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The interface between the arbuscular mycorrhizal fungus Glomus intraradices and root cells of Panax quinquefolius: a Paris-type mycorrhizal association

     Department of Botany, University of Guelph, Guelph, Ontario, N1G 2W1 Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Two major types of arbucular mycorrhizal associations, the Arum-type and the Paris-type, have been identified based on morphological features. Although the Paris-type is the most common, it is the Arum-type that has been most intensively studied in terms of structure/function because of its prevalence in agronomically important plant species. In this study, the interface between the host cell cytoplasm and intracellular hyphae (extensive hyphal coils and arbusculate coils), which typify the Paris-type mycorrhiza, was studied. Using immunofluorescence techniques combined with laser scanning confocal microscopy, dramatic changes in the cytoskeleton in colonized cells were observed. Changes in the positioning of both host cell microtubules and actin filaments occurred in colonized plant cells. Both microtubules and actin filaments were associated with the hyphal coils and the arbusculate coils. An interfacial matrix, of host origin, was demonstrated between hyphal coils and arbusculate coils using various affinity techniques. It formed an apoplastic compartment consisting of cellulose and pectins between the fungus and host cell cytoplasm. There was less labelling adjacent to the fine branches of arbusculate coils compared to the hyphal coils. These observations show some similarities to those seen with Arum-type mycorrhizas.

Key words: actin filaments, arbuscules, hyphal coils, interfacial matrix, microtubules

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Two types of arbuscular mycorrhizal (AM) associations, the Arum-type and the Paris-type, have been recognized based on morphological characteristics of the colonization process (Gallaud 1905). The Arum-type, in which the spread of colonization within the root is largely by intercellular hyphae, has been the most frequently studied in terms of morphology and physiology mainly because it occurs in most agronomically important plant species (Smith and Smith 1996, 1997). The Paris-type, in which spread of colonization is mainly through the growth of hyphae from cell to cell, is also characterized by extensive intracellular coiling of hyphae prior to arbuscule formation from these coils (Gallaud 1905, Smith and Smith 1996, Whitbread et al 1996). The quantification of the colonization process has been studied for one Paris-type mycorrhizal association (Cavagnaro et al 2001). These authors showed that hyphal coils develop within 5 d and arbuscules (referred to as arbusculate coils) within 11 d when seedlings of Asphodelus fistulosus were planted into pot cultures of Allium porrum colonized by Glomus coronatum. Dickson and Kolesik (1999) compared the surface area and volume of intracellular coils in Lilium sp. and arbuscules in Allium porrum, and found that the coils had higher values for both measurements. This is indicative of the potentially large interface for transfer of nutrients between coils and host cell cytoplasm.

In contrast to the detailed studies of the interface between intracellular fungal structures, particularly arbuscules, and root cells in Arum-type mycorrhizas, there is limited comparable information for Paris-type mycorrhizas. In Arum-type mycorrhizas, a host-derived membrane surrounds arbuscule branches, and an apoplastic space (interface) forms between this membrane and hyphal cell walls (Bonfante and Perotto 1995). This apoplastic space consists of a complex of host-derived cell wall molecules including cellulose and hydroxyproline-rich glycoprotein (Balestrini et al 1994), other glycoproteins (Perotto et al 1994), and pectins (Bonfante-Fasolo et al 1990). There is some variation in results for cellullose. In Zea mays L., cellulose is abundant around hyphal coils but is present in low amounts around fine arbuscule branches (Balestrini et al 1994) whereas in Allium porrum L., considerable cellulose is present in the interface around arbuscule branches (Bonfante-Fasolo et al 1999). Regardless of differences in the nature of this interface, nutrients must pass through this region from the fungus into the host cell.

The interaction between intracellular hyphae and the cytoskeleton of root cells has been studied fairly extensively in Arum-type mycorrhizas (Genre and Bonfante 1997, 1998, Matsubara et al 1999). Both major components of the plant cytoskeletal system, microtubules (Mts) and actin filaments, become closely associated with intracellular hyphae (see Peterson et al 2000 for a review). Similar studies have not been undertaken with Paris-type mycorrhizas.

