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Cytochemical aspects of fungal penetration, haustorium formation and interfacial material in rose leaves infected by Sphaerotheca pannosa var. rosae
Physiological and Molecular Plant Pathology (1991) 39, 341-355
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Cytochemical aspects of fungal penetration, haustorium formation and interfacial material in rose leaves infected by Sphaerotheca p a n n o s a var. rosae MOHAMED R. HAJLAOUI, NICOLE BENHAMOU and RICHARD R. BI~LANGER'~ Dipartement de ph.ytologie, Facultgdes sciencesde l'agriculture et de l'alirnentation, UniversitgLoyal, Sainte-Foy, Quibec, Canada GIA" 7P4 (Acceptedfor publication April 1991)
Fungal development and host reactions induced in rose leaf epidermal ceils by the powdery mildew fungus, Sphaerothecapannosa var. rosae,were examined on the basis ofultrastructure and of cytochemlstry of chitin, pectin, and cellulose subunits. Fungal growth in the epidermis was associated with the formation of haustoria which appeared muhilobed and delimited by an extrahaustorlal membrane probably originating from the host plasmalemma. The extrahaustorlal matrix was free of chitin as judged by the absence of labelling following incubation with a wheat germ agglutinin/ovomucoid-gold complex. Similarly, neither pectin nor cellulose was detected in this matrix, whereas these compounds were abundant in host cell walls. Various host reactions were observed at sites of fungal penetration. The formation of papillae was often associated with a restricted development of the infection peg. The most striking reaction was the occurrence of successive layers of a fibrillar material, forming a collar around the haustorial neck. Our cytochemical observations demonstrated that the collar was made of an amorphous material surrounded by fibrillar layers, the outermost ones being cellulose-rich. The possible nature and origin of this structure is discussed in relation to its possible involvement in resistance to fungal attack.
INTRODUCTION T h e process o f p l a n t tissue i n f e c t i o n by p o w d e r y m i l d e w fungi i n v o l v e s specific e v e n t s including appressorium formation, penetration and development of a haustorium s u r r o u n d e d b y a n e x t r a h a u s t o r i a l m a t r i x [8, 20, 26, 29]. E a c h e v e n t has b e e n well d o c u m e n t e d in t e r m s o f i n d u c e d host reactions. F o r instance, a t t e m p t e d p e n e t r a t i o n o f host e p i d e r m a l cells is o f t e n associated w i t h t h e f o r m a t i o n o f p a p i l l a e a r o u n d t h e site o f p e n e t r a t i o n [1, 24], p r e s u m a b l y to restrict f u n g a l i n v a s i o n a n d g r o w t h [16, 20]. U p o n p e n e t r a t i o n , w i t h i n the host cell, a n o t h e r r e a c t i v e m a t e r i a l , the collar, c a n d e p o s i t a l o n g the h a u s t o r i a l neck. I n s o m e cases, this c o l l a r m a y d e v e l o p to such a n e x t e n t t h a t the e n t i r e h a u s t o r i u m is encased thus r e d u c i n g the h a u s t o r i a l f u n c t i o n b y Abbreviations used in text: AGL, Aplysia gonad lectin; PBS, phosphate buffered saline; PEG, polyethylene glycol; WGA, wheat germ agglutinin. 1" To whom correspondence should be addressed. Paper no. CRH-6 from Le Centre de Recherche en Horticulture. 0885-5765/91/050341 + 15 $03.00]0
© 1991 Academic Press Limited
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acting as a barrier to nutrient uptake [16, 20, 32]. The collar has been well studied and characterized in relation to its possible role in defence mechanisms [23,26, 29], although its origin and chemical composition remain poorly understood. Considerable attention has also been paid to the interfacial matrix formed between host and parasite cells in biotrophic infections. The rationale for such an interest was that this matrix could be involved not only in nutrient uptake, but it could also be of key importance in the survival of penetrated host cells by providing sites for potential cell-cell recognition a n d / o r by interfering with deleterious fungal toxins and enzymes [9, 10, 18]. In this context, efforts have been directed towards elucidating the origin and chemical composition of the extrahaustorial matrix [12, 13, 14]. However, the exact nature of the interfacial material is still unknown and is debated in the literature. Although the occurrence ofpolysaccharides in the matrix is now generally accepted, their exact nature and origin is open to question. Some authors favour the hypothesis that the haustorial material is plant-derived and probably plays a role in resistance by preventing further pathogen ingress [28, 31]. Support for this speculation is drawn from the recent finding that both cellulose and pectin occur in the interracial material formed between Allium porrum L. root cells and the vesicular-arbuscular mycorrhizal fungus, Glomus versiforme (Nicol. & Gerd.) Gerdemann & Trappe [5]. Another possibility that has been proposed is that the extrahaustorial matrix may be composed of a mixture of plant and fungus-derived compounds [6, 13]. In view of the little information available on the nature of the collar and of the divergent results reported on the extrahaustorial matrix, tile object of this study was to bring further insight into the interfaciaI materials formed between both partners. For this purpose, we used the interaction between Sphaerotheca pannosa (Wallr: Fr) L6v. var rosae Woronichin and rose which has been little studied in terms ofpathogen action and host reactions [29]. Cytochemical techniques based on the use of specific colloidal goldlabelled probes were used to locate fungal and plant cell wall components.
MATERIALS AND METHODS
Plant material and inoculation Rose plants (Rosa hybrida L.) cv. Ruiredro, purchased from a local breeder in Q u6bec, were maintained in a greenhouse at 21 °C under a 16 h photoperiod. One day before inoculation, inoculum-donor plants were shaken to remove old spores; newly produced conidia from plants were used as inoculum. Donor plants were shaken over test plants to inoculate them. Inoculated plants were kept in the greenhouse and leafsamples were collected 5 days after inoculation.
Tissue processing for electron microscopy For electron microscopy, samples (1 mm 2) from young rose leaves infected with the powdery mildew fungus were immersed in 3 °/o (v/v) glutaraldehyde in 0"1 ~[ sodium cacodylate at pH 7"2 for 2 h at 4 °(3, rinsed with the same buffer, and post-fixed with 1 °/o (w/v) osmium tetroxide in sodium cacodylate buffer. They were then dehydrated in a series of ethanol solutions graded in I 0 % steps and embedded in Epon 812.
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Uhrathin sections were collected on Formvar-coated nickel grids and processed for cytochemical labelling. Experiments were repeated four times. For each experiment, five leaves from five different infected plants were collected. Samples were examined with a J E O L 1200 EX electron microscope at 80 kV.
Preparation of gold complexes Gold particles ofapprox. 14-16 nm in diameter were prepared according to Frens [17]. The pH of the colloidal gold solution was adjusted according to the isoelectric point of the lcctin or enzyme used [2]. Tile exoglucanase, a fl-l,4-cellobiohydrolase purified from a cellulase produced by Trichoderma harzianum Rifai [3], was used to locate cellulose subunits. The lectin from Aplysia depilans [4] was used to locate pectin because of its demonstrated affinity for polygalacturonic acids. They were directly complexed to colloidal gold at the appropriate pH. Because of its low molecular weight, the wheat germ agglutinin (WGA), a lectin with N-acetylglucosamine-binding sites, could not be complexed directly to gold since flocculation occurred. Ovomucoid, a protein with high affinity for WGA, was complexed to gold at pH 4"8 and used as a second step reagent as previously described [2].
Cytochemieal labelling For direct labelling, sections were first incubated for 5 min on a drop of PBS at the pH of optimal protein activity (6"0 for the exoglucanase and 8"0 for the AGL) containing 0"02% of PEG 20000 in PBS buffer. They were then transferred to a drop of the protein-gold complex and incubated for 30 min at room temperature in a moist chamber. Grids were then washed with PBS, rinsed with distilled water and stained with uranyl acetate and lead citrate. For indirect labelling, sections were incubated on a drop of WGA (I0 lag m1-1) in PBS pH 7"2 for 30 min at room temperature. Grids were rinsed with PBS and incubated with gold-complexed ovomucoid for 30 min. The were washed with PBS and distilled water, stained with uranyl acetate and lead citrate and examined under the electron microscope.
Reagents Tetrachloroauric acid was obtained from BDH Chemicals, Montr6al, Canada, and PEG 20000 from Fisher Scientific, Q udbec, Canada. Ovomucoid and WGA were purchased from Sigma Chemical Co., St. Louis, Missouri, USA. The exoglucanase was provided by Dr C. Breuil, Forintek Canada, Ottawa, and the AGL by Dr N. GilboaGarber, Bar Ilan University, Israel.
