Microtubules of the mycorrhizal fungus Glomus intraradices in ...

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Microtubules of the mycorrhizal fungus Glomus intraradices in symbiosis with tomato roots S. Timonen, F.A. Smith, and S.E. Smith

Abstract: In this study the presence and orientation of fungal microtubules were recorded in arbuscular mycorrhizal symbiosis for the first time. Visualization of the fungal microtubules was achieved by using a protocol specifically labelling only fungal tubulins. Microtubules of external mycelium, intraradical hyphae, arbuscules, and vesicles of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith were examined when in symbiosis with tomato (Lycopersicon esculentum Mill.). Microtubules were organized as bundles in both external and intraradical hyphae. The bundles of microtubules extended directly from intraradical hyphae into the arbuscules, where the microtubules remained as bundles in the larger hyphae. In the fine fungal branches of the arbuscules, microtubules were seen as thinner filaments. Fungal microtubules were seen to connect the intraradical hyphae and arbuscules. In addition, microtubules of adjacent arbuscules could continue directly from one arbuscule to another. Microtubules reached to the basal cone of each vesicle, but the live vesicles, containing many nuclei, seemed devoid of any microtubular labelling. Key words: cytoskeleton, endomycorrhiza, filamentous fungi, tomato, tubulin, Zygomycota. Résumé : Les auteurs ont, pour la première fois, inscrit la présence et l’orientation des microtubules fongiques chez une symbiose mycorhizienne arbusculaire. Ils ont pu visualiser les microtubules fongiques en utilisant un protocole spécifique qui ne marque que les tubulines fongiques. Ils ont examiné le mycélium externe, les hyphes intra-racinaires, les arbuscules et les vésicules du champignon mycorhizien arbusculaire Glomus intraradices Schenck & Smith, vivant en symbiose avec la tomate (Lycopersicon esculentum Mill.). Les microtubules sont organisés en faisceaux dans les hyphes externes aussi bien qu’intra-racinaires. Les faisceaux de microtubules s’étendent directement des hyphes intra-racinaires dans les arbuscules, où les microtubules demeurent en faisceaux dans les hyphes les plus grosses. Dans les fines ramifications des arbuscules, on retrouve les microtubules sous forme de filaments plus minces. On observe des microtubules fongiques qui connectent les hyphes intra-racinaires et les arbuscules. De plus, les microtubules d’arbuscules adjacents peuvent être en continuité d’un arbuscule à un autre. Les microtubules se rendent jusqu’au cône basal de chaque vésicule mais les vésicules vivante, contenant plusieurs noyaux, semblent dépourvues de microtubules marqués. Mots clés : cytosquelette, endomycorhize, champignons filamenteux, tomate, tubuline, Zygomycota. [Traduit par la Rédaction]

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Introduction Cytoskeletal elements are key factors regulating cell morphology and polarity as well as transfer of compounds within cytoplasmic cells. In arbuscular mycorrhizal symbiosis, both fungal and plant partners undergo changes in their metabolism and morphology (Gianinazzi-Pearson 1986). Moreover, for an active mutualistic symbiosis, based on bidirectional nutrient transfer, the modifications of cellular structures of both partners are likely to be related to efficient exchange and translocation of metabolites. Thus far, only the plant side of the cytoskeletal network of arbuscular mycorrhizal root systems has been studied (Genre and Bonfante 1997; Genre and Bonfante 1998; Genre and Bonfante 1999; Matsubara et al. 1999). In these indirect immunofluorescence microscopical studies, extensive

changes in organization of plant microtubules have been shown to take place (Genre and Bonfante 1997; Matsubara et al. 1999). In other endomycorrhizal symbioses, only Uetake and Peterson (1998) have demonstrated fungal microtubules in symbiotic hyphae of Ceratobasidium cornigerum in orchid mycorrhizas, but that study had a strong emphasis on the plant microtubular structures. In ectomycorrhizal symbiosis, changes in both fungal and plant microtubular structures during mycorrhizal development have been observed (Timonen et al. 1993). Plant microtubules were shown to become sparse and less distinct in the cortical cells of the mycorrhizal regions, whereas in the vascular tissue cells containing cytoplasm retained frequent microtubular bundles. The fungal microtubules formed longitudinal bundles in the external mycelium and mantle but fine networks in the