Since the function of the extensive coils in Paris-type mycorrhizas is not known (Smith and Smith 1996), it is important to determine the nature of the interface between them and root cell cytoplasm and to determine how these coils interact with the root cell cytoskeleton before hypotheses concerning their function can be put forward. The objectives of this study were to determine the nature of the interface between hyphal coils and arbusculate coils with root cell cytoplasm and to determine the effect that these structures have on Mts and actin filaments in host cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Vernalized Panax quinquefolius L. (ginseng) seeds with protruding radicles were transplanted into Conetainers (120 mL capacity; Bay Leach Nursery, Oregon) that were prepared as follows: Each Conetainer was 3/4 filled with Turface^TM. Inoculum, consisting of chopped roots of Allium porrum L. (leek) previously colonized by an isolate of Glomus intraradices Schenck and Smith, was placed on the surface and covered with 1 cm Turface. One seed was then placed on this surface and covered with 1 cm Turface. Control plants were grown in Turface lacking root inoculum. Twenty five Conetainers with inoculum and twenty five controls were arranged randomly in a controlled environment growth chamber under fluorescent and incandescent light (250 µ·m^-2·s^-1), with a 14-h photoperiod and a day/night temperature of 20/15 C. They were watered daily with distilled water and, after seedling emergence, were fertilized once a week with Long Ashton's nutrient solution (Hewitt 1966). Lateral roots were harvested between 8–11 wk post-seedling emergence.

Root clearing and staining – Roots were severed from seedlings at 11 wk, fixed in 50% ethanol for a minimum of 24 h, cleared in 10% (w/v) KOH by boiling for 30 min, rinsed in distilled water, and stained with 0.1% (w/v) chlorazol black E for 1 h at 55 C (Brundrett et al 1984). A random sub-sample of stained lateral roots was destained in glycerine and then mounted in glycerine on microscope slides for microscopic observation of the colonization process.

Tissue preparation for immunofluorescence microscopy – Methods used for observation of Mts and actin filaments in control and colonized roots were similar to those used by Uetake et al (1997) and Uetake and Peterson (1997). Briefly, roots were fixed for 1 h at room temperature in a mixture of freshly prepared 2% or 4% (w/v) paraformaldehyde and 2.5% or 1.0% (v/v) glutaraldehyde in PME (50 mM PIPES, 1 mM MgSO₄·7H₂O, 2 mM EGTA, pH 6.9) buffer for preparation of tissue for labelling Mts or actin filaments, respectively. Fixed roots were then hand-sectioned longitudinally with a sharp two-sided razor blade, treated for two 5-min periods with 0.1% NaBH₄ in 20 mM Sörenson's phosphate buffer with 150 mM NaCl (PBS, pH 7.0), blocked for 10 min in 1% (w/v) bovine serum albumin (BSA) in PBS, incubated overnight at 4 C with primary antibody (monoclonal mouse anti-chick ß-tubulin for labelling Mts and monoclonal mouse anti-actin for labelling actin ; Amersham International plc, Bucks, UK) and then treated for 3 h at room temperature with the secondary antibody (Cy3^TM -conjugated goat anti-mouse IgG, F (ab¹)₂ fragment; Jackson Immunoresearch Laboratories, Inc., West Grove, Pennsylvania, USA). Both the primary and secondary antibodies were diluted 1:50 in the blocking solution. Control sections were prepared in the absence of primary antibody. Root sections were rinsed twice in PBS, once in equilibration buffer (pH 9.0) (Molecular Probes Inc. Eugene, Oregon, USA) and observed with a BIORAD MRC-600 laser scanning confocal microscope (LSCM) mounted onto a Nikon Optiphot-2 microscope and equipped with a krypton-argon mixed gas laser.

Alternate method for labelling actin filaments – Unfixed tissue was cut directly into 0.2 µM BODIPY®-phallicidin (Molecular Probes Inc., Eugene, Oregon, USA) in PME buffer and left in the dark at room temperature for 1 h. Sections were rinsed in equilibration buffer, pH 9.0, mounted and observed in the same manner used for antibody labelled sections.

Tissue preparation for immunocytochemistry and enzyme-gold labelling – Colonized and uncolonized lateral roots were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for 1.5 h at room temperature, rinsed several times in the same buffer, cut into 1 mm pieces and then post-fixed in 1% osmium tetroxide in phosphate buffer for 1 h. Root pieces were then rinsed in buffer for 1 h and dehydrated in a graded ethanol series over 2 d and embedded in LR White resin (London Resin Co., Basingstoke, UK). Thin sections of gold interference color were cut with a Reichert ultramicrotome and picked up on either 100 mesh formvar coated grids or 200 mesh uncoated grids in preparation for labelling. Observations were made with either a Philips 300 or a JEOL-100 CX transmission electron microscope.

