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Microinjected antisense Inf24 oligonucleotides inhibit appressorium development in Uromyces
Mycol. Res. 102 (12) : 1513–1518 (1998)
1513
Printed in the United Kingdom
Microinjected antisense Inf24 oligonucleotides inhibit appressorium development in Uromyces
F R A N Ç O I S B A R JA1, A R Y C O R R E< A J R2, R. C. S T A P L ES3 A N D H. C. H O C H3 " Laboratoire de Microbiologie geT neT rale, Sciences III, 30 quai E-Ansemet, 1211 GeneZ ve 4, Switzerland # Departamento de Microbiologia, UFMG, Av. AntoV nio Carlos 6627, Belo Horizonte-M.G., C.P. 486, CEP-31270-901, Brazil $ Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456, U.S.A.
Germlings of Uromyces appendiculatus respond to topographical stimuli and develop appressoria. During this period several genes are expressed, one of which is Inf24. Germlings were microinjected with a sense and antisense fragment of the ORF of Inf24. Sense (Inf24-S), 5h-ATG AGT GCT TCA TCG CAG CAG CAG-3h fragment did not affect germling response to the topographical stimuli and the developed appressoria. Microinjection of antisense (Inf24-RC), 5h-CTG CTG CTG CGA TGA AGC ACT CAT-3h blocked gene transcription and appressoria were rarely formed. Injection of Inf24-RC into already formed appressoria did not inhibit continued development of subsequent infection structures, e.g. penetration peg, sub-stomatal vesicle.
Urediospore germlings of Uromyces appendiculatus (Pers. : Pers) Unger, like those of other rust fungi, invade their hosts after developing a series of specialized infection structures. Unique to most urediospore germlings is the fastidious requirement that these fungi must first penetrate through host stomates in order to infect. The thigmosensing mechanism that is utilized to locate and respond to the stomates is particularly unique to these germlings, namely, the germling apex senses topographical features inherent in the architecture of the stomatal complex (Wynn, 1976 ; Terhune, Bojko, & Hoch, 1993). More specifically, the germling senses abrupt elevation changes in topography in the order of 0n4–0n7 µm (Hoch et al., 1987), and as a result the germling ceases forward polarized growth and develops an appressorium, the first in a series of infection structures. Following appressorium formation other morphological structures associated with the infection process are developed, e.g. a penetration peg, a sub-stomatal vesicle, infection hyphae, and haustorium, through a pre-programmed sequence of gene regulated transcription events. Such thigmosensing responses have not been noted for other (nonrust) fungi. The process by which urediospore germlings sense and respond to topographical features has been the subject of considerable research. Also of interest is the process by which these cells proceed through the pre-programmed sequence of gene regulation. Previous studies identified a series of unique genes (Inf genes) that are, in general, expressed later in the course of infection structure development, viz., after mitosis and septum formation in the appressorium and before substomatal vesicle development is completed. One such gene, Inf24, is expressed between 3 and 4n5 h following initiation of urediospore germination, a time that corresponds to maturation of the appressorium and the elevated levels of mRNA
are maintained at least until maturation of the sub-stomatal vesicle (Bhairi et al., 1989). Little is known about this gene ; it does, however, have an open reading frame of 450 nucleotides and encodes a 16n4 kDa polypeptide (Bhairi et al., 1989). The first 35 amino acid residues at the N-terminus have some homology to a human helix destabilizing protein at its Cterminus. The gene product (expressed in E. coli) was located immunocytochemically within the ER, and on western blots corresponded to the 24 kDa differentiation-related protein which arises in mature appressoria, i.e. 40 min after initiation of appressorium formation. Gene expression persists through vesicle development (Huang & Staples, 1982). No additional information regarding putative function or identification of this gene has been made, in part because Uromyces, like most obligate fungal pathogens, are difficult cell systems to transform with current methodologies, and thus it is difficult to perform DNA insertion or ‘ knock-out ’ experiments. One approach that may lend some insight into the function of these genes in obligate systems is to microinject sense and antisense oligonucleotides into germ tubes. Thus, we report here the results of a study in which Inf24-antisense was microinjected into Uromyces germlings that were subsequently allowed to grow onto topographical features inductive for appressorium initiation. MATERIALS AND METHODS Urediospores of U. appendiculatus, race 0, were harvested from infected, greenhouse-grown bean leaves (Phaseolus vulgaris L. cv. Pinto) and stored at 4 mC until used for experimentation. Prior to use, the urediospores were exposed to vapours of βionone (4-[2,6,6-trimethyl-1-cyclohexen-1-yl]-3-buten-2-one) (Sigma) for at least 20 min as described by French et al. (1977)