Received October 24, 2000. Published on the NRC Research Press Web site on March 8, 2001. S. Timonen,1,2 F.A. Smith and S.E. Smith. Department of Soil and Water and the Centre for Plant Root Symbiosis, The University of Adelaide, Waite Campus, Private Bag 1, Glen Osmond, South Australia 5064. 1 2

Corresponding author (e-mail: [email protected]). Present address: Department of Biosciences, Division of General Microbiology, P.O. Box 56, 00014 University of Helsinki, Finland.

Can. J. Bot. 79: 307–313 (2001)

J:\cjb\cjb79\cjb-03\B01-005.vp Friday, March 02, 2001 2:16:02 PM

DOI: 10.1139/cjb-79-3-307

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Hartig net region within the root, where the fungus forms complex lamella structures in close association with plant cells. Arbuscular mycorrhizal fungi have been shown to contain microtubules, but so far the only work has involved Glomus mosseae hyphae originating from spores germinated in vitro, in the absence of host tissue (Åström et al. 1994). The interesting question is whether the location, frequency, and orientation of the fungal microtubules change in active symbiosis, as do those of the plant partner. This might be expected as changes in the fungal cytoskeleton during penetration of fungal plant pathogens into roots have been observed (Kwon et al. 1991a, 1991b). In this paper we show how fungal microtubules react during penetration of fungus into root tissue in the less aggressive interaction of arbuscular mycorrhizal symbiosis.

Materials and methods Root material Mycorrhizal tomatoes (Lycopersicon esculentum Mill. cv. Rio Grande 76R; Peto Seed Co., Calif.) were obtained by using a nurse pot inoculating system, with low phosphate conditions (Rosewarne et al. 1997). In short, 10 cm diameter nurse pots were set up with 8 leek seedlings (Allium porrum L. cv Vertina) as host plant and Glomus intraradices Schenck & Smith as the arbuscular mycorrhizal fungus. These nurse pots were grown for 6 weeks. Tomato seedlings were first germinated and grown for 4 weeks in sterilized 1:9 soil–sand mix and then six seedlings were transplanted into each fully mycorrhizal nurse pot that contained an extensive external mycelial network. Pots were watered three times weekly to 7% of soil dry mass. A nutrient solution containing 8 mM NaNO3, 4 mM (NH4)2SO4, 3 mM CaCl2, 2 mM K2SO4, 1.5 mM MgSO4, 109 µM FeEDTA, 46.2 µM H3BO3, 9.2 µM MnCl2, 0.77 µM ZnSO4, 0.32 µM CuSO4, and 0.1 µM Na2MoO4 was applied at the rate of 7 mL/kg dry soil per week. Harvesting of the material was carried out when the tomato seedlings were estimated to be fully mycorrhizal after 3 or 4 weeks of growth in the nurse pots. A sample of each root system was taken to confirm the degree of mycorrhizal colonization. Clearing and staining of the samples was carried out by a method modified from Phillips and Hayman (1970). Briefly, roots were cleared in 10% KOH for 2 days at room temperature, neutralized in 0.1 M HCl, stained with 0.05% trypan blue in 50:50 (v/v) lactic acid – glycerol and mounted on slides in 50:50 (v/v) lactic acid – glycerol. Fungal colonization (arbuscules, vesicles, and (or) intraradical hyphae) was then estimated by the magnified intersects method at 160× magnification, using a minimum of 100 random hairline intersects from each root system (McGonigle et al. 1990). Nonmycorrhizal tomato root material was produced and checked for mycorrhization in a similar way, omitting mycorrhizal inoculum from the nurse pots.

