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Involvement of fimbriae in fungal host-mycoparasite interaction
Physiological and Molecular Plant Pathology (1992) 41, 139-148
139
Involvement of fimbriae in fungal host-mycoparasite interaction N . A . RGHEI, A . J . CASTLE *
and M .
S . MANOCHA
Department of Biological Sciences, Brock University St. Catharines, Ontario L2S 3A1, Canada (Accepted for publication August 1992)
Germ tubes of the biotrophic haustorial mycoparasite, Piptocephalis virginiana, show directed growth towards hyphae of the mucoraceous fungi Mortierella pusilla, Phascolomyces articulosus and Al . candelabrum. The mycoparasite will subsequently attach to cell walls of both the susceptible, M . pusilla, and resistant, P . articulosus, hosts but not to the non-host M. candelabrum . Fimbriae were observed by electron microscopy on the surfaces of the host and non-host species but not the mycoparasite . Polyclonal antiserum prepared against the fimbrial protein of Ustilago violacea crossreacted with 64 kDa proteins from both M. pusilla and P . articulosus and 60 and 57 kDa proteins from M . candelabrum. These proteins were electroeluted from polyacrylamide gels and were shown subsequently to form fibrils with the same diameter as the cell surface fimbriae . To ascertain the role of fimbriae in host-mycoparasite interactions, the antiserum was incubated with P. virginiana and M. pusilla, and with P . virginiana and P. articulosus . Contacts between mycoparasite and host were blocked significantly by the antiserum . This inhibition was not due to any effect of the antiserum on the linear growth rate of mycoparasite germ tubes . Thus, it was proposed that the recognition of fimbriae by the mycoparasite leads to directed growth and contact between mycoparasite germ tubes and the hyphae of a potential host .
INTRODUCTION
Fungal fimbriae are cell surface filaments described first on Ustilago violacea (Pers .) Fuckel [23, 24] . Polyclonal antiserum raised against U. violacea fimbrial protein was used to survey other fungi for the presence of fimbriae by agglutination and immunofluorescence techniques . These fibrils were found to be very widespread [5, 7, 12, 13, 25] . Fimbriae have been implicated in different functions : adhesion of Candide albicans to host tissue [9], flocculation of Saccharomyces cerevisiae [8], and conjugation in U. violacea [6] . A possible role of fimbriae in host-parasite interaction is still a matter of conjecture . This study was undertaken to investigate the role of fimbriae in mycoparasitism . The zygomycetous fungus, Piptocephalis virginiana Leadbeater and Mercer, is a biotrophic haustorial mycoparasite with a host range limited to the members of the order Mucorales . Germ tubes of P . virginiana are capable of directed growth towards other *To whom correspondence should be addressed . Abbreviations used in text : AU, polyclonal antiserum to Ustilago violacea fumbrial protein ; PBS, phosphate buffered saline ; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis . 0885-5765/92/080139+ 10 $08 .00/0 10
© 1992 Academic Press Limited MPP 41
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fungi, however, they attach to the hyphal surfaces of both susceptible (Mortierella pusilla Oudeman) and resistant hosts (Phascolomvces articulosus Boedijn ex Benny and Benjamin) but not of the non-host (Mortierella candelabrum V . "l'eigh and Le Morn) [16, 201 . Attachment of P . virginiana to host cell walls has been shown to be dependent upon the presence of two host specific glycoproteins . These glycoproteins agglutinate mycoparasite spores and prior incubation of my coparasite germ tubes with the glycoproteins prevents attachment of the germ tubes to host hyphae [19] . Attachment is followed by the formation of an appressorium and a penetration peg by the mycoparasite . In the susceptible host, penetration leads to the formation of a haustorium . In the resistant host, however, penetration is impeded by thickening of hyphal wall . Occasionally, penetration is successful and a haustorium is developed . When this occurs, a thick sheath develops around the haustorium thus preventing the establishment of a nutritional relationship with the host protoplast [17] . Implicit in these observations is that these events require recognition between host and mycoparasite at several levels : in the cell wall vicinity, at the cell wall, and at the host plasma membrane [21] . The objectives of this investigation were : (a) to determine iffimbriae are present on the surfaces of the host, non-host and mycoparasite species mentioned previously ; (b) to characterize partially the physical and chemical nature of fimbriae on these species : (e) to determine if a polyelonal antiserum prepared against U. violacea fimbrial protein cross-reacts with fimbrial proteins of these species ; and (d) to see if the antiserum, if cross-reactive, has any effect on the interaction between the hosts M . pusilla and P . articulosus and the mycoparasite P . virginiana .
MATERIALS AND METHODS
Cultures and growth conditions Cultures of Mortierella pusilla (susceptible host), Phascolomyces articulosus (resistant host) and Mortierella candelabrum (non-host) were maintained at 22±1 °C on Malt Yeast (MY) agar consisting of malt extract (20 g), yeast extract (2 g), and agar (20 g) in 1 litre of distilled water . Cultures of the mycoparasite, Piptocephalis virginiana, were maintained on its susceptible host Choanephora cucurbitarum (Berk and Rav .) Thaxter on MY medium . To obtain an axenic population of P . virginiana spores, the mycoparasite was grown on C . cucurbitarum at 22± I °C in darkness in order to inhibit the sporulation of the host while allowing the mycoparasite to sporulate normally [16] . Protein isolation M . pusilla, P . articulosus and M. candelabrum were grown in a liquid MY medium for 2-3 days . P . virginiana spores were germinated in a liquid medium containing nutrient broth (8 g), yeast extract (2 . 5 g), and glycerol (10 ml) in 1 litre of distilled water for 24--72 h [1] . Cultures were harvested by filtration through cheese cloth and rinsed repeatedly with distilled water prior to freeze drying . Proteins were isolated by grinding the frozen sample in a mortar and pestle with two parts silica gel and one part cold TEPI (10 mm Tris, 1 mm EDTA, 1 µM phenylmethylsulfonyl fluoride, and 1 mrvt iodoacetamide ; pH 6 . 8) . The slurry was centrifuged (10000 g ; 10 min ; 4 ° C) and the supernatant was shaken vigorously with two parts cold n-butanol to remove lipids [22 J . The aqueous layer was collected after centrifugation (1000g ; 10 min ; 4 ° C) and
141 dialysed against several changes of TE (10 mm Tris, 1 mm EDTA ; pH 7 . 5) for 2 h at 4 °C . Samples were lyophilized and suspended in minimal volumes of TEPI . Protein concentrations of the samples were determined by the Bradford [2] assay . Fungal host-mycoparasite interaction
