbreviations: er, endoplasmic reticulum; h, host; IA, interaction apparatus; M, m, mitochondrion; MB, microbody; Nu, nu, nucleus; pl, chloroplast.

Discussion

General remarks

High-pressure freezing and freeze substitution generally yield better structural preservation of biological specimens than conventional fixation (Hoch, 1991; Mendgen et al., 1991; Hippe-Sanwald, 1993; Berg, 1994; Hippe-Sanwald et al., 1994; Mims et al., 2002). Yet few plant pathologists actually use these techniques (Mims et al., 2002). Furthermore, they have been applied primarily on rust fungi (Swann & Mims, 1990; Mendgen et al., 1991;Roberson, 1993; Voegele et al., 2001 Mims et al., 2002). Only a small number of reports are available for other fungal pathogens (Hippe-Sanwald et al., 1994; Bauer et al., 1995; Ouellette et al., 1995; O’Connell et al., 1996; Simon et al., 2004). However, in combination with specific embedding resins (e.g. LR White, Lowicryls) these methods not only keep samples nearer to their state in nature (Bourett et al., 1999), they also result in good preservation and accessibility of various molecules (Hippe-Sanwald, 1993; Hess, 2003) – a prerequisite for immunocytochemical studies. Therefore, different embedding media were assessed in the present study. While Epon yielded an overall good preservation of carbohydrates such as cellulose, β-1,3-glucans, homogalacturonans and chitin, sufficient preservation of other antigens was only achieved in Lowicryl HM20 and in LR White. The ultrastructural appearance was acceptable in all cases, although slight differences were found. For example, the bubble triggered by the pathogen inside the host cell appeared much more electron dense in LR White- and HM20-embedded specimens than in Epon-embedded specimens, possibly owing to a stronger contraction during the embedding process (H. Schwarz, pers. comm.). The IA and the cell walls of both plant and fungus were weakly stained after HM 20 treatment, which might result from the omission of OsO4 during the substitution procedure (Hippe-Sanwald et al., 1994). Generally, the decision about which resin to take should be case-specific, since especially Lowicryls pose a potential health threat to the researcher.

A problem of immunolocalization of cell wall antigens is their frequently uneven distribution. Several authors have pointed out that some plant cell wall components are probably present in discrete domains (Lynch & Staehelin, 1992; Freshour et al., 1996; Freshour et al., 2003). Such incoherence makes it difficult to provide definite evidence for a cell wall area as small as 300–400 nm, the diameter of the tube. Yet because of the great number of interactions studied for each antibody, our data are based on solid grounds.

Ultrastructural observations of the interaction between C. trifolii and clover

Ultrastructural details of this interaction have been discussed by Simon et al. (2004). In the context of the results presented here, special emphasis is placed on the altered electron density of the plant cell wall within the tube, for which changes of wall chemistry were held responsible by Simon et al. (2004). Similar observations and speculations have been made in other plant–pathogen interactions, for example in Cucumis melo infected by Colletotrichum lindemuthianum (Vian et al., 1996). The electron translucency of parts of the host wall appositions parallels findings of Golotte et al. (1993) in a pea mutant which blocks mycorrhizal infections.

Immunocytochemical labelling

The cell wall of the fungus and the interaction apparatus  The photomicrographs presented in this paper document the existence of β-1,3-glucans and chitin in the cell wall of C. trifolii. Both polymers are known to be the most important structural components of ascomycetous cell walls (Kuhn et al., 1990; Ruiz-Herrera, 1992). The fact that they are also present in the IA, as shown by dense labelling in either case, provides evidence for the wall-like nature of that structure (Simon et al., 2004). It is worth noting the different labelling patterns of the various methods employed to detect chitin. While WGA in combination with an ovomucoid–gold complex reliably labelled the wall and the IA, it was almost impossible to avoid gold clusters. Furthermore, large areas of the hyphal walls remained unlabelled, although it is unlikely that chitin should not be present at these places.

This method only worked with Epon-embedded samples. We therefore tested biotinylated WGA, various secondary or even tertiary antibodies. Not all of the WGAs and antibodies worked well (not shown), but the ones referred to in here resulted in very neat labelling. Although WGA from Sigma-Aldrich consistently labelled the fungal wall, only Vector WGA regularly labelled the IA. Furthermore, the latter showed no preference for any of the tested embedding resins, while the former resulted in only weak labelling of Epon-embedded samples. Therefore we recommend to try more than one procedure when using WGA to locate chitin.