RESULTS
The cytology of infection of rose leaves by S. pannosa var rosae was similar in most respects to that previously described in other plants infected by biotrophic fungi
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F]o. 1. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerolheca pannosa var. rosae. (a) The thick, electron-lucent wall of an elongated germ tube is intensely labelled by the WGA/ovomucoid-gold complex. By contrast, the host cell wall is unlabelled. Bar = 0"5 lam. (b) At the site of attempted penetration, the cuticle and epidermal cell wall undergo a downward deformation (arrow). Labelling with exoglucanase-gold complex results in regular distribution of gold particles. Bar = 1 I.tm. (c) The appressorium is fixed to the host cell surface by a slime layer. The host cell wall is heavily labelled by the exoglucanase-gold complex even in the areas sho~ing obvious signs of deformation (arrow). Some gold particles occur in the area between the retracted plasmalemma and the host cell wall (double arrow, and c'). (c) Bar = 0"5 lam. (c') Bar = 2'25 lam. (d). Penetration is characterized by the formation of a narrow infection peg. A hemispherical protuberance made ofgranular and opaque materials accumulates
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[16, 27]. Conidia germinated on the leaf surface and produced one or two germ tubes surrounded by a thick, electron-lucent wall which labelled densely with the W G A / o v o m u c o i d - g o l d complex [Fig. 1 (a)]. Appressoria, formed at the end of germ tubes, were usually characterized by a large, centrally-located vacuole [Fig. 1 (b), (d)]. T h e wall of the appressorium was thinner than that of the germ tubes, and was also intensely labelled following incubation with the W G A / o v o m u c o i d - g o l d complex (not illustrated). At the sites of a t t e m p t e d penetration, appressoria a p p e a r e d to be closely fixed to the host cell surface by a fine, slime layer [Fig. l(c)]. At these sites, a d o w n w a r d deformation of both the cuticle and the underlying epidermal wall was usually observed [Fig. 1 (b), (c), arrows]. Incubation of sections with the exoglucanase-gold complex resulted in the deposition of gold particles over the locally deformed host cell walls [Fig. 1 (b) and (c)]. Some gold particles lining the innermost host wall layers were detected in the space between the deformed host wall and the invaginated host p l a s m a l e m m a [Fig. 1 (c) and (c')]. Successful wall penetration was characterized by the elaboration of a narrow penetration peg which emerged through a pore of the appressorium and entered the cuticle and the underlying epidermal cell wall [Fig. 1 (d)]. Invasion of host cells by penetration pegs was associated with the unusual occurrence of a fibrill-granular material between the host wall and the invaginated p l a s m a l e m m a [Fig. I (d)]. This material consisted of two areas which differed by their texture and electron-density. T h e first area, near the infection peg, was amorphous and electron-opaque whereas the second area, close to the p l a s m a l e m m a was more fibrillar and of lighter electron opacity [Fig. 1 (d)]. V a r y i n g amounts of reaction material were detected in areas neighbouring infection pegs [Fig. 2 (a)]. Most intracellular cells of the pathogen were not restricted by this material, but in some cases, the penetration peg was found to be totally encased in it [Fig. 2(a)]. In the lumen of epidermal cells, infection pegs enlarged to form an elongated structure from which arose the haustorial body [Fig. 2(b) and (c)]. Incubation with the W G A / o v o m u c o i d - g o l d complex resulted in an uneven deposition of gold p a r t i c l e s o v e r the wall of this elongated structure, referred to as a neck [Fig. 2 (b) and (c)]. O v e r the haustorial neck, labelling was either mainly concentrated over the apical area [Fig. 2(b), arrow] or more uniformly distributed [Fig. 2 (c)]. It is noteworthy that in most haustorial necks, the proximal region was usually devoid of any significant labelling [Fig. 2 (c), arrow]. A laminated material occurred around the neck forming a collar which was unlabelled by the W G A / o v o m u c o i d - g o l d complex [Fig. 2 (c)]. T h e neck usually had enlarged to form a typical ellipsoidal structure, the haustorial body, which appeared multilobed [Fig. 2 (d); Fig. 3 (a), (b)]. Connections between the lobes and the main body were seldom seen in transverse sections [Fig. 2 (d)], but could be easily detected in more tangential sections [Fig. 3 (a) and (b)]. around the infection peg. The host plasmalemma surrounds the hemispherical protuberance. Bar = 0"5 lam. Abbreviations: Ap = appressorium; C = collar; Ch = chloroplast; Cy = cytoplasm; EHMa = extrahaustofial matrix; EHMe = extrahaustorial membrane; F = fungus; GM=granular material; GTW=germ tube wall; HB=haustorial body; HCy=host cytoplasm; HGW = host cell wall; HN = haustorial neck; HW = haustorial wall; IP = infection peg; L=lobe; M=mitochondrlon; N=nucleus; OM=opaque material; P=papilla; PL = plasmalemma; S = septum; SL = slime layer; Va = vacuole. 2~
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Fzo. 2. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerotheca pannosa var. rosae. (a) The infection peg is encased in an electron-opaque papilla. Incubation with the WGA/ovomucoid-gold complex results in the deposition of gold particles over the fungus cell wall. B a r = 0"5 lam. (b) T h e apical region of a haustorial neck is labelled with the WGA/ovomucoid-gold complex (arrow). Bar = 0"25 lain. (c) T h e distal region of the haustorial neck is unlabelled (arrow). Bar = 0"5 lam. (d) A typical haustorium is formed in an epidermal cell and is separated from the neck by a septum. T h e ellipsoldal body contains a nucleus, several mitochondria and small vacuoles. A large number of lobes are found close to the main body. T h e haustorium is surrounded by an extrahaustorial membrane. Bar = 1 tam. For abbreviations see Fig. 1.
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Haustorial lobes were found to be highly polymorphic, some of them being curved [Fig. 3 (c)] or highly contorted. The haustorial cytoplasm usually appeared densely packed with organelles embedded in a ribosome-rich matrix. The haustorium contained a large nucleus and elongated mitochondria [Fig. 2 (d)]. Another feature of interest was the presence of small vacuoles that were filled with a large number of vesicular structures. Nuclei and mitochondria were never seen to occur in the neck portion. Following incubation with the WGA/ovomucoid-gold complex, the wall of the haustorium body and the lobes was intensely labelled (Fig. 3). By contrast, cytoplasm, organelles and vacuoles were devoid of gold particles. The septum delimiting the neck from the main body was also heavily labelled [Fig. 3 (a)]. This labelling pattern was observed in all haustoria with, however, some variations probably due to the stage of development. In some haustoria, the number of lobes surrounded by a labelled wall was much higher [Fig. 3(a), double arrow] than in others where plasmolysis and distortion of the lobes was frequently observed [Fig. 3(a)]. The extrahaustorial membrane, resulting from an invagination of the host plasmalemma [Fig. 3 (a)] was unlabelled [Fig. 3 (b)]. Similarly, the extrahaustorial matrix was free of WGA-binding sites [Fig. 3 (b)]. Following incubation of infected rose leaf samples with the fl-exoglucanase-gold complex used for the localization of cellulose subunits, an intense and regular deposition of gold particles was observed over host cell walls [Fig. 4 (a)]. By contrast, neither the haustorial matrix nor the haustorial membrane exhibited labelling. Similarly, the haustorial body as well as the lobes were free oflabelling [Fig. 4 (a)]. Preadsorption of the enzyme-gold complex with fl-l,4-glucans from barley [3] prior to section incubation resulted in an absence of host cell wall labelling [Fig. 6 (b)]. When sections were incubated with the AGL-gold complex for the localization of polygalacturonic acid-containing molecules [4], a few scattered gold particles were associated with host cell walls, but not with the extrahaustorial matrix and the extrahaustorial membrane [Fig. 4 (b)]. Fungal structures were also devoid of labelling. All control tests including previous adsorption o f the gold-complex with polygalacturonic acids yielded negative results [not shown]. Colonization of the host epidermis by S. pannosa var. rosae was associated with striking host reactions. One noticeable feature was the necrotic appearance ofsome uncolonized palisade mesophyll ceils neighbouring infected ones [Fig. 4 (c), arrows]. By contrast, other mesophyll cells adjacent to necrotic ones remained apparently alive as judged by the normal appearance of tile cytoplasm and organelles. In infected epidermal cells, large amounts of reactive material along the haustorial neck were often detected [Fig. 5 (a)]. This material, which was either amorphous in areas adjacent to the haustorial neck or fibrillar and laminated towards the extrahaustorial membrane [Fig. 5 (a)], was often associated with aborted haustorium development. The amorphous material was usually separated from the haustorial neck by a narrow channel [Fig. 5 (a) and (b), arrows]. It could be easily distinguished from the adjacent host cell wall by its texture and electron density. The outer fibrillar material resembled the host cell wall in terms of texture, but it appeared to be derived from the successive deposition of layers of different electron density [Fig. 5(b)]. Extensive development ofthis material caused severe distortion ofthe |lost plasmalemma and prevented close association between the extrahaustorial membrane and the neck wall.