Preparation of samples for visualisation of cytoskeleton and nuclei Each root system was fixed by gently removing the seedling from the pot and immediately plunging it into –80°C acetone. The shoot was cut off and the loose sand from the frozen roots was quickly washed off in –80°C ethanol. The washed roots were then fixed for 2 days in 3% formaldehyde in ethanol at –80°C. Fixed roots were gradually warmed to room temperature over a period of 6 h. Following rehydration of the roots in an ethanol – phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4 (pH 7.3)) series (2:1, 1:1, 1:2, 0:1), the remainder of the clinging sand particles were removed, and the roots were cut into ca. 1-cm pieces. The root segments were em-

Can. J. Bot. Vol. 79, 2001 bedded in gelatine blocks and cryosectioned into 90 µm sections, which were placed in PBS. The freely floating sections were observed under a binocular microscope and intact sections containing cortical root tissue were chosen for staining. Sections of this thickness contained intact cells suitable for observation by confocal microscopy. Sections for visualizing fungal microtubules were digested for 30 min at room temperature in fungal cell wall digesting buffer containing proteinase inhibitor (PBS; prepared as before with the pH adjusted to 5.5 with HCl, 0.2% Novozyme (Sigma, St. Louis, Mo.), 0.5 mM AEBSF (Calbiochem®, San Diego)) and washed with PBS. Sections for labelling of plant microtubules were incubated in a plant cell wall digesting buffer, pH 7 (50 mM PIPES, 2 mM EGTA, 2 mM MgSO4, 0.05% TritonX-100, 0.4 M mannitol, 0.05% pectolyase Y23 (Seishin Corp., Tokyo); Sugimoto et al. 2000) and washed three times for 5 min with the same buffer without mannitol and pectolyase. All samples were further permeabilized in –20°C methanol for 10 min and rehydrated in PBS for 10 min. Cytoskeletal staining was carried out by first blocking the sections with 0.1% bovine serum albumine (BSA) in PBS for 30 min, washing and incubating in α-tubulin antibody solution (1:250, N356, Amersham, Sydney) with gentle agitation for 1 h at room temperature. After washing with 0.1% BSA in PBS, sections were incubated with Cy3-conjugated F(ab1)2 fragment goat anti-mouse IgG (H+L) 1:500 in PBS (Jackson Immunochemicals, West Baltimore Pike, Pa.). Labelled sections were then washed in PBS, and the nuclei were stained with 1 µg/mL DAPI (Sigma) in PBS pH 8.5 for 15 min. Sections were dropped on glass slides in 50:50 PBS – glycerol, and a coverslip was added on top and sealed with nail polish. Triple staining with trypan blue was not attempted. The sections were viewed with a Bio-Rad MRC-1000UV laser scanning confocal microscope system in combination with an argon laser and Nikon Diaphot 300 microscope in fluorescent mode. Excitation and emission wavelengths were: DAPI, excitation 363/32 and emission 460 nm long pass; Cy3, excitation 541 and emission 605 nm. Objective lens 40× water immersion NA 1.15 was used to produce a series of x–y optical slices, each at 1 µm interval on the z axis. In one case (Fig. 10), only every other slice is shown of the observed nuclei in vesicles to reduce the number of figures. The images were captured as digital computer files. Stacked images were constructed by Confocal Assistant© (Todd Clark Brelje, http://confocal.med.unc.edu/wwwleica/ ImageHandling/ih_cas_overview.html). The fields were also viewed and photographed in bright field mode to confirm the types and positions of the fungal and plant structures.

Results The root systems used to investigate microtubules and nuclei were variably colonized. The percentage of the root length containing arbuscules, vesicles, and (or) intraradical hyphae were in the ranges 14–66, 6–36, and 50–90% for the different structures, respectively, as determined by trypan blue staining. The sectioned material used for confocal imaging was not stained with trypan blue to avoid overlap of fluorescence by the fungal stain and Cy3. However, bright field images of the sections, although not very clear because of the thickness of the sections, allowed us to locate the positions of fungal structures. In these thick sections we were able to observe fungal microtubules in an arbuscular mycorrhizal symbiosis for the first time by specifically permeabilizing only fungal cell walls and thus avoiding the labelling of microtubules in plant cells. Cortical cells of plant roots without mycorrhizal colonization (Fig. 1a) have a © 2001 NRC Canada



Timonen et al. Fig. 1. Uncolonized cortical cells of tomato root: (a) bright field; (b) plant microtubules; (c) plant nuclei (arrow).