The plant cell wall and its alterations within the tube  Most antibodies against plant cell wall constituents used in the present work gave reliable and reproducible results. There were, however, some exceptions: Three polyclonal antibodies, directed against xyloglucans (XGs), rhamnogalacturonan-I (RG-I)/polygalacturonic acids (PGAs) and extensins (Moore et al., 1986; Moore et al., 1991; Lynch & Staehelin, 1992), heavily labelled the clover cell wall. Unfortunately, they also had a very high affinity for unknown epitopes in the wall of the fungus and in the IA, and were therefore useless for the present study. Such cross-labelling has been reported for Trichoderma cellobiohydrolase-I recognizing β-1,4-glucans in the cell walls of Calluna vulgaris and Hymenoscyphus ericae (Bonfante, 1994). Very little to no labelling was obtained with the monoclonal antibody JIM 5, which recognizes pectin epitopes with a low degree of esterification (Knox et al., 1990), and CCRC-M2 with a high affinity for an unknown epitope in RG-I (Puhlmann et al., 1994). The respective epitopes might have been masked since, especially in the former case, it is unlikely that the middle lamella and cell corners, which in many plants and plant organs have been shown to be reactive with JIM 5 (VandenBosch et al., 1989; Knox et al., 1990; Rioux et al., 1995; Wisnieswski & Davies, 1995; Majewska-Sawka & Münster, 2003), were almost completely devoid of such homogalacturonans.

The labelling pattern of those antibodies that worked gave a sufficiently detailed overview of the changes occurring in the tube linking IA and the host bubble. Unlike intracellular fungi, which must break through the host wall and therefore need to dissolve its cellulose microfibrils and xyloglucan network, those polymers are apparently not attacked by C. trifolii. The consistent labelling obtained for cellulose and xyloglucan at the interaction zone suggests that these main structural elements of dicotyledonous plant cell walls (Carpita & Gibeaut, 1993; Schindler, 1993) are left unaltered by the fungus.

The antibody used against xyloglucan, CCRC-M1, was primarily generated against RG-I, but exhibited a much higher affinity for several XGs (Puhlmann et al., 1994). It cannot be ruled out that CCRC-M1 and CCRC-M7, the latter binding to epitopes in RG-I present in the inner half of the plant cell wall and, to a smaller degree, plant membrane proteins (Puhlmann et al., 1994), may have recognized similar antigens. Yet, the slightly different labelling patterns of CCRC-M1 and CCRC-M7 in our study, and the absence of CCRC-M7 antigens within the tube indicate that CCRC-M1 has not recognized RG-I but xyloglucan. Other authors have also proven the predominant xyloglucan affinity of CCRC-M1 (Sherrier & VandenBosch, 1994; Rodriguez-Galvez & Mendgen, 1995).

Furthermore, two pectins were successfully analysed: RG-I and homogalacturonans with a medium to high degree of esterification. These polysaccharides represent the bulk part of the matrix in most dicotyledonous plant cell walls (Carpita & Gibeaut, 1993; Schindler, 1993). Yet, even though the unaltered cell wall was consistently labelled by the respective antibodies, CCRC-M7 and JIM 7, we never found any labelling in the tube. Consequently, it has to be assumed that the pectin matrix is degraded there by the fungus. As one piece of evidence to support this point we could detect the presence of a polygalacturonase (PGase) in functioning but not in degenerated IAs using an antibody against a PGase isolated from Fusarium oxysporum f. sp. radicis-lycopersici (Blais, 1991; Charest et al., 2004). Apparently, this enzyme is not needed at this site once the interaction has come to an end. The slight anti-PGase labelling along the fungal wall may be explained by the intercellular spread of the pathogen. On its way through the plant tissue, the pectinaceous middle lamella has to be dissolved, otherwise C. trifolii would be restricted to naturally occurring air chambers.

The importance of pectinolytic enzymes in plant–pathogen interactions is rather controversial (Mann, 1962; Puhalla & Howell, 1975; Collmer & Keen, 1986; Durrands & Cooper, 1988; Scott-Craig et al., 1990; Annis & Goodwin, 1997; Baayen et al., 1997; Murdoch et al., 1999). For Claviceps purpurea, Tenberge et al. (1996) showed that PGase, probably in synergy with other enzymes such as pectin methylesterase, was responsible for complete breakdown of galacturonans in rye ovary cell walls. Applying a gene-replacement approach, Oeser et al. (2002) could demonstrate that the product of two endopolygalacturonase genes, which are expressed in all stages of infection, are crucial pathogenicity factors for the development of this fungus on rye ovaries. Claviceps purpurea is a biotrophic pathogen like C. trifolii. It might well be that in such fungi PGases are much more important than in necrotrophic pathogens. Consequently, the suggestion that ‘pectinase is not very important in fungal pathogenesis’ (Walton, 1994) cannot be sustained. The high homology of fungal PGases (Cervone et al., 1990; Bussink et al., 1992; Tenberge et al., 1996) might explain why an antibody generated against a Fusarium PGase worked so well in our system. Although we have examined only polygalacturonase, other enzymes such as pectin methylesterase may be significant as well. Further work is needed to clarify whether such enzymes are produced by C. trifolii.