INF24 in Uromyces to overcome the activity of natural germination self-inhibitors. The β-ionone treated urediospores were dispersed onto specially prepared 0n15 mm thick polystyrene supports bearing 0n5 µm high topographical ridges spaced 30 µm apart arranged in a grid pattern, prepared by heat-pressing pieces of polystyrene against a silicon template bearing negative images of the topographical pattern prepared by microlithography (Hoch et al., 1987 ; Allen et al., 1991 ; Kwon & Hoch, 1991). The urediospore-laden polystyrene supports were attached to the bottom of a stainless steel holder with high vacuum silicone grease (971 V, Dow Corning) to form an observation chamber with an open top (Corre# a & Hoch, 1993). The urediospores were misted lightly with distilled deionised water (ddH O) and left in a humidified chamber for # 15 min, after which time they were briefly air dried. This procedure helped to adhere the urediospores to the substratum (Terhune & Hoch, 1993). Next a U-shaped piece of Plexiglas (3n5 mm thick) was positioned on the top of the holder straddling the opening, followed by a coverglass on top of the Plexiglas. All were held in place with a light application of silicone grease. The chamber was filled with ddH O and the # entire apparatus was placed in a 17m incubator for 1–2 h until the germlings were at an appropriate stage of development for experimentation. Subsequently, the chambers were positioned on a modified IMT-2 Olympus inverted microscope stage through which coolant was recirculated to achieve a 17m surface (Corre# a & Hoch, 1993). Microinjection of the urediospore germlings was similar to that previously reported by Corre# a & Hoch (1993), except that micropipettes were prepared from Drummond Pyrex tubing 1n0 mm OD and 0n8 mm ID (Drummond Scientific Company, Broomal, PA). The micropipettes were pulled with a Flaming\Brown puller (model P-87) (Sutter Instrument Company, Novato, CA) to yield tapered tips 0n4–0n5 µm OD. The micropipettes were generally ‘ front filled ’ with the solutions to be injected, ddH O or oligonucleotides diluted in # ddH O. The oligonucleotides were injected into germlings at # either 5 or 20 µg µl−" at various times prior to and after germling recognition of the inductive ‘ signal ’ (ridge) for appressorium formation. That material was injected was noted as an abrupt momentary movement of the cytoplasm distally. The exact volume injected was not determined, but based on previous studies by us, it was generally between 30 and 100 fl. In all instances where there appeared to be an inhibition of appressorium formation following injection, the germlings were allowed to grow for an additional 3–6 h or to encounter a minimum of six additional inductive ridges. In some instances, a second injection was performed on the same germling. Also, in such instances, non-injected control germlings in the immediate surrounding area were assessed for response to the same or similar ridges. The injection process and subsequent response of the germlings were recorded on video tape and\or as time-lapsed images captured using MetaMorph Imaging software (Universal Imaging, West Chester, PA) in which the light source was shuttered to 1 s exposure times during image capture (Corre# a & Hoch, 1993) and stored on optical disc media (Corre# a & Hoch, 1995). Composite images were prepared using Photoshop software (Adobe Systems Incorporated). Synthetic oligonucleotides
1514 were prepared at either Cornell University’s Centre for Advanced Technology or DNA International (Lake Oswego, OR), and were : sense (Inf24-S), 5h-ATG AGT GCT TCA TCG CAG CAG CAG-3h ; and antisense (Inf24-RC), 5h-CTG CTG CTG CGA TGA AGC ACT CAT-3h. The oligonucleotides were derived from nt 388 to 411 (GenBank M29256). RESULTS Control non-injected urediospore germlings grown on polystyrene substrata bearing ridges 0n5 µm high and 2n0 µm wide initiated appressorium formation within minutes following contact with the inductive topographical feature as has been reported previously (Hoch et al., 1987 ; Allen et al., 1991). Furthermore, the efficiency of appressorium induction by the ridges was high ( 90 %), and they were generally induced by at least the second ridge encountered (Fig. 1). Appressoria were mature, namely, having completed septum formation and in post-mitotic stages, 60–80 min following initial contact of the germling apex with the ridge. Following microinjection with either 5 or 20 µg µl−" of Inf24-S oligonucleotide, germlings developed normal appressoria upon contact with the first or second ridge, and within the time-frame noted for the control germlings (Figs 2 a–c). The time interval between Figs 2 a and 2 c is 65 min. Overall, 11 of 12 germlings microinjected with Inf24-S developed appressoria. Microinjection of solutions into the germlings not already in contact with a ridge was generally difficult since the ridges were 30 µm apart, a spacing that allows a sufficient number of encounters during a given time period before the germling depletes its endogenous nutrient reserve. In addition, germlings could not be easily injected when the germ tubes were much less than 30 µm long since they were not well adhered to the substratum, a prerequisite for optimal insertion of the micropipette. Microinjection of ddH O yielded similar results, e.g. appressorium formation # over the next ridge or two. Microinjection of the antisense oligonucleotide, Inf24-RC, exhibited profound effects on the germling’s ability to complete appressorium development. Frequently, germlings that were injected with 20 µg µl−" Inf24-RC after contact with a ridge, but before appressorium formation, continued to swell and develop a normally-shaped outline of an appressorium ; within 30–40 min after initial contact, however, a germ tube usually emerged from the ‘ appressorium ’ and continued growth as a typically shaped germ tube, namely, with a broadrounded apex (Figs 3, 4) as opposed to a narrower and tapered profile characteristic of a penetration peg and substomatal vesicle that develops much later (Staples et al., 1985). Such ‘ appressorial ’ outgrowth germ tubes displayed two nuclei when stained with DAPI, indicative that mitosis had not occurred (Kwon & Hoch, 1991) and that they were not penetration pegs which would have had four nuclei. Often, the renewed germ tube swelled slightly upon subsequent encounters with ridges (Figs 4 d–f). Inf24-RC injected at rates of 20 µg µl−" almost always resulted in no appressoria. Inf24RC (20 µg µl−") injected germlings failed to develop appressoria in 16 of 19 injections after contacting the next six ridges. As a check for the inducability of the ridge, germlings