Immunoblot analysis offimbrial proteins Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Laemmli [15] . An 11 ;~ separating gel and a 4 ° stacking gel were used . Protein samples were heated for 4 min at 95 °C in SDS reducing buffer (0 . 05 M Tris pH 6 . 8, 10 % glycerol, 2 °/0 SDS, 5 °/0 2-mercaptoethanol, and 0 . 001 % bromophenol blue) . The electrophoresis buffer was 7 mm Tris, 1 . 44 % glycine and 0 . 1 % SDS (pH 8 . 3) and protein samples were separated at 200 V . Proteins were stained with 0 . 1 °,,/o Coomassie brilliant blue R-250 in 40 °/,, methanol, 10 °"0 acetic acid and destained with 40 % methanol, 10 % acetic acid . Proteins separated by SDS-PAGE were transferred by electrophoresis to nitrocellulose membranes . Transfer buffer consisted of 25 mm Tris, 192 mm glycine and 20 °.o methanol, pH 8 . 3 [26] . Proteins were transferred overnight at room temperature at 30V with one change of voltage to 60 V for the last hour . Fimbrial proteins were detected by an indirect immunoblot procedure with polyclonal antiserum (AU) which recognizes the fimbrial protein of Ustilago violacea [4, 10, 13] . Electroelution of proteins Protein samples were separated by SDS-PAGE and stained with Coomassie brilliant blue R-250 . Gels were destained until protein bands were apparent . Target bands were excised for subsequent electroelution according to the method described in Hanaoka et al. [14] . Eluted proteins were dialysed against several changes of TE . Dialysed protein samples were lyophilized and resuspended in a minimal volume of distilled water . Electron microscopy M. pusilla, P. articulosus and M . candelabrum cultures grown in liquid medium for 24-72 h were harvested by centrifugation . P . virginiana germ tubes were prepared by inoculating a spore suspension into liquid MY medium or onto semi-solid MY medium overlaid with sterile dialysis tubing and incubating for 19-72 h . Samples were resuspended in 15 °-0 acetone, mixed vigorously and then left in the solution for 10 min prior to centrifugation . The samples were washed twice with distilled water and then left to stand in distilled water for 2 h . A drop of sample was placed on a formvar coated, carbon reinforced copper grid and left to settle on the grids for 5 min . Excess fluid was removed with filter paper . Grids were shadowed with gold palladium oxide at an angle of 19-21° . Alternatively, grids were negative stained . A drop of either 1 % (w/v) uranyl acetate or 3 % (w/v) ammonium molybdate was placed on the sample grid for 5-10 min . Excess stain was removed with filter paper and the samples were viewed with a Philips 300 electron microscope . Measurement of attachment of mycoparasite to host Sporangiospores of M. pusilla, P . articulosus, and P. virginiana were obtained from culture plates by adding sterile distilled water and gently scraping the cultures . The suspension was filtered through cheese cloth . Spores of both the mycoparasite and of a host species 10-2
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N . A . Rghei et al. were mixed to a total volume of I ml . Final spore concentrations were : 10' spores ml - ' for P . virginiana and 10 4 spores ml -' for a host species . The antiserum AU was added to the mycoparasite-host mixture to achieve the following final concentrations : 0 . 045, 0 . 087 and 0 . 125 tg protein pl - ' . In control experiments, pre-immune serum was added in the same concentrations . In a second set of control experiments, a 2 : 1 ratio of fimbrial protein from U . violacea and AU antiserum was incubated for 1 h at room temperature then overnight at 4 ° C . This pre-absorbed antiserum was then used as above . Three trials for each treatment in all experiments were analysed . The mycoparasite-host--antiserum mixtures were mixed well and left to stand for 1 h at room temperature . One drop was spread on dialysis membrane placed on semi-solid MY medium . The plates were incubated for 19-24 h at 22 ± 1 °C . The dialysis membrane was removed and placed on a microscope slide and the germinated spores were stained with cotton blue '1 °,, methylene blue in lactophenol) for light microscopic examination . At least 300 spores of the mycoparasite were counted in their relation to host hyphae : non-contact, contact, appressorium formation and relative percentage of appressoria were recorded . Germinated spores of P . virginiana observed to have no apparent physical contact with host hyphae were scored as non-contact events . Germ tubes of P . virginiana in contact along any part of the host hyphae were placed in the contact category . Germ tubes which showed contact and appressorium formation were considered as separate attachment events .
Effect of antiserum on linear growth of the mycoparasite The effect of the antifimbrial antiserum on linear growth of P . virginiana germ tubes was determined by incubating spore suspensions (10 - ` spores ml -') with (1) AU ; (2) pre-immune serum at concentrations of 0 . 1 tg protein pl - ' ; or (3) phosphate buffered saline (PBS) . The suspensions were incubated on sterile dialysis membrane on MY agar at room temperature for 20 h . The membranes were placed on a microscope slide for light microscopic examination . Germ tube lengths were measured with a calibrated ocular micrometer .
RESULTS
Fimbriae were observed on the surfaces of the hyphae of M . pusilla, P. articulosus and M . candelabrum (Figs 1-4) . They were distributed non-uniformly along the cell wall . For all three species, fimbriae were estimated to be up to 25 µm in length as determined from direct observations with the electron microscope and from electron micrographs . Diameters of the fimbriae did not vary significantly amongst the three species . M . candelabrum fimbriae had a mean diameter (+ standard error) of 9-1 ±0 . 4 nm, M. pusilla fimbriae were 9 . 4±0. 5 nm and P . articulosus fimbriae were 8 . 6±0 . 6 nm . These measurements were determined from electron micrographs of the negatively stained samples . With P . virginiana, fimbriae were not observed on germinating sporangiospores or on 19-72 h germ tubes grown in liquid or on semi-solid medium . Germ tubes of P . virginiana in this age range are capable of contacting and forming appressoria on host species on semi-solid medium .
Fungal host-mycoparasite interaction
1 43
Fits 1-2 . Shadow cast replica of the outer surface of the hyphae : Fig. 1, M . pusilla, Fig . 2, P. articulosus. Note the long fibrillar structures, fimbriae, (arrows) arising from the hyphal surface . 60 000 x . Fics 3-4 . Negatively stained preparations of hyphae : Fig . 3, M . pusilla, Fig. 4, M. candelabrum . Note distinct fimbriae (arrows) at the cell surface . 61875 x and 52 300 x , respectively .
