several important plant pathogens (Turgeon et al. 1985, 1987; Parsons et al. 1987; Wang et al. 1988;. Cooley et al. 1988; Kisler & Benny 1988; Royer et al.
Canadian Journal of Plant Pathology Revue canadienne de phytopathologie Published by
Publiée par
The Canadian Phytopathological Society
La Société Canadienne de Phytopathologie
Volume 15(1): 1-64
March
1993
mars
ISSN 0706-0661
CANADIAN JOURNAL OF PLANT PATHOLOGY 15:1-6, 1993
Transient expression of the beta-glucuronidase gene delivered into urediniospores of Uromyces appendiculatus by particle bombardment Aimin Li, Illimar Altosaar, Michèle C. Heath, and Paul A. Horgen Department of Biochemistry, University of Ottawa, 40 Marie Curie, Ottawa, Ontario K1A 0C6; (M.C.H.) Centre for Plant Biotechnology, Department of Botany, University of Toronto, 25 Willcocks St., Toronto, Ontario M5S 3B2; (P.A.H.) Centre For Plant Biotechnology, Department of Botany, Erindale Campus, University of Toronto, Mississauga, Ontario L5L 1C6. Send correspondence and offprint requests to: Dr. Aimin Li, Centre For Plant Biotechnology, Department of Botany, Erindale Campus, University of Toronto, Mississauga, Ontario Canada L5L 1C6. Tel. (416) 828-3877, Fax (416) 828-3792. Accepted for publication 1992 08 31 Transient expression of Escherichia colt beta-glucuronidase (GUS) gene is reported in Uromyces appendiculatus var. appendiculatus following bombardment of urediniospores with gold particles coated with plasmid DNA. The plasmid contained the chimaeric GUS gene driven by the cauliflower mosaic virus (CaMV) 35S promoter fragment. GUS activity could be detected both histochemically and spectrophotometrically after particle introduction of the plasmid into the urediniospores. Li, A., I. Altosaar, M.C. Heath, and P.A. Horgen. 1993. Transient expression of the beta-glucuronidase gene delivered into urediniospores of Uromyces appendiculatus by particle bombardment. Can. J. Plant Pathol. 15:1-6. On rapporte l'expression transitoire du gène rapporteur beta-glucuronidase (GUS) transmis à des urediniospores de Uromyces appendiculatus par bombardement de particules d'or recouvertes de l'ADN plasmidique. Le plasmide contenait le gène chimérique GUS ainsi que le fragment du promoteur 35S du virus de la mosaïque du chou-fleur (CaMV). L'expression de ce gène a été démontrée soit par des tests histochimiques, soit par des tests spectrophotométriques, après sa transmission aux urediniospores de ce champignon.
Genetic transformation of organisms has been widely used to study gene function and regulation. Transgenic plants are routinely produced by several alternative methods, including Agrobacteriummediated transformation, electroporation, microinjection, and protoplast fusion. The introduction of microprojectile bombardment has facilitated the transformation of some eukaryotic cells, particularly in plant species that could not be transformed using Agrobacterium-mediated transformation. This novel technique has also led to the transformation of subcellular organelles such as chloroplasts and mitochondria (Boynton et al. 1988, Johnston et al. 1988). Stable integrative transformation systems have been developed for a growing number of filamentous fungi (Fincham 1989) based on the procedures developed for Neurospora crassa by Case et al. (1979). Among the list of fungi transformed are several important plant pathogens (Turgeon et al. 1985, 1987; Parsons et al. 1987; Wang et al. 1988;