François Barja and others
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Fig. 1. Urediospore germlings of Uromyces appendiculatus grown on a polystyrene substratum bearing 0n5 µm high ridges 30 µm apart in a grid pattern. All germlings shown developed an appressorium upon contact with either the first or second ridge. Bar, 30 µm. Fig. 2. Microinjection of sense Inf24 oligonucleotide (20 µg µl−") into a germling of Uromyces appendiculatus. Upon contact with the next 0n5 µm high ridge the germling initiated and developed an appressorium. Time interval between Fig. 2 a and 2 c is 65 min. The site of injection is indicated by the arrowheads. Fig. 2 c shows a cytoplasmic wound response, which is common upon insertion of the micropipette. Bar, 30 µm. Fig. 3. A germling of Uromyces appendiculatus that had been microinjected with 20 µg µl−" of antisense Inf24 oligonucleotide. At the time of injection the germling apex was in contact with the ridge and an ‘ appressorium ’ (‘ Ap ’) developed ; however, neither mitosis nor septum formation followed and a ‘ new ’ germ tube emerged from the aborted appressorium. Total time after injection was 80 min. Injection site is indicated by arrowhead. Sp, urediospore. Bar, 30 µm.
INF24 in Uromyces
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4
Fig. 4. Microinjection of antisense Inf24 oligonucleotide into Uromyces appendiculatus germling. (a) Injection (arrowhead) when germling was just contacting an inductive ridge (ridge encounter site not shown). (b) The cell subsequently initiated and formed a nearly complete appressorium. (c) As appressorium development ceased, the cell initiated a new apical domain and continued normal growth (arrowhead l injection site with cytoplasmic leakage). (d–f) Same cell as in (a–c) showing three additional encounters with inductive topographies, and subsequent outgrowths. Each encounter with a ridge normally would have induced a mature appressorium and substomatal vesicle. Bar, 30 µm. Fig. 5. Photographic montage of a Uromyces appendiculatus germling that had been microinjected with antisense Inf24 oligonucleotide prior to contact with the inductive ridge. Seven of nine non-injected germlings in the immediate area developed appressoria (Ap) (two are shown). Arrowhead, injection site. Bar, 30 µm.
François Barja and others in the immediate vicinity were assessed for appressorium development. For example, the injected germling depicted in Fig. 5 that did not develop an appressorium was closely surrounded by nine germlings, seven which developed appressoria upon contact with the first ridge, and two upon contact with the second ridge. Germlings injected at 5 µg µl−" sometimes developed appressoria after 2–3 h additional growth, e.g. upon encountering the sixth or later ridge. Also, injection of Inf24-RC into maturing appressoria (n l 6) (not shown), e.g. those appressoria in which mitosis had likely commenced (later than 20 min following ridge contact), no germ tube outgrowths occurred and normal penetration peg and substomatal vesicles formed. DISCUSSION Contact between the urediospore germling and a chemically inert substratum bearing 0n5 µm high topographies triggers appressorium formation. From northern blots it was determined that Inf24 was first expressed between 3 and 4n5 h after initiation of urediospore germination, a time when appressoria had begun to mature, albeit the precise morphological stage of appressorium development was difficult to ascertain because RNA was derived from a population of germlings. Nevertheless, the gene was not expressed in nondifferentiated germlings. The function of genes in obligate fungi such as U. appendiculatus is difficult to assess, since a transformation protocol useful for gene analysis does not yet exist for these fungi even though protocols for expression of heterologous genes have been demonstrated (Bhairi & Staples, 1992 ; Li et al., 1993). To gain some insight into gene function, we approached the problem of gene analysis by introducing an antisense fragment of the ORF of Inf24 by microinjection in order to block gene transcription. We expected, since gene transcription is strongly upshifted as the appressorium matures and the penetration peg emerges, that microinjected germlings would develop the immature appressoria that they have. Clearly, microinjection of the sense Inf24-S had no effect on germling growth or response to inductive signals for appressorium development. Antisense Inf24-RC, however, generally inhibited development of appressoria as well as development of subsequent infection structures when it was introduced into the cell prior to signal reception or early in appressorium development. Injection into ‘ mature ’ appressoria did not prevent the next morphological stage of development, namely, penetration peg and sub-stomatal vesicle. While we do not know the exact time during appressorium development when Inf24-RC no longer is effective, it appears to correspond to a time period of 20–30 min after signal reception. From previous studies this relates to a time when the two nuclei have begun to enter the swelling germling apex and start mitotic prophase (Kwon & Hoch, 1991). This is also a time that corresponds closely to the 20 minute period during which the inductive signal must remain present at the germling apex for appressorium formation to continue to completion (Corre# a & Hoch, 1995). Furthermore, it is close to the time when the immunologically associated 40 kDa differentiation related protein appears in appressoria (Huang & Staples, 1982). Thus,
