Changes in actin filament arrays in protocorm cells of the orchid species, Spiranthes sinensis, induced by the symbiotic fungus Ceratobasidium cornigerum Can. J. Bot. Downloaded from www.nrcresearchpress.com by BATES COLLEGE on 05/28/13 For personal use only.
Yukari Uetake and R. Larry Peterson
Abstract: Seeds of the terrestrial orchid, Spirnnthes sirzensis, were germinated in vitro in association with the synlbiotic fungus, Ceratobasidiurn corrzigerur~z.Resulting colonized protocorms were prepared for light microscopy, transmission electron microscopy, and fluorescence labelling of actin filaments for examination with laser scanning confocal microscopy. Fungal hyphae invaded the suspensor end of embryos, formed typical hyphal coils (pelotons) within parenchyma cells, and then underwent lysis resulting in degraded hyphal masses. Hyphae and hyphal masses were enveloped by host-derived membrane. Changes in actin filament arrays accompanied fungal colonization. Uncolonized cells had a network of actin filaments and actin bundles (cables) located in the cortical region of the cell cytoplasm; some of these were associated with the nucleus and amyloplasts. Although actin filament arrays were still prescnt in protocorm cell cytoplasm during fungal entry and peloton formation, most of the cortical network disappeared and instead actin filaments radiated from the periphery of developing pelotons towards the cell wall. Degraded hyphal masses also had actin filament arrays associated with them, again radiating toward the cell periphery; a network of cortical actin filaments reappeared in the protocorm cell cytoplasm at this stage. Actin filaments did not appear to have a close physical association with fungal hyphae except in the epidermal hairs that developed from protocorms; this differs from our previous observations on microtubules in this system.
Key words: actin, actin filaments, orchids, mycorrhizas, laser scanning confocal microscopy.
R6sum6 : Les auteurs ont obtenu la germination in vitro des graines de I'orchidCe terrestre Spirnrzthes sinensis, en association avec le champignon symbiotique Cerntobrrsidiurn corniger~crn.Les protocormes ainsi obtenus ont CtC prCparCs pour l'examen en microscopie photonique et Clectronique par transmission, ainsi que par marquage fluorescent des filaments d'actine pour I'examen en microscopie confocale au laser. Lcs hyphes fongiqucs envahissent I'extrCmitC suspenseur de l'embryon, forment des pelotons d'hyphes typiques dans les cellules de parenchyme, puis subissent la lyse conduisant B des masses d'hyphes dCgradCes. Lcs hyphes et les masses d'hyphes sont ainsi enveloppCes dans des membranes dCrivCes de l'hbte. Des changements au niveau des filaments d'actine accompagnent la colonisation fongique. Les cellules non-colonisCes montrent un rCseau de filaments d'actine et de faisceaux (cables) d'actine IocalisCs dans la region corticale du cytoplasme cellulaire; certains de ces rCseaux sont associCs avec le noyau et les amyloplastes. Bien que I'ensemble des filaments d'actine soit encore prCsent dans le cytoplasme des cellules de protocorme au cours de la pinitration du champignon et la formation des pelotons, la majeure partie du rCseau cortical disparait et les filaments d'actine s'irradient plutbt de la pCriphCrie des pelotons en dCveloppement vers la paroi cellulaire. Les masses d'hyphes dCgradCes posskdent Cgalement des ensembles de filaments d'actine qui leur sont associCs, et qui s'irradient de nouveau vers la pCriphCrie de la cellule; un rCseau de filaments corticaux d'actine scmble ~Capparaitredans le cytoplasme dcs cellules du protocorme B ce stade. Les filaments d'actine ne seniblent pas montrer d'association physique Ctroite avec des hyphes fongiques except6 dans les poils Cpidermiques qui se dCveloppent des protocormes; ceci diffkre des observations antCcCdentes des auteurs sur les microtubules, effectuCes avec ce mCme systkme. Mots clks : actine, filaments d'actine, orchidCes, mycorhizes, microscopie confocale par balayage au laser. [Traduit par la rCdaction] 4
Introduction Most plants, including orchid species, are associated with 'ymbionts to form and
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ing on the plant and fungal species involved, specific structural changes occur in the symbionts (Peterson and Farquhar 1994). Some of these changes are undoubtedly mediated by alterations to the cytoskeleton (Martin and Tagu 1995). Actin filaments (AFs), which are integral components of the plant cy toskeleton (Parthasarathy et al. 1985), have received considerable attention in terms of functions in plant cells (see Seagull et al. 1987; Lloyd 1991). To date, however, limited
I Received February 17, 1997.
Y. Uetake and R.L. Peterson.' Department of Botany,
University of Guelph, Guelph, ON N I G 2W1, Canada.
Author to whom all correspondence should be addressed. e-mail:
[email protected]
Can. J. Bot. 75: 1661-1669 (1997)
either information microtubules has been(MTs) published or AFs dealing in plant with cells changes during in
O 1997 NRC
Canada
Can. J. Bot. Vol. 75, 1997
Can. J. Bot. Downloaded from www.nrcresearchpress.com by BATES COLLEGE on 05/28/13 For personal use only.
