1 United States Department of Agriculture, Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945, USA
2 Indian River Research and Education Center, IFAS, University of Florida, Fort Pierce, FL 34945, USA
Correspondence
Robert G. Shatters, Jr
rshatters{at}ushrl.ars.usda.gov
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ABSTRACT |
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A figure showing begomovirus detection in plants and vectors by real-time RT-PCR is available as Supplementary material in JGV Online.
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INTRODUCTION |
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Recent research, including the effect of virus on insect fecundity, apparent sexual transmission of virus in the insect and non-quantitative detection of virus coat protein and genome, has lead to the speculation that Tomato yellow leaf curl virus (TYLCV), a ssDNA plant virus that induces severe symptoms in tomato, may replicate in its whitefly vector (Czosnek et al., 2001). However, this idea is controversial due to a lack of definitive proof and some conflicting data: Ghanim et al. (1998)
versus Bosco et al. (2004)
. Definitive determination of the genetic activity of TYLCV awaits quantitative detection of de novo synthesized transcripts and viral genomes. Also, analysis of the genetic activity of other begomoviruses compared with that of TYLCV in the insect vector will provide information on the frequency of occurrence of virus genetic activity in the insect among this group of plant viruses.
In this report, work was performed to compare the transcriptional activity in Bemisia tabaci (Gennadius) biotype B of two different begomoviruses that represent two major subgroups. Tomato mottle virus (ToMoV) and TYLCV are two whitefly-transmitted ssDNA circular genome plant-pathogenic viruses that belong to the genus Begomovirus within the family Geminiviridae (Rybicki, 1994; Padidam et al., 1995
; Stanley et al., 2004
). ToMoV is a New World bipartite begomovirus (Fig. 1a
), whereas TYLCV is a monopartite Old World begomovirus (Fig. 1b
). These viruses have emerged as agricultural problems, with TYLCV being a serious threat to worldwide crop production (Polston & Anderson, 1997
; Moffat, 1999
).
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METHODS |
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Cotton plants (Gossypium hirsutum L. cv. Delta Pine 5415) were obtained from seed sown directly into 15-cm pots. Seeds were allowed to germinate, thinned to six plants per pot and fertilized weekly with 20-10-20 Peters Professional Plant Starter Product (Scotts-Sierra Horticultural Products). Plants with three to four fully expanded leaves were used for whitefly infestations.
In 1997, a ToMoV-viruliferous whitefly colony was established by obtaining tomato plants infected with ToMoV from P. Stansly, University of Florida, Immokalee, FL, USA, and infesting them with whiteflies from a non-viruliferous colony. In 2001, a TYLCV-viruliferous whitefly colony was established by obtaining cuttings from field-grown tomatoes infected with TYLCV from D. Schuster, University of Florida, Bradenton, FL, USA. Rooted cuttings were planted in 15-cm pots and infested with whiteflies from a non-viruliferous colony. After their establishment, serial transfers on dwarf cherry tomato cultivars maintained both virus colonies. The presence of the viruses was assessed by visual identification of symptom development and PCR amplification with virus-specific primers (Pico et al., 1998
; Sinisterra et al., 1999
) (Table 1
).
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After a total of 7 days on cotton, the remaining whiteflies were harvested and stored at 80 °C. At both 4 and 7 days, cotton leaf samples were taken and used as controls in quantitative PCR experiments. Furthermore, the cotton plants were held for an additional 30 days and then tested for the presence of virus. At no time during this 30 day period were virus symptoms observed and, after 30 days, no virus DNA could be detected by PCR analysis (see Supplementary figure available in JGV Online). This experiment was repeated three times.
Nucleic acid extraction.
Total DNA was extracted from groups of 10 whiteflies by grinding whole insects in liquid nitrogen and using the AquaPure Genomic DNA isolation kit (Bio-Rad) according to the manufacturer's instructions. DNA from 0·1 g tomato leaf tissue was extracted following the DNA extraction protocol described by Edwards et al. (1991). For cotton samples, 70 mg leaf tissue was processed as described by Kobayashi et al. (1998)
.