1517 the findings that Inf24-RC is effective prior to ca 30 min after induction of appressoria, and the start of mitosis (Kwon & Hoch, 1991) and signal perturbation (Corre# a & Hoch, 1995) is further evidence that beyond this time period appressorium development is at a point of no return and is irreversible. Always a concern when introducing ‘ foreign ’ materials into cells is the possibility of a non-specific effect on the cell that may perturb cell function such that normal developmental processes are interrupted. There are several criteria that one would like to use to indicate that the introduced material is specific in action. Optimally, one would prefer to monitor gene product (protein) production and mRNA levels ; however, this is difficult on single cells, if not impossible. For Uromyces germlings to sense surface topography they must be in intimate contact with the substratum, and we have been attentive to the status of the microinjected germlings. Germlings in contact with the substratum grow with slight angular profiles and with an uneven diameter of the germ tube, especially where it has grown over a ridge, namely, it is often slightly wider at that point (Fig. 5). Germlings in poor contact with the substratum have slightly thinned diameter germ tubes and their profile is smooth and gently curving. All injected germlings exhibited profiles indicative of intimate contact with the substratum, thus we feel that the cells were in thigmoreceptive positions. This is the first report in which antisense gene constructs have been microinjected into a fungal cell and shown to block an inducable morphological change. Injection of antisense oligonucleotides has been a proven approach for studying mammalian cell function (Murry & Crockett, 1992), in part, because the cells are considerably easier to work with, namely they have near zero turgor pressure and they are considerably larger. While the time course for the expression of Inf24 has been determined based on analysis of northern blots (Bhairi et al., 1989), it has not been possible to extract sufficient RNA from single microinjected germlings to allow an estimation of transcription of Inf24 after injection of the antisense fragment. Thus we have little knowledge of the mechanism of action of the antisense gene. It is encouraging, however, that the development of microinjected germlings was blocked prior to the time when gene transcription would be expected to have occurred. It was not expected that germlings injected with Inf24-RC would partially form appressoria and then revert to germ tube growth as we have noted in this study although this is not necessarily surprising since initial signal reception and morphological changes are likely influenced by protein signalling, e.g. phosphorylation. We acknowledge the help and advice of Brian Shaw during the course of this study as well as financial support from NRIUSDA (HCH\RCS) and Ciba-Geigy-Jubila$ ums-Stiftung, Socie! te! Acade! mique de Gene' ve and Fonds Topalini (FB).
REFERENCES Allen, E. A., Hoch, H. C., Stavely, R. J. & Steadman, J. R. (1991). Uniformity among races of Uromyces appendiculatus in response to topographic signaling for appressorium formation. Phytopathology 81, 883–887.
INF24 in Uromyces Bhairi, S. M. & Staples, R. C. (1992). Transient expression of the βglucuronidase gene introduced into Uromyces appendiculatus uredospores by particle bombardment. Phytopathology 82, 986–989. Bhairi, S. M., Staples, R. C., Freve, P. & Yoder, O. C. (1989). Characterization of an infection structure-specific gene from the rust fungus, Uromyces appendiculatus. Gene 81, 237–243. Corre# a Jr, A. & Hoch, H. C. (1993). Microinjection of urediospore germlings of Uromyces appendiculatus. Experimental Mycology 17, 241–252. Corre# a Jr, A. & Hoch, H. C. (1995). Identification of thigmoresponsive loci for cell differentiation in Uromyces germlings. Protoplasma 186, 34–40. French, R. C., Graham, C. L., Gale, A. W. & Long, R. K. (1977). Structural and exposure time requirements for chemical stimulation of germination of uredospores of Uromyces phaseoli. Journal of Agricultural Food Chemistry 25, 84–88. Hoch, H. C., Staples, R. C., Whitehead, B., Comeau, J. & Wolf, E. D. (1987). Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235, 1659–1662. Huang, B.-F. & Staples, R. C. (1982). Synthesis of proteins during differentiation of the bean rust fungus. Experimental Mycology 6, 7–14. Kwon, Y. & Hoch, H. C. (1991). Temporal and spatial dynamics of (Accepted 3 March 1998)
1518 appressorium development in Uromyces appendiculatus. Experimental Mycology 15, 116–131. Li, A., Altosaar, I., Heath, M. C. & Horgen, P. A. (1993). Transient expression of the β-Glucuronidase gene delivered into urediniospores of Uromyces appendiculatus by particle bombardment. Canadian Journal of Plant Pathology 15, 1–6. Murry, J. A. H. & Crockett, N. (1992). Antisense technology : and overview. In Antisense RNA and DNA (ed. J. A. H. Murry), pp. 1–49. Wiley-Liss, Inc. : U.S.A. Staples, R. C., Hoch, H. C., Epstein, L., Laccetti, L. & Hassouna, S. (1985). Recognition of host morphology by rust fungi ; responses and mechanisms. Canadian Journal of Plant Pathology 7, 314–322. Terhune, B. T. & Hoch, H. C. (1993). Substrate hydrophobicity and adhesion of Uromyces urediospores and germlings. Experimental Mycology 17, 253–273. Terhune, B. T., Bojko, R. J. & Hoch, H. C. (1993). Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology 17, 70–78. Wynn, W. K. (1976). Appressorium formation over stomates by the bean rust fungus : Response to a surface contact stimulus. Phytopathology 66, 136–146.
