Department of Plant Pathology, University of California, 1 Shields Avenue, Davis, CA 95616, USA1
Author for correspondence: Adib Rowhani. Fax +1 530 752 2132. e-mail akrowhani{at}ucdavis.edu
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Abstract |
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Introduction |
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The level of genomic variation suggests that GFLV genomes may consist of a genetically diverse collection of mutants, the dominant members of which may vary during shifts among successive host varieties, in the manner of a quasispecies (Roossinck, 1997 ; Schneider & Roossinck, 2000
). We have attempted a qualitative characterization of the GFLV genome, on the assumption that it does exist as a collection of sequences varying around its own consensus sequence. Gross variation in the viral RNA was visualized by restriction fragment length polymorphism (RFLP) analysis of sections of the unfractionated viral genome amplified by PCR. The variability was compared to variation found by sequence analysis of the same sections of the viral genome purified by cloning. GFLV isolates recovered from eight California vineyards were compared, and gross changes associated with the passage of these isolates in alternative hosts were visualized by this method.
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Methods |
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Inoculations.
Chenopodium quinoa plants were used as a systemic, herbaceous host for GFLV. Plants were grown from seed to the six-leaf stage before inoculation. Five-hundred mg of grapevine shoot tip and leaf were ground in 5 ml 50 mM phosphate buffer, pH 6·5, containing 2% nicotine. All C. quinoa leaves were dusted with carborundum and inoculated with grape tissue extract using a gloved finger. Inoculated leaves were washed immediately with DI water.
RTPCR.
A modification of the method of Rowhani et al. (1995) was used for immunocapture of the virions into the wells of microtitre plates, after precoating plates with GFLV-specific
-globulin. After washes with phosphate-buffered salineTween 20 (PBST) and phosphate-buffered saline (PBS), 3050 µl of warm 1% Triton X-100 was added to the wells, mixed by shaking, and then incubated at 65 °C for 10 min in a water-bath for virion disruption. Complementary DNA strand was synthesized by adding 5 µl of the above solution to 2·5 µl of 10 µM (RT) primer [5' AAAAATTTGCATAACAGTAAAAAG 3' (binding at positions 37543775 in the 3' UTR of viral RNA 2)] in a cocktail composed of 2 µl 10 mM dNTP mix, 2 µl 100 mM dithiothreitol, 4 µl 5xRTase buffer (250 mM TrisHCl pH 8·3, 375 mM KCl, 13 mM MgCl2), 0·25 µl M-MLV RTase (200 Units/µl) (Life Technologies) and 4·25 µl DEPC-treated sterile water. The reverse transcription reaction was carried out using a top-heating thermal cycler for 1 h at 37 °C, and stopped by an incubation of 5 min at 99 °C, followed by 5 min at 5 °C.
Eighty µl of PCR reaction cocktail containing 10 µM C primer [5' CAAGGCAAGTGTGTCCAAA 3' (binding at positions corresponding to 37653724 in the viral coat protein (CP) gene sequence)], 2·5 µl 10 µM V primer [5' TGATGCTTATAATCGGATAACTA 3' (binding at genomic positions 22572270)], 0·25 µl Taq DNA polymerase (5 U/µl), 8 µl 10x PCR buffer, 2·5 µl 50 mM MgCl2 and 44 µl of sterile water were added to each cDNA reaction for a final PCR reaction volume of 100 µl.
The PCR used a 2 min heating step at 95 °C, followed by ten cycles of 30 s melting at 95 °C, 1 min annealing at 63 °C, and 2 min elongation at 72 °C. These ten cycles were followed by 25 cycles of 30 s melting at 95 °C, 1 min annealing at 63 °C, and 2 min elongation at 72 °C with an incremental addition of 5 s of elongation per cycle to ensure full amplification.
RFLP analysis protocol.
After amplification, 5 µl of each reaction was run on a 1·5% agarose gel in 1xTrisacetate buffer (40 mM Trisacetate, 1 mM EDTA, pH 8·0) to confirm product synthesis and estimate DNA concentration by comparison with standards. The PCR product was ethanol precipitated. Half of the product was digested with 0·5 U AvaII restriction endonuclease (New England Biolabs) and the RFLP fragments were separated on a 20 cm 10% polyacrylamide gel in 1xTAE buffer overnight at constant 60 V (3 V/cm) using a ProteanII polyacrylamide gel system (Bio-Rad). DNA was visualized using a UV transilluminator following incubation of gels in 5 µl/ml ethidium bromide.
Cloning of 1557 bp products.
The 1557 bp product was gel-purified using the Prep-A-Gene DNA purification system (Bio-Rad) according to the manufacturers specifications. The fragment was cloned into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen) as per instructions.
Sequencing.
Sequence data were obtained with a Perkin Elmer/Applied Biosystems ABI 377 automated sequencer.
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Results |
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GFLV isolates were inoculated from the grapevine sources to C. quinoa, and serially passaged into healthy C. quinoa 1 day after the onset of symptoms, on a 1016 day cycle. RTPCR and RFLP analysis was performed on extracts from these plants, which showed systemic symptoms (grapevines grown under greenhouse conditions showed much reduced symptoms although they were also RTPCR positive for GFLV). The 1557 bp RTPCR product was obtained from each plant. In most cases, the RFLP banding pattern was simplified during passage. The RFLP profiles from isolate A infecting C. quinoa in sequential passages are shown separately in Fig. 3. A comparison of these profiles shows that the first passage from grapevine into C. quinoa did not cause much change in the RFLP pattern derived from the 1557 bp PCR product. On the second passage a simplified RFLP pattern was observed. This new pattern remained fairly stable through five passages. Bands appearing at approximately the 500 bp position in the second through fifth C. quinoa passages were not observed in the original host.
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The sequence data showed a variation from 11 to 13%, and from 4 to 9% at the nucleotide and amino acid level, respectively (data not shown). Changes in the AvaII sites predicted by these sequences correlated with the RFLP patterns observed. Specific regions of high variability (hypervariable regions) were not apparent.
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Discussion |
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The RFLP bands cover a range of intensities broad enough to suggest that much of the complexity may reside with quasispecies members at concentrations below detectable thresholds. The immediacy of appearance, upon passage, of novel bands with no visible correlates in the original inoculum (Fig. 3) suggests that the newly appearing sequences were present in the original mixture, but at concentrations below the minimum threshold of visibility in RFLP analysis. The reproducibility of the patterns in each of the three replications of the procedure suggests that the variation does not arise from inaccuracies in the RTPCR analysis.
The primary bands in the RFLP patterns show wholesale changes between isolates, and upon passage of a given isolate. This is consistent with the variation seen in cDNA sequences of subclones analysed here, or from the database. Members of the quasispecies mix isolated by cloning differed by 11 to 13% in the 1557 bp genomic sequence amplified in this study. On statistical average, the GGA/TCC AvaII recognition site would occur three times in a 1557 bp sequence, producing four digestion fragments. The AvaII footprint would cover only 15 bp in that sequence. If the sequence were to vary randomly by 15%, approximately two positions in those 15 would change, destroying two of the three sites, and changing three of the four original restriction fragments. (Two new sites would, by chance, be created, further obscuring the original RFLP pattern.)
The number of bands from the California isolates visualized by RFLP analysis was seen to diminish upon serial passage within an herbaceous host. For example, banding patterns of isolates AD were simplified upon passage within C. quinoa (Fig. 3), a general host commonly used to culture plant viruses. This points up the extent to which diversity can be lost from a population of genomic sequences upon passage through a novel host background, suggesting that genomic variability should be characterized directly in the primary host.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 November 2000;
accepted 7 March 2001.