Soluble proteins isolated from M. pusilla, P. articulosus, M . candelabrum and P . were separated by SDS-PAGE and were transferred to nitrocellulose membranes for immunoblot identification of fimbrial proteins . Polyclonal antiserum raised against fimbriae of U. violacea virginiana
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species . From the non-host, Al . candelabrum, two proteins with molecular masses of 60 and of 57 kDa were visualized . In contrast, the presumptive fimbrial proteins of both the susceptible and resistant hosts, .l. pusilla and P. articulosus, respectively, had masses of 64 kDa (Fig . 5) . The antiserum AU cross-reacted with two P . iirginiana
Mc
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Pa
Pa
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Mc
Us
106 80 49
32
(a )
5
(b)
6
FIG . 5 . Immunoblot analyses of fimbrial proteins obtained from hosts A1L pusilla (Mp) and P . articulosus (Pa) and non-host M. candelabrum (Mc) . (a) Immunoblot treated with the anti-fimbrial antiserum AU . Note cross-reaction of AU with M r 64 kDa proteins of both M . pusilla and P. articulosus, but with two proteins, M, 57 and 60 kDa, of a-1 . candelabrum . (b) Duplicate immunoblot reacted with pre-immune serum did not show any bands . The marker lane was that ofpre-stained low molecular range protein standards from Bio-Rad . FIG . 6 . Immunoblot analysis of proteins from P . rirginiana Pv ; . AU cross-reacted with two proteins, M 5 94 and 91 kDa, even though no fimbriac were observed at the surface of the mycoparasite . Right lane shows cross-reaction with fimbrial protein of Fittings riola(ea . Marker lane was that of pre-stained high molecular range protein standards from Bio-Rad . Ftos 7 8 . Negatively stained ,'1 ° uranyl acetate) preparations of electroeluted fimbrial proteins from Fig. 7, .14 . ptsilla and Fig . 8, 51 . candelabrum showing distinct fibrils . 57600 x .
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Fungal host-mycoparasite interaction
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point represents the mean of three independent tests . Top : Percentage contact (number of mycoparasite germ tubes in contact with a host hypha x 100°/total number of mycoparasite germ tubes examined) ; Middle : percentage appressorium formation (number of mycoparasite germ tubes with an appressorium x 100%/total number of mycoparasite germ tubes examined) ; Bottom : The relative percentage of appressorium formation (No . of appressoria x 100%/No . Of contacts) . Treatments are : AU ), preimmune serum (-E-) and antiserum previously treated with fimbrial protein (---0-) . Each data point followed by the letter 9 is significantly different (Kruskal-Wallis test) from the 0 concentration point and from the pre-immune serum point at the same protein concentration .
proteins with molecular masses of 94 and 91 kDa (Fig . 6) . These proteins were not likely to be fimbrial subunits since fimbriae had not been observed on P . virginiana . Their relationship to fimbrial protein, if any, remains unknown . Proof that AU cross-reacted with fimbrial subunits from host and non-host species was obtained by reconstitution of the immunoreactive proteins into fibrils in vitro . Proteins recognized by AU were electroeluted from the gel matrix and the SDS was removed . Fibrils were formed spontaneously by the electroeluted proteins of M . pusilla, M. candelabrum and P . articulosus . Figures 7 and 8 show the fibrils obtained from M . pusilla and M . candelabrum . The diameters of the reformed fibrils were approximately the same as those of the fimbriae attached to the hyphal surfaces . Fibril diameter means
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N . A . Rghei et al. ±Standard error) of 9 . 7±0 . 3 run, 8-4 ±0 . 6 urn and 9 . 2±0 . 5 urn für A1 . candelabrum, .11 . pusilla, and P. articulosus, respectively, were observed . In our previous experiences, fibrils are not observed : (1) when the SDS is not displaced from the immuno-reactive proteins or (2) when other regions of these gels are treated as above . Thus, the immunoblot results coupled with the observations from the electroelution tests indicate that AU cross-reacted with fimbrial proteins from host and non-host species with high specificity . To determine if fimbriae are required for events in mycoparasitism, spores of the mycoparasite, P . virginiana, and the susceptible host, M. pusilla, or P . virginiana and the resistant host, P . articulosus, were incubated with AU in various concentrations . Incubation with AU resulted in a significant inhibition ofcontact between mycoparasite and host . Once contact was achieved, however, appressorium formation was apparently normal . Hence, the relative percentage of appressoria formed (No . of appressoria x 100° 0 /No . of contacts) did not change (Figs 9a and b) . The specificity of this inhibition was confirmed in control tests with pre-immune serum or AU pre-incubated with purified Ustilago violacea fimbrial protein . Neither treatment inhibited host---parasite contact significantly . The observed inhibition of contact could conceivably have been due to an inhibition of growth of germ tubes of the mycoparasite . Thus, the effects of AU, pre-immune serum or PBS on germ tube growth were determined . Spore germination in all three treatments was high : AU, 84"1 0 .. pre-immune serum, 74 11 0 ; PBS, 73 11,,( , . The mean ± standard error of 20 germ tubes treated with AU in two independent tests was 42 ± 6 . 4 µm . The length of germ tubes treated with pre-immune serum was 54 ± 9 . 5 Am and germ tubes treated with PBS had a mean length of 51±9 . 1 µm . Analysis of variance indicated that these values were not significantly different . DISCUSSION The present demonstration of fimbriae on mucoraceous species, M. pusilla, P . articulosus and M . candelabrum, adds to an increasing list of fungi from all major classes possessing non-flagellar cell surface filaments [5, 7, 12, 13, 25] . Morphological characteristics, as observed by electron microscopy, of fimbriae from other fungal species are similar to those reported here . Most fungi have fimbriae with diameters of 6-10 nm [10] . The length of fimbriae reported varies from as short as 1 Am in several ascomycetous yeast species to 20 Am in length in U . violacea [13] . The apparent lack of fimbriae on Piptocephalis virginiana is not surprising . In addition to this species, fimbriae were not detected by electron microscopy and immunological techniques on isolates of several basidiomycetous and ascomycetous species [13] . Whether these fungi produce fimbriac under different environmental conditions is unknown . At present it appears that the distribution of fimbriae on fungal species is very broad but not universal . Fimbriae of M . pusilla, M . candelabrum, and P . articulosus consist of monomeric subunits which cross-react with polyclonal antiserum produced against Ustilago violacea fimbrial protein . From these studies and similar investigations, it appears that the molecular mass of fimbrial protein is quite variable between species . The masses of fimbrial subunits range from 74 kDa in U. violacea [4, 5, 11 ] to 37 kDa in Goprinus
Fungal host-mycoparasite interaction
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cinereus [3] . These size differences may result in different functional roles for fimbriae in these species, although evidence for such a relationship is lacking . The observation that the fimbrial specific antiserum AU inhibited contact between P . virginiana and M . pusilla or P . articulosus suggests that host fimbriae serve as an initial point of interaction between mycoparasite and host . The subsequent parasitic event of appressorium formation did not appear to be inhibited . Parasitism by P . virginiana is initiated by directed growth, over short distances, of the parasite germ tube towards a hypha of a potential host . The phenomenon of directed growth does not apparently involve host/non-host differentiation since directed growth of P . virginiana towards non-host (M . candelabrum) hyphae also occurs [17] . Contact, the physical abuttment of cell walls, occurs with equal frequency in parasite-host or parasite-non-host interactions [20] and is a consequence of directed growth . Prior to this study it was proposed that directed growth is likely to be promoted by a physical or diffusible chemical stimulus produced by the potential host [17] . It is proposed that fimbriae serve as such a stimulus : the mycoparasite encounters fimbriae extending from the host hyphal wall and directed growth along these structures ensues . The observed inhibition of contact is due, therefore, to a disruption of directed growth . This hypothesis leads to the idea that the mycoparasite has a mechanism for reception of the fimbrial stimulus and recognition, therefore, of a potential host . Studies which directly examine the effect of AU on the phenomenon of directed growth should be undertaken to test this hypothesis . After contact, the mycoparasite adheres to the host but not to the non-host hyphae . The attachment of P . virginiana to host cell walls has been shown to be dependent upon the presence of two host-specific glycoproteins [19] . It should be noted that the attachment proteins are not fimbrial proteins : they have different masses (glycoproteins M r 100 and 85 kDa ; fimbrial protein Mr 64 kDa) and antiserum AU does not recognize either glycoprotein, M r 100 or 85 kDa, on immunoblots (data not shown) . Attachment is followed closely by appressorium formation and penetration . It is likely that fimbrial protein and the two attachment proteins are not involved in appressorium formation . Appressorium formation can be inhibited by heat treatment without disrupting attachment [21] and in this study appressoria developed in the presence of anti-fimbrial protein antiserum . Thus, it is likely that yet another host structure is involved at this level of interaction between host and mycoparasite . In summary, we propose that the interaction between host and parasite appears to occur at several levels : (1) initially through fimbriae, resulting in directed growth and contact ; (2) through attachment of the mycoparasite to the host cell surface mediated by two host cell wall glycoproteins [19] ; (3) during appressorium formation ; and (4) at the host plasmalemma as indicated by the resistant host response to the mycoparasite, formation of a papilla which inhibits penetrations [21] . This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada to A . C . and M . S . M . REFERENCES 1 . Balasubramanian R, Manocha MS. 1986 . Proteinase, chitinase, chitosanase activities in germinating spores of Piptocephalis virginiana . Mycologia 78 : 157-163 .