1513
Printed in the United Kingdom
Microinjected antisense Inf24 oligonucleotides inhibit appressorium development in Uromyces
F R A N Ç O I S B A R JA1, A R Y C O R R E< A J R2, R. C. S T A P L ES3 A N D H. C. H O C H3 " Laboratoire de Microbiologie geT neT rale, Sciences III, 30 quai E-Ansemet, 1211 GeneZ ve 4, Switzerland # Departamento de Microbiologia, UFMG, Av. AntoV nio Carlos 6627, Belo Horizonte-M.G., C.P. 486, CEP-31270-901, Brazil $ Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456, U.S.A.
Germlings of Uromyces appendiculatus respond to topographical stimuli and develop appressoria. During this period several genes are expressed, one of which is Inf24. Germlings were microinjected with a sense and antisense fragment of the ORF of Inf24. Sense (Inf24-S), 5h-ATG AGT GCT TCA TCG CAG CAG CAG-3h fragment did not affect germling response to the topographical stimuli and the developed appressoria. Microinjection of antisense (Inf24-RC), 5h-CTG CTG CTG CGA TGA AGC ACT CAT-3h blocked gene transcription and appressoria were rarely formed. Injection of Inf24-RC into already formed appressoria did not inhibit continued development of subsequent infection structures, e.g. penetration peg, sub-stomatal vesicle.
Urediospore germlings of Uromyces appendiculatus (Pers. : Pers) Unger, like those of other rust fungi, invade their hosts after developing a series of specialized infection structures. Unique to most urediospore germlings is the fastidious requirement that these fungi must first penetrate through host stomates in order to infect. The thigmosensing mechanism that is utilized to locate and respond to the stomates is particularly unique to these germlings, namely, the germling apex senses topographical features inherent in the architecture of the stomatal complex (Wynn, 1976 ; Terhune, Bojko, & Hoch, 1993). More specifically, the germling senses abrupt elevation changes in topography in the order of 0n4–0n7 µm (Hoch et al., 1987), and as a result the germling ceases forward polarized growth and develops an appressorium, the first in a series of infection structures. Following appressorium formation other morphological structures associated with the infection process are developed, e.g. a penetration peg, a sub-stomatal vesicle, infection hyphae, and haustorium, through a pre-programmed sequence of gene regulated transcription events. Such thigmosensing responses have not been noted for other (nonrust) fungi. The process by which urediospore germlings sense and respond to topographical features has been the subject of considerable research. Also of interest is the process by which these cells proceed through the pre-programmed sequence of gene regulation. Previous studies identified a series of unique genes (Inf genes) that are, in general, expressed later in the course of infection structure development, viz., after mitosis and septum formation in the appressorium and before substomatal vesicle development is completed. One such gene, Inf24, is expressed between 3 and 4n5 h following initiation of urediospore germination, a time that corresponds to maturation of the appressorium and the elevated levels of mRNA
are maintained at least until maturation of the sub-stomatal vesicle (Bhairi et al., 1989). Little is known about this gene ; it does, however, have an open reading frame of 450 nucleotides and encodes a 16n4 kDa polypeptide (Bhairi et al., 1989). The first 35 amino acid residues at the N-terminus have some homology to a human helix destabilizing protein at its Cterminus. The gene product (expressed in E. coli) was located immunocytochemically within the ER, and on western blots corresponded to the 24 kDa differentiation-related protein which arises in mature appressoria, i.e. 40 min after initiation of appressorium formation. Gene expression persists through vesicle development (Huang & Staples, 1982). No additional information regarding putative function or identification of this gene has been made, in part because Uromyces, like most obligate fungal pathogens, are difficult cell systems to transform with current methodologies, and thus it is difficult to perform DNA insertion or ‘ knock-out ’ experiments. One approach that may lend some insight into the function of these genes in obligate systems is to microinject sense and antisense oligonucleotides into germ tubes. Thus, we report here the results of a study in which Inf24-antisense was microinjected into Uromyces germlings that were subsequently allowed to grow onto topographical features inductive for appressorium initiation. MATERIALS AND METHODS Urediospores of U. appendiculatus, race 0, were harvested from infected, greenhouse-grown bean leaves (Phaseolus vulgaris L. cv. Pinto) and stored at 4 mC until used for experimentation. Prior to use, the urediospores were exposed to vapours of βionone (4-[2,6,6-trimethyl-1-cyclohexen-1-yl]-3-buten-2-one) (Sigma) for at least 20 min as described by French et al. (1977)