Cytochemistry of the rose---Sphaerotheca pannosa interaction
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F~o. 4. Transmission electron micrographs of rose leaf cells infected by Sphaerotheca pannosa var. rosae. (a) A section treated with exoglucanase-gold complex for the localization of the fl-l,4-glucan residues. An intense and regular deposition of gold particles over the host cell wall is seen. In contrast, the extrahaustorial membrane, extrahaustorial matrix, haustorial body and lobes are free of labelling. Bar = 0"5 lam. (b) A section incubated with AGL-gold complex for localization ofgalacturonic acids. Scattered gold particles are associated with the host cell wall but not with the extrahaustorial membrane, the extrahaustorial matrix, the haustorlal body or the lobes. Bar = 0"5 ~tm. (c) T h e necrosis of some uncolonized palisade mesophyll cells neighbouring the infected epidermal cell is observed (arrows). Some cells adjacent to the damaged ones remained apparently alive. Bar = 1 lam. For abbreviations see Fig. 1.
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FIO. 5. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerotheca pannosa var. rosae. (a) A prominent collar occurs around tile haustorial neck. This reactive material is amorphous in areas adjacent to the haustorial neck and fibrillar and laminar towards tile extrahaustorial membrane. Bar = 1 lain. (b) T h e walls of the haustorial body and the appressorium are uniformly labelled following incubation with the WGA/ovomucoid-gold complex. T h e collar is free of labelling. Bar = 1 lam. For abbreviations see Fig. 1.
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Fro. 6. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerotheca pannosa var. rosae. Labelling with exoglucanase--gold complex for localization of fl-l,4-glucan residues. (a) Labelling accumulates mainly over the host cell wall and the more external layers of the laminated material surrounding the haustorial neck. A few scattered gold particles are seen over the amorphous material of the collar. Neither the haustorial neck nor the haustorial body are labelled• Bar = 0"25 tam. (b) Control test. Incubation with the exoglucanase-gold complex to which was previously added /~-l,4-glucans from barley (1 m g ml -x) results in an absence of labelling•
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Both the amorphous and the stratified materials were unlabelled following incubation with the WGA/ovomucoid-gold complex [Fig. 5 (b)]. By contrast, gold particles were associated with the haustorial neck. When sections were incubated with the exoglueanase-gold complex, labelling accumulated mainly over the host cell wall as well as over the more external layers of the laminated material surrounding the haustorial neck [Fig. 6 (a)]. Only a few scattered gold particles could be seen over the amorphous material. The intensely labelled fibrillar layers were in apparent connection with the host cell wall. Neither the haustorial neck nor the haustorial body were labelled. Control tests including the adsorption of the gold-complexed exoglueanase with fl-1,4-glucans prior to section labelling resulted in a near absence of gold particles [Fig. 6 (b)]. DISCUSSION
Results of the present study confirm that S. pannosa vat. rosae is capable of producing haustoria and associated structures typical ofother powdery mildew fungi [27, 28]. The formation of multilobed haustoria is a feature of members of the Erysiphaceae [7, 22]. The occurrence of a large number of mitochondria associated with a dense polyribosome-rich matrix in the haustorial main body as well as in the lobes may be interpreted as indicating an intense activity related to fungal growth and development within the host epidermis [8]. These observations are in agreement with those reported by Bushnell & Gay [9], who suggested that marked accumulation of organelles in haustoria was probably related to a highly active metabolism. The present cytochemical results revealed that walls of the haustorial body and lobes were easily distinguishable from the extrahaustorial matrix by their relative abil!ty to react with the WGA/ovomucoid-gold complex; no WGA receptors (chitin) were detected in the surrounding matrix whereas they were commonly found in fungal walls. The occurrence of chitin in the neck wall may also be related to cell-cell recognition phenomena. Rohringer et al. [30] pointed out that key determinants of compatibility and incompatibility in the interaction between wheat and Puccinia graminis Pers. : Pers. f. sp. tritici Eriks. & E. Henn. were associated with the haustorial neck wall. The origin and chemical composition of the extrahaustorial matrix has" been the subject of debate for many years [6, 7, 11, 12, 34]. In the present study, we chose to study the localization of the most widely distributed components in fungal (i.e., chitin) and plant (i.e., cellulose and pectin) cell walls. Several investigators have noted the amorphous nature of the extrahaustorlal matrix and suggested that it may display fluid properties. In our study, the use of two specific pi'obes conjugated to colloidal gold indicated the absence of cellulose and pectin in the extrahaustorial matrix. Although such negative results have to be taken with caution since they may indicate inaccessibility of the probes to their corresponding receptors, the intense positive reaction noted in the host cell wall within the same sections supports the view that these components do not occur in the extrahaustorial matrix. Gil & Gay [19] reported that the extrahaustorial matrix formed in pea leaf cells infected with Erysiphe pisi DC. ex Mdrat was a fluid that reacted less intensely than the extrahaustorial membrane with polysaccharide reagents and was removed by enzymes degrading the host cell wall.
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Sphaerotheca pannosa interaction
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Chong [11], Chong et al. [12, 13] and Rohringer et al. [30] reported that the matrix around haustoria of rust fungi contained a mixture of lipids and large amounts of polysaccharides and proteins. In the flax rust infections, the extrahaustorial matrix was also shown to contain bound sugars, probably glycoproteins [15]. In view of the high activity ofdictyosomes usually observed in haustorium-infected cells, Gil & Gay [19] and Rohringer etal. [30] speculated that powdery mildew and rust haustoria can disturb cell wall metabolism so that pectic and hemicellulosie substances are secreted into the host matrix. The absence of pectin and cellulose in the extrahaustorial matrix raises again the question as to what extent the extrahaustorial matrix may be derived from the adjacent host cell wall, or may result from a de novo synthesis in response to fungal attack. Whatever its origin, the nature of this material remains an unsolved, challenging puzzle. The attempted penetration of rose epidermal cells by S. pannosa var. rosae resulted in the deposition of heterogeneous materials, resulting in wall appositions between the plasma membrane and the host cell wall at the point directly facing the fungal penetration peg. Aist & Isra~l [1] suggested that the effectiveness of papilla formation as a defence mechanism against powdery mildew fungi may be dependent on the ability of epidermal cells to complete papilla deposition and consolidation before the penetration becomes too advanced. In barley infected with Erysiphe graminis DC. f. sp. hordei Era. Marchal, papillae were initiated in advance of the formation of penetration pegs in resistant isolines, whereas they appeared later in susceptible ones [21]. In hop leaves expressing resistance to powdery mildew fungi, lignin-like material and callose were detected in the papillae formed in penetrated cells [20]. Lignin and callose were also found to occur at the point of penetration in cultivars of muskmelon resistant to Sphaerothecafuliginea (Sehlechtend.:Fr.) Pollacci [16]. Colonization of epidermal ceils of rose leaves by S. pannosa var. rosae was associated with striking modifications of host cell walls. Our observations showed that a fibrillargranular material accumulated around and along the haustorial neck, which prevented a close association between the extrahaustorial membrane and the neck wall [23]: This material, called a collar by others [29], was made of two distinct.areas, one being amorphous and the other one fibrillar. Neither the amorphous nor the fibrillar material reacted with the WGA/ovomucoid-gold complex, thus indicating the absence of chitin. Cellulosic fl-l,4-glucans were found to be restricted to the outermost fibrillar layers whereas the innermost fibrillar layers as well as the amorphous material were nearly unlabelled, although a few scattered gold particles were present. This labelling pattern indicates the relatively iow content of cellulose in .the collar. Whether the presence of cellulose in the outermost layers results from the release of fragments from the adjacent host cell wall through the action of fungal hydrolases [24, 25], or reflects a new synthesis in the final stages of collar development remains to be determined. The presence of collars was usually associated with poor development of the haustoria, perhaps because close association between the extrahaustorial membrane and the haustorial neck was ruptured. This junction is normally impermeable to solutes within the extrahaustorial matrix and acts as a barrier to apoplastic flow in rust fungi [9, 33]. In some cases, the collar extends to the haustorial body and can encase it completely. Heath [23] postulated that such encasement may be a resistance mechanism against rust fungi. Manners & Gay [26] suggested that the collar may develop from
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p a p i l l a e t h a t f o r m b e f o r e o r d u r i n g host cell p e n e t r a t i o n . I n h o p p l a n t s infected w i t h
S. fuliginea, the c o l l a r was f o u n d to c o n t a i n callose-like deposits [17]. F u r t h e r studies d i r e c t e d t o w a r d s e l u c i d a t i n g the o r i g i n a n d c h e m i c a l c o m p o s i t i o n o f the c o l l a r f o r m e d in host cells c h a l l e n g e d b y S. pannosa var. rosae s h o u l d stress t h e l o c a l i z a t i o n o f f l - l , 3 g l u c a n s as well as o t h e r c o m p o u n d s i n c l u d i n g lignin a n d phenolics. W e a r e g r a t e f u l to S. Nofil for e x c e l l e n t t e c h n i c a l assistance. T h i s p r o j e c t was s u p p o r t e d b y a g r a n t f r o m Le M i n i s t b r e d e l ' A g r i c u l t u r e , des Pficheries et de l ' A l i m e n t a t i o n d u Q u6bec a n d f r o m the N a t u r a l Sciences a n d E n g i n e e r i n g R e s e a r c h Council of Canada.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17.
