Protoplasma (1988) 142:1-4

PROTOR.ASMA 9 by Springer-Verlag 1988

Intracellular Junctional Structures in Germinating Ascopores of Venturia inaequMis K. J. SMEREKA 1, A. P. KAUSCH*, and W. E. MACHARDY2 1University of Vermont, Hills Building, Burlington, Vermont 2University of New Hampshire, Nesmith Hall, Durham, New Hampshire Received January 16, 1987 Accepted August 17, 1987

Summary An ultrastructural study of the fungal plant pathogen, Venturia inaequalis (Cke)Wint., has revealed the presence ofjunctions that form within germinated ascospores as they penetrate the cuticle of apple leaves. This is the first report of junctional structures in fungi or in any organism with a cell wall. Morphologically they resemble septate junctions of invertebrate tissues, but developmentally they differ in origin. Junctions in V. inaequalis form intracellularly during formation of the infection sac by an invagination and subsequent folding back of the plasma membrane around the penetration pore. The apposed plasma membranes form junctions and result in a junctional belt around the penetration pore. The plasma membrane and the junctional structures stain positively with phosphotungstic acid and the confluent infection sac membrane remains tmstained. As the infection sac grows, additional junctions form between the infection sac membrane and plasma membrane. The intermembrane space of these junctions measures 17 nm, and the septa occur at regular 2.5 nm intervals. The junctions appear to provide adhesive support and structural rigidity for the developing infection sac and may also facilitate differentiation between the infection sac membrane and the plasma membrane. Similarities and differences between these junctions and invertebrate septate junctions are discussed. Keywords: Apple scab; FungaI junction; Infection sac; Septate junction; Venturia inaequalis. Abbrevation: A, ascospore; cu, cuticle;fw, fungal wall; IS, infection sac; mu, mucilage; P, Penetration pore; pl, plasma membrane; pw,

plant wall; w, epicuticular wax.

1. Introduction Cell junctions are regions o f m e m b r a n e specialization in animal tissues, which f o r m a close association between plasma membranes o f adjacent cells (STAEHELIN * Correspondence and Reprints: Mt. Holyoke College, Biological Sciences, S. Hadely, MA 01075, U.S.A.

1974, STAEHELIN and HULL 1978). Various types o f cell junctions have been described and are characterized by: (a) the width o f the intercellular space between the membranes; (b) the appearance and composition o f material in the intercellular space; (c) the extent o f m e m b r a n e surface involved in the junction; and (d) the presumed function(s) o f the tissues in contact (STAEHELIN 1974, STAEHELIN and HULL 1978). Tight and gap junctions o f vertebrate tissues and septate junctions o f invertebrates, represent areas o f physical contact between plasma membranes o f adjacent cells. These junctions are multiffinctional and provide a highly specific structural barrier to diffusion between cells (STAEHELIN 1974, STAEHELIN and HULL 1978), intercellular c o m m u n i c a t i o n and ion transport (STAEHELIN 1978), maintanence o f m e m b r a n e polarity (DRAGSTEN et aL 1981, NOIROT-TIMOTHEE and NOIROT 1980), and cell-to-cell adhesion and structural rigidity (STAEHELIN 1974), STAEVlELIN and HULL 1978). Evidence indicates that junctions are formed when specific integral proteins in two interacting plasma m e m b r a n e leaflets m a k e direct contact across the intercellular space (STAEHELIN 1974, VANMEER etal. 1986). M e m b r a n o u s cell junctions have never been reported in plants, algae, or fungi. However, a developmental study of host penetration by the fungal pathogen Venturia inaequalis (SMEREKAe t al. 1987) revealed the presence of intracellular junctions. They resemble invertebrate septate junctions. The present study was undertaken to describe the morphological and cytochemical characteristics of these junctions.