INF24 in Uromyces to overcome the activity of natural germination self-inhibitors. The β-ionone treated urediospores were dispersed onto specially prepared 0n15 mm thick polystyrene supports bearing 0n5 µm high topographical ridges spaced 30 µm apart arranged in a grid pattern, prepared by heat-pressing pieces of polystyrene against a silicon template bearing negative images of the topographical pattern prepared by microlithography (Hoch et al., 1987 ; Allen et al., 1991 ; Kwon & Hoch, 1991). The urediospore-laden polystyrene supports were attached to the bottom of a stainless steel holder with high vacuum silicone grease (971 V, Dow Corning) to form an observation chamber with an open top (Corre# a & Hoch, 1993). The urediospores were misted lightly with distilled deionised water (ddH O) and left in a humidified chamber for # 15 min, after which time they were briefly air dried. This procedure helped to adhere the urediospores to the substratum (Terhune & Hoch, 1993). Next a U-shaped piece of Plexiglas (3n5 mm thick) was positioned on the top of the holder straddling the opening, followed by a coverglass on top of the Plexiglas. All were held in place with a light application of silicone grease. The chamber was filled with ddH O and the # entire apparatus was placed in a 17m incubator for 1–2 h until the germlings were at an appropriate stage of development for experimentation. Subsequently, the chambers were positioned on a modified IMT-2 Olympus inverted microscope stage through which coolant was recirculated to achieve a 17m surface (Corre# a & Hoch, 1993). Microinjection of the urediospore germlings was similar to that previously reported by Corre# a & Hoch (1993), except that micropipettes were prepared from Drummond Pyrex tubing 1n0 mm OD and 0n8 mm ID (Drummond Scientific Company, Broomal, PA). The micropipettes were pulled with a Flaming\Brown puller (model P-87) (Sutter Instrument Company, Novato, CA) to yield tapered tips 0n4–0n5 µm OD. The micropipettes were generally ‘ front filled ’ with the solutions to be injected, ddH O or oligonucleotides diluted in # ddH O. The oligonucleotides were injected into germlings at # either 5 or 20 µg µl−" at various times prior to and after germling recognition of the inductive ‘ signal ’ (ridge) for appressorium formation. That material was injected was noted as an abrupt momentary movement of the cytoplasm distally. The exact volume injected was not determined, but based on previous studies by us, it was generally between 30 and 100 fl. In all instances where there appeared to be an inhibition of appressorium formation following injection, the germlings were allowed to grow for an additional 3–6 h or to encounter a minimum of six additional inductive ridges. In some instances, a second injection was performed on the same germling. Also, in such instances, non-injected control germlings in the immediate surrounding area were assessed for response to the same or similar ridges. The injection process and subsequent response of the germlings were recorded on video tape and\or as time-lapsed images captured using MetaMorph Imaging software (Universal Imaging, West Chester, PA) in which the light source was shuttered to 1 s exposure times during image capture (Corre# a & Hoch, 1993) and stored on optical disc media (Corre# a & Hoch, 1995). Composite images were prepared using Photoshop software (Adobe Systems Incorporated). Synthetic oligonucleotides
1514 were prepared at either Cornell University’s Centre for Advanced Technology or DNA International (Lake Oswego, OR), and were : sense (Inf24-S), 5h-ATG AGT GCT TCA TCG CAG CAG CAG-3h ; and antisense (Inf24-RC), 5h-CTG CTG CTG CGA TGA AGC ACT CAT-3h. The oligonucleotides were derived from nt 388 to 411 (GenBank M29256). RESULTS Control non-injected urediospore germlings grown on polystyrene substrata bearing ridges 0n5 µm high and 2n0 µm wide initiated appressorium formation within minutes following contact with the inductive topographical feature as has been reported previously (Hoch et al., 1987 ; Allen et al., 1991). Furthermore, the efficiency of appressorium induction by the ridges was high ( 90 %), and they were generally induced by at least the second ridge encountered (Fig. 1). Appressoria were mature, namely, having completed septum formation and in post-mitotic stages, 60–80 min following initial contact of the germling apex with the ridge. Following microinjection with either 5 or 20 µg µl−" of Inf24-S oligonucleotide, germlings developed normal appressoria upon contact with the first or second ridge, and within the time-frame noted for the control germlings (Figs 2 a–c). The time interval between Figs 2 a and 2 c is 65 min. Overall, 11 of 12 germlings microinjected with Inf24-S developed appressoria. Microinjection of solutions into the germlings not already in contact with a ridge was generally difficult since the ridges were 30 µm apart, a spacing that allows a sufficient number of encounters during a given time period before the germling depletes its endogenous nutrient reserve. In addition, germlings could not be easily injected when the germ tubes were much less than 30 µm long since they were not well adhered to the substratum, a prerequisite for optimal insertion of the micropipette. Microinjection of ddH O yielded similar results, e.g. appressorium formation # over the next ridge or two. Microinjection of the antisense oligonucleotide, Inf24-RC, exhibited profound effects on the germling’s ability to complete appressorium development. Frequently, germlings that were injected with 20 µg µl−" Inf24-RC after contact with a ridge, but before appressorium formation, continued to swell and develop a normally-shaped outline of an appressorium ; within 30–40 min after initial contact, however, a germ tube usually emerged from the ‘ appressorium ’ and continued growth as a typically shaped germ tube, namely, with a broadrounded apex (Figs 3, 4) as opposed to a narrower and tapered profile characteristic of a penetration peg and substomatal vesicle that develops much later (Staples et al., 1985). Such ‘ appressorial ’ outgrowth germ tubes displayed two nuclei when stained with DAPI, indicative that mitosis had not occurred (Kwon & Hoch, 1991) and that they were not penetration pegs which would have had four nuclei. Often, the renewed germ tube swelled slightly upon subsequent encounters with ridges (Figs 4 d–f). Inf24-RC injected at rates of 20 µg µl−" almost always resulted in no appressoria. Inf24RC (20 µg µl−") injected germlings failed to develop appressoria in 16 of 19 injections after contacting the next six ridges. As a check for the inducability of the ridge, germlings