Cooley et al. 1988; Kisler & Benny 1988; Royer et al. 1991). Although the protoplast transformation system gives rise to a high frequency of transformants (Fincham 1989), it has not been successful in all systems tried and may be impractical for some fungi. Recently, a rapid transformation method has been developed by direct electroporation of germinating conidia of N. crassa and Pénicillium urticae (Chakraborty & Kapoor 1990). Biolistic nuclear transformation has also been reported in Saccharomyces cerevisiae and other fungi (Armaleo et al. 1990). Electroporation and particle bombardment transformation technologies offer considerable potential for use in difficult fungal systems. Rust fungi are major pathogens of a number of agronomically important crops and forest tree species, resulting in high yield loss. Classical genetic data has demonstrated the presence of gene-for-gene interactions between rust fungi and host plants (Flor 1971). Gene-for-gene interactions also have been l
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 15, 1993
demonstrated in several other plant-microbe interactions. Such a gene-for-gene system has been confirmed by transgenic technology in bacterial pathogens and bacterial genes for avirulence have been cloned (Staskawicz et al. 1984). More recently, a fungal gene for avirulence also has been cloned (Van Kan et al. 1991). However, such gene cloning has not been achieved in pathogenic rust fungi because of the lack of a successful transformation technique. In this paper, we report the transient expression of the E. coli beta-glucuronidase gene in urediniospores of a rust fungus (Uromyces appendiculatus) following particle bombardment. These experiments represent an important first step in the development of a transformation system for this important group of plant pathogens. Materials and methods Plant and fungal materials. Plants (Phaseolus vulgaris L. cv. Pinto) were grown in Pro-Mix (Premier Brands Inc., New Rochelle, NY) in growth chambers maintained at 22-24°C, with a 16-hour photoperiod of about 250 \mvo\ rrrV 1 provided by tungsten and cool-white fluorescent lamps. Primary leaves from 12-day-old P. vulgaris plants were inoculated with the bean rust fungus (BR) (Uromyces appendiculatus (Pers.) linger var. appendiculatus). Urediniospores of BR were produced and stored as described previously (Heath 1977). Preparation of urediniospores for bombardment. For the urediniospores used in the histochemical assay of beta-glucuronidase (GUS) expression , 1 x 2-cm bean leaf pieces were excised 1 week after inoculation and placed directly on solid MS salt (Murashige & Skoog 1962) medium containing 3% sucrose, 1 mg/L NAA, 1 mg/L kinetin, and 0.8% agar. Urediniospore pustules on the infected leaf tissue were then bombarded. For spectrophotometrical detection of transient expression of GUS, urediniospores were washed with 0.01% Tween 20 and suspended in sterile doubledistilled water. Approximately 2.8 x 104 spores were pipetted on to a nylon filter paper, 2.5 cm in diameter (Millipore, 8.0 jjm) which was then laid on 3 layers of Whatman #1 filter paper (soaked in sterile distilled water) in a petri dish. Urediniospores were incubated at 25°C in the dark (wrapped in aluminium foil) for 2-3 hours prior to bombardment. Plasmid DNA. The plasmid pKANGUS was kindly provided by M. Molony (University of Calgary, Alberta, Canada). It carries a kanamycin resistance gene cassette and a beta-glucuronidase (GUS) gene cassette (Fig. 1). The direction of transcription is indicated in Figure 1. A promoter-less GUS gene vector, pGUS (Fig. 1), was constructed by
