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The unique cellular interaction between the leaf pathogen Cymadothea trifolii and Trifolium repens

     Universität Tübingen, Lehrstuhl Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Cellular interactions between the ascomycete Cymadothea trifolii and Trifolium repens (white clover) were analyzed using high-pressure freezing and freeze substitution. Cymadothea trifolii, a biotrophic leaf pathogen, forms a unique structure within its own hyphae, presumably for nutrient uptake from its host. This structure, called an interaction apparatus, consists of long, thin, often net-like cisternae surrounded by a membrane continuous with the fungal plasma membrane. The plant plasmalemma opposite the interaction apparatus invaginates to produce a host bubble. The interaction apparatus and host bubble are apoplastic and are linked by a tube with an electron dense sheath that may channel nutrients from the host to the pathogen. Within the tube, the cell walls of host and parasite appear altered. The interaction apparatus and host bubble may be analogous to haustoria in other obligately biotrophic fungi while the electron dense sheath of the tube may be equivalent to the haustorial neckband.

Key words: cell wall, clover, freeze substitution, high-pressure freezing, host-parasite interaction, interaction apparatus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Biotrophic plant parasitic fungi have numerous methods for obtaining nutrients from their hosts. For example, Blumeria graminis covers the leaf surface and attacks the host epidermal cells via haustoria (Hippe-Sanwald et al 1992). The rust fungus Uromyces fabae enters the leaf through stomata and grows intercellularly before forming haustoria in mesophyll cells (Mendgen and Deising 1993). Phytophthora infestans also grows between host cells before the production of haustoria (Shimony and Friend 1975), as does the smut fungus Ustacystis waldsteiniae (Bauer et al 1995). The anthracnose fungus Colletotrichum lindemuthianum lives intracellularly during its biotrophic phase but does not form specialized haustoria. It later switches to a necrotrophic mode of nutrition and proliferates both intracellularly and intramurally (O’Connell et al 1996). These examples document the range of ecological, physiological and structural adaptations of biotrophic pathogenic fungi for extracting nutrients from their plant hosts and illustrate that haustoria have developed independently in ascomycetes, basidiomycetes and oomycetes (for a comprehensive comparison of characteristics of infection hyphae and haustoria, see Perfect and Green 2001). Structures comparable to haustoria also have been reported in arbuscular mycorrhizal fungi (Smith and Read 1997).

The objective of the present study was to examine the ultrastructural characteristics of the cellular interaction between the biotrophic leaf pathogen Cymadothea trifolii (Dothideales, Ascomycota), the cause of sooty blotch of clover, and Trifolium repens. Because earlier observations of the interaction of C. trifolii and its clover host were based on conventionally fixed samples (Camp and Whittingham 1972), we applied high-pressure freezing and freeze substitution to gain a better insight into the structural features of the disease. Our results provide evidence for the evolution of a unique interaction structure in this biotrophic plant pathogen.

	MATERIALS AND METHODS

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Cryofixation and freeze substitution. – Disks 1 mm wide and approximately 0.5 mm thick were punched out of the leaves of Trifolium repens and infiltrated with 8% (2.5 M) aqueous methanol for about 5 min at room temperature to remove intercellular air. Single disks were placed in the 0.3 mm hollow of one-half of an aluminium holder and 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 by Mendgen et al (1991) and stored in liquid nitrogen.

Immediately before substitution, the sandwiches were opened in liquid nitrogen and the holders containing the samples were transferred to Eppendorf tubes filled with 2 mL of 2% OsO₄ in acetone. 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 over 3 d, after which the aluminium holders were removed, the fixative discarded and the samples rinsed three times in pure acetone. Infiltration with resin (Epon or Spurr) in acetone was done according to this schedule: 20% resin for 1 h followed by 33% resin for 9 h at –40 C, 50% resin for 2 h on ice and finally pure resin for 1 d at room temperature.

