Tuija Kekarainen and Jari P. T. Valkonen, Department of Plant Biology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7080, SE-75007 Uppsala, Sweden
Particle bombardment is a physical method widely used for gene transfer into plants, mammals, fungi and bacteria (Armaleo et al. 1990, Klein et al. 1992, Shark et al. 1991, Williams et al. 1991, Zelenin et al. 1989). The hand-held Helios gene gun provides wider possibilities for gene transfer to plant tissue both in vivo and in vitro, because it is not limited by the large size of the target, and bombardment does not need to be carried out under vacuum.
The particle bombardment technique has been used to infect plants with DNA viruses, RNA viruses and cloned RNA viruses (Gal-On et al. 1995, Gilbertson et al. 1991, Klein et al. 1992 ) . The PDS-1000/He system is efficient for inoculation of detached leaves, but the Helios gene gun enables inoculation of an intact plant. In this study we have used the Helios gene gun to inoculate whole tobacco and potato plants with the cDNA of an RNA virus, potato virus A (PVA; Puurand et al. 1996). We wanted to optimize bombardment parameters to achieve 100% efficiency of PVA infection in susceptible tobacco plants. In the case of potato plants, we used potato clone A6 (Solanum demissum Lindl. x S . tuberosumcv. Aquila), which develops necrotic lesions at the site of PVA infection (Valkonen et al. 1995 ), and thus enables counting of the number of initial infection sites. The number of lesions can be used as a readily observed measure for optimal inoculation conditions. The bombardment parameters examined in this study were helium pressure, distance of target tissue from the s pacer, polyvinylpyrrolidone (PVP) concentration, viral DNA and RNA amounts, and the size and number of gold particles (micro carriers ) .
PVA belongs to the family Potyviridae, the largest and economically the most important group of plant viruses (Bartels 1971). PVA is widely distributed in potato-growing areas and decreases the yield of infected potato plants by up to 40% (Brunt 1992). A full-length cDNA clone of the RNA genome of PVA has been constructed under the control of a T7 promoter (PVA - T 7 ), f rom which infectious transcripts can be produced in vitro for inoculation (Puurand et al. 1996). The same construct is also available under the control of a cauliflower mosaic virus (CMV) 35S promoter (PVA-35S) that can be used directly for inoculation (. Puurand pers. comm., Rajamaki and Valkonen 1999).
Plant Material. Tobacco (Nicotiana tabacum L. cv. Samsun) is susceptible to PVA, whereas the test potato clone A6 reacts with the development of necrotic lesions (hypersensitive response) at the sites of infection (Valkonen et al. 1995 ). Tobacco and potato plants were grown in a growth chamber under an 18 hr photoperiod (250 E s-1 m- 2) at 19/17 C (day/night) and a relative humidity of 40%. Tobacco plants were inoculated by particle bombardment when the first true leaf was fully expanded. Potato plants propagated by taking cuttings were grown in the growth chamber for four weeks before bombardment.
Preparation of DNA or RNA for Bombardment. The PVA-T7 construct was linearized with AgeI prior to in vitro synthesis of the 5'-capped transcripts according to the manufacturers instructions (RiboMax, Promega). Trans cript quality and concentration were determined by electrophoresis in an agarose gel. RNA was precipitated on gold particles by addition of 1/10 volume of 3 M NaAc (pH 7.4) and 3 volumes of 99.5% ethanol. The suspension was mixed by tapping the tube and placing it at -20 C for 30 min. After a quick centrifugation (~5 sec), the supernatant was discarded and the gold pellet washed three times with 1 ml 99.5% ethanol. The gold particles were then resuspended in 200 l of ethanol containing the appropriate concentration of PVP. The suspension was transferred to a 15 ml polypropylene centrifuge tube, and 2.8 ml ethanol/PVP solution was added. The suspension was immediately used for preparation of cartridges.
P VA-35S plasmid was isolated from an E. coli XL1-Blue culture using the QIAGEN Plasmid Maxi Kit (Q IAGEN Ltd., UK) according to the manufacture rs instructions. PVA-35S was linearized prior to precipitation of the DNA on gold particles , as described above for RNA. Cartridges were prepared as described in the Helios gene gun instruction manuals (catalog # 165-2431 and 165-2432, Bio-Rad).
Inoculation. For each tobacco plant, the first full-grown true leaf was inoculated with one bombardment. For the potato clone A6, one or two full-grown leaves were bombarded either one or multiple times on different sides of the midvein. The latter arrangement permitted comparison of two or more different bombardment parameters on the same A6 leaf (Figure 1). Before bombardment, plants were kept in the dark for at least five hr. After bombardment, plants were sprayed with water and placed in a transparent box in dim light overnight . The next day, the plants were returned to the growth chamber, and the ex tent of mechanical damage on bombarded areas of the leaves was observed. Symptoms of PVA infection in A6 (necrotic lesions) were observed four days after bombardment. In tobacco, PVA infection was detected twelve days after bombardment of upper uninoculated leaves.
The assay was a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using monoclonal antibodies and alkaline phosphatase-conjugated monoclonal antibodies to PVA (MAb 58/0; Rajamaki et al. 1998) obtained from Adgen, UK.