AtST,J. R. & ISRAEL,H. W. (1977). Papilla formation: timing and significance during penetration of barley coleoptiles by Er_)'siphegraminis f. sp. hordei. Ph)'topathology67, 455-461. BE.NIIAMOU,N. (1989). Preparation and application of lectln-gold complexes. In Colloidal Gold: Principles, Methods, and Applications, Ed. by M. A. Hayer, Vol. 1, pp. 95-143, Academic Press, New York. BENHAMOU,N., CHAMBERLAND,H., OUELLETTE, G.B. & PAUZ#.., F.J. (1987). Uhrastructural localization of fl-l,4-glucans in two pathogenic fungi and in their host tissues by means of an exoglucanase-gold complex. Canadian o7ournal of Microbiology 33, 405--417. BENIIAMOU,N., GILBoA-GARBER,N., TRUDEL,J. & ASSELIN,A. (1988). A new lectin-gold complex for the uhrastructural localization of galacturonic acids. Journal of Histoehemistry and C.)'tochcmistO' 36, 1403-1411. BONFANTE-FAsoLo,P., VIAN, B., PEROTTO,S., FACCIO,A. & K.~ox, J. P. (1990). Cellulose and pectin localization in roots of mycorrhlzal Allium porrum: labelling continuity between host cell wall and interfacial material. Planta 180, 537-547. BRACKER,C.E. & LIT'rLErIELD, L.J. (1973). Structural concepts of host-pathogen interfaces. In Fungal Pathogenidty & the Plant's Response, Ed. by R . J . W . Byrde & C.V. Cutting, pp. 159-318. Academic Press, London. BUSHNELL,W.R. (1971). The haustorium of Er_)'siphegraminis. An experimental study by light microscopy. In ~lIorphologieal and Biochemical Events in Plant-Parasite Interaction, Ed. by S. Akai & S. Ouchi, pp. 229-254. Phytopathological Society of Japan, Tokyo. BCSIIXELL,W. R. (1972). Physlology of fungal haustoria. Annual Review of Ph.ytopathology 10, 151-176. BUSltNELL,"~V.R. & GAY, J. L. (1978). Accumulation of solutes in rclatlon to the structure and function of haustoria of powdei3" mildews. In The Powdery Mildews, Ed. by D. Spencer, pp. 183-235. Academic Press, London. CIIARD,J. 3.1. & GAY,J. L. (1986). Cytochemical investigations ofphospholipase and lipase activity at the parasitic interface between ErA'siphe pisi and Pisum sativum. Physiological and 3lolecular Plant Pathology 28, 33-39. CHOXO,J. (1981). Ontogeny of mono- and dicaryotic haustoria of Puccinia eoronata: uhrastructure, cytochemistry and electron-probe-X-ray analysis. Ph.D. thesis, University of.Manitoba. CHoxo, J., HARDER, D. E. & ROtmX.~GER, R. (1984). Cytochemical studies on Puccinia gramins f. sp. tr#ici in a compatible wheat host. I. Walls ofintercellular hyphal cells and haustorlum mother cells. Canadian Journal of Botany 83, 1713-1724. CHO.XO,J., HARDER, D.E. & ROFIRINGER,R. (1985). Cytochemical studies on Puccinia graminis f. sp. tritid in a compatible wheat host. I I. Haustorium mother cell walls at the host cell penetration site, haustorial walls and the extrahaustorial matrix. Canadian Journal of Botany 64, 2561-2575. COFFEY,M. D. & ALLEN, F. H. E. (1983). A quantitative histological and uhrastructural analysis of interactions between the flax rust and near-isogenic host lines varying in their degree on incompatibility. Canadian Journal of Botany 61, 1831-1850. COFFEY,M. D. (1982). Cytochemical specialization at the haustorial interface ofa blotrophlc fungal parasite, Albugo eandida. Canadian Journal of Botany 61, 2004-2014. Co~tLx,Y., EYAL, H. & HA.NA.~IA,J. (1990). Ultrastructure, autofluorescence, callose deposition and lignification in the susceptible and resistant muskmelon leaves infected with the powdery mildew fungus Sphaerothecafuliginea. Physiologicaland Molecular Plant Pathology 36, 191-204. FRE.~S,G. (1973). Controlled nucleation for the regulation of the particle size in monodisperse gold solutions. Nature Physkal Science 241, 20-22.
Cytochemistry of the rose~Sphaerotheca pannosa interaction 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 3t.
355
GAY,J. L. & .MANNERS,J. M. (1987). Permeability of the haustorium-host interface in powdery mildews. Physiological and Molecular Plant Pathology 30, 389-399. GxL, F. & GAY, J. L. (1977). Ultrastructural and physiological properties of the host interracial components ofhaustoria of Erysiphe pisi in vivo and in vitro. Physiologicaland Molecular Plant Pathology 10, 1-12. Gom~aN,J. R., MANSFIELD,J. W. & DARBY, P. (1987). Microscopical studies ofresistanee to powdery mildew disease in the hop cuhivar Wye Target. Plant Pathology 36, 21-32. GOLD,R. E., AIST, J. R., HAZEN,B. H., STOLZENBURO,.XI.C., .~[ARSHALL,.X|. R. & ISRAEL,H. W. (1986). Effects of calcium nitrate and chlortetracycIine on papilla formation, ml-o resistance and susceptibility of barley to powder" mildew. Phyffological and Molecular Plant Pathology 29, 115-129. HARDER,D. E. & CIIO.XG,J. (1984). Structure and physiology ofhaustoria. In The Cereal Rusts, Ed. by W. R. Bushnell & A. P. Roelfs, Vol. I, pp. 430-475. Academic Press, London. HEATII,M. C. (1976). Ultrastructural and functional similarity of the haustorial neckband of rust fungi and the Casparian strip of vascular plants. Canadian Journal of Botany 54, 2484-2489. KENjI,T., AtST,J. R. & ISRAEL, H. W. (1985). Distribution ofhydrolytlc enzymes at barley powdery mildew encounter sites: implications for resistance associated with papilla formation in a compatible system. Physiological Plant Pathology 27, 167-184. KEoN, J. P. R., BYDRE, R . j . W . & COOPER, R.M. (1989). Some aspects of fungal enzymes that degrade plant cell walls. In Fungal Infection of Plants, Ed. by G. F. Pegg & Peter G. Ayers, pp. 132-157. Cambridge University Press. MANNERS,J. M. &GAY, j . L. (1983). The host-paraslte interface and nutrient transfer in biotrophic parasitism. In Biochemical Plant Pathology, Ed. byJ. A. Callow, pp. 163-195. j o h n Wiley, New York. MA.XXERS,J. M. & GAY,J. L. (1977). The morphology of haustorial complexes isolated from apple, barley, beet and vine infected with powdery mildews. Physiological Plant Pathology 11,261-266. .XIENDGEN,K. & HEITEFUSS,R. (1975). Micro-autoradiographic studies on host-parasite interactions. I. The infection of Phaseolus rulgaris with tritium-IabelIed uredospores of Uromycesphaseoli. Archives of ,llicrobiology 105, 193-199. PERERA,R. G. & GAY,J. L. (1976). The ultrastructure of haustoria ofSphaerothecapannosa (Wallroth ex Fries) Ldveill6, and changes in infected and associated cells of rose. Physiological Plant Pathology 9, 57-65. ROHRLNGER,R., CHONO,J., KBq W. K. & HARDER, D. E. (1982). Cytochemistry of the host/parasite interface and the use of gold markers in cereal/rust interactions, jVA TO A&'anced Study of the Institute of Serology, Serology A 37, 349-350. SPENCER-PmLLIPS, P . T . N . & GAY, J . L . (1980). Electron microscope autoradiography of HC photosynthate distribution at the haustorium-host interface in powdery mildew of Pisum sativum. Protoplasma 103, 131-154. STENZEL,K., STEINER, U. & SClIONBECK, F. (1985). Effect of induced resistance on the efficiency of powdery mildew haustoria in wheat and barley. PIo'siological Plant Pathology 27, 357-367. STU.MPF,M. A. &GAY, J. L. (1989). The haustorial interface in a resistant interaction of Erysiphepisi with Pisum sath'um. Physiological and ,lloletular Plant Pathology 35, 519-533. ZI.xI.X:ER,D. E. (1978). Fine structure ofPuerinia rarthami and the uhrastructural nature ofexclusionary seedling-rust resistance of safflower. Phytopathology 68, 1157-1163.