Cutting and poststaining of samples. – Semithin sections (50–70 µm) were cut with a glass knife using an ultramicrotome (Ultracut Reichert-Jung, Austria) and examined for thoroughly infected and well preserved leaf tissue with a light microscope. Ultrathin sections (60–90 nm) were cut from selected samples using a diamond knife (Diatome, Switzerland), placed on copper grids coated with formvar and poststained with 1% uranyl acetate for 1 h and Reynold’s lead citrate for 20 min. Sections were rinsed with distilled water between and after poststaining. Observations were made with a Zeiss EM 109 transmission electron microscope at 80 kV.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our ultrastructural analysis of the interaction between Cymadothea trifolii and Trifolium repens revealed that the pathogen grew intercellularly in clover leaves and produced an intricate, electron opaque structure within its own hyphae (FIGS. 1, 2). This structure, which resembles the interaction apparatus (IA) described by Bauer et al (1997) for Exobasidium, consisted of a system of elongate, highly branched, electron-dense cisternae (FIGS. 2, 3) that were fused to form a trunk; the base of the trunk was attached to the fungal cell wall (FIG. 2). Mitochondria were numerous between the cisternae and in the vicinity of the IA (FIGS. 2–4). The fungal cytoplasm contained many glycogen particles, especially near the IA (FIG. 3). The IA was surrounded by a membrane that was continuous with the plasmalemma of the fungus (FIGS. 6, 7 and 16–19) making it a solely apoplastic compartment. The tripartite nature of this membrane is clearly visible in FIGS. 4 and 5. Small, electron dense vesicles that elongated into cisternae were present (FIGS. 11–13) and the fusion of these vesicles with IA cisternae was observed at later stages of the development of the IA (FIGS. 14, 15).

View larger version (157K): [in this window] [in a new window]   FIGS. 1–5. In all figures, the plant organelles are marked by lowercase letters and the fungal organelles by capital letters. Abbreviations are: apw = altered plant cell wall; bl = host bubble; er = endoplasmatic reticulum; FW = fungal cell wall; IA = interaction apparatus; M, m = mitochondrion; MB = microbody; Nu, nu = nucleus; pl = chloroplast; pw = plant cell wall; s = starch grain; v = vacuole. Intercellular proliferation of C. trifolii and interaction of the pathogen with the host cell. FIG. 1. The pathogen produces an interaction apparatus (IA) within its hyphae that consists of long cisternae (arrows). ER-cisternae (arrowheads) are clearly recognizable within the host cell close to the site of interaction. FIG. 2. The IA cisternae (arrows) extend far into the fungal cell and fuse to form a trunk (asterisk) that contacts the fungal cell wall. Opposite the IA, the plasma membrane of the host cell forms a host bubble (bl). A tube with an electron dense sheath (arrowheads) connects the IA and host bubble. Numerous mitochondria (M) are found between the cisternae and in the vicinity of the IA. A crystal containing microbody (MB) that is producing a Woronin body is located in the fungal cell. FIG. 3. IA cisternae (arrows) in close contact with mitochondria (M). Glycogen particles (arrowheads) are present between the cisternae. FIG. 4. Higher magnification showing the intimate association between the IA-cisternae and mitochondria (M). FIG. 5. The tripartite membrane of the IA-cisternae (arrows). Bars: FIGS. 1, 2 = 1 µm; FIG. 3, 5 = 0.2 µm; FIG. 4 = 0.1 µm.

View larger version (154K): [in this window] [in a new window]   FIGS. 6–10. Interaction zone of C. trifolii and host bubble. FIG. 6. The membrane of the IA of C. trifolii is continuous with the plasmalemma of the hypha (arrows). The sheath of the tube between the IA and host bubble (bl) connects the membranes of both organisms. Within the tube, the fungal wall (FW) is much more electron dense and the plant cell wall (apw) appears nearly translucent. Wall appositional material (arrowhead) is deposited to the side of the invaginated host membrane. FIG. 7. The host bubble (bl) is continuous with the plasma membrane of the plant cell (small arrows) and the membrane surrounding the IA is continuous with the plasmalemma of the hypha (large arrows). Arrowheads indicate the enlarging wall appositions. FIG. 8. Oblique section through the tube. FIG. 9. Membranes do not surround the electron dense sheath of the tube (arrowheads). A clathrin coated vesicle (arrow) indicates endocytosis. FIG. 10. Detail of the host bubble (bl) showing a clathrin coated vesicle (arrow) attached to the bubble membrane. Bar: FIGS. 6–10 = 0.1 µm.

View larger version (158K): [in this window] [in a new window]   FIGS. 16–19. Later stages of the interaction between C. trifolii and T. repens. FIG. 16. Wall appositions (arrowheads) with electron dense and electron translucent parts that nearly encase the host bubble (asterisk). The IA consists of fine protrusions (arrows) and mitochondria are present in the vicinity of the IA. FIG. 17. Detail of FIG. 16. FIG. 18. The host bubble (asterisk) has been encased by wall appositional material. The IA is degenerating and only remnants of the cisternae remain (arrow). Mitochondria now are almost absent from the site of interaction. FIG. 19. The final stage of interaction. The membrane surrounding the IA and the plasmalemma of the hypha are continuous (large arrows). The host bubble (asterisk) has completely collapsed and its membrane (small arrowheads) is now separated from the plasma membrane of the plant cell (small arrows). Remnants of fungal plasmalemma (large arrowheads) are connected to the tube. Bars: FIGS. 16, 18, 19 = 0.2 µm; FIG. 17 = 0.1 µm.