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2 . Bradford M . 1976 . A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding . Analytical Biochemistry 72 : 248-251 . 3 . Castle AJ, Boulianne RB . 1991- Fimbrial proteins of 1'stilago riolacca, Coprinus cinereus and Sdcizophyllum comrnune . 11S:A ,;Veu, sletter 42 : 8 (Abstr . ; . 4 . Castle AJ, Boulianne R, Xu J, Day AW . 1992 . Post-translational modification of fimbrial protein from Ustilago violacea. Canadian Journal oj' .116robiology (in press . 5 . Day AW, Gardiner RB . 1988 . Surface proteinaceous fibrils fimbriae) on filamentous fungi . Canadian Journal of Botany 66 : 2474-2484 . 6 . Day AW, Poon NH . 1975 . Fungal fimbriae II : Their role in conjugation in t,'stilago riolacea . Canadian Journal of .tilicrobiology 21 : 547-557 . 7 . Day AW, Gardiner RB, Smith R, Svircev AM, McKeen WE . 1986 . Detection of fungal fimbriae by protein A-gold immunocytochemical labelling in host plants infected with t'stilago heufleri or Peronospora hyosoami f.sp . tabacina . Canadian Journal of Microbiology 32 : 577-584 . 8 . Day AW, Poon NH, Stewart GG. 1975 . Fungal fimbriae III : The effect on flocculation in Saccharomyces . Canadian 7ownal of Microbiology 21 : 558- 564 . 9 . Douglas LJ, Houston JG, McCourtie J . 1981 . Adherence of Candida albicans to human buccal epithelial cells after growth on different carbon sources . FEBS Microbiology Letters 12 : 241-243 . 10 . Gardiner RB . 1985 . Fungal Fimbriae : Their Structure and Distribution . Ph .D . Thesis, The University of Western Ontario . 11 . Gardiner RB, Day AW . 1985 . Fungal fimbriae IV : Composition and properties of finibriae from U. violacea . Experimental Mycology 9 : 334--350 . 12 . Gardiner RB, Canton M, Day AW . 1981 . Fimbrial variation in smuts and heterobasidiomvcete fungi . Botanical Gazette 142 : 147 150, 13 . Gardiner RB, Podgorski C, Day AW . 1982 . Serological studies on the fimbriac of ycasts and yeastlike species . Botanical Gazette 143 : 534- 541 . 14 . Hanoaka F, Shaw JL, Mueller GC . 1979 . Recovery of functional proteins from sodium dodecyl sulfate-polyacrylamide gels . Analytical Biochemistry 99 : 170-174 . 15 . Laemmli UK . 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . N, ature 227 : 680-685 . 16 . Manocha MS . 1985 . Specificity of mvcoparasite attachment to the host cell surface . Canadian ,fournal of Botany 63 : 772-778 . 17 . Manocha MS . 1988 . Biotrophic mycoparasitism : a model system for investigations in hostparasite interactions. In : Agnihotri VP, Sarbhoy AK, Kumar D, eds . Perspectives in Mycology and Plant Pathology, New Delhi : Malhotra Publishing . 462 495 . 18 . Manocha MS . 1990 . Cell-cell interaction in fungi . Journal of Plant Diseases and Protection 97 : 655-669 . 19 . Manocha MS, Chen Y . 1991 . Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite . Canadian Journal of Microbiology 37 : 377-383 . 20 . Manocha MS, Balasubramanian R, Enskat S . 1986 . Attachment of a mycoparasite with host but not with nonhost Vortierella species . In : .NATO ASI Series Vol . 41 . Biology and Molecular Biology of Plant-Pathogen Interactions, by Bailey, J . Ed . Berlin : Springer-Verlag, 59-69 . 21 . Manocha MS, Chen Y, Rao N . 1990 . Involvement of cell surface sugars in recognition, attachment, and appressorium formation by a mycoparasite . Canadian Journal of Microbiology 36 : 771 778 . 22 . Penevsky HS, Tzagoloff A . 1971 . Extraction of water-soluble enzymes and proteins from membranes . I1-lethods in Enztrology 22 : 204 219 . 23 . Poon NH, Day AW . 1974 . Fimbriae in the fungus Ustilago violaces . .Nature 250 : 648-649 . 24 . Poon NH, Day AW . 1975 . Fungal fimbriae I : structure, origin and synthesis . Canadian Journal of Microbiology 21 : 537-546 . 25 . Svircev A, Gardiner RB, Day AW, Smith R . 1986 . Detection by protein a-gold immunocytochemical labelling of antigens to Botrytis cinerea in cytoplasm of infected Vicia faba. Phytopathology 76 : 622-626 . 26 . Towbin H, Staehelin T, Gordon J . 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets : Procedures and some applications . Proceedings of the .National Academy of Sciences of the US-1 76 : 4350-4354 .
139
Involvement of fimbriae in fungal host-mycoparasite interaction N . A . RGHEI, A . J . CASTLE *
and M .