Enzyme-gold labelling – Cellobiohydralase (CBH II) (Megazyme International, Bray, Co., Wicklow, Ireland) was conjugated to colloidal gold (12 nM) using a protocol modified from Slot and Geuze (1985) and Wang et al (1985). Briefly, the pH of 10 mL of the gold sol solution was adjusted to pH 8.5 and 125 µL of the enzyme was added and stirred for 10 min. Two mL of 5% BSA were added to the solution to act as a stabilizer and the enzyme-gold complex was centrifuged in a Beckman J-221 ultracentrifuge for 25 min at 16 000 rpm. The pellet was resuspended in 0.5 M NaCl and 0.05% sodium azide (TBS, pH 8.2) containing 1% BSA. Uncoated 200 mesh grids with root sections were then incubated for 10 min in 1% BSA diluted in 0.05 M citrate-phosphate buffer, pH 4.5 with 0.1 M NaCl and 0.2% Tween 20. Following this, they were incubated in a 1:100 dilution of the enzyme-gold complex in the blocking solution for 30 min. The grids were rinsed 3 times in the citrate-phosphate buffer and several times in distilled water, allowed to dry and then stained with uranyl acetate and lead citrate.

Immunogold labelling – Immunogold labelling of pectins was done according to the protocol of Bonfante et al (1990). JIM 5 and JIM 7 monoclonal antibodies were used for the localization of non-esterified and methyl-esterified pectins, respectively. Colonized and uncolonized root sections on 100 mesh formvar-coated copper grids were treated as follows: sections were incubated in a 1:30 dilution of normal goat serum in 0.05 M Tris-HCL buffer, pH 7.5 with 0.2% BSA for 20 min. They were then incubated overnight at 4 C in a 1:2 dilution of the primary antibody (JIM 5 or JIM 7) with 1% BSA in 0.05 M Tris-HCl buffer. Sections were then washed 3 times in 0.2 M Tris-HCl buffer, pH 8.2 and incubated at room temperature for 1 h in a 1:20 dilution of the secondary anti-rat (goat) IgG (Jackson ImmunoResearch Laboratories, Inc., Westgrove Pennsylvania, USA) conjugated to colloidal gold (12 nM) in 0.1% BSA in Tris-HCl buffer, pH 8.2). Sections were then rinsed 2 times in the buffer and several times in distilled water. Control samples were treated in the same manner but were incubated with secondary antibody only. Sections were stained with uranyl acetate and lead citrate.

	RESULTS

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Cleared root material – The usual colonization process of the Paris-type mycorrhizal association was observed between Glomus intraradices and ginseng roots. External hyphae contacted the root epidermis and formed appressoria from which penetration hyphae developed (Fig. 1). At 11 wk, many arbusculate coils and vesicles were present (Fig. 1). At 8 wk post-seedling emergence cleared roots showed cortical cells with typical hyphal coils (Fig. 2) some of which formed small lateral branches to become arbusculate coils (Fig. 3). It was these latter two stages that were of interest in determining the nature of the interface between the fungus and the root cell cytoplasm.

View larger version (104K): [in this window] [in a new window]    Plate I. FIGS. 1–3. Cleared roots of Panax quinquefolius colonized with Glomus intraradices stained with chlorazol black E. Fig. 1. Segment of root 11 wk after seedling emergence showing an appressorium (arrowhead), vesicles (v) and arbusculate coils (double arrowheads). Scale bar = 100 µm Fig. 2. Cortical cells in root processed 8 wk after seedling emergence showing hyphal coils (arrowheads). Scale bar = 250 µm Fig. 3. Cortical cell showing the development of fine branches (arrowheads) from a coiled hypha (double arrowheads) to form an arbusculate coil. Scale bar = 50 µm (Photo from Whitbread et al 1996, with permission)

View larger version (164K): [in this window] [in a new window]    Plate II. FIGS. 4–12. Immunofluorescence microscopy of uncolonized Panax quinquefolius roots (Figs. 4, 5) or roots colonized with Glomus intraradices (Figs. 6–12). Fig. 4. Cortical microtubules (arrowheads) in cortical cells. Scale bar = 25 µm. Fig. 5. Actin filaments (arrowheads) and cables (double arrowhead), some associated with the nucleus (n). Scale bar = 100 µm. Fig. 6. Actin filaments mostly associated with a hyphal coil (arrowheads) with a few remaining in the peripheral cytoplasm (double arrowhead). Scale bar = 25 µm. Fig. 7. A network of actin filaments (arrowheads) associated with a hyphal coil. Scale bar = 250 µm. Fig. 8. Short actin filaments (arrowheads) associated with arbusculate coil. Scale bar = 250 µm. Fig. 9. Microtubules forming a network around hyphal coils. A few microtubules are present in the peripheral cytoplasm (arrowheads). Scale bar = 25 µm. Fig. 10. Microtubules associated with hyphal coils and arbusculate coils (arrowheads). Scale bar = 25 µm. Fig. 11. A collapsed arbusculate coil with microtubules (arrowhead). Cortical microtubules (double arrowheads) are present at this stage. Scale bar = 25 µm. Fig. 12. Control sample with primary antibody only. There is some primary fluorescence of the hyphal coils. Scale bar = 50 µm