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K.J. SMEREKAet al. : Intracellular Junctional Structures in Germinating Ascospores of Venturia inaequalis

2. Methods Naturally infected Mcintosh apple leaves with immature pseudothecia were collected in April 1985 from the Mast Road research orchard in Durham, NH. Leaves were fiozen for storage and used throughout the duration of this study. When ascospores were needed, moistened leaves were placed in a high humidity chamber at 20 ~ until a majority of the spores had matured. Collection of a concentrated solution of ascospores has been described elsewhere (SMEREKAetal. 1987). Inoculum drops (200gl) containing 1.8 x 106 spores/ml were applied to leaves on potted suckers from Malling rootstockVII in a humidity chamber, or to excised leaves in Petri dishes with moistened no. 3 Whatman paper. Samples were incubated 5, 10, 15 and 20hrs at 20~ to obtain suitable developmental stages as estimated by TURNERet al. (1986). For microscopicexamination, leaf discs were excised and fixed in 3 percent glutaraldehyde in phosphate buffer (0.1 M pH 7.4) for 4hrs at room temperature. The leaf discs were then rinsed in four changes of buffer, postfixed in 2 percent OsO4 (same buffer) for 1.5hrs, rinsed again in 4 changes of buffer and two changesof double distilled water and dehydratedin an ascending ethanol series. After dehydration, leaf discs were infiltrated with either propylene oxide and embedded in Spurr's medium (SPuRa 1969) or with acetone and embedded in Mollenhauer's resin (MoLLENHAUER

1964).

Cytochemical procedures were conducted on thin sections of Mollenhauer's resin embedded material on 200 mesh gold grids. Selective staining of plasma membranes was done with the periodic acidchromic acid-phosphotungsticacid (PACP) procedure described by ROLANDet al. (1972), and carbohydrateswith vicinalhydroxylswere stained with the periodic acid-thiocarbohydrazide-silverproteinate (PA-TCH-SP) procedure (THIERY1967). Details of these procedures are described by SMEREKAetal. (1987).

3. Results During penetration of the apple leaf cuticle by ascospores of V. inaequalis, an infection sac develops within the ascopore in the region of the penetration pore (Fig. 1). The infection sac is a large, membranous structure unique to this fungus. It's formation is initiated by invagination or "pushing up" of the fungal plasma membrane over the penetration pore. Growth of this membrane results in folding of it upon itself around the border of the penetration pore (Fig. 2). Where the plasma membrane interfaces around the margin of the pore, junctional structures are observed (Fig. 2) and a junctional-belt is produced that circumscribes the penetration pore within the ascospore. Higher magnifications of the junctions (Figs. 3 and 4), illustrate that the apposed plasma membrane is separated by an intermembrane space of approximately 17nm and spanned by septa 5 n m wide that occur at 2.5 nm intervals. The septa are not always distinct and often the junctions appear non-septate (Fig. 3), however, when the septa are visible the junctions have a ladder-like appearance (Fig. 4). The septa extend from

the inner leaflet of one membrane to the inner leaflet of the adjacent membrane (Fig. 4). In later stages of penetration, additional junctions form as the infection sac extends along the fungal wall (Fig. 5). The PA-TCH-SP reaction for carbohydrate moieties revealed that the spore plasma membrane and the infection sac membrane stain positively with approximately equal intensity (Fig. 6). These fungal membranes reacted much more intensely in comparison to the host plasma membrane in the same section (not shown). The junctional structures stain more intensely than he adjacent plasma membrane or infection sac membrane (Fig. 6). A slight deposition of silver grains occurs in the intermembrane space; however, the septa are never visualized by the PA-TCH-SP reaction. The junctional structures reacted intensely to the PACP stain (Figs. 7 and 8). With this stain the junctions appear as three parallel membranes and the septa are not stained (Fig. 7). The plasma membrane of the ascospore also stains with PACP (Figs. 7 and 8) but the infection sac membrane remains unstained throughout its' development after formation of the junctions (Fig. 8). 4. Discussion Intracellular junctions that form within ascospores of V. inaequalis during penetration have a remarkable similarity to pleated septate junctions of invertebrates. The septate junctions of invertebrates are composed of apposed plasma membranes of adjacent cells, separated by an intermembrane cleft of 15-20 nm (GREEN and BERGQUIST 1982, LANE and SKAER 1980, STAEHEHN 1974). The regularly spaced septa of invertebrate septate junctions are the constituents of their characteristic ladder-like appearance (LANE and SKAER 1980, NOIROT-TIMOTHEE and NOIROT 1980, STAEHELINand HULL 1978) but there is considerable variation in the size and spacing of septa amongst the invertebrates (GREEN and BERGQUIST 1982). The images of transverse sections of V. inaequalis junctions are consistant with transverse profiles of the invertebrate septate junctions and measure 17nm across the intermembrane cleft. Our investigation represents the first report of a unicellular junction and contrasts with multicellular junctions in animals that form by a close association of adjacent cells, usually in an epithelial lining. This is also the first report of a junctional structure in fungi. The PA-TCH-SP and P A C P reactions have been used to cytochemically characterize animal cell junctions (GREEN and BERGQUIST 1982, REINHARDT and HECKER 1973, ROLAND etal. 1972). The visualization of membrane carbohydrate moieties by PA-TCH-SP

Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8.

Transverse section of an ascopore penetrating the apple leaf cuticle. (10-15 hrs post-inoculation). Bar = 1.0gm The infection sac over the penetration pore. Arrows indicate junctional structures. Bar = 0.2gm A transverse section of a junction that doesn't visualize septa. Bar = 0.1pm A junction showing regularily spaced septa. Bar = 0.05~tm Junction that forms later in penetration during extension of the infection sac (15-20 hrs post-inoculation). Bar = 0.1 pm PA-TCH-SP staining of junctions. Outer membranes stain positively, however, septa remain unstained. Bar=0.1gm PACP staining of junctions that give them a tripartite image. Bar = 0.2gm PACP reaction that shows unstained infection sac membrane and a stained plasma membraneand junctional area (arrow). Bar = 0.5gm

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K.J. SMEREKAet al. : Intracellular Junctional Structures in Germinating Ascospores of Venturia inaequalis

demonstrates that the fungal junctions stain more intensely than the infection sac membrane or the plasma membrane and that the septa did not have carbohydrates with vicinal hydroxyls. The membrane leaflets of septate junctions in Aedes midgut epithelium react positively for carbohydrate with this reaction while the septa do not stain (REINHARDTand HECKER1973). It should be recognized that even though the septa are not clearly discerned in oblique sections stained with lead and uranyl salts, their presence would still be cytochemically detectable with PA-TCH-SP. Hence, the observation that the septa are not visualized with this reaction reflects their composition. The PACP reaction is specific for the plasma membrane, coated vesicles and all cell junctions (ROLAND etal. 1972). The fungal and plant host plasma membranes stain positively with PACP and serve as internal controls for the reaction. The membrane leaflets of the junctions stain more intensely with PACP than the spore plasma membrane. The septa are not reactive. These features are consistent with e-PTA staining characteristics of Aedes septate junctions (REINHARDT and HECKER 1973). The cytochemical reactivity of the junctions may reflect differences in membrane composition of the leaflets comprising the junctions and the membranes from which they are derived. For example, the infection sac membrane, even though derived from the invaginated plasma membrane, did not stain with PACP, and at least by this criterion, is differentiated from the plasma membrane subsequent to the formation of the junctional complex at the penetration pore. Membrane differentiation, as indicated by PACP staining, is maintained throughout growth of the infection sac; presumably by the junctions since differential staining occurs only after formation of the junctions. Membrane differentiation after formation of the junctions may render the infection sac membrane specialized for recognition, transport and compartmentalization of penetration specific enzymes. In previous ultrastructural investigations of this fungus (MAEDA 1970, NICHOLSONetal. 1972, and SMEREKAetal. 1987), micrographs suggest that the infection sac contains enzymes and there is evidence of some enzyme activity in the host tissues below the infection sac in later stages of penetration.

Acknowledgements The authors gratefully acknowledge BRUCEWAGNER (University of Iowa, Ames, Iowa) for technical assistance and Drs. WAYNE FA-

GERBERG and CHARLES WALKER (University of New Hampshire, Durham, NH) for their suggestions and discussion of results. This study was supported by grants to KAREN J. SMEREKA from Sigma Xi and from the Central University Research Fund. Scientific contribution 1470 number from the University of New Hampshire Agricultural Experiment Station.

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