S . MANOCHA
Department of Biological Sciences, Brock University St. Catharines, Ontario L2S 3A1, Canada (Accepted for publication August 1992)
Germ tubes of the biotrophic haustorial mycoparasite, Piptocephalis virginiana, show directed growth towards hyphae of the mucoraceous fungi Mortierella pusilla, Phascolomyces articulosus and Al . candelabrum. The mycoparasite will subsequently attach to cell walls of both the susceptible, M . pusilla, and resistant, P . articulosus, hosts but not to the non-host M. candelabrum . Fimbriae were observed by electron microscopy on the surfaces of the host and non-host species but not the mycoparasite . Polyclonal antiserum prepared against the fimbrial protein of Ustilago violacea crossreacted with 64 kDa proteins from both M. pusilla and P . articulosus and 60 and 57 kDa proteins from M . candelabrum. These proteins were electroeluted from polyacrylamide gels and were shown subsequently to form fibrils with the same diameter as the cell surface fimbriae . To ascertain the role of fimbriae in host-mycoparasite interactions, the antiserum was incubated with P. virginiana and M. pusilla, and with P . virginiana and P. articulosus . Contacts between mycoparasite and host were blocked significantly by the antiserum . This inhibition was not due to any effect of the antiserum on the linear growth rate of mycoparasite germ tubes . Thus, it was proposed that the recognition of fimbriae by the mycoparasite leads to directed growth and contact between mycoparasite germ tubes and the hyphae of a potential host .
INTRODUCTION
Fungal fimbriae are cell surface filaments described first on Ustilago violacea (Pers .) Fuckel [23, 24] . Polyclonal antiserum raised against U. violacea fimbrial protein was used to survey other fungi for the presence of fimbriae by agglutination and immunofluorescence techniques . These fibrils were found to be very widespread [5, 7, 12, 13, 25] . Fimbriae have been implicated in different functions : adhesion of Candide albicans to host tissue [9], flocculation of Saccharomyces cerevisiae [8], and conjugation in U. violacea [6] . A possible role of fimbriae in host-parasite interaction is still a matter of conjecture . This study was undertaken to investigate the role of fimbriae in mycoparasitism . The zygomycetous fungus, Piptocephalis virginiana Leadbeater and Mercer, is a biotrophic haustorial mycoparasite with a host range limited to the members of the order Mucorales . Germ tubes of P . virginiana are capable of directed growth towards other *To whom correspondence should be addressed . Abbreviations used in text : AU, polyclonal antiserum to Ustilago violacea fumbrial protein ; PBS, phosphate buffered saline ; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis . 0885-5765/92/080139+ 10 $08 .00/0 10
© 1992 Academic Press Limited MPP 41
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fungi, however, they attach to the hyphal surfaces of both susceptible (Mortierella pusilla Oudeman) and resistant hosts (Phascolomvces articulosus Boedijn ex Benny and Benjamin) but not of the non-host (Mortierella candelabrum V . "l'eigh and Le Morn) [16, 201 . Attachment of P . virginiana to host cell walls has been shown to be dependent upon the presence of two host specific glycoproteins . These glycoproteins agglutinate mycoparasite spores and prior incubation of my coparasite germ tubes with the glycoproteins prevents attachment of the germ tubes to host hyphae [19] . Attachment is followed by the formation of an appressorium and a penetration peg by the mycoparasite . In the susceptible host, penetration leads to the formation of a haustorium . In the resistant host, however, penetration is impeded by thickening of hyphal wall . Occasionally, penetration is successful and a haustorium is developed . When this occurs, a thick sheath develops around the haustorium thus preventing the establishment of a nutritional relationship with the host protoplast [17] . Implicit in these observations is that these events require recognition between host and mycoparasite at several levels : in the cell wall vicinity, at the cell wall, and at the host plasma membrane [21] . The objectives of this investigation were : (a) to determine iffimbriae are present on the surfaces of the host, non-host and mycoparasite species mentioned previously ; (b) to characterize partially the physical and chemical nature of fimbriae on these species : (e) to determine if a polyelonal antiserum prepared against U. violacea fimbrial protein cross-reacts with fimbrial proteins of these species ; and (d) to see if the antiserum, if cross-reactive, has any effect on the interaction between the hosts M . pusilla and P . articulosus and the mycoparasite P . virginiana .
MATERIALS AND METHODS
Cultures and growth conditions Cultures of Mortierella pusilla (susceptible host), Phascolomyces articulosus (resistant host) and Mortierella candelabrum (non-host) were maintained at 22±1 °C on Malt Yeast (MY) agar consisting of malt extract (20 g), yeast extract (2 g), and agar (20 g) in 1 litre of distilled water . Cultures of the mycoparasite, Piptocephalis virginiana, were maintained on its susceptible host Choanephora cucurbitarum (Berk and Rav .) Thaxter on MY medium . To obtain an axenic population of P . virginiana spores, the mycoparasite was grown on C . cucurbitarum at 22± I °C in darkness in order to inhibit the sporulation of the host while allowing the mycoparasite to sporulate normally [16] . Protein isolation M . pusilla, P . articulosus and M. candelabrum were grown in a liquid MY medium for 2-3 days . P . virginiana spores were germinated in a liquid medium containing nutrient broth (8 g), yeast extract (2 . 5 g), and glycerol (10 ml) in 1 litre of distilled water for 24--72 h [1] . Cultures were harvested by filtration through cheese cloth and rinsed repeatedly with distilled water prior to freeze drying . Proteins were isolated by grinding the frozen sample in a mortar and pestle with two parts silica gel and one part cold TEPI (10 mm Tris, 1 mm EDTA, 1 µM phenylmethylsulfonyl fluoride, and 1 mrvt iodoacetamide ; pH 6 . 8) . The slurry was centrifuged (10000 g ; 10 min ; 4 ° C) and the supernatant was shaken vigorously with two parts cold n-butanol to remove lipids [22 J . The aqueous layer was collected after centrifugation (1000g ; 10 min ; 4 ° C) and
141 dialysed against several changes of TE (10 mm Tris, 1 mm EDTA ; pH 7 . 5) for 2 h at 4 °C . Samples were lyophilized and suspended in minimal volumes of TEPI . Protein concentrations of the samples were determined by the Bradford [2] assay . Fungal host-mycoparasite interaction