digesting pKANGUS with restriction enzyme BamHI, religating with T4 DNA ligase, and selecting a recombinant that contained only the coding region of GUS and the 3' nos-ter sequence. Plasmid DNA was amplified through transformed Escherichia coli DH-alpha cells (genotype), isolated and purified as described by Maniatis et al. (1982). DNA/gold particle complex and bombardment. Gold particles (average diameter 1.0 \\m) were prepared as described by the manufacturer (Du Pont Biolistic Delivery System PDS-1000/He). To 50 ^L of a 60 mg/mL gold particle suspension were added 10 ,uL plasmid DNA (1 \\%J\\L\ 50 jiL of 2.5M CaCl, 20 ^AL of 0.1 M spermidine. The mixture was vortexed for 3 minutes and then centrifuged at 10 000 rpm for 10 seconds. The gold particle pellet was washed with 250 jiL of 100% ethanol, centrifuged, and resuspended in 60 ^L of 100% ethanol. Ten ^L of the particle suspension were applied on to the macrocarrier and dried at room temperature. Urediniospores were bombarded using the PDS1000/Helium system at a pressure of 1550 psi. The distance between the stopping screen and the sample was 2.5 cm. Vacuum in the bombardment chamber was about 70 cm Hg. Histochemical assay of GUS in urediniospores. Following bombardment, the sporulating leaf tissue (with urediniospore pustules) was incubated overnight under light with a 16-hour photoperiod. After incubation, leaf tissues were transferred to 1.5 mL microcentrifuge tubes and stained for approximately 8 h at 37°C in 1.0 mL of GUS reaction buffer consisting of 100 mM NaH 2 P0 4 , 0.1% formaldehyde, 0.1% Triton-X 100, 0.1% betamercaptoethanol, 1 mM 5-bromo-4-chloro-3-indoylbeta-glucuronic acid (X-Gluc), pH 7.0. Leaf tissues were then fixed for 10-15 min in 20% formaldehyde, 5% ethanol, and 5% acetic acid. Urediniospores washed off the leaf tissue were centrifuged in a microcentrifuge tube at 10 000 rpm for 10 seconds and resuspended in 100 jiL of Hoyer's medium (Cunningham 1972). For light microscopy, urediniospores were directly mounted in Hoyer's medium and observed using a Carl Zeiss light microscope. Leaf tissues were decolored with 95% ethanol, cleared in saturated chloral hydrate and mounted in Hoyer's medium. Photographs were taken using a M35W camera attached to the microscope. Spectrophotometric assay of GUS activity. A spectrophotometric assay of GUS activity was performed using p-nitrophenyl beta-D-glucuronide (PNPG) as the GUS substrate as described by Jefferson (1987), with minor modifications. GUS was extracted from bombarded urediniospores on nylon membranes with extraction buffer consisting of 50
LI ET AL.: GUS GENE EXPRESSION IN UROMYCES
mM NaP0 4 , pH 7.0, 10 mM DTT, lmM Na2EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton X100 (Jefferson, 1987) at 0, 4, 8, 16, and 24 h after bombardment. Bombarded urediniospores on nylon membranes were washed with 1 mL extraction buffer into a 3 mL-volume glass homogenizer (Corning Glass). To 1 mL of extraction buffer was added about 20 mg of glass beads (prepared by grinding a glass pipette in a mortar). The mixture was homogenized on ice for 3-5 min (Con-Torque Power unit, Eberbach Co., Ann Arbor, Michigan, USA), transferred into a 1.5 mL microcentrifuge tube, and centrifuged for 1 min at 12 000 g at 4°C. The supernatant (GUS extract) was transferred to a sterile microtube and stored at -70°C. One hundred \*L of GUS extract from each sample was used for determination of protein concentration. To 900 \xL of GUS extract was added 90 ^L of fresh GUS extraction buffer and 10 ^L of 100 mM PNPG stock prepared in water. The reaction was carried out for 7.5 h at 37°C and stopped by adding 400 ^iL of 2.5 M 2-amino-2-methyl propanediol (Sigma A-9754). Absorbance was measured at 415 nm against a substrate blank. Protein assay. Protein concentrations in GUS extracts were determined using the Bio-Rad protein assay kit as described by the manufacturer. Bovine serine albumin (BSA) (Sigma Chem. Co.) was used as a standard. Results and discussion When urediniospore pustules of BR on infected bean leaves were bombarded with gold particles coated with pKANGUS plasmid DNA, transient
Figure 1. Restriction map of plasmids pKANGUS (9.8 kilobases) direction of transcription.