341
Cytochemical aspects of fungal penetration, haustorium formation and interfacial material in rose leaves infected by Sphaerotheca p a n n o s a var. rosae MOHAMED R. HAJLAOUI, NICOLE BENHAMOU and RICHARD R. BI~LANGER'~ Dipartement de ph.ytologie, Facultgdes sciencesde l'agriculture et de l'alirnentation, UniversitgLoyal, Sainte-Foy, Quibec, Canada GIA" 7P4 (Acceptedfor publication April 1991)
Fungal development and host reactions induced in rose leaf epidermal ceils by the powdery mildew fungus, Sphaerothecapannosa var. rosae,were examined on the basis ofultrastructure and of cytochemlstry of chitin, pectin, and cellulose subunits. Fungal growth in the epidermis was associated with the formation of haustoria which appeared muhilobed and delimited by an extrahaustorlal membrane probably originating from the host plasmalemma. The extrahaustorlal matrix was free of chitin as judged by the absence of labelling following incubation with a wheat germ agglutinin/ovomucoid-gold complex. Similarly, neither pectin nor cellulose was detected in this matrix, whereas these compounds were abundant in host cell walls. Various host reactions were observed at sites of fungal penetration. The formation of papillae was often associated with a restricted development of the infection peg. The most striking reaction was the occurrence of successive layers of a fibrillar material, forming a collar around the haustorial neck. Our cytochemical observations demonstrated that the collar was made of an amorphous material surrounded by fibrillar layers, the outermost ones being cellulose-rich. The possible nature and origin of this structure is discussed in relation to its possible involvement in resistance to fungal attack.
INTRODUCTION T h e process o f p l a n t tissue i n f e c t i o n by p o w d e r y m i l d e w fungi i n v o l v e s specific e v e n t s including appressorium formation, penetration and development of a haustorium s u r r o u n d e d b y a n e x t r a h a u s t o r i a l m a t r i x [8, 20, 26, 29]. E a c h e v e n t has b e e n well d o c u m e n t e d in t e r m s o f i n d u c e d host reactions. F o r instance, a t t e m p t e d p e n e t r a t i o n o f host e p i d e r m a l cells is o f t e n associated w i t h t h e f o r m a t i o n o f p a p i l l a e a r o u n d t h e site o f p e n e t r a t i o n [1, 24], p r e s u m a b l y to restrict f u n g a l i n v a s i o n a n d g r o w t h [16, 20]. U p o n p e n e t r a t i o n , w i t h i n the host cell, a n o t h e r r e a c t i v e m a t e r i a l , the collar, c a n d e p o s i t a l o n g the h a u s t o r i a l neck. I n s o m e cases, this c o l l a r m a y d e v e l o p to such a n e x t e n t t h a t the e n t i r e h a u s t o r i u m is encased thus r e d u c i n g the h a u s t o r i a l f u n c t i o n b y Abbreviations used in text: AGL, Aplysia gonad lectin; PBS, phosphate buffered saline; PEG, polyethylene glycol; WGA, wheat germ agglutinin. 1" To whom correspondence should be addressed. Paper no. CRH-6 from Le Centre de Recherche en Horticulture. 0885-5765/91/050341 + 15 $03.00]0
© 1991 Academic Press Limited
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acting as a barrier to nutrient uptake [16, 20, 32]. The collar has been well studied and characterized in relation to its possible role in defence mechanisms [23,26, 29], although its origin and chemical composition remain poorly understood. Considerable attention has also been paid to the interfacial matrix formed between host and parasite cells in biotrophic infections. The rationale for such an interest was that this matrix could be involved not only in nutrient uptake, but it could also be of key importance in the survival of penetrated host cells by providing sites for potential cell-cell recognition a n d / o r by interfering with deleterious fungal toxins and enzymes [9, 10, 18]. In this context, efforts have been directed towards elucidating the origin and chemical composition of the extrahaustorial matrix [12, 13, 14]. However, the exact nature of the interfacial material is still unknown and is debated in the literature. Although the occurrence ofpolysaccharides in the matrix is now generally accepted, their exact nature and origin is open to question. Some authors favour the hypothesis that the haustorial material is plant-derived and probably plays a role in resistance by preventing further pathogen ingress [28, 31]. Support for this speculation is drawn from the recent finding that both cellulose and pectin occur in the interracial material formed between Allium porrum L. root cells and the vesicular-arbuscular mycorrhizal fungus, Glomus versiforme (Nicol. & Gerd.) Gerdemann & Trappe [5]. Another possibility that has been proposed is that the extrahaustorial matrix may be composed of a mixture of plant and fungus-derived compounds [6, 13]. In view of the little information available on the nature of the collar and of the divergent results reported on the extrahaustorial matrix, tile object of this study was to bring further insight into the interfaciaI materials formed between both partners. For this purpose, we used the interaction between Sphaerotheca pannosa (Wallr: Fr) L6v. var rosae Woronichin and rose which has been little studied in terms ofpathogen action and host reactions [29]. Cytochemical techniques based on the use of specific colloidal goldlabelled probes were used to locate fungal and plant cell wall components.
MATERIALS AND METHODS
Plant material and inoculation Rose plants (Rosa hybrida L.) cv. Ruiredro, purchased from a local breeder in Q u6bec, were maintained in a greenhouse at 21 °C under a 16 h photoperiod. One day before inoculation, inoculum-donor plants were shaken to remove old spores; newly produced conidia from plants were used as inoculum. Donor plants were shaken over test plants to inoculate them. Inoculated plants were kept in the greenhouse and leafsamples were collected 5 days after inoculation.
Tissue processing for electron microscopy For electron microscopy, samples (1 mm 2) from young rose leaves infected with the powdery mildew fungus were immersed in 3 °/o (v/v) glutaraldehyde in 0"1 ~[ sodium cacodylate at pH 7"2 for 2 h at 4 °(3, rinsed with the same buffer, and post-fixed with 1 °/o (w/v) osmium tetroxide in sodium cacodylate buffer. They were then dehydrated in a series of ethanol solutions graded in I 0 % steps and embedded in Epon 812.
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Uhrathin sections were collected on Formvar-coated nickel grids and processed for cytochemical labelling. Experiments were repeated four times. For each experiment, five leaves from five different infected plants were collected. Samples were examined with a J E O L 1200 EX electron microscope at 80 kV.
Preparation of gold complexes Gold particles ofapprox. 14-16 nm in diameter were prepared according to Frens [17]. The pH of the colloidal gold solution was adjusted according to the isoelectric point of the lcctin or enzyme used [2]. Tile exoglucanase, a fl-l,4-cellobiohydrolase purified from a cellulase produced by Trichoderma harzianum Rifai [3], was used to locate cellulose subunits. The lectin from Aplysia depilans [4] was used to locate pectin because of its demonstrated affinity for polygalacturonic acids. They were directly complexed to colloidal gold at the appropriate pH. Because of its low molecular weight, the wheat germ agglutinin (WGA), a lectin with N-acetylglucosamine-binding sites, could not be complexed directly to gold since flocculation occurred. Ovomucoid, a protein with high affinity for WGA, was complexed to gold at pH 4"8 and used as a second step reagent as previously described [2].
Cytochemieal labelling For direct labelling, sections were first incubated for 5 min on a drop of PBS at the pH of optimal protein activity (6"0 for the exoglucanase and 8"0 for the AGL) containing 0"02% of PEG 20000 in PBS buffer. They were then transferred to a drop of the protein-gold complex and incubated for 30 min at room temperature in a moist chamber. Grids were then washed with PBS, rinsed with distilled water and stained with uranyl acetate and lead citrate. For indirect labelling, sections were incubated on a drop of WGA (I0 lag m1-1) in PBS pH 7"2 for 30 min at room temperature. Grids were rinsed with PBS and incubated with gold-complexed ovomucoid for 30 min. The were washed with PBS and distilled water, stained with uranyl acetate and lead citrate and examined under the electron microscope.
Reagents Tetrachloroauric acid was obtained from BDH Chemicals, Montr6al, Canada, and PEG 20000 from Fisher Scientific, Q udbec, Canada. Ovomucoid and WGA were purchased from Sigma Chemical Co., St. Louis, Missouri, USA. The exoglucanase was provided by Dr C. Breuil, Forintek Canada, Ottawa, and the AGL by Dr N. GilboaGarber, Bar Ilan University, Israel.