Immunoblot analysis offimbrial proteins Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method of Laemmli [15] . An 11 ;~ separating gel and a 4 ° stacking gel were used . Protein samples were heated for 4 min at 95 °C in SDS reducing buffer (0 . 05 M Tris pH 6 . 8, 10 % glycerol, 2 °/0 SDS, 5 °/0 2-mercaptoethanol, and 0 . 001 % bromophenol blue) . The electrophoresis buffer was 7 mm Tris, 1 . 44 % glycine and 0 . 1 % SDS (pH 8 . 3) and protein samples were separated at 200 V . Proteins were stained with 0 . 1 °,,/o Coomassie brilliant blue R-250 in 40 °/,, methanol, 10 °"0 acetic acid and destained with 40 % methanol, 10 % acetic acid . Proteins separated by SDS-PAGE were transferred by electrophoresis to nitrocellulose membranes . Transfer buffer consisted of 25 mm Tris, 192 mm glycine and 20 °.o methanol, pH 8 . 3 [26] . Proteins were transferred overnight at room temperature at 30V with one change of voltage to 60 V for the last hour . Fimbrial proteins were detected by an indirect immunoblot procedure with polyclonal antiserum (AU) which recognizes the fimbrial protein of Ustilago violacea [4, 10, 13] . Electroelution of proteins Protein samples were separated by SDS-PAGE and stained with Coomassie brilliant blue R-250 . Gels were destained until protein bands were apparent . Target bands were excised for subsequent electroelution according to the method described in Hanaoka et al. [14] . Eluted proteins were dialysed against several changes of TE . Dialysed protein samples were lyophilized and resuspended in a minimal volume of distilled water . Electron microscopy M. pusilla, P. articulosus and M . candelabrum cultures grown in liquid medium for 24-72 h were harvested by centrifugation . P . virginiana germ tubes were prepared by inoculating a spore suspension into liquid MY medium or onto semi-solid MY medium overlaid with sterile dialysis tubing and incubating for 19-72 h . Samples were resuspended in 15 °-0 acetone, mixed vigorously and then left in the solution for 10 min prior to centrifugation . The samples were washed twice with distilled water and then left to stand in distilled water for 2 h . A drop of sample was placed on a formvar coated, carbon reinforced copper grid and left to settle on the grids for 5 min . Excess fluid was removed with filter paper . Grids were shadowed with gold palladium oxide at an angle of 19-21° . Alternatively, grids were negative stained . A drop of either 1 % (w/v) uranyl acetate or 3 % (w/v) ammonium molybdate was placed on the sample grid for 5-10 min . Excess stain was removed with filter paper and the samples were viewed with a Philips 300 electron microscope . Measurement of attachment of mycoparasite to host Sporangiospores of M. pusilla, P . articulosus, and P. virginiana were obtained from culture plates by adding sterile distilled water and gently scraping the cultures . The suspension was filtered through cheese cloth . Spores of both the mycoparasite and of a host species 10-2
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N . A . Rghei et al. were mixed to a total volume of I ml . Final spore concentrations were : 10' spores ml - ' for P . virginiana and 10 4 spores ml -' for a host species . The antiserum AU was added to the mycoparasite-host mixture to achieve the following final concentrations : 0 . 045, 0 . 087 and 0 . 125 tg protein pl - ' . In control experiments, pre-immune serum was added in the same concentrations . In a second set of control experiments, a 2 : 1 ratio of fimbrial protein from U . violacea and AU antiserum was incubated for 1 h at room temperature then overnight at 4 ° C . This pre-absorbed antiserum was then used as above . Three trials for each treatment in all experiments were analysed . The mycoparasite-host--antiserum mixtures were mixed well and left to stand for 1 h at room temperature . One drop was spread on dialysis membrane placed on semi-solid MY medium . The plates were incubated for 19-24 h at 22 ± 1 °C . The dialysis membrane was removed and placed on a microscope slide and the germinated spores were stained with cotton blue '1 °,, methylene blue in lactophenol) for light microscopic examination . At least 300 spores of the mycoparasite were counted in their relation to host hyphae : non-contact, contact, appressorium formation and relative percentage of appressoria were recorded . Germinated spores of P . virginiana observed to have no apparent physical contact with host hyphae were scored as non-contact events . Germ tubes of P . virginiana in contact along any part of the host hyphae were placed in the contact category . Germ tubes which showed contact and appressorium formation were considered as separate attachment events .
Effect of antiserum on linear growth of the mycoparasite The effect of the antifimbrial antiserum on linear growth of P . virginiana germ tubes was determined by incubating spore suspensions (10 - ` spores ml -') with (1) AU ; (2) pre-immune serum at concentrations of 0 . 1 tg protein pl - ' ; or (3) phosphate buffered saline (PBS) . The suspensions were incubated on sterile dialysis membrane on MY agar at room temperature for 20 h . The membranes were placed on a microscope slide for light microscopic examination . Germ tube lengths were measured with a calibrated ocular micrometer .
RESULTS
Fimbriae were observed on the surfaces of the hyphae of M . pusilla, P. articulosus and M . candelabrum (Figs 1-4) . They were distributed non-uniformly along the cell wall . For all three species, fimbriae were estimated to be up to 25 µm in length as determined from direct observations with the electron microscope and from electron micrographs . Diameters of the fimbriae did not vary significantly amongst the three species . M . candelabrum fimbriae had a mean diameter (+ standard error) of 9-1 ±0 . 4 nm, M. pusilla fimbriae were 9 . 4±0. 5 nm and P . articulosus fimbriae were 8 . 6±0 . 6 nm . These measurements were determined from electron micrographs of the negatively stained samples . With P . virginiana, fimbriae were not observed on germinating sporangiospores or on 19-72 h germ tubes grown in liquid or on semi-solid medium . Germ tubes of P . virginiana in this age range are capable of contacting and forming appressoria on host species on semi-solid medium .
Fungal host-mycoparasite interaction
1 43
Fits 1-2 . Shadow cast replica of the outer surface of the hyphae : Fig. 1, M . pusilla, Fig . 2, P. articulosus. Note the long fibrillar structures, fimbriae, (arrows) arising from the hyphal surface . 60 000 x . Fics 3-4 . Negatively stained preparations of hyphae : Fig . 3, M . pusilla, Fig. 4, M. candelabrum . Note distinct fimbriae (arrows) at the cell surface . 61875 x and 52 300 x , respectively .
