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Short Communication |
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
Correspondence
Marcel Prins
marcel.prins{at}wur.nl
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ABSTRACT |
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A table of primer sequences used in fusion-PCR is available as supplementary material in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Originally, sense- or antisense-mediated RNA silencing resulted in a maximum resistance frequency of 20 %, but often far lower frequencies were obtained (Smith et al., 2000). Moreover, not all viral genes used in transgenic constructs rendered plants resistant. A significant improvement in RNA silencing could be obtained by introducing inverted repeat transgenes, resulting in double-stranded RNA transcripts (dsRNAs). Compared to the originally used (single) sense transgenes, these dsRNAs render the system very efficient in that a much higher frequency of transformed plant lines expressing such dsRNA constructs will display efficient gene knockdown or virus resistance (Smith et al., 2000; Waterhouse & Helliwell, 2003). It is perceived that this is due to the dsRNAs being fed directly into the silencing pathway at the level of the RNaseIII-like enzyme Dicer and therefore there is no reliance on the action of plant-encoded RNA-dependent RNA polymerase proteins, such as RDR6, required for the production of dsRNAs in (single) sense transgene-induced RNA silencing (Beclin et al., 2002). Using inverted repeats, it has proved possible to use transgenes that had previously seemed unsuitable for sense RNA-mediated silencing (Chen et al., 2004).
Tospoviruses rank among the most detrimental plant viruses worldwide, causing significant economic losses in the cultivation of vegetables and ornamental crops (Prins & Goldbach, 1998). Previous work in transgenic plants has shown that from all genes of Tomato spotted wilt virus (TSWV) only the N and NSM gene constructs resulted in resistance, albeit at low frequencies (Prins et al., 1996). Subsequent work (Pang et al., 1997; Jan et al., 2000) showed that sequences as short as 110 nt from the TSWV N gene were sufficient to efficiently induce RNA silencing, and therefore virus resistance, but only when fused to a carrier mRNA such as green fluorescent protein (GFP).
To obtain broad (tospo)virus resistance at a high frequency, a new approach was taken, which is described here. We aspired to use N gene sequence fragments of the four major tomato-infecting tospoviruses, TSWV, Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV) and Watermelon silver mottle virus (WSMoV), in a single small chimaeric hairpin (hp) RNA construct. As the overall homologies of the N genes of the tospoviruses used in this study range between 40 and 80 % (Prins & Goldbach, 1998), effective RNA silencing against one virus is insufficient to protect against the other viruses (De Haan et al., 1992). Primers were designed to amplify sequential 150 bp N gene segments of TSWV, GRSV, TCSV and WSMoV and to overlap where the segments of the cassette should be fused (see Table S1, available as supplementary material in JGV Online). After a first round of PCR, the products were used in another round of PCR, resulting in the fusion of the two segments. Using this technique, the four segments were fused, resulting in a chimaeric N gene cassette of 600 bp. Successive parts of the tospoviral N genes were chosen to reduce the risk of intramolecular homologous recombination either in bacteria or plants. To prevent possible complications due to translatability of the transgene, the start codon sequence was omitted in the amplified sequence, while subsequent potential start codons were out-of-frame. Primers MH17–MH20 (Table S1, available as supplementary material in JGV Online) were used to amplify the 500 bp intron sequence of the Arabidopsis thaliana actin2 gene, and the quadruple chimaeric tospovirus cassettes, flanked by BamHI and NotI sites, were cloned into inverted repeat (IR) arrays around the intron as described previously (Chen et al., 2004), resulting in the sense-antisense IR-IN and the antisense-sense IR-OUT (Fig. 1a). The intron was inserted to stabilize the construct during cloning and to enhance the effectiveness of the transcript in inducing hpRNA-mediated silencing (Smith et al., 2000). The IR constructs were cloned into a modified pBIN19 binary vector containing the restriction sites AscI and PacI between the right and left border. Finally the binary vector was transformed into Agrobacterium tumefaciens LBA 4404. Plasmid DNA was rescued from A. tumefaciens and sequenced. A. tumefaciens clones were finally used to transform small leaf explants of Nicotiana benthamiana, as described by Horsch et al. (1985). Each resulting transgenic plant line originated from a single independent callus.