Total RNA extractions were performed using 500 mg tissue from previously frozen samples of tomato or cotton plants or approximately 2500 whiteflies. Samples were ground to a fine powder using mortar and pestle in the presence of liquid nitrogen, then processed with the RNeasy midiprep kit (Qiagen), following the manufacturer's protocol for isolation of total RNA. Trace DNA contamination was removed from total RNA preparations by a double extraction with one volume acid phenol : chloroform (5 : 1, pH 4·7; Ambion), followed by precipitation with two volumes 95 % ethanol and 1/10 volume 2 M sodium acetate (pH 4·0) overnight at 20 °C. RNA was pelleted by centrifugation for 5 min at 12 000 g and resuspended in water. RNA (10 µg) was digested with 2 U DNase I (Ambion) in a 25 µl reaction mix for 1 h at 37 °C. After a second precipitation, the sample was resuspended in water and digested with 30 U RecJ (New England Biolabs) ssDNA-specific exonuclease, 2 U DNase I, 1x RecJ reaction buffer in a 25 µl reaction mix for 1 h at 37 °C. This sample was used directly for quantitative PCR applications.
Virus DNA and RNA quantification.
A Rotor-Gene RG-3000 (Corbett Research) real-time PCR machine coupled with the DNA minor groove binding fluorescent dye SYBR Green I were used for quantitative PCR methods. Specific primers were designed to amplify segments of <200 bases from transcripts containing the following genes: ToMoV AV1, BC1 and BV1; TYLCV V1, V2 and C3; tomato -actin; whitefly
-actin; and cotton
-actin (Table 1
). AV1 and V1 encode coat proteins of ToMoV and TYLCV, respectively, BC1 and V2 encode proteins believed to be involved in cell-to-cell movement, and BV1 encodes a nuclear shuttle protein, which has no direct counterpart in the TYLCV genome, but was chosen to determine if a transcript encoding a protein with this function was expressed in the insect vector. The C3 gene, encoding a replication enhancer, was chosen to monitor complementary strand transcripts. Prior to initiation of the experiment, viral PCR amplicons were verified by sequence analysis (data not shown) and amplification of viral sequences was shown to be specific to whiteflies viruliferous for the virus being tested.
Real-time RT-PCR (RRT-PCR) was conducted using 300 ng total RNA from every sample in a 25 µl reaction mix using the Quantitect SYBR Green RRT-PCR kit (Qiagen) under recommended reaction conditions. Reverse transcription was performed for 30 min at 50 °C followed by a 15 min denaturation at 95 °C and 40 cycles of 40 s at 95 °C, 40 s at 58 °C and 40 s at 72 °C. For each sample, RRT-PCR quantification was based on relative abundance, as determined by Ct value compared with either plant or whitefly -actin Ct. Simultaneously, the retention of plant transcripts in all ToMoV- and TYLCV-viruliferous whitefly samples was monitored using primers specific for tomato ribulose-bisphosphate carboxylase (Rubisco, 4.1.1.39) (Table 1
).
In order to detect DNA contamination in the RNA samples, RRT-PCR was conducted with 300 ng total RNA from all plant and insect samples. The Quantitect SYBR PCR kit (Qiagen) was used for RT-PCR. For detection of viral genomic sequences in total DNA samples, 300 ng total DNA was used following the above cycling profile using primer sets for the selected virus genes and -actin. The relative titres of ToMoV and TYLCV in infected tomato and in all whitefly samples were determined using RT-PCR with primers for AV1 and V1 genes, respectively.
All real-time experiments (RRT-PCR and RT-PCR) were conducted in triplicate for each sample and melting curve and agarose gel analyses were performed to verify single product formation. Relative quantification analysis was performed using a dynamic amplification efficiency determination for each amplification run as provided in the comparative quantification function with the Rotor-Gene RG-3000 software (described in the technical bulletin entitled An Explanation of the Comparative Quantification Technique Used in the Rotor-Gene Analysis Software, Matthew Herrmann, Corbett Research, Mortlake, Australia). Briefly, the following exponential growth model represents the increase in fluorescence (R) during the amplification: Rn+1=Rn*(A), where n is the cycle number and A is the amplification value, a measure of reaction efficiency. The first differential was taken to remove the fluorescence background. Fluorescence increase during the exponential phase was monitored by a rearrangement of the above formula to give an observed amplification (An) at each point within the exponential phase of a reaction: (An)=Rn+1/Rn. The mean amplification over these points produced an amplification value for the sample (As). The mean amplification of all samples (A) was then determined and the variance was used to provide a measure of error. The amount of gene product in any given sample relative to a designated reference sample was then calculated using the formula: (A)^(control take-off pointsample take-off point). Error coefficient was determined with a 95 % confidence interval.