Soluble proteins isolated from M. pusilla, P. articulosus, M . candelabrum and P . were separated by SDS-PAGE and were transferred to nitrocellulose membranes for immunoblot identification of fimbrial proteins . Polyclonal antiserum raised against fimbriae of U. violacea virginiana
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species . From the non-host, Al . candelabrum, two proteins with molecular masses of 60 and of 57 kDa were visualized . In contrast, the presumptive fimbrial proteins of both the susceptible and resistant hosts, .l. pusilla and P. articulosus, respectively, had masses of 64 kDa (Fig . 5) . The antiserum AU cross-reacted with two P . iirginiana
Mc
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Pa
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MP
PV
Mc
Us
106 80 49
32
(a )
5
(b)
6
FIG . 5 . Immunoblot analyses of fimbrial proteins obtained from hosts A1L pusilla (Mp) and P . articulosus (Pa) and non-host M. candelabrum (Mc) . (a) Immunoblot treated with the anti-fimbrial antiserum AU . Note cross-reaction of AU with M r 64 kDa proteins of both M . pusilla and P. articulosus, but with two proteins, M, 57 and 60 kDa, of a-1 . candelabrum . (b) Duplicate immunoblot reacted with pre-immune serum did not show any bands . The marker lane was that ofpre-stained low molecular range protein standards from Bio-Rad . FIG . 6 . Immunoblot analysis of proteins from P . rirginiana Pv ; . AU cross-reacted with two proteins, M 5 94 and 91 kDa, even though no fimbriac were observed at the surface of the mycoparasite . Right lane shows cross-reaction with fimbrial protein of Fittings riola(ea . Marker lane was that of pre-stained high molecular range protein standards from Bio-Rad . Ftos 7 8 . Negatively stained ,'1 ° uranyl acetate) preparations of electroeluted fimbrial proteins from Fig. 7, .14 . ptsilla and Fig . 8, 51 . candelabrum showing distinct fibrils . 57600 x .
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Fungal host-mycoparasite interaction
U O C O U C a) U a) a-
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Antiserum concentration (mg protein ml -1 ) Ftc . 9 . Inhibition of P . virginiana contact and appressorium formation at the surface of (a) AILL pusilla and (b) P . articulosus by various concentrations of anti-fimbrial protein antiserum . Each
point represents the mean of three independent tests . Top : Percentage contact (number of mycoparasite germ tubes in contact with a host hypha x 100°/total number of mycoparasite germ tubes examined) ; Middle : percentage appressorium formation (number of mycoparasite germ tubes with an appressorium x 100%/total number of mycoparasite germ tubes examined) ; Bottom : The relative percentage of appressorium formation (No . of appressoria x 100%/No . Of contacts) . Treatments are : AU ), preimmune serum (-E-) and antiserum previously treated with fimbrial protein (---0-) . Each data point followed by the letter 9 is significantly different (Kruskal-Wallis test) from the 0 concentration point and from the pre-immune serum point at the same protein concentration .
proteins with molecular masses of 94 and 91 kDa (Fig . 6) . These proteins were not likely to be fimbrial subunits since fimbriae had not been observed on P . virginiana . Their relationship to fimbrial protein, if any, remains unknown . Proof that AU cross-reacted with fimbrial subunits from host and non-host species was obtained by reconstitution of the immunoreactive proteins into fibrils in vitro . Proteins recognized by AU were electroeluted from the gel matrix and the SDS was removed . Fibrils were formed spontaneously by the electroeluted proteins of M . pusilla, M. candelabrum and P . articulosus . Figures 7 and 8 show the fibrils obtained from M . pusilla and M . candelabrum . The diameters of the reformed fibrils were approximately the same as those of the fimbriae attached to the hyphal surfaces . Fibril diameter means
146
N . A . Rghei et al. ±Standard error) of 9 . 7±0 . 3 run, 8-4 ±0 . 6 urn and 9 . 2±0 . 5 urn für A1 . candelabrum, .11 . pusilla, and P. articulosus, respectively, were observed . In our previous experiences, fibrils are not observed : (1) when the SDS is not displaced from the immuno-reactive proteins or (2) when other regions of these gels are treated as above . Thus, the immunoblot results coupled with the observations from the electroelution tests indicate that AU cross-reacted with fimbrial proteins from host and non-host species with high specificity . To determine if fimbriae are required for events in mycoparasitism, spores of the mycoparasite, P . virginiana, and the susceptible host, M. pusilla, or P . virginiana and the resistant host, P . articulosus, were incubated with AU in various concentrations . Incubation with AU resulted in a significant inhibition ofcontact between mycoparasite and host . Once contact was achieved, however, appressorium formation was apparently normal . Hence, the relative percentage of appressoria formed (No . of appressoria x 100° 0 /No . of contacts) did not change (Figs 9a and b) . The specificity of this inhibition was confirmed in control tests with pre-immune serum or AU pre-incubated with purified Ustilago violacea fimbrial protein . Neither treatment inhibited host---parasite contact significantly . The observed inhibition of contact could conceivably have been due to an inhibition of growth of germ tubes of the mycoparasite . Thus, the effects of AU, pre-immune serum or PBS on germ tube growth were determined . Spore germination in all three treatments was high : AU, 84"1 0 .. pre-immune serum, 74 11 0 ; PBS, 73 11,,( , . The mean ± standard error of 20 germ tubes treated with AU in two independent tests was 42 ± 6 . 4 µm . The length of germ tubes treated with pre-immune serum was 54 ± 9 . 5 Am and germ tubes treated with PBS had a mean length of 51±9 . 1 µm . Analysis of variance indicated that these values were not significantly different . DISCUSSION The present demonstration of fimbriae on mucoraceous species, M. pusilla, P . articulosus and M . candelabrum, adds to an increasing list of fungi from all major classes possessing non-flagellar cell surface filaments [5, 7, 12, 13, 25] . Morphological characteristics, as observed by electron microscopy, of fimbriae from other fungal species are similar to those reported here . Most fungi have fimbriae with diameters of 6-10 nm [10] . The length of fimbriae reported varies from as short as 1 Am in several ascomycetous yeast species to 20 Am in length in U . violacea [13] . The apparent lack of fimbriae on Piptocephalis virginiana is not surprising . In addition to this species, fimbriae were not detected by electron microscopy and immunological techniques on isolates of several basidiomycetous and ascomycetous species [13] . Whether these fungi produce fimbriac under different environmental conditions is unknown . At present it appears that the distribution of fimbriae on fungal species is very broad but not universal . Fimbriae of M . pusilla, M . candelabrum, and P . articulosus consist of monomeric subunits which cross-react with polyclonal antiserum produced against Ustilago violacea fimbrial protein . From these studies and similar investigations, it appears that the molecular mass of fimbrial protein is quite variable between species . The masses of fimbrial subunits range from 74 kDa in U. violacea [4, 5, 11 ] to 37 kDa in Goprinus
Fungal host-mycoparasite interaction