François Barja and others
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Fig. 1. Urediospore germlings of Uromyces appendiculatus grown on a polystyrene substratum bearing 0n5 µm high ridges 30 µm apart in a grid pattern. All germlings shown developed an appressorium upon contact with either the first or second ridge. Bar, 30 µm. Fig. 2. Microinjection of sense Inf24 oligonucleotide (20 µg µl−") into a germling of Uromyces appendiculatus. Upon contact with the next 0n5 µm high ridge the germling initiated and developed an appressorium. Time interval between Fig. 2 a and 2 c is 65 min. The site of injection is indicated by the arrowheads. Fig. 2 c shows a cytoplasmic wound response, which is common upon insertion of the micropipette. Bar, 30 µm. Fig. 3. A germling of Uromyces appendiculatus that had been microinjected with 20 µg µl−" of antisense Inf24 oligonucleotide. At the time of injection the germling apex was in contact with the ridge and an ‘ appressorium ’ (‘ Ap ’) developed ; however, neither mitosis nor septum formation followed and a ‘ new ’ germ tube emerged from the aborted appressorium. Total time after injection was 80 min. Injection site is indicated by arrowhead. Sp, urediospore. Bar, 30 µm.
INF24 in Uromyces
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Fig. 4. Microinjection of antisense Inf24 oligonucleotide into Uromyces appendiculatus germling. (a) Injection (arrowhead) when germling was just contacting an inductive ridge (ridge encounter site not shown). (b) The cell subsequently initiated and formed a nearly complete appressorium. (c) As appressorium development ceased, the cell initiated a new apical domain and continued normal growth (arrowhead l injection site with cytoplasmic leakage). (d–f) Same cell as in (a–c) showing three additional encounters with inductive topographies, and subsequent outgrowths. Each encounter with a ridge normally would have induced a mature appressorium and substomatal vesicle. Bar, 30 µm. Fig. 5. Photographic montage of a Uromyces appendiculatus germling that had been microinjected with antisense Inf24 oligonucleotide prior to contact with the inductive ridge. Seven of nine non-injected germlings in the immediate area developed appressoria (Ap) (two are shown). Arrowhead, injection site. Bar, 30 µm.
François Barja and others in the immediate vicinity were assessed for appressorium development. For example, the injected germling depicted in Fig. 5 that did not develop an appressorium was closely surrounded by nine germlings, seven which developed appressoria upon contact with the first ridge, and two upon contact with the second ridge. Germlings injected at 5 µg µl−" sometimes developed appressoria after 2–3 h additional growth, e.g. upon encountering the sixth or later ridge. Also, injection of Inf24-RC into maturing appressoria (n l 6) (not shown), e.g. those appressoria in which mitosis had likely commenced (later than 20 min following ridge contact), no germ tube outgrowths occurred and normal penetration peg and substomatal vesicles formed. DISCUSSION Contact between the urediospore germling and a chemically inert substratum bearing 0n5 µm high topographies triggers appressorium formation. From northern blots it was determined that Inf24 was first expressed between 3 and 4n5 h after initiation of urediospore germination, a time when appressoria had begun to mature, albeit the precise morphological stage of appressorium development was difficult to ascertain because RNA was derived from a population of germlings. Nevertheless, the gene was not expressed in nondifferentiated germlings. The function of genes in obligate fungi such as U. appendiculatus is difficult to assess, since a transformation protocol useful for gene analysis does not yet exist for these fungi even though protocols for expression of heterologous genes have been demonstrated (Bhairi & Staples, 1992 ; Li et al., 1993). To gain some insight into gene function, we approached the problem of gene analysis by introducing an antisense fragment of the ORF of Inf24 by microinjection in order to block gene transcription. We expected, since gene transcription is strongly upshifted as the appressorium matures and the penetration peg emerges, that microinjected germlings would develop the immature appressoria that they have. Clearly, microinjection of the sense Inf24-S had no effect on germling growth or response to inductive signals for appressorium development. Antisense Inf24-RC, however, generally inhibited development of appressoria as well as development of subsequent infection structures when it was introduced into the cell prior to signal reception or early in appressorium development. Injection into ‘ mature ’ appressoria did not prevent the next morphological stage of development, namely, penetration peg and sub-stomatal vesicle. While we do not know the exact time during appressorium development when Inf24-RC no longer is effective, it appears to correspond to a time period of 20–30 min after signal reception. From previous studies this relates to a time when the two nuclei have begun to enter the swelling germling apex and start mitotic prophase (Kwon & Hoch, 1991). This is also a time that corresponds closely to the 20 minute period during which the inductive signal must remain present at the germling apex for appressorium formation to continue to completion (Corre# a & Hoch, 1995). Furthermore, it is close to the time when the immunologically associated 40 kDa differentiation related protein appears in appressoria (Huang & Staples, 1982). Thus,