RESULTS
The cytology of infection of rose leaves by S. pannosa var rosae was similar in most respects to that previously described in other plants infected by biotrophic fungi
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F]o. 1. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerolheca pannosa var. rosae. (a) The thick, electron-lucent wall of an elongated germ tube is intensely labelled by the WGA/ovomucoid-gold complex. By contrast, the host cell wall is unlabelled. Bar = 0"5 lam. (b) At the site of attempted penetration, the cuticle and epidermal cell wall undergo a downward deformation (arrow). Labelling with exoglucanase-gold complex results in regular distribution of gold particles. Bar = 1 I.tm. (c) The appressorium is fixed to the host cell surface by a slime layer. The host cell wall is heavily labelled by the exoglucanase-gold complex even in the areas sho~ing obvious signs of deformation (arrow). Some gold particles occur in the area between the retracted plasmalemma and the host cell wall (double arrow, and c'). (c) Bar = 0"5 lam. (c') Bar = 2'25 lam. (d). Penetration is characterized by the formation of a narrow infection peg. A hemispherical protuberance made ofgranular and opaque materials accumulates
Cytochemistry of the
rose~Sphaerotheca pannosa interaction
345
[16, 27]. Conidia germinated on the leaf surface and produced one or two germ tubes surrounded by a thick, electron-lucent wall which labelled densely with the W G A / o v o m u c o i d - g o l d complex [Fig. 1 (a)]. Appressoria, formed at the end of germ tubes, were usually characterized by a large, centrally-located vacuole [Fig. 1 (b), (d)]. T h e wall of the appressorium was thinner than that of the germ tubes, and was also intensely labelled following incubation with the W G A / o v o m u c o i d - g o l d complex (not illustrated). At the sites of a t t e m p t e d penetration, appressoria a p p e a r e d to be closely fixed to the host cell surface by a fine, slime layer [Fig. l(c)]. At these sites, a d o w n w a r d deformation of both the cuticle and the underlying epidermal wall was usually observed [Fig. 1 (b), (c), arrows]. Incubation of sections with the exoglucanase-gold complex resulted in the deposition of gold particles over the locally deformed host cell walls [Fig. 1 (b) and (c)]. Some gold particles lining the innermost host wall layers were detected in the space between the deformed host wall and the invaginated host p l a s m a l e m m a [Fig. 1 (c) and (c')]. Successful wall penetration was characterized by the elaboration of a narrow penetration peg which emerged through a pore of the appressorium and entered the cuticle and the underlying epidermal cell wall [Fig. 1 (d)]. Invasion of host cells by penetration pegs was associated with the unusual occurrence of a fibrill-granular material between the host wall and the invaginated p l a s m a l e m m a [Fig. I (d)]. This material consisted of two areas which differed by their texture and electron-density. T h e first area, near the infection peg, was amorphous and electron-opaque whereas the second area, close to the p l a s m a l e m m a was more fibrillar and of lighter electron opacity [Fig. 1 (d)]. V a r y i n g amounts of reaction material were detected in areas neighbouring infection pegs [Fig. 2 (a)]. Most intracellular cells of the pathogen were not restricted by this material, but in some cases, the penetration peg was found to be totally encased in it [Fig. 2(a)]. In the lumen of epidermal cells, infection pegs enlarged to form an elongated structure from which arose the haustorial body [Fig. 2(b) and (c)]. Incubation with the W G A / o v o m u c o i d - g o l d complex resulted in an uneven deposition of gold p a r t i c l e s o v e r the wall of this elongated structure, referred to as a neck [Fig. 2 (b) and (c)]. O v e r the haustorial neck, labelling was either mainly concentrated over the apical area [Fig. 2(b), arrow] or more uniformly distributed [Fig. 2 (c)]. It is noteworthy that in most haustorial necks, the proximal region was usually devoid of any significant labelling [Fig. 2 (c), arrow]. A laminated material occurred around the neck forming a collar which was unlabelled by the W G A / o v o m u c o i d - g o l d complex [Fig. 2 (c)]. T h e neck usually had enlarged to form a typical ellipsoidal structure, the haustorial body, which appeared multilobed [Fig. 2 (d); Fig. 3 (a), (b)]. Connections between the lobes and the main body were seldom seen in transverse sections [Fig. 2 (d)], but could be easily detected in more tangential sections [Fig. 3 (a) and (b)]. around the infection peg. The host plasmalemma surrounds the hemispherical protuberance. Bar = 0"5 lam. Abbreviations: Ap = appressorium; C = collar; Ch = chloroplast; Cy = cytoplasm; EHMa = extrahaustofial matrix; EHMe = extrahaustorial membrane; F = fungus; GM=granular material; GTW=germ tube wall; HB=haustorial body; HCy=host cytoplasm; HGW = host cell wall; HN = haustorial neck; HW = haustorial wall; IP = infection peg; L=lobe; M=mitochondrlon; N=nucleus; OM=opaque material; P=papilla; PL = plasmalemma; S = septum; SL = slime layer; Va = vacuole. 2~
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Fzo. 2. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerotheca pannosa var. rosae. (a) The infection peg is encased in an electron-opaque papilla. Incubation with the WGA/ovomucoid-gold complex results in the deposition of gold particles over the fungus cell wall. B a r = 0"5 lam. (b) T h e apical region of a haustorial neck is labelled with the WGA/ovomucoid-gold complex (arrow). Bar = 0"25 lain. (c) T h e distal region of the haustorial neck is unlabelled (arrow). Bar = 0"5 lam. (d) A typical haustorium is formed in an epidermal cell and is separated from the neck by a septum. T h e ellipsoldal body contains a nucleus, several mitochondria and small vacuoles. A large number of lobes are found close to the main body. T h e haustorium is surrounded by an extrahaustorial membrane. Bar = 1 tam. For abbreviations see Fig. 1.
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M.R. Hajlaoui et aL
Haustorial lobes were found to be highly polymorphic, some of them being curved [Fig. 3 (c)] or highly contorted. The haustorial cytoplasm usually appeared densely packed with organelles embedded in a ribosome-rich matrix. The haustorium contained a large nucleus and elongated mitochondria [Fig. 2 (d)]. Another feature of interest was the presence of small vacuoles that were filled with a large number of vesicular structures. Nuclei and mitochondria were never seen to occur in the neck portion. Following incubation with the WGA/ovomucoid-gold complex, the wall of the haustorium body and the lobes was intensely labelled (Fig. 3). By contrast, cytoplasm, organelles and vacuoles were devoid of gold particles. The septum delimiting the neck from the main body was also heavily labelled [Fig. 3 (a)]. This labelling pattern was observed in all haustoria with, however, some variations probably due to the stage of development. In some haustoria, the number of lobes surrounded by a labelled wall was much higher [Fig. 3(a), double arrow] than in others where plasmolysis and distortion of the lobes was frequently observed [Fig. 3(a)]. The extrahaustorial membrane, resulting from an invagination of the host plasmalemma [Fig. 3 (a)] was unlabelled [Fig. 3 (b)]. Similarly, the extrahaustorial matrix was free of WGA-binding sites [Fig. 3 (b)]. Following incubation of infected rose leaf samples with the fl-exoglucanase-gold complex used for the localization of cellulose subunits, an intense and regular deposition of gold particles was observed over host cell walls [Fig. 4 (a)]. By contrast, neither the haustorial matrix nor the haustorial membrane exhibited labelling. Similarly, the haustorial body as well as the lobes were free oflabelling [Fig. 4 (a)]. Preadsorption of the enzyme-gold complex with fl-l,4-glucans from barley [3] prior to section incubation resulted in an absence of host cell wall labelling [Fig. 6 (b)]. When sections were incubated with the AGL-gold complex for the localization of polygalacturonic acid-containing molecules [4], a few scattered gold particles were associated with host cell walls, but not with the extrahaustorial matrix and the extrahaustorial membrane [Fig. 4 (b)]. Fungal structures were also devoid of labelling. All control tests including previous adsorption o f the gold-complex with polygalacturonic acids yielded negative results [not shown]. Colonization of the host epidermis by S. pannosa var. rosae was associated with striking host reactions. One noticeable feature was the necrotic appearance ofsome uncolonized palisade mesophyll ceils neighbouring infected ones [Fig. 4 (c), arrows]. By contrast, other mesophyll cells adjacent to necrotic ones remained apparently alive as judged by the normal appearance of tile cytoplasm and organelles. In infected epidermal cells, large amounts of reactive material along the haustorial neck were often detected [Fig. 5 (a)]. This material, which was either amorphous in areas adjacent to the haustorial neck or fibrillar and laminated towards the extrahaustorial membrane [Fig. 5 (a)], was often associated with aborted haustorium development. The amorphous material was usually separated from the haustorial neck by a narrow channel [Fig. 5 (a) and (b), arrows]. It could be easily distinguished from the adjacent host cell wall by its texture and electron density. The outer fibrillar material resembled the host cell wall in terms of texture, but it appeared to be derived from the successive deposition of layers of different electron density [Fig. 5(b)]. Extensive development ofthis material caused severe distortion ofthe |lost plasmalemma and prevented close association between the extrahaustorial membrane and the neck wall.