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RESULTS AND DISCUSSION |
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The apparent increase of TYLCV transcripts is based on relative abundance of the viral transcripts compared with the whitefly -actin transcript and therefore could be due to an increase in total number of viral transcripts or to a decline in the abundance of the whitefly
-actin transcript, possibly a result of whitefly colony ageing or changing the whitefly host plant and thus nutritional status. Relative determination of whitefly
-actin abundance to whitefly 18S rRNA indicated that, after 4 days of feeding on cotton, there was no change in whitefly
-actin abundance relative to the 18S rRNA, but there was still a significant increase in virus transcripts relative to this rRNA (data not shown). The increase in viral transcripts relative to either whitefly 18S rRNA or
-actin was surprising, since it was previously shown that begomovirus DNA reached a steady-state level within the whitefly after approximately 10 h of feeding on a single virus-infected host (Czosnek et al., 2001
). Our results, indicating an increase in virus transcript abundance after transferring whiteflies to a new host, can only be explained by TYLCV transcriptional activity within the whitefly. This result may suggest a dynamic control over TYLCV transcription or at least changes in viral transcript stability in response to changes in whitefly physiology (lack of ingested virus, whitefly ageing or altered nutritional status).
Comparison of virus genome titre and viral transcript abundance in viruliferous whiteflies and infected tomato
Virus genomic DNA (ssDNA viral genome and replicative dsDNA) abundance was determined using RT-PCR and primers for V1 (TYLCV) and AV1 (ToMoV) DNA detection. The ToMoV titre in whiteflies sampled directly from infected tomato was about three times higher than the virus titre in TYLCV-viruliferous whiteflies similarly sampled and, after 4 days of feeding on cotton, a significant decrease in ToMoV titre to the level equal to the TYLCV titre was observed (Fig. 4a). Conversely, TYLCV titre in viruliferous whiteflies remained constant even after 7 days of feeding on cotton plants (Fig. 4a
). The rapid decline in ToMoV DNA abundance in whiteflies transferred to cotton compared with the lack of a similar drop in TYLCV suggests a higher ToMoV DNA load present in the gut contents that was cleared during feeding on cotton. Since whiteflies are strict phloem-feeding insects, a higher abundance of ToMoV DNA compared with TYLCV DNA in the phloem would account for this difference and, in support of this hypothesis, quantification of viral DNA titre in tomato indicated a ToMoV DNA titre more than 10 times higher than the TYLCV DNA titre (Fig. 4b
) in total leaf tissue. Furthermore, the finding that TYLCV transcripts are less abundant in leaf tissue than ToMoV transcripts (at least in comparisons of V1 and V2 to AV1 and BV1) (Fig. 4c
) is also supportive evidence showing that the stable detection of TYLCV transcripts in the whitefly is not the result of differences in bulk loading of these transcripts from feeding (i.e. whiteflies are not incorporating more TYLCV transcript from their diet than they are ToMoV transcripts). One caveat to these observations is that our quantification of TYLCV and ToMoV DNA and RNA transcripts was performed on whole leaf samples and tissue specificity of TYLCV is still uncertain in view of published observations of phloem-specific localization (Morilla et al., 2004
) and localization to both mesophyll and phloem (Michelson et al., 1997
). Tissue specificity of ToMoV is also uncertain. Therefore, it is not clear if the relative abundance relationship between the two viruses as measured in whole leaves would be maintained in phloem to phloem comparisons (the feeding location of the whitefly). Since the relative distribution of virus DNA and transcripts within different leaf tissues is uncertain, differences in whitefly feeding uptake as opposed to tissue distribution of virus cannot accurately be assessed.
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Previous reports have shown that TYLCV appears to reduce whitefly fitness, whereas ToMoV does not. It has been speculated that this may be the result of the ability of TYLCV to replicate in the whitefly (Czosnek et al., 2001). Whether this occurs or not, our data show interesting differences in transcriptional activity of the two viruses that may be related to the reported influences of these viruses on whitefly biology. Czosnek et al. (2001)
have further hypothesized that begomoviruses may represent a genus with species that are undergoing an evolutionary divergence in the mechanisms by which they interact with their insect vectors.
In that context, it is interesting to speculate that the differences in accumulation of genomes and transcripts in the plant and the insect may represent two different strategies for maintaining a virus titre within the vector sufficient to assure transmission to other host plant species. ToMoV, a virus that does not replicate within the whitefly, accumulates to a high titre in the plant, allowing rapid saturation of the insect with virus particles and rapid replenishment of this pool as the whitefly feeds. TYLCV, a virus displaying genetic activity (transcription and, by inference, replication) in the whitefly, does not require production of such high virus titres in the plant, as it would be maintained in the insect vector by virus replication. This implies that the ability to replicate in the insect may obviate the insect's need to constantly take in high levels of virus to assure future virus transmission.
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Received 5 October 2004;
accepted 4 February 2005.
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