Optimization of Bombardment Parameters. We tested various bombardment conditions in order to identify those that resulted in the largest number of necrotic lesions on A6 leaves and in infection of all inoculated tobacco plants. First, different helium pressures (80, 100, 150, 200, 250 and 300 psi) were tested using 2 g PVA-35S or PVA-T7 per mg gold (1.0 m), 0.05 mg PVP per ml 99.5% ethanol, and 0.5 mg gold per bombardment as recommended for starting conditions in the Helios gene gun instruction manual. Bombardments were done with 0 or 2 cm distance between the spacer and the target. The first experiments showed that bombardment at 250 and 300 psi damaged leaves extensively, so these pressures were not included in further experiments.
Next, PVP concentrations were varied (0, 0.05 and 0.1 mg per ml 99.5% ethanol). Third, using the previously determined optimal helium pressure and PVP concentration, different sizes of microcarriers (0.6, 1.0 and 1.6 m) were tested. In the last experiment, various amounts of microcarriers per shot (the microcarrier loading quantity, or MLQ) and various amounts of PVA cDNA coated on microcarriers (the DNA loading ratio, or DLR) were tested .
In each optimization step, three cartridges of PVA-T7 transcript (RNA)
or PVA-35S (cDNA) were bombarded onto tobacco and potato plants. Each
test was carried out at least three times. After each optimization step,
the optimal parameter was used in further experiments, as explained above.
Significance of observed differences was tested using analysis of variance
and calculation of the least significant differences (Steel and Torrie
Pressure and Distance. In tobacco, 100% infection was achieved using a helium pressure of 150 or 200 psi at a distance of 0 cm, but extensive death of the cells in the middle of the bombarded area was observed (Figure 2). At the higher pressure of 250 or 300 psi, leaves tended to be ripped. No infection was detected in tobacco plants bombarded at the low pressures of 80 and 100 psi, and at these pressures the bombarded areas usually remained undamaged. Results were similar for PVA-T7 (RNA) and PVA-35S (cDNA).
In the potato clone A6, infection initiation sites could be visually identified by the development of necrotic lesions as a result of the hypersensitive response to PVA. A few necrotic lesions characteristic of PVA infection (Valkonen et al. 1995) were observed at 80 psi, and their numbers increased with increased pressure (Figure 3). At lower pressures (80 and 100 psi), the bombarded area of leaf was usually undamaged and just a few necrotic lesions were observed. Bombardment with higher pressures (150 and 200 psi) led to cell death in the middle part of the bombarded tissue, but numerous necrotic lesions were still observed in the surrounding surviving tissue (Figure 4).
The pressure of 200 psi and the distance of 0 cm were optimal for A6, differing significantly (p< 0.01) from the other pressures used. The higher pressures of 250 and 300 psi ripped the leaves and are thus not recommended for use. Although bombardment at a 2 cm distance from the target tissue resulted in spreading of the micro carriers (and subsequently the necrotic lesions) over a larger area on the leaf, the total number of necrotic lesions was no greater than after bombardment at a distance of 0 cm. No differences were observed between bombardment using PVA RNA or cDNA (data not shown).
PVP Concentration and Micro c a rrier Size. PVP serves as an adhesive during the coating of DNA/(or RNA)/micro carrier suspension to the walls of the Goldcoat tubing. With both DNA and RNA of PVA, the optimal concentration was shown to be 0.05 mg PVP/ml 99.5% ethanol. This resulted in significantly more lesions (p< 0.01) in A6 leaves than at PVP concentrations of 0 or 0.1 mg/ml (Figure 5).
Three sizes of micro carriers were used: 0.6, 1.0 and 1.6 m. The 0.6 m carriers were significantly more efficient in initiation of infection (p< 0.01) than the other two sizes used (Figure 6).
Ratios of Microcarrier and DNA Amounts. Various ratios of microcarriers and viral cDNA (Table 1) were tested by bombarding A6 leaves. The numbers of infection sites observed with different ratios of microcarriers and DNA were not significantly different statistically. As little as 0.1 g viral cDNA per bombardment was sufficient to trigger development of necrotic lesions in A6.
The Helios gene gun is being used in several laboratories f or plant transformation and also virus inoculation. We have optimized bombardment parameters for a cloned RNA virus (PVA) using an experimental host widely used for many plant v i ruses (N. tabacum L. cv. Samsun) and also using a host that reacts with a hypersensitive response to virus infection (potato clone A6). The latter type of host is useful for an optimization experiment because the sites of infection can easily be identified visually after only a few days. Because the aim of our study was to inoculate a cloned virus (RNA transcripts or linearized viral cDNA), it was inferred that the optimal bombardment parameters could be different from those used for plant transformation. For this reason, optimization of parameters was needed.
For successful infection, it is important to optimize the condition of plants before and after inoculation. We noticed that shading the plants overnight before bombardment and keeping them under humid conditions in dim light after bombardment improved initiation of infection considerably. Such treatments are known to increase susceptibility of plants to mechanical inoculation (Matthews 1995).
This study showed that it is important to determine the optimal pressure, PVP concentration and micro carrier size, because differences in these parameters provided varying success in infection. These parameters may also vary for different host plants. In our study, the low pressure of 80 psi caused a few necrotic lesions in A6 leaves, whereas in tobacco no PVA infection was detected following inoculation at a pressure of 80 or 100 psi. In contrast, at a pressure of 150 or 200 psi, all tobacco plants became infected. PVP concentration of 0.05 mg/ml and micro carrier size of 1.6 m were the best for both plants. The number of micro carriers and amount of viral cDNA tested did not affect infection efficiency.
We suggest the starting conditions indicated in Table 2 to optimize viral RNA or cDNA inoculation of tobacco and potato plants. They have been used successfully for many more recent studies in our laboratory (Hamalainen et al. in press, Rajamaki and Valkonen 1999).
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