Taken together, these data indicate that C. trifolii has developed a cunning strategy to gain nutrients from its host. Cellulose and xyloglucan, the main skeletal elements of the plant cell wall, are left intact, while the pectin matrix is broken down (Table 2) by a polygalacturonase and possibly other enzymes. This process is restricted to a wall penetrating tube which links the IA and the host bubble and probably functions as a channel for nutrient transfer from the host bubble into the IA. The result of such a differential degradation of the host cell wall would be a significant increase of wall porosity within the tube. Similar effects of pectinolytic enzymes have been demonstrated by Baron-Epel et al. (1988), Fujino & Itoh (1998) and McCann et al. (1990). Since nutrient molecules like oligopeptides or oligosaccharides are small enough to easily pass through the plant cell wall, it is unlikely that the assumed nutrient flow is much increased by larger pores. Conversely, fungal molecules triggering the formation of the host bubble or facilitating nutrient transfer across the bubble membrane may be required to travel through the host wall and into the bubble. In this respect, we have preliminary data indicating that a protein of C. trifolii becomes integrated into the bubble membrane.

Table 2.  Presence or absence of the respective plant cell wall polymers within the tube
Target moleculeAntibody/enzymePresence in tube
β-1,4-Glucans (cellulose)Exoglucanase+
XyloglucanCCRC-M1+
Rhamnogalacturonan I/arabinogalactan proteinsCCRC-M7
Medium to highly esterified homogalacturonansJIM 7

To obtain nutrients from a host cell without disrupting its wall may decrease the efficacy of possible defence mechanisms such as a hypersensitive reaction or phytoalexin production. Our studies show that one plant cell can be attacked by more than one IA from the same or different hyphae (unpublished). There are no signs of degradation in the hyphal walls as, for example, in F. oxysporum f. sp. radicis-lycopersici in tomato roots (Benhamou et al., 1990). This indicates that no enzymatic or toxin-producing machinery is turned on after aggression towards the host cell has begun. Yet, although there is no apparent enzymatic fight-back on the host side, the plant cell does react. Wall appositional material is transported to the site of the interaction until the bubble is completely encased and finally collapses. Similar defence mechanisms have been reported in many plant–pathogen interactions, for example against Pseudomonas in bean (Brown & Mansfield, 1991), Xanthomonas in cassava (Boher et al., 1996), F. oxysporum in tomato (Charest et al., 1984) or cotton (Rodriguez-Galvez & Mendgen, 1995), Ophiostoma ulmi in elm trees (Rioux & Biggs, 1994), Glomus mosseae in a nonmycorrhizal pea mutant (Golotte et al., 1993) and Uromyces vigniae on broad bean (Xu & Mendgen, 1997). Many authors have shown that these deposits contain glycoproteins and/or PR-1 proteins (Brown & Mansfield, 1991; Golotte et al., 1995; Rodriguez-Galvez & Mendgen, 1995; Cordier et al., 1998) and callose (Golotte et al., 1993; Rodriguez-Galvez & Mendgen, 1995; Boher et al., 1996; Xu & Mendgen, 1997; Cordier et al., 1998). Rodriguez-Galvez & Mendgen (1995) have furthermore proven the occurrence of fucosylated xyloglucans along the ridges of wall deposits in cotton cells to ward off Fusarium, while their centre remained unlabelled. They also noted that labelling for callose seemed to be denser in more electron-translucent areas. In addition, they observed that the appositions contained pectic epitopes. These results are mostly mirrored by our observations on clover cells infected with C. trifolii. Contrary to Rodriguez-Galvez & Mendgen (1995), we could not see any labelling of wall appositions after incubation with JIM 5 or 7. Instead, CCRC-M7 consistently labelled such deposits. Therefore, the wall appositions in the system studied here contain at least callose, RG-I and XG, but no epitopes reactive with JIM 5 or JIM 7. This also means that the Rodriguez-Galvez & Mendgen's hypothesis (1995) stating that ‘the absence of … callose seems to characterize compatible biotrophic interactions’ cannot be corroborated.

In conclusion, we were able to demonstrate that in the interaction between C. trifolii and Trifolium repens the wall of the plant cell is partly but not completely degraded at a very localized area. This alteration very likely leads to an increased porosity. Although the reasoning behind this remains unclear at present, a possible explanation, the transfer of fungal proteins into the host bubble and its membrane, is under investigation. The interaction apparatus produced by the pathogen for nutrient uptake contains components characteristic of ascomycete walls and is therefore considered apoplastic, confirming ultrastructural observations made earlier (Simon et al., 2004). The wall appositions loaded onto the host bubble by the plant cell are made of at least callose, rhamnogalacturonan-I and xyloglucan and appear to end the interaction.

Acknowledgements

We express our gratitude to R. Kirschner for specimens of C. trifolii, H. Schwarz, Y. Stierhof and P. Walther for their help with cryofixation and freeze-substitution and Y. Stierhof, I. Kottke, H. Schwarz, R. Voegele, U. Nehls and G. B. Ouellette for many inspiring discussions and ideas. We are grateful to S. Hippe-Sanwald, I. Kottke and R. Voegele for critically reading the manuscript. Furthermore, we are indebted to C. Breuil, P.-M. Charest, M. Hahn, K. Roberts, H. Schwarz, A. Staehelin and Y. Stierhof for the kind gifts of primary or secondary antibodies. The technical assistance of M. Wagner-Eha, F. Albrecht, D. Ripper and H. Steigele is very much appreciated. This work has been partly funded by a DFG-fellowship to U. S. (Graduate College ‘Infection Biology’, 685).

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