Figs. 1-3. Microscopy of embedded and sectioned Spiranthes sinensis protocorms colonized by the symbiotic fungus Ceratobnsicliurn corrligerurn. Fig. 1. Longitudinal section viewed with bright field optics. Fungal hyphae have entered the suspensor end (double arrowhead) and colonized parenchyma cells. Cells with developing pelotons (arrowheads) and others with degraded hyphae (white asterisk) are evident. Cells at the chalaza1 end (black asterisk) and epidermal (E) cells are not colonized by hyphae. Nuclei (N) and vacuoles (V) are evident in protocorm cells. Scale bar = 100 pm. Fig. 2. Higher magnification of protocorm cells showing subcellular organization during colonization. As pelotons (double arrowheads) develop, host cell cytoplasm aggregates around hyphae and several vacuoles (V) appear. A collapsed mass of hyphae (:*) is present in a cell that has been reinvaded. A hypha (arrowheads) is evident between adjacent cells. N, nucleus. Scale bar = 50 pm. Fig. 3. Portion of colonized parenchyma cell viewed with transmission electron microscopy. A mass of degenerated hyphae ($:) and sections of a second peloton (F) are evident. The peloton hypha is surrounded by membrane (arrowheads) as is the mass of lysed hyphae (arrowhead). Numerous organelles including mitochondria (M), microbodies (Mb), amyloplasts (Ap), and rough endosplasmic reticulum (rE) are present in the host cell cytoplasm. Fungal cytoplasm is dense and elongated mitochondria (FM) are evident. Scale bar = I pm.
mycorrhiza establishment. Timonen et al. (1993) reported the loss of MTs and AFs from pine root cells during ectomycorrhiza formation. However, using immunoblotting of two-dimensional gels with anti-tubulin and anti-actin antibodies, Niini et al. (1996) suggested that the short roots of Pirzus sylvestris have two distinct a-tubulins and that an increase in plant actin occurs during early mycorrhiza formation. Dearnaley and McGee (1996) showed the loss of MTs in protocorm cells of the orchid Microtis par-viflor-a colonized by a mycorrhizal fungus, but recent work in our laboratory with the orchid Spiranthes sinensis has localized MTs in protocorm cells during mycorrhiza formation (Uetake et al. 1997). There are no reports in the literature, however, of A F arrays in orchid protocorm cells either when uncolonized or colonized by fungi. One of the incentives to study interactions between mutualistic symbiotic fungi and plant cells in terms of the cytoskeleton is the reports of changes in MTs and AFs of plant cells induced by pathogenic fungi (Kobayashi et al. 1992, 1994, 1995) and the general question as to whether mycorrhizal associations share characteristics with plantpathogen interactions (Bonfante-Fasolo and Perotto 1990; Smith and Smith 1990). The objective of this study, therefore, was to determine the effect that fungi have on the AFs of orchid protocorm cells during all stages in establishment of the mutualistic association and to compare this with information on MTs in the same system (Uetake et al. 1997).
Materials and methods Plant and fungal materials The method used to obtain colonized protocorms of Spirantl~es sinensis (Pers.) Ames was identical to that described in Uetake et al. (1997). Briefly, seeds were surface sterilized and placed on 0.3% oatmeal agar medium solidified with 1 % agar in a Petri dish or on a disc of agar medium placed on a microscope slide and covered with a cover glass. An agar plug from the periphery of a rapidly growing culture of the fungus Ceratobasicliltm cornigerlrrn (Bourdot) Rogers (binucleate Rhizoctonia AG-C) was positioned centrally on the surface of the agar in each Petri dish or on a slide.
Light and electron microscopy for basic structural features Protocorms were fixed in 2.5 % glutaraldehyde -2% paraformaldehyde in phosphate buffer, pH 7.0, for 2 h at room temperature, rinsed in buffer, and postfixed for 1 h in 1% OsO, (for Spurr's resin embedding tissue only). Tissue was then rinsed in buffer, dehydrated in a graded ethanol series, and embedded in either LR-White resin or Spurr's resin. Sections (0.5 - 1.0 pm) of LR-White resin-embedded tissue were mounted on glass slides, stained with
0.05% toluidine blue-0 in 1 % sodium borate, and viewed with bright field optics. Thin sections of Spurr's embedded tissues were picked up on copper grids and stained with saturated uranyl acetate in water diluted 1: 1 in acetone prior to staining for 5 min followed by lead citrate for 1 min prior to viewing with a JEOL-IOOCX transmission electron microscope.
Fluorescence microscopy for basic structural features Protocorms were fixed as described for actin labelling below but for 1 h, rinsed in PME buffer (50 mM PIPES, 2 mM Mg, 1 mM EGTA, pH 7.0), sectioned in this buffer, treated with 5 % Triton X-100 for 1 h, rinsed in PME buffer and then in phosphate buffer, treated with 0.1 % NaBH, in phosphate buffer, and then stained in 0.1 % aqueous acridine orange diluted 1:9 with phosphate buffer for 20 min - 1 h. Acridine orange was found to be a good general stain for fungal hyphae as well as for nuclei. Tissue was rinsed, mounted in phosphate buffer, and viewed using the same methods for laser scanning confocal microscopy described below but using photomultiplier (PMT) detector 2 (