View larger version (167K): [in this window] [in a new window]   FIGS. 11–15. Formation of the interaction apparatus. FIGS. 11–13. Serial sections showing electron dense vesicles with dark cores (arrows) that elongate into IA-cisternae (arrowheads). FIGS. 14–15. Fusion of vesicles (arrows) with already developed IA-cisternae. Bars: FIGS. 11–14 = 0.1 µm; FIG. 15 = 0.05 µm.

The host cell added wall appositional material of different electron density to the site of interaction in response to the fungal attack (FIGS. 6, 7, and 16, 17). This deposition eventually led to the encasement of the host bubble, which thereafter separated from the host cytoplasm and collapsed. At this stage the cisternae had begun to disintegrate and mitochondria were almost completely absent from the vicinity of the IA (FIG. 18, 7). Finally, only the trunk of the IA remained (FIG. 19). The electron-dense sheath of the tube, to which remnants of the fungal plasma membrane were attached, still was visible at this stage (FIG. 19). No differences were noted between samples embedded in Epon and in Spurr.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
High-pressure freezing and freeze substitution are used infrequently by plant pathologists studying the interactions of parasitic fungi and their host plants (Mims et al 2002). Although these techniques generally improve the preservation of structures of biological specimens compared to conventional fixation (Berg 1994, Bonfante-Fasolo et al 1994, Hippe-Sanwald 1993, Hippe-Sanwald et al 1992, Mendgen et al 1991, Mims et al 2002), they have been applied primarily to the study of the rust fungi (Mendgen et al 1991, Mims et al 2002, Roberson 1993, Swann and Mims 1990, Voegele et al 2001). Few studies employing high-pressure freezing and freeze substitution have been reported for other fungal pathogens (Bauer et al 1995, Hippe-Sanwald et al 1992, O’Connell et al 1996, Ouellette et al 1995).

Haustoria facilitate nutrient exchange between biotrophic fungi and their hosts (Hall and Williams 2000, Perfect and Green 2001) and occur in almost all groups of fungi (Beckett et al 1974, Perfect and Green 2001). Cymadothea trifolii does not form haustoria or invade the host cells, but it produces a complex structure that we believe is involved in the transfer of nutrients. This structure appears to develop from vesicles that elongate into cisternae. The IA consists of branched and often net-like cisternae that protrude deeply into the fungal cell. The membrane surrounding these protrusions is continuous with the plasmalemma. The resulting apoplastic compartment, with its greatly enlarged surface area, resembles the wall ingrowths of transfer cells as illustrated by Pate and Gunning (1972). Mitochondria found in large numbers around and between the cisternae might provide the energy required for transport processes in the membrane lining the IA, a role suggested for the numerous mitochondria present near the wall ingrowths in transfer cells (Pate and Gunning 1972). Structures resembling the IA have been reported in Arthuriomyces peckianus, a species that develops an appressorial cone at the site of attachment to a dialysis membrane (Swann and Mims 1991), and from the appressoria of Venturia inaequalis, which produces a structure called an infection sac above the penetration pore (Smereka et al 1987). However, both the appressorial cone and infection sac have been found in fungal cells outside host tissues and probably are related to the penetration process. In the system described here, C. trifolii alters but does not penetrate the wall of the host.

Opposite the site at which the IA contacts the fungal wall, the host membrane invaginates to form another apoplastic compartment called the host bubble. Clathrin-clad vesicles can be observed near the host bubble, but whether these vesicles contain fungal material that is transferred to the host remains unclear. A tube that passes through the cell walls of the plant and the fungus links the IA and host bubble. The electron-dense sheath of the tube connects the membranes of the interaction partners and might play a role in preventing the leakage of nutrients that are transferred from plant to parasite.