Cytochemistry of the rose---Sphaerotheca pannosa interaction
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F~o. 4. Transmission electron micrographs of rose leaf cells infected by Sphaerotheca pannosa var. rosae. (a) A section treated with exoglucanase-gold complex for the localization of the fl-l,4-glucan residues. An intense and regular deposition of gold particles over the host cell wall is seen. In contrast, the extrahaustorial membrane, extrahaustorial matrix, haustorial body and lobes are free of labelling. Bar = 0"5 lam. (b) A section incubated with AGL-gold complex for localization ofgalacturonic acids. Scattered gold particles are associated with the host cell wall but not with the extrahaustorial membrane, the extrahaustorial matrix, the haustorlal body or the lobes. Bar = 0"5 ~tm. (c) T h e necrosis of some uncolonized palisade mesophyll cells neighbouring the infected epidermal cell is observed (arrows). Some cells adjacent to the damaged ones remained apparently alive. Bar = 1 lam. For abbreviations see Fig. 1.
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FIO. 5. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerotheca pannosa var. rosae. (a) A prominent collar occurs around tile haustorial neck. This reactive material is amorphous in areas adjacent to the haustorial neck and fibrillar and laminar towards tile extrahaustorial membrane. Bar = 1 lain. (b) T h e walls of the haustorial body and the appressorium are uniformly labelled following incubation with the WGA/ovomucoid-gold complex. T h e collar is free of labelling. Bar = 1 lam. For abbreviations see Fig. 1.
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Fro. 6. Transmission electron micrographs of rose leaf epidermal cells infected by Sphaerotheca pannosa var. rosae. Labelling with exoglucanase--gold complex for localization of fl-l,4-glucan residues. (a) Labelling accumulates mainly over the host cell wall and the more external layers of the laminated material surrounding the haustorial neck. A few scattered gold particles are seen over the amorphous material of the collar. Neither the haustorial neck nor the haustorial body are labelled• Bar = 0"25 tam. (b) Control test. Incubation with the exoglucanase-gold complex to which was previously added /~-l,4-glucans from barley (1 m g ml -x) results in an absence of labelling•
352
M.R. Hajlaoui et aL
Both the amorphous and the stratified materials were unlabelled following incubation with the WGA/ovomucoid-gold complex [Fig. 5 (b)]. By contrast, gold particles were associated with the haustorial neck. When sections were incubated with the exoglueanase-gold complex, labelling accumulated mainly over the host cell wall as well as over the more external layers of the laminated material surrounding the haustorial neck [Fig. 6 (a)]. Only a few scattered gold particles could be seen over the amorphous material. The intensely labelled fibrillar layers were in apparent connection with the host cell wall. Neither the haustorial neck nor the haustorial body were labelled. Control tests including the adsorption of the gold-complexed exoglueanase with fl-1,4-glucans prior to section labelling resulted in a near absence of gold particles [Fig. 6 (b)]. DISCUSSION
Results of the present study confirm that S. pannosa vat. rosae is capable of producing haustoria and associated structures typical ofother powdery mildew fungi [27, 28]. The formation of multilobed haustoria is a feature of members of the Erysiphaceae [7, 22]. The occurrence of a large number of mitochondria associated with a dense polyribosome-rich matrix in the haustorial main body as well as in the lobes may be interpreted as indicating an intense activity related to fungal growth and development within the host epidermis [8]. These observations are in agreement with those reported by Bushnell & Gay [9], who suggested that marked accumulation of organelles in haustoria was probably related to a highly active metabolism. The present cytochemical results revealed that walls of the haustorial body and lobes were easily distinguishable from the extrahaustorial matrix by their relative abil!ty to react with the WGA/ovomucoid-gold complex; no WGA receptors (chitin) were detected in the surrounding matrix whereas they were commonly found in fungal walls. The occurrence of chitin in the neck wall may also be related to cell-cell recognition phenomena. Rohringer et al. [30] pointed out that key determinants of compatibility and incompatibility in the interaction between wheat and Puccinia graminis Pers. : Pers. f. sp. tritici Eriks. & E. Henn. were associated with the haustorial neck wall. The origin and chemical composition of the extrahaustorial matrix has" been the subject of debate for many years [6, 7, 11, 12, 34]. In the present study, we chose to study the localization of the most widely distributed components in fungal (i.e., chitin) and plant (i.e., cellulose and pectin) cell walls. Several investigators have noted the amorphous nature of the extrahaustorlal matrix and suggested that it may display fluid properties. In our study, the use of two specific pi'obes conjugated to colloidal gold indicated the absence of cellulose and pectin in the extrahaustorial matrix. Although such negative results have to be taken with caution since they may indicate inaccessibility of the probes to their corresponding receptors, the intense positive reaction noted in the host cell wall within the same sections supports the view that these components do not occur in the extrahaustorial matrix. Gil & Gay [19] reported that the extrahaustorial matrix formed in pea leaf cells infected with Erysiphe pisi DC. ex Mdrat was a fluid that reacted less intensely than the extrahaustorial membrane with polysaccharide reagents and was removed by enzymes degrading the host cell wall.
Cytochemistry of the rose
Sphaerotheca pannosa interaction
353
Chong [11], Chong et al. [12, 13] and Rohringer et al. [30] reported that the matrix around haustoria of rust fungi contained a mixture of lipids and large amounts of polysaccharides and proteins. In the flax rust infections, the extrahaustorial matrix was also shown to contain bound sugars, probably glycoproteins [15]. In view of the high activity ofdictyosomes usually observed in haustorium-infected cells, Gil & Gay [19] and Rohringer etal. [30] speculated that powdery mildew and rust haustoria can disturb cell wall metabolism so that pectic and hemicellulosie substances are secreted into the host matrix. The absence of pectin and cellulose in the extrahaustorial matrix raises again the question as to what extent the extrahaustorial matrix may be derived from the adjacent host cell wall, or may result from a de novo synthesis in response to fungal attack. Whatever its origin, the nature of this material remains an unsolved, challenging puzzle. The attempted penetration of rose epidermal cells by S. pannosa var. rosae resulted in the deposition of heterogeneous materials, resulting in wall appositions between the plasma membrane and the host cell wall at the point directly facing the fungal penetration peg. Aist & Isra~l [1] suggested that the effectiveness of papilla formation as a defence mechanism against powdery mildew fungi may be dependent on the ability of epidermal cells to complete papilla deposition and consolidation before the penetration becomes too advanced. In barley infected with Erysiphe graminis DC. f. sp. hordei Era. Marchal, papillae were initiated in advance of the formation of penetration pegs in resistant isolines, whereas they appeared later in susceptible ones [21]. In hop leaves expressing resistance to powdery mildew fungi, lignin-like material and callose were detected in the papillae formed in penetrated cells [20]. Lignin and callose were also found to occur at the point of penetration in cultivars of muskmelon resistant to Sphaerothecafuliginea (Sehlechtend.:Fr.) Pollacci [16]. Colonization of epidermal ceils of rose leaves by S. pannosa var. rosae was associated with striking modifications of host cell walls. Our observations showed that a fibrillargranular material accumulated around and along the haustorial neck, which prevented a close association between the extrahaustorial membrane and the neck wall [23]: This material, called a collar by others [29], was made of two distinct.areas, one being amorphous and the other one fibrillar. Neither the amorphous nor the fibrillar material reacted with the WGA/ovomucoid-gold complex, thus indicating the absence of chitin. Cellulosic fl-l,4-glucans were found to be restricted to the outermost fibrillar layers whereas the innermost fibrillar layers as well as the amorphous material were nearly unlabelled, although a few scattered gold particles were present. This labelling pattern indicates the relatively iow content of cellulose in .the collar. Whether the presence of cellulose in the outermost layers results from the release of fragments from the adjacent host cell wall through the action of fungal hydrolases [24, 25], or reflects a new synthesis in the final stages of collar development remains to be determined. The presence of collars was usually associated with poor development of the haustoria, perhaps because close association between the extrahaustorial membrane and the haustorial neck was ruptured. This junction is normally impermeable to solutes within the extrahaustorial matrix and acts as a barrier to apoplastic flow in rust fungi [9, 33]. In some cases, the collar extends to the haustorial body and can encase it completely. Heath [23] postulated that such encasement may be a resistance mechanism against rust fungi. Manners & Gay [26] suggested that the collar may develop from
M . R . Hajlaoui et aL
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p a p i l l a e t h a t f o r m b e f o r e o r d u r i n g host cell p e n e t r a t i o n . I n h o p p l a n t s infected w i t h
S. fuliginea, the c o l l a r was f o u n d to c o n t a i n callose-like deposits [17]. F u r t h e r studies d i r e c t e d t o w a r d s e l u c i d a t i n g the o r i g i n a n d c h e m i c a l c o m p o s i t i o n o f the c o l l a r f o r m e d in host cells c h a l l e n g e d b y S. pannosa var. rosae s h o u l d stress t h e l o c a l i z a t i o n o f f l - l , 3 g l u c a n s as well as o t h e r c o m p o u n d s i n c l u d i n g lignin a n d phenolics. W e a r e g r a t e f u l to S. Nofil for e x c e l l e n t t e c h n i c a l assistance. T h i s p r o j e c t was s u p p o r t e d b y a g r a n t f r o m Le M i n i s t b r e d e l ' A g r i c u l t u r e , des Pficheries et de l ' A l i m e n t a t i o n d u Q u6bec a n d f r o m the N a t u r a l Sciences a n d E n g i n e e r i n g R e s e a r c h Council of Canada.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17.