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expression of the GUS gene was indicated by the blue-staining urediniospores after the histochemical assay (Fig. 2b, 2c, and 2d). No blue-staining urediniospores were observed following bombardment with particles coated with pGUS plasmid DNA (Fig. 2a) or particles without DNA (data not shown). The estimated frequency of urediniospores expressing GUS was 8.2 x 10~4 per 1.6 ng DNA per bombardment. Transient expression of the GUS gene was also detected in epidermal as well as mesophyll cells (data not shown) of bean leaves following bombardment. The average number of blue spots per leaf piece was 52 per 1.6 ^g DNA per bombardment (Table 1). When isolated urediniospores on nylon membranes were bombarded with particles coated with pKANGUS DNA, an increase in GUS activity was observed when compared to controls (particles only). This increase was detected as early as 4 h and decreased 16 h after bombardment and thereafter, presumably as a result of a decrease in the metabolic activity of urediniospores after prolonged incubation in water. The presence of blue-staining urediniospores of BR following bombardment of rust-infected leaf tissues suggests that a high level of GUS activity could be detected in metabolically active urediniospores. Extracts from urediniospores bombarded with particles without DNA exhibited low background "GUS" activity. The transient expression of the GUS gene in urediniospores of Uromyces appendiculatus demonstrates the capability of particle bombardment to deliver foreign DNA into urediniospores of rust fungi. Although we have not provided direct physical
pGUS (5.1 kilobases) used for bombardment. Arrow indicates the
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 15, 1993
Figure 2. Histochemical assay of GUS activity in urediniospores of Uromyces appendiculatus following bombardment. GUS expression was detected (as indicated by the blue staining) in both mature (B, x 625 and D, x 625) and immature (C, x 1150) BR urediniospores following bombardment with particles coated with pKANGUS plasmid DNA. No GUS activity was observed in urediniospores following bombardment with particles coated with pGUS DNA (A, x 625).
LI ET AL.: GUS GENE EXPRESSION IN UROMYCES
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Table 1. Histochemical assay of GUS activity in urediniospores of Uromyces appendiculatus and in rust-infected bean leaf tissue following particle bombardment Aa
B
C
D
29±7.7e 24±5.8 27±7.1 28±7.3
52±5.2 0 0 0
24±6.4 0 0 0
8.2 x W4 0 0 0
Treatment BR-infected bean leaves bombarded with pKANGUS bombarded with pGUS bombarded with gold particle unbombarded a
A = total number of pustules on each leaf piece. B = number of blue spots (1- 6 epidermal or mesophyll cells) on each leaf piece per bombardment. C = number of urediniospores per leaf piece showing GUS activity.per bombardment. D - frequency of urediniospores expressing GUS e Mean and standard deviation were obtained from two separate experiments in which two bombardments were performed for each treatment.
evidence for the presence of foreign DNA, we have shown that the potential exists for the development of a stable integrative transformation system for rust fungi by using the GUS gene as a reporter gene. The utility of the GUS system has been shown for other plant pathogenic fungi (Roberts et al. 1989, Bunkers 1991). The intact CaMV-35S promoter has been reported to confer a high level of expression of chimeric genes in most cells of transgenic plants (Odell et al. 1985, Jefferson 1987, Kay et al. 1987), although dissection into subdomains that are able to confer tissue-specific gene expression has demonstrated that the promoter has a modular organization (Benfey & Chua 1990). Nevertheless, the fact that its expression is independent of viral trans-acting factors (Odell et al. 1985) has made the CaMV-35S promoter one of the most commonly used promoters in plant biotechnology. In
the present study, the expression of the GUS gene driven by the CaMV-35S promoter in urediniospores of rust fungi suggests that the transcription factors required for the expression of the CaMV-35S promoter are present in the urediniospores of rust fungi. Based on our histochemical assay, the level of GUS gene expression in urediniospores was similar to that in bean leaf cells. Following a similar bombardment approach with Uromyces appendiculatus, but using a homologous promoter, similar histochemical evidence was found for comparable transient expression of the GUS gene in urediniospores (Bhairi & Staples 1992). Further work is required examining the effect of promoter sequences on expression levels. This study reports the transient expression of the GUS gene in urediniospores of the bean rust fungus Uromyces appendiculatus. We also provide evidence that suggests that the CaMV-35S promoter and the beta-glucuronidase gene can function as a reporter gene system in studying rust fungi. Currently, we are attempting to develop a stable integrative transformation system for this important group of plant pathogens. This work was partially supported by the Ontario Ministry of Agriculture and Food.
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Figure 3. Transient expression of GUS activity in urediniospores of Uromyces appendiculatus. Aproximately 2.8 x 10 4 urediniospores were bombarded with gold particles without DNA (x—x) or coated with pKANGUS DNA ( — ). GUS activity was measured as nanomolar product released per mg protein per hour. The mean and standard deviation (error bar) obtained from two separate experiments are shown in which duplicate bombardments were performed for each time point.
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