Fig. 2. Cortical cells of tomato root colonized by Glomus intraradices: (a) arbuscule (arrow), bright field; (b) plant microtubules; (c) nuclei of the plant (arrow) and the fungus (arrowheads).

dense microtubular network arranged mostly as long filaments in the peripheral cytoplasm close to the cell walls (Fig. 1b). Plant cells containing highly branched fungal arbuscules (Fig. 2a) have an even denser microtubular network that tightly envelops the fungal structures (Fig. 2b). Thus, the plant microtubules, if labelled, would critically obscure the view of the fungal microtubules in arbuscules growing within the plant cell walls. Nuclear DAPI staining shows the amount and positioning of nuclei in the image fields. The presence of nuclei in plant and fungal cells indicates the presence of cytoplasm and vitality of the cells. It also helps in visualizing the fungal and plant structures in the relatively transparent tissues, which can be only partially visualized in the two-dimensional bright field images (Figs. 1–11c).

309 Fig. 3. External mycelium of tomato root colonized by Glomus intraradices: (a) hypha (arrow), plant tissue (p), bright field; (b) microtubules in external mycelium (arrows); (c) fungal nuclei.

Fig. 4. Intraradical intercellular hyphae: (a) hypha (arrow), vesicle (v), bright field; (b) fungal microtubules in intercellular hyphae (arrows), vesicle (v); (c) fungal and plant nuclei.

Frequency and orientation of microtubules of both extraand intra-radical fungal hyphae as well as those of arbuscules and vesicles of G. intraradices were recorded. In extraradical hyphae of G. intraradices (Fig. 3a), microtubules were positioned in clear longitudinal bundles (Fig. 3b). In intraradical intercellular hyphae (Fig. 4a) the orientation and frequency of microtubules were similar to those of the external mycelium (Fig 4b). In arbuscules (Fig. 5a), microtubules were observed in the large trunk hyphae as well as in the finest branches (Fig. 5b). With the accuracy of the preparation and microscope used, the microtubules were seen to be arranged in bundles or fine filaments in the arbuscular branch hyphae; no netlike structures were observed. In some arbuscules the microtubules of the wider hyphae formed strikingly thick bundles (Fig. 6a). Intercellular intraradical hyphae (Fig. 7a) appeared to have direct cytoplasmic connection with the arbuscules, with microtubules extending through connecting hyphae between the two structures (Fig. 7b). Moreover, adjacent arbuscules © 2001 NRC Canada



310 Fig. 5. Arbuscules in tomato cortical tissue colonized by Glomus intraradices: (a) arbuscule (arrow), bright field; (b) fungal microtubules in the trunk hyphae (arrow) and fine branches of arbuscules (arrowheads); (c) fungal and plant nuclei.

Fig. 6. Tomato root tissue heavily colonized by Glomus intraradices: (a) fungal microtubules as thick bundles in an arbuscule (arrow); (b) fungal and plant nuclei.

Can. J. Bot. Vol. 79, 2001 Fig. 7. Intercellular hyphae connected to an arbuscule of tomato root colonized by Glomus intraradices: (a) connecting hypha not in focus (arrow), bright field; (b) fungal microtubules connecting an intercellular hypha with an arbuscule (arrow), and microtubules coinciding with nuclei are marked in two places (arrowheads); (c) fungal and plant nuclei. Faintly autofluorescing connecting hypha marked with arrow. The nuclei coinciding with fungal microtubules marked in Fig. 7b are marked with arrowheads here.