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cinereus [3] . These size differences may result in different functional roles for fimbriae in these species, although evidence for such a relationship is lacking . The observation that the fimbrial specific antiserum AU inhibited contact between P . virginiana and M . pusilla or P . articulosus suggests that host fimbriae serve as an initial point of interaction between mycoparasite and host . The subsequent parasitic event of appressorium formation did not appear to be inhibited . Parasitism by P . virginiana is initiated by directed growth, over short distances, of the parasite germ tube towards a hypha of a potential host . The phenomenon of directed growth does not apparently involve host/non-host differentiation since directed growth of P . virginiana towards non-host (M . candelabrum) hyphae also occurs [17] . Contact, the physical abuttment of cell walls, occurs with equal frequency in parasite-host or parasite-non-host interactions [20] and is a consequence of directed growth . Prior to this study it was proposed that directed growth is likely to be promoted by a physical or diffusible chemical stimulus produced by the potential host [17] . It is proposed that fimbriae serve as such a stimulus : the mycoparasite encounters fimbriae extending from the host hyphal wall and directed growth along these structures ensues . The observed inhibition of contact is due, therefore, to a disruption of directed growth . This hypothesis leads to the idea that the mycoparasite has a mechanism for reception of the fimbrial stimulus and recognition, therefore, of a potential host . Studies which directly examine the effect of AU on the phenomenon of directed growth should be undertaken to test this hypothesis . After contact, the mycoparasite adheres to the host but not to the non-host hyphae . The attachment of P . virginiana to host cell walls has been shown to be dependent upon the presence of two host-specific glycoproteins [19] . It should be noted that the attachment proteins are not fimbrial proteins : they have different masses (glycoproteins M r 100 and 85 kDa ; fimbrial protein Mr 64 kDa) and antiserum AU does not recognize either glycoprotein, M r 100 or 85 kDa, on immunoblots (data not shown) . Attachment is followed closely by appressorium formation and penetration . It is likely that fimbrial protein and the two attachment proteins are not involved in appressorium formation . Appressorium formation can be inhibited by heat treatment without disrupting attachment [21] and in this study appressoria developed in the presence of anti-fimbrial protein antiserum . Thus, it is likely that yet another host structure is involved at this level of interaction between host and mycoparasite . In summary, we propose that the interaction between host and parasite appears to occur at several levels : (1) initially through fimbriae, resulting in directed growth and contact ; (2) through attachment of the mycoparasite to the host cell surface mediated by two host cell wall glycoproteins [19] ; (3) during appressorium formation ; and (4) at the host plasmalemma as indicated by the resistant host response to the mycoparasite, formation of a papilla which inhibits penetrations [21] . This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada to A . C . and M . S . M . REFERENCES 1 . Balasubramanian R, Manocha MS. 1986 . Proteinase, chitinase, chitosanase activities in germinating spores of Piptocephalis virginiana . Mycologia 78 : 157-163 .
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2 . Bradford M . 1976 . A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding . Analytical Biochemistry 72 : 248-251 . 3 . Castle AJ, Boulianne RB . 1991- Fimbrial proteins of 1'stilago riolacca, Coprinus cinereus and Sdcizophyllum comrnune . 11S:A ,;Veu, sletter 42 : 8 (Abstr . ; . 4 . Castle AJ, Boulianne R, Xu J, Day AW . 1992 . Post-translational modification of fimbrial protein from Ustilago violacea. Canadian Journal oj' .116robiology (in press . 5 . Day AW, Gardiner RB . 1988 . Surface proteinaceous fibrils fimbriae) on filamentous fungi . Canadian Journal of Botany 66 : 2474-2484 . 6 . Day AW, Poon NH . 1975 . Fungal fimbriae II : Their role in conjugation in t,'stilago riolacea . Canadian Journal of .tilicrobiology 21 : 547-557 . 7 . Day AW, Gardiner RB, Smith R, Svircev AM, McKeen WE . 1986 . Detection of fungal fimbriae by protein A-gold immunocytochemical labelling in host plants infected with t'stilago heufleri or Peronospora hyosoami f.sp . tabacina . Canadian Journal of Microbiology 32 : 577-584 . 8 . Day AW, Poon NH, Stewart GG. 1975 . Fungal fimbriae III : The effect on flocculation in Saccharomyces . Canadian 7ownal of Microbiology 21 : 558- 564 . 9 . Douglas LJ, Houston JG, McCourtie J . 1981 . Adherence of Candida albicans to human buccal epithelial cells after growth on different carbon sources . FEBS Microbiology Letters 12 : 241-243 . 10 . Gardiner RB . 1985 . Fungal Fimbriae : Their Structure and Distribution . Ph .D . Thesis, The University of Western Ontario . 11 . Gardiner RB, Day AW . 1985 . Fungal fimbriae IV : Composition and properties of finibriae from U. violacea . Experimental Mycology 9 : 334--350 . 12 . Gardiner RB, Canton M, Day AW . 1981 . Fimbrial variation in smuts and heterobasidiomvcete fungi . Botanical Gazette 142 : 147 150, 13 . Gardiner RB, Podgorski C, Day AW . 1982 . Serological studies on the fimbriac of ycasts and yeastlike species . Botanical Gazette 143 : 534- 541 . 14 . Hanoaka F, Shaw JL, Mueller GC . 1979 . Recovery of functional proteins from sodium dodecyl sulfate-polyacrylamide gels . Analytical Biochemistry 99 : 170-174 . 15 . Laemmli UK . 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . N, ature 227 : 680-685 . 16 . Manocha MS . 1985 . Specificity of mvcoparasite attachment to the host cell surface . Canadian ,fournal of Botany 63 : 772-778 . 17 . Manocha MS . 1988 . Biotrophic mycoparasitism : a model system for investigations in hostparasite interactions. In : Agnihotri VP, Sarbhoy AK, Kumar D, eds . Perspectives in Mycology and Plant Pathology, New Delhi : Malhotra Publishing . 462 495 . 18 . Manocha MS . 1990 . Cell-cell interaction in fungi . Journal of Plant Diseases and Protection 97 : 655-669 . 19 . Manocha MS, Chen Y . 1991 . Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite . Canadian Journal of Microbiology 37 : 377-383 . 20 . Manocha MS, Balasubramanian R, Enskat S . 1986 . Attachment of a mycoparasite with host but not with nonhost Vortierella species . In : .NATO ASI Series Vol . 41 . Biology and Molecular Biology of Plant-Pathogen Interactions, by Bailey, J . Ed . Berlin : Springer-Verlag, 59-69 . 21 . Manocha MS, Chen Y, Rao N . 1990 . Involvement of cell surface sugars in recognition, attachment, and appressorium formation by a mycoparasite . Canadian Journal of Microbiology 36 : 771 778 . 22 . Penevsky HS, Tzagoloff A . 1971 . Extraction of water-soluble enzymes and proteins from membranes . I1-lethods in Enztrology 22 : 204 219 . 23 . Poon NH, Day AW . 1974 . Fimbriae in the fungus Ustilago violaces . .Nature 250 : 648-649 . 24 . Poon NH, Day AW . 1975 . Fungal fimbriae I : structure, origin and synthesis . Canadian Journal of Microbiology 21 : 537-546 . 25 . Svircev A, Gardiner RB, Day AW, Smith R . 1986 . Detection by protein a-gold immunocytochemical labelling of antigens to Botrytis cinerea in cytoplasm of infected Vicia faba. Phytopathology 76 : 622-626 . 26 . Towbin H, Staehelin T, Gordon J . 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets : Procedures and some applications . Proceedings of the .National Academy of Sciences of the US-1 76 : 4350-4354 .