1517 the findings that Inf24-RC is effective prior to ca 30 min after induction of appressoria, and the start of mitosis (Kwon & Hoch, 1991) and signal perturbation (Corre# a & Hoch, 1995) is further evidence that beyond this time period appressorium development is at a point of no return and is irreversible. Always a concern when introducing ‘ foreign ’ materials into cells is the possibility of a non-specific effect on the cell that may perturb cell function such that normal developmental processes are interrupted. There are several criteria that one would like to use to indicate that the introduced material is specific in action. Optimally, one would prefer to monitor gene product (protein) production and mRNA levels ; however, this is difficult on single cells, if not impossible. For Uromyces germlings to sense surface topography they must be in intimate contact with the substratum, and we have been attentive to the status of the microinjected germlings. Germlings in contact with the substratum grow with slight angular profiles and with an uneven diameter of the germ tube, especially where it has grown over a ridge, namely, it is often slightly wider at that point (Fig. 5). Germlings in poor contact with the substratum have slightly thinned diameter germ tubes and their profile is smooth and gently curving. All injected germlings exhibited profiles indicative of intimate contact with the substratum, thus we feel that the cells were in thigmoreceptive positions. This is the first report in which antisense gene constructs have been microinjected into a fungal cell and shown to block an inducable morphological change. Injection of antisense oligonucleotides has been a proven approach for studying mammalian cell function (Murry & Crockett, 1992), in part, because the cells are considerably easier to work with, namely they have near zero turgor pressure and they are considerably larger. While the time course for the expression of Inf24 has been determined based on analysis of northern blots (Bhairi et al., 1989), it has not been possible to extract sufficient RNA from single microinjected germlings to allow an estimation of transcription of Inf24 after injection of the antisense fragment. Thus we have little knowledge of the mechanism of action of the antisense gene. It is encouraging, however, that the development of microinjected germlings was blocked prior to the time when gene transcription would be expected to have occurred. It was not expected that germlings injected with Inf24-RC would partially form appressoria and then revert to germ tube growth as we have noted in this study although this is not necessarily surprising since initial signal reception and morphological changes are likely influenced by protein signalling, e.g. phosphorylation. We acknowledge the help and advice of Brian Shaw during the course of this study as well as financial support from NRIUSDA (HCH\RCS) and Ciba-Geigy-Jubila$ ums-Stiftung, Socie! te! Acade! mique de Gene' ve and Fonds Topalini (FB).
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INF24 in Uromyces Bhairi, S. M. & Staples, R. C. (1992). Transient expression of the βglucuronidase gene introduced into Uromyces appendiculatus uredospores by particle bombardment. Phytopathology 82, 986–989. Bhairi, S. M., Staples, R. C., Freve, P. & Yoder, O. C. (1989). Characterization of an infection structure-specific gene from the rust fungus, Uromyces appendiculatus. Gene 81, 237–243. Corre# a Jr, A. & Hoch, H. C. (1993). Microinjection of urediospore germlings of Uromyces appendiculatus. Experimental Mycology 17, 241–252. Corre# a Jr, A. & Hoch, H. C. (1995). Identification of thigmoresponsive loci for cell differentiation in Uromyces germlings. Protoplasma 186, 34–40. French, R. C., Graham, C. L., Gale, A. W. & Long, R. K. (1977). Structural and exposure time requirements for chemical stimulation of germination of uredospores of Uromyces phaseoli. Journal of Agricultural Food Chemistry 25, 84–88. Hoch, H. C., Staples, R. C., Whitehead, B., Comeau, J. & Wolf, E. D. (1987). Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235, 1659–1662. Huang, B.-F. & Staples, R. C. (1982). Synthesis of proteins during differentiation of the bean rust fungus. Experimental Mycology 6, 7–14. Kwon, Y. & Hoch, H. C. (1991). Temporal and spatial dynamics of (Accepted 3 March 1998)
1518 appressorium development in Uromyces appendiculatus. Experimental Mycology 15, 116–131. Li, A., Altosaar, I., Heath, M. C. & Horgen, P. A. (1993). Transient expression of the β-Glucuronidase gene delivered into urediniospores of Uromyces appendiculatus by particle bombardment. Canadian Journal of Plant Pathology 15, 1–6. Murry, J. A. H. & Crockett, N. (1992). Antisense technology : and overview. In Antisense RNA and DNA (ed. J. A. H. Murry), pp. 1–49. Wiley-Liss, Inc. : U.S.A. Staples, R. C., Hoch, H. C., Epstein, L., Laccetti, L. & Hassouna, S. (1985). Recognition of host morphology by rust fungi ; responses and mechanisms. Canadian Journal of Plant Pathology 7, 314–322. Terhune, B. T. & Hoch, H. C. (1993). Substrate hydrophobicity and adhesion of Uromyces urediospores and germlings. Experimental Mycology 17, 253–273. Terhune, B. T., Bojko, R. J. & Hoch, H. C. (1993). Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology 17, 70–78. Wynn, W. K. (1976). Appressorium formation over stomates by the bean rust fungus : Response to a surface contact stimulus. Phytopathology 66, 136–146.