(Fig. 8a) were occasionally connected directly to each other via hyphae containing intact microtubular filaments (Fig. 8b). Nuclei could also be seen in the connecting hyphae (Fig. 8c). Vesicles (Fig. 9a), although full of nuclei (Fig. 9c), were devoid of microtubular structures, although microtubules in the subtending hyphae did reach the basal cone of each vesicle (Fig. 9b, arrow). In the uncolonized cortical cells of the plant roots the nuclei were seen adjacent to the cell walls (Fig. 1c), but in most cells containing arbuscules the plant nuclei had moved into the center of the cells (Figs. 2c, 5c, 6c, and 7c). Both extra- (Fig. 3c) and intra-radical hyphae (Figs. 2c, 4c–7c, and 9c) contained large numbers of nuclei. Abundant nuclei were also observed in most arbuscules (Figs. 5c–7c) where they appeared to be lined along the microtubules (e.g., Figs. 7b and 7c). In vesicles the nuclei were mostly situated along the inner wall, but in a few places, nuclear clusters were observed in deeper regions of the vesicles (Figs. 10a– 10e). In contrast to other fungal structures, in vesicles, microtubules were not seen in areas where nuclei were present (Figs 4a–4c). Control preparations without the first antibody showed only slight autofluorescence of the fungus and plant cell walls both in the fungal and the plant microtubule staining procedures (Figs. 11a–11c; compare Fig. 11b with Figs. 1b–9b). © 2001 NRC Canada



Timonen et al. Fig. 8. Mycorrhizal tissue of tomato root colonized by Glomus intraradices. (a) two adjacent arbuscules in tomato roots (arrowheads), connected by a hypha passing directly between the two cortical cells (arrow), bright field; (b) fungal microtubules in the hyphae interconnecting one arbuscule to another (arrow); (c) fungal nuclei in the connecting hyphae (arrow).

Discussion Because of the practical problems of preserving and visualizing cytoskeletal filaments in cells with complex cell walls, there are still only a few studies of cytoskeletal structures in filamentous fungi. Only one study of arbuscular mycorrhizal fungi (which belong to the class Zygomycetes) showing microtubules of G. mosseae in axenic culture has been published previously (Åström et al. 1994). However, arbuscular mycorrhizal fungi only thrive and grow in symbiosis and form many specialized structures in association with plant roots, which are not seen when grown axenically in vitro. We have studied the microtubules in the different types of structures formed by G. intraradices when in symbiosis with tomato roots in pot culture. The staining of the microtubules in the thinner arbuscule branches appeared to be weaker than in the larger hyphae. This could be due either to the fact that they occur in finer bundles or to weaker staining caused by reduced penetration of antisera across the interface material that surrounds the branches. The orientation and organization of fungal microtubules was, however, surprisingly similar in the external mycelium and in the different intraradical fungal structures. The microtubules were mostly oriented in longitudinal filaments and bundles as shown here and previously reported in the axenic culture of G. mosseae by Åström et al. (1994). Even in the arbuscule branch hyphae the microtubules occurred either in bundles or relatively straight filaments and no netlike or other specialized arrangements were seen as in the fungus Suillus bovinus forming Hartig net in ecto-

311 Fig. 9. Tomato root colonized by Glomus intraradices: (a) vesicle (v), and subtending hypha (arrow), bright field; (b) vesicle (v) is devoid of microtubules which reach only to the basal cone (arrow); (c) plant nuclei and fungal nuclei in both vesicle (v) and subtending hypha.

mycorrhizal symbiosis (Timonen et al. 1993). The relatively unchanged organization of fungal microtubules contrasted strikingly with the dramatic change of microtubular organization in plant cells containing arbuscules, whose microtubules were seen to closely envelop the fungal structures (Genre and Bonfante 1997; Matsubara et al. 1999; see Fig. 2b). It may be that the differences in the extent of changes in plant and fungal cytoskeletal structures reflect the dissimilar roles of the symbionts. The fungus develops most of the extra- and intra-radical structures through normal apical branching and growth, whereas the plant needs to react to the invaginations of membrane caused by the fungus in arbuscule-containing cells. Some changes in microtubular structures might be seen during close inspection of developing appressoria or in aborted appressoria in plants where the fungus is unable to penetrate into the roots. Considerable morphogenetic changes have been observed in these situations both where direct interaction is prevented by a barrier (e.g., Giovannetti et al. 1993) or in mycorrhiza-defective mutant plants (e.g., Barker et al. 1998), which could well be associated with changes in the microtubule organization. It is possible that the lack of major change in microtubule organization reflects the fact that functional changes in the uptake and translocational activity in the extra- and intra-radical fungal structures are minor. Both phases have transport activities, and although different substrates are absorbed, translocated, or lost by the different parts of the hyphal network, the differences might not necessitate major changes in the microtubular network. Whether the seemingly unchanged microtubular organization in symbiotic fungal structures is due to the lack of resolution, to the relatively small diameter © 2001 NRC Canada