View larger version (151K): [in this window] [in a new window]    Plate III. FIGS. 13–20. Sections of Panax quinquefolius roots either uncolonized (Figs. 13, 18, 19) or colonized with Glomus intraradices (Figs. 14–17, 20) embedded in LR White resin, sectioned and labelled with either cellobiohydrolase (CBH II) complexed to gold (Figs. 13–17 or JIM 5 primary antibodies followed by secondary antibodies complexed to gold (Figs. 18–20). Scale bars for all figures = 5.0 µm. Figure 13. Labelling for cellulose occurs in the primary cell walls of cortical cells (arrowheads) but not in the middle lamella (*). Figure 14 and 15. Transverse sections of portions of hyphal coils. The interfacial matrix surrounding the coil (*) is labelled for cellulose. The fungal cell wall (arrowheads) is not labelled. Fig. 16. Sections of fine branches of an arbusculate coil. Very little labelling (arrowheads) is present in the interfacial matrix material. Fig. 17. Collapsed arbusculate coil showing diffuse labelling for cellulose (arrowheads) in the vicinity of the collapsed hyphae. Fig. 18. Labelling for non-esterified pectins has occurred in the primary cell walls (arrowheads) and middle lamella (double arrowhead) of cortical cells. Fig. 19. Junction of cortical cells showing heavy labelling for non-esterified pectins in the middle lamella material (*) and less labelling in the primary cell walls (arrowheads). Fig. 20. Portion of a hyphal coil (*) showing labelling for non-esterified pectins in the interfacial matrix (arrowheads). The fungal cell wall (double arrowhead) is not labelled

View larger version (189K): [in this window] [in a new window]    Plate IV. FIGS. 21–26. Sections of Panax quinquefolius roots either uncolonized (Fig. 23) or colonized with Glomus intraradices (Figs. 21, 22, 24–26) embedded in LR White resin and treated with either JIM 5 primary antibodies (Figs. 21, 22) or JIM 7 primary antibodies (Figs. 23–25) prior to treatment with secondary antibodies complexed to gold. Scale bars for all figures = 5.0 µm. Figure 21. Glancing section of hyphal coil showing labelling for non-esterified pectins in the interfacial matrix (*). Fig. 22. Sections of fine branches of an arbusculate coil with very limited labelling (arrowhead) of the interfacial matrix. Fig. 23. Primary cell walls of cortical cells with labelling for methyl esterified pectins (arrowheads). Fig. 24. Fine branches of an arbusculate coil with very low labelling (arrowheads) in interfacial matrix. Fig. 25. Collapsed branch of arbusculate coil with some labelling for methyl esterified pectins in the vicinty of the hypha (arrowheads). Fig. 26. Tissue treated with secondary antibody-gold complex showing lack of background labelling

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A typical Paris-type mycorrhizal association developed when Panax quinquefolius roots were colonized by Glomus intraradices, i.e., hyphal coils formed in numerous cortical cells from which fine lateral branches developed to form arbusculate coils. Panax quinquefolius plants grown on commercial farms and colonized by unidentified indigenous AM fungi form similar structures (Whitbread et al 1996, McGonigle et al 1999). Processing of roots at various times after seedling emergence allowed us to study the interface between root cell cytoplasm and either hyphal coils or arbusculate coils. As shown by Whitbread et al (1996) and Cavagnaro et al (2001), hyphal coils form fine lateral branches to form what the latter authors called arbusculate coils.

The development of hyphal coils and arbusculate coils altered the arrangement of both Mts and actin filaments in P. quinquefolius root cells when compared to uncolonized cells. Mts and actin filaments were associated with hyphal coils and arbusculate coils. Some cortical Mts and actin filaments remained but these were fewer in number and much shorter than in uncolonized cells. The Arum-type mycorrhizas formed between Gigaspora margarita and roots of Nicotiana tabacum L. are similar in this respect (Genre and Bonfante 1997, 1998) but actin filaments were rarely associated with the arbuscule trunk (Genre and Bonfante 1998). The close association of Mts with the arbuscule trunk in N. tabacum is similar to that observed between Mts and the thick coiled hyphae in P. quinquefolius and the coiled hyphae (pelotons) of orchid mycorrhizas (Uetake et al 1997).