Although the function of the IA in C. trifolii has yet to be confirmed, the enormous surface area of the cisternae that would allow a high rate of nutrient transfer supports the suggestion that this structure plays a role in nutrient transfer. By remaining outside the host cell, the pathogen avoids attack by the host’s intracellular defense mechanisms. The success of this strategy becomes apparent when the long-term interaction between plant and pathogen is considered. Even though the invaginated host membrane eventually is encased by wall appositions, the fungus remains undamaged. There is no evidence of degradation of the cell walls of C. trifolii by plant-defense enzymes, as has been documented for Fusarium oxysporum f. sp. radicis-lycopersici in susceptible and resistant tomato cultivars (Benhamou et al 1990). The only visible affect on the fungus is the disintegration of the IA cisternae. Furthermore, the cells of T. repens generally survive, even when attacked by more than one IA (Simon et al unpubl), a fact that suggests that C. trifolii successfully maintains the balance between nutrient transfer from the host and host defense reactions.

The apoplastic compartment created in the host cell is probably analogous to the extrahaustorial matrix around haustoria (Beckett et al 1974). The electron-dense sheath of the tube formed between the IA and host bubble might be equivalent to the haustorial neckband found in the rusts and in the members of the genus Erysiphe or to the Casparian strip of endodermis cells. According to Heath (1976), Casparian strips and haustorial neckbands represent "unusual examples of parallel evolution at the structural and functional level." The tube described in the C. trifolii-T. repens system might represent another such example. The diameter of the tube (0.3–0.4 µm) is similar to dimensions of the haustorial necks of Albugo candidans (0.4 µm), Blumeria graminis (0.5 µm), Erysiphe pisi (0.8 µm), Melampsora lini (0.4 µm), Peronospora manshurica (0.2 µm), Peronospora parasitica (0.9 µm), Phythophthora infestans (0.7 µm) and Uromyces phaseolus (0.7 µm), and to the penetration pegs of Colletotrichum lindemuthianum (0.4 µm) as measured from micrographs of Beckett et al (1974), Heath (1976), O’Connell (1987), Peyton and Bowen (1963), Shimony and Friend (1975) and Spencer-Phillips and Gay (1981) (numbers are rounded up or down). This suggests that the width of haustorial necks, penetration pegs and the tube connecting the IA and the host bubble might have an optimal range.

One notable difference between the IA and haustoria concerns the route of nutrients from the plant to the pathogen. Host nutrients that move to haustoria have to pass the extrahaustorial membrane, the extrahaustorial matrix and the fungal cell wall. If the IA is involved in nutrient transfer, then the host nutrients also must pass through the plant cell wall. The plant wall in the tube appears altered, and preliminary data based on immunocytochemistry (Simon et al unpubl) indicate that structural elements of the plant wall within the tube remain intact while the pectin matrix is degraded.

An interaction structure resembling the IA of C. trifolii has been documented in the basidiomycete genus Exobasidium based on the examination of conventionally fixed material (Bauer et al 1997, Bauer et al 2001, Mims 1982, Mims and Nickerson 1986). The interaction structures of the Exobasidiales are of considerable taxonomic value for delimiting genera within this order (Bauer et al 1997) and the examination of high-pressure frozen and freeze substituted samples might yield new information pertaining to their formation. The fact that similar interaction structures are found in such distantly related fungi also points toward the convergent evolution of these presumably nutrition-related structures. Because very little is known regarding the morphological and ultrastructural characteristics of the Dothideales, the order to which C. trifolii belongs (Lumbsch and Lindemuth 2001, Ríos and Grube 2000), one of our objectives is to examine other species that are related closely to Cymadothea to test whether this interaction structure (or any others that are discovered) can be used as systematic markers for specific groups within the Dothideales.

	ACKNOWLEDGMENTS

 
We would like to express our gratitude to R. Kirschner for specimens of C. trifolii, H. Schwarz for his help with cryofixation and freeze substitution and Y. Stierhof and U. Nehls for many inspiring discussions. The valuable technical assistance of E. Wagner-Eha, F. Albrecht and H. Steigerle is appreciated greatly. We also thank the reviewers for their helpful suggestions. This work was financed by a DFG-fellowship to U. Simon (Graduate College "Infection Biology" 685).

	FOOTNOTES

 
Accepted for publication June 28, 2004.

¹ Corresponding author. Phone: + 49 7071 29 766 89. Fax: + 49 7071 29 53 44. E-mail: uwe.simon{at}uni-tuebingen.de

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Simon, U. K. Articles by Oberwinkler, F. Search for Related Content PubMed Articles by Simon, U. K. Articles by Oberwinkler, F. Agricola Articles by Simon, U. K. Articles by Oberwinkler, F.