312 Fig. 10. (a–e) Tomato root colonized by Glomus intraradices, dark field. Optical slices at 2-µm intervals through a vesicle showing the peripheral and uneven distribution of fungal nuclei. Nuclear clusters extending deeper into the vesicle are marked with arrows.

of the hyphae, or truly to lack of differentiation in organization remains to be seen. Microtubules continued without disjunction from the intraradical hyphae via cytoplasmic links to the adjacent arbuscules. These tracks may provide one route of translocation of substrates to and from the arbuscules, which are believed to be the main sites of phosphate (P) transfer to the plant. Microtubule inhibition studies in ectomycorrhizal systems by Ashford et al. (1998) indicate that fungal microtubules may be the key factors in translocation of nutrients, such as P and K transported in vacuoles in the fungal hyphae. The observed microtubular connections between arbuscules may also provide translocational routes and demonstrate that arbuscules can be actively directly linked to each other by living hyphae. Cross wall development between the arbuscules and trunk hyphae, associated with loss of vitality of arbuscules, has been recently demonstrated by S. Dickson (personal communication). In the future, a time course study of arbuscules and the response of nuclei and microtubules to the cross wall formation may give insight into the regulation of arbuscular senescence.

Can. J. Bot. Vol. 79, 2001 Fig. 11. Cortical tissue of tomato root colonized by Glomus intraradices: (a) arbuscule (arrow), bright field; (b) control preparation of labelling protocol omitting primary antibody; (c) fungal and plant nuclei.

Nuclei and microtubules appeared to be associated with each other in the fungal structures, but since the relatively thin hyphae were crowded with both nuclei and microtubules, this may be due to coincidence. However, nuclei have been shown to be associated with microtubules in G. mosseae in an indirect immunofluorescence study by Åström et al. (1994). At the electron microscopical level, nucleus-associated organelles have been shown to be associated with cytoplasmic microtubules in other fungi belonging to the class Zygomycetes (Heath and Rethoret 1982). In our study, nuclei were also abundant in vesicles, indicating vitality and presence of cytoplasm, although microtubules could not be seen. The movement of the nuclei into the developing vesicles is most likely to be assisted by microtubules, which have been shown to be involved in nuclear movement in several types of fungi including other Zygomycetes (e.g., see McKerracher and Heath 1985; Oakley and Rinehart 1985; McKerracher and Heath 1986; Raudaskoski et al. 1988). However, the nuclei do not appear to move via direct interaction with microtubules, although selective destruction of microtubules has been demonstrated to affect the speed and direction of nuclear movement in hyphae of Basidiobolus magnus (Zygomycetes) (McKerracher and Heath 1986). It is not clear at what stage the microtubules disappear from the vesicles nor whether the disappearance of microtubules is linked with the accumulation of large numbers of nuclei and lipids into the mature vesicles. Again, a developmental study would be valuable. © 2001 NRC Canada



Timonen et al.

In conclusion, a novel preparation technique has allowed us to visualize microtubules in arbuscular mycorrhizal structures within plant roots. We have shown that the orientation of the tubules appears relatively unaffected in the different phases (intercellular arbuscules and intercellular hyphae) compared with the extraradical mycelium. The lack of change may well reflect the relatively unaltered apical growth pattern as well as common roles of the different phases in nutrient translocation.

Acknowledgements We wish to thank Dr. Peter Kolesik for his outstanding technical support with the confocal imaging. We are also very thankful for Dr. Sandy Dickson’s help in image processing and many discussions, as well as for Ms. Debbie Miller’s expert technical help. The Academy of Finland and Australian Research Council are thanked for financial support.

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