The profound reorganization of the cytoskeleton in AM mycorrhizal systems (Genre and Bonfante 1997, 1998) and in an orchid mycorrhizal system (Uetake et al 1997, Uetake and Peterson 1997), both of which can be classified as endomycorrhizas, suggested to Genre and Bonfante (1999) that this may influence nutrient exchange by being involved in the repositioning of the host plasma membrane to form the perifungal membrane and the synthesis of the interfacial material. Using antibodies to -tubulin, Genre and Bonfante (1999) have shown that microtubule organizing centres (MTOC) show a dramatic change from being associated with the nucleus and peripheral plasma membrane in uncolonized cells to being associated with the nucleus and the perifungal membrane surrounding the fine branches of arbuscules in colonized cells. The authors suggest that the location of the MTOC along the fine arbuscule branches indicates the sites of Mt assembly. In their study Genre and Bonfante (1999) also used an antibody to clathrin and found that this protein coats the perifungal membrane and other endomembranes in colonized cells. They suggest that the cytoskeleton and clathrin may be working in concert in the formation of the interfacial matrix between the arbuscule branches and the perifungal membrane. Their results and recent results with orchid mycorrhizas using a fluorecent probe for endoplasmic reticulum in combination with laser scanning confocal microscopy (Uetake and Peterson 2000) indicate the complex interaction between the cytoskeleton and the endomembrane system in endomycorrhizas.

Labelling for cellulose and pectins showed that there is an interfacial matrix formed between hyphal coils of G. intraradices and cortical cell cytoplasm that consists of host-derived cell wall molecules. Although we did not do an exhaustive study to determine the precise nature of this interfacial matrix, it is obvious that it does resemble that described for Arum-type mycorrhizas in terms of the presence of cellulose and pectin molecules (Bonfante-Fasolo et al 1990, Balestrini et al 1994). It is of interest that in the Arum-type mycorrhiza, formed between Glomus versiforme (Karst) Berch and Zea mays L roots, the interfacial matrix around large intracellular hyphal branches labelled heavily for cellulose whereas the finer arbuscular branches showed far less labelling (Balestrini et al 1994). We found a similar labelling pattern in the Paris-type mycorrhizal association between G. intraradices and P. quinquefolius in that there was heavy labelling in the extensive interfacial matrix material surrounding hyphal coils but very light labelling around the fine branches of arbusculate coils. In both systems, there was a diffuse labelling in the vicinity of collapsed arbuscules, suggesting that some residual cellulose remains in this area. Labelling with JIM 5, an antibody against non-esterified epitopes of pectin, showed that cell walls and middle lamellae of uncolonized cells contained non-esterified pectins, with the latter being particularly rich; similar results were obtained in Arum-type mycorrhizas (Bonfante-Fasolo et al 1990). In the Paris-type studied here, the heaviest labelling in colonized cells was in the interfacial matrix surrounding hyphal coils; less labelling was apparent in the interface between arbuscule branches and the cytoplasm and around collapsed hyphae. This differed from the labelling reported by Bonfante-Fasolo et al (1990).

Although this study has shown that the hyphal coils in a Paris-type mycorrhiza interact with the host cell cytoskeleton and are separated from the host cell cytoplasm by an interfacial matrix forming an apoplastic compartment showing similarities to Arum-type mycorrhizas (Bonfante and Perotto 1995), it has yet to be shown that there is transfer of carbon compounds from host to fungus and phosphorus (and other nutrients) from fungus to host cells. This question can only be answered through the use of radioactive tracers and methods to localize enzyme activity on the perifungal membrane surrounding the coils. Other mycorrhizal systems such as those found in ericoid and orchid species only form hyphal coils (hyphal complexes) and it is presumably through these that nutrients are passed (Smith and Smith 1997). It is likely, therefore, that the hyphal coils in Paris-type AM mycorrhizas play a similar role.

	ACKNOWLEDGMENTS

 
We thank Dr. John Proctor for the vernalized seeds of Panax quinquefolius, Dr. John Klironomos for the Glomus intraradices inoculum, Dr. Paul Knox for the JIM 5 and JIM 7 antibodies, Dr. John Greenwood for advice on gold labelling, Lewis Melville for help with the plates, Ryan Geil for comments on the manuscript and the Natural Sciences and Engineering Research Council of Canada for financial support.

	FOOTNOTES

 
¹ Corresponding author, Email: lpeterso{at}uoguelph.ca

Accepted for publication December 1, 2001.

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