AtST,J. R. & ISRAEL,H. W. (1977). Papilla formation: timing and significance during penetration of barley coleoptiles by Er_)'siphegraminis f. sp. hordei. Ph)'topathology67, 455-461. BE.NIIAMOU,N. (1989). Preparation and application of lectln-gold complexes. In Colloidal Gold: Principles, Methods, and Applications, Ed. by M. A. Hayer, Vol. 1, pp. 95-143, Academic Press, New York. BENHAMOU,N., CHAMBERLAND,H., OUELLETTE, G.B. & PAUZ#.., F.J. (1987). Uhrastructural localization of fl-l,4-glucans in two pathogenic fungi and in their host tissues by means of an exoglucanase-gold complex. Canadian o7ournal of Microbiology 33, 405--417. BENIIAMOU,N., GILBoA-GARBER,N., TRUDEL,J. & ASSELIN,A. (1988). A new lectin-gold complex for the uhrastructural localization of galacturonic acids. Journal of Histoehemistry and C.)'tochcmistO' 36, 1403-1411. BONFANTE-FAsoLo,P., VIAN, B., PEROTTO,S., FACCIO,A. & K.~ox, J. P. (1990). Cellulose and pectin localization in roots of mycorrhlzal Allium porrum: labelling continuity between host cell wall and interfacial material. Planta 180, 537-547. BRACKER,C.E. & LIT'rLErIELD, L.J. (1973). Structural concepts of host-pathogen interfaces. In Fungal Pathogenidty & the Plant's Response, Ed. by R . J . W . Byrde & C.V. Cutting, pp. 159-318. Academic Press, London. BUSHNELL,W.R. (1971). The haustorium of Er_)'siphegraminis. An experimental study by light microscopy. In ~lIorphologieal and Biochemical Events in Plant-Parasite Interaction, Ed. by S. Akai & S. Ouchi, pp. 229-254. Phytopathological Society of Japan, Tokyo. BCSIIXELL,W. R. (1972). Physlology of fungal haustoria. Annual Review of Ph.ytopathology 10, 151-176. BUSltNELL,"~V.R. & GAY, J. L. (1978). Accumulation of solutes in rclatlon to the structure and function of haustoria of powdei3" mildews. In The Powdery Mildews, Ed. by D. Spencer, pp. 183-235. Academic Press, London. CIIARD,J. 3.1. & GAY,J. L. (1986). Cytochemical investigations ofphospholipase and lipase activity at the parasitic interface between ErA'siphe pisi and Pisum sativum. Physiological and 3lolecular Plant Pathology 28, 33-39. CHOXO,J. (1981). Ontogeny of mono- and dicaryotic haustoria of Puccinia eoronata: uhrastructure, cytochemistry and electron-probe-X-ray analysis. Ph.D. thesis, University of.Manitoba. CHoxo, J., HARDER, D. E. & ROtmX.~GER, R. (1984). Cytochemical studies on Puccinia gramins f. sp. tr#ici in a compatible wheat host. I. Walls ofintercellular hyphal cells and haustorlum mother cells. Canadian Journal of Botany 83, 1713-1724. CHO.XO,J., HARDER, D.E. & ROFIRINGER,R. (1985). Cytochemical studies on Puccinia graminis f. sp. tritid in a compatible wheat host. I I. Haustorium mother cell walls at the host cell penetration site, haustorial walls and the extrahaustorial matrix. Canadian Journal of Botany 64, 2561-2575. COFFEY,M. D. & ALLEN, F. H. E. (1983). A quantitative histological and uhrastructural analysis of interactions between the flax rust and near-isogenic host lines varying in their degree on incompatibility. Canadian Journal of Botany 61, 1831-1850. COFFEY,M. D. (1982). Cytochemical specialization at the haustorial interface ofa blotrophlc fungal parasite, Albugo eandida. Canadian Journal of Botany 61, 2004-2014. Co~tLx,Y., EYAL, H. & HA.NA.~IA,J. (1990). Ultrastructure, autofluorescence, callose deposition and lignification in the susceptible and resistant muskmelon leaves infected with the powdery mildew fungus Sphaerothecafuliginea. Physiologicaland Molecular Plant Pathology 36, 191-204. FRE.~S,G. (1973). Controlled nucleation for the regulation of the particle size in monodisperse gold solutions. Nature Physkal Science 241, 20-22.
Cytochemistry of the rose~Sphaerotheca pannosa interaction 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 3t.
355
GAY,J. L. & .MANNERS,J. M. (1987). Permeability of the haustorium-host interface in powdery mildews. Physiological and Molecular Plant Pathology 30, 389-399. GxL, F. & GAY, J. L. (1977). Ultrastructural and physiological properties of the host interracial components ofhaustoria of Erysiphe pisi in vivo and in vitro. Physiologicaland Molecular Plant Pathology 10, 1-12. Gom~aN,J. R., MANSFIELD,J. W. & DARBY, P. (1987). Microscopical studies ofresistanee to powdery mildew disease in the hop cuhivar Wye Target. Plant Pathology 36, 21-32. GOLD,R. E., AIST, J. R., HAZEN,B. H., STOLZENBURO,.XI.C., .~[ARSHALL,.X|. R. & ISRAEL,H. W. (1986). Effects of calcium nitrate and chlortetracycIine on papilla formation, ml-o resistance and susceptibility of barley to powder" mildew. Phyffological and Molecular Plant Pathology 29, 115-129. HARDER,D. E. & CIIO.XG,J. (1984). Structure and physiology ofhaustoria. In The Cereal Rusts, Ed. by W. R. Bushnell & A. P. Roelfs, Vol. I, pp. 430-475. Academic Press, London. HEATII,M. C. (1976). Ultrastructural and functional similarity of the haustorial neckband of rust fungi and the Casparian strip of vascular plants. Canadian Journal of Botany 54, 2484-2489. KENjI,T., AtST,J. R. & ISRAEL, H. W. (1985). Distribution ofhydrolytlc enzymes at barley powdery mildew encounter sites: implications for resistance associated with papilla formation in a compatible system. Physiological Plant Pathology 27, 167-184. KEoN, J. P. R., BYDRE, R . j . W . & COOPER, R.M. (1989). Some aspects of fungal enzymes that degrade plant cell walls. In Fungal Infection of Plants, Ed. by G. F. Pegg & Peter G. Ayers, pp. 132-157. Cambridge University Press. MANNERS,J. M. &GAY, j . L. (1983). The host-paraslte interface and nutrient transfer in biotrophic parasitism. In Biochemical Plant Pathology, Ed. byJ. A. Callow, pp. 163-195. j o h n Wiley, New York. MA.XXERS,J. M. & GAY,J. L. (1977). The morphology of haustorial complexes isolated from apple, barley, beet and vine infected with powdery mildews. Physiological Plant Pathology 11,261-266. .XIENDGEN,K. & HEITEFUSS,R. (1975). Micro-autoradiographic studies on host-parasite interactions. I. The infection of Phaseolus rulgaris with tritium-IabelIed uredospores of Uromycesphaseoli. Archives of ,llicrobiology 105, 193-199. PERERA,R. G. & GAY,J. L. (1976). The ultrastructure of haustoria ofSphaerothecapannosa (Wallroth ex Fries) Ldveill6, and changes in infected and associated cells of rose. Physiological Plant Pathology 9, 57-65. ROHRLNGER,R., CHONO,J., KBq W. K. & HARDER, D. E. (1982). Cytochemistry of the host/parasite interface and the use of gold markers in cereal/rust interactions, jVA TO A&'anced Study of the Institute of Serology, Serology A 37, 349-350. SPENCER-PmLLIPS, P . T . N . & GAY, J . L . (1980). Electron microscope autoradiography of HC photosynthate distribution at the haustorium-host interface in powdery mildew of Pisum sativum. Protoplasma 103, 131-154. STENZEL,K., STEINER, U. & SClIONBECK, F. (1985). Effect of induced resistance on the efficiency of powdery mildew haustoria in wheat and barley. PIo'siological Plant Pathology 27, 357-367. STU.MPF,M. A. &GAY, J. L. (1989). The haustorial interface in a resistant interaction of Erysiphepisi with Pisum sath'um. Physiological and ,lloletular Plant Pathology 35, 519-533. ZI.xI.X:ER,D. E. (1978). Fine structure ofPuerinia rarthami and the uhrastructural nature ofexclusionary seedling-rust resistance of safflower. Phytopathology 68, 1157-1163.