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and Heinze et al. (1995), respectively. To prevent the occurrence of symptom-attenuating defective viruses (Inoue-Nagata et al., 1997), tospovirus isolates were regularly transmitted by thrips and maintained on N. benthamiana plants for no more than five sequential mechanical passages. Inoculum for infection of the transgenic plants was obtained by grinding systemically infected N. benthamiana leaves in 0.5 % Na2SO3 and was applied to the two top leaves of four-leaf-stage plants with carborundum powder. In mixed inoculation experiments equal amounts of diluted sap of systemically infected plants were combined. Non-transformed control plants were inoculated following the inoculation of the transgenic plants. To avoid escapes, inoculations of young emerging leaves were repeated after a week. The plants were then monitored for 30 days for symptom development, and analysed by ELISA (de Ávila et al., 1991) to exclude symptomless infections, using four times the background value as a cut-off point. While all non-transgenic plants readily developed virus symptoms, 81 % of all IR-OUT lines showed high resistance (Fig. 1b
and Table 1). In nearly all cases where an IR-OUT line was resistant to a single tested virus, it was also resistant to the other three viruses. Moreover, most plants showed resistance to infection using a mixture of the four different tospoviruses. Interestingly, the resistance incidence for the IR-IN lines, though still high, was lower than for the IR-OUT lines, as 63 % of the lines were found to be resistant to all four viruses. This phenomenon of lower silencing induction by inwardly oriented IR constructs has also been observed by others (Chen et al., 2004; K. Kalantidis, personal communication). It is possible that ribosome scanning or shunting of the sense RNA prevents proper folding of the RNA into perfect dsRNA and thereby suppresses effective Dicer function. For both IR-IN and IR-OUT lines, progeny generations of resistant plants (S2 and S3) all remained 100 % virus-free, as determined by ELISA, despite a repeated inoculation using a mixed inoculum of all four viruses.
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; Bucher et al., 2004). Twenty micrograms of total small RNA per sample was separated on a 15 % PAGE gel and analysed by Northern blotting using specific digoxigenin (Roche)-labelled double-stranded DNA probes derived from each of the four 150 bp segments of the transgene cassette. As these fragments represent non-homologous subsequent parts of the N gene, no cross-hybridization to segments of the other viruses occurred (results not shown). siRNAs originating from the different parts of the transgene could readily be detected, but exclusively in virus-resistant transgenic lines (Fig. 1c). Similar to the non-transgenic controls, none of the susceptible plants accumulated detectable amounts of siRNAs from any part of the transgene, even though the presence of the genomic DNA copy of the transgene was demonstrated by PCR (data not shown). This might indicate transcriptional gene silencing of the transgene.
The feasibility of obtaining high-frequency resistance based on RNA silencing against multiple viruses simultaneously has been demonstrated. The data presented show that by using RNA silencing, virus resistance frequencies of over 80 % of all transformed plant lines can be achieved. It was shown that the plants became resistant to four different tospoviruses at once, when producing abundant siRNAs originating from each segment of the cassette. It has been shown before that the detection of specific siRNAs in transgenic plant lines expressing an hpRNA construct correlated with virus resistance (Kalantidis et al., 2002; Chen et al., 2004). Since our constructs are entirely hpRNA-based, they do not rely on the inclusion of random carrier sequences such as GFP RNA, as used by Jan et al. (2000). Using the constructs presented here, the size limitation problem (Pang et al., 1997) can be overcome by linking fragments of as many viral sequences as required. Transgenic mRNAs did not accumulate in plant lines expressing hpRNA constructs (results not shown) as these were apparently efficiently diced to siRNAs, thereby minimizing the risk of recombination or complementation events in the field.
Overall, the work presented here demonstrates a simple procedure to obtain broad virus resistance at a high frequency by RNA silencing, using a single transgene construct of limited size. By extending the transgene construct with additional viral sequences, the broadness of the resistance can be extended further. Due to the high frequency of this multiple virus resistance, the approach can be applied first and foremost to tomato in which the four tospoviruses of this study present a major problem. In addition, other susceptible plant species can thus be protected. As many of the tospoviruses have a broad host range, this presents a major advantage.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bucher, E., Hemmes, H., De Haan, P., Goldbach, R. & Prins, M. (2004). The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J Gen Virol 85, 983–991.
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Received 6 June 2006;
accepted 28 July 2006.
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