Department of Plant Pathology, Cornell University, NYSAES, Geneva, NY 14456, USA1
Author for correspondence: Dennis Gonsalves. Fax +1 315 787 2389. e-mail dg12{at}cornell.edu
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Abstract |
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Introduction |
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Post-transcriptional gene silencing (PTGS) is considered to be one of the dominant mechanisms of RNA-mediated protection in transgenic plants (Dawson, 1996 ; Goodwin et al., 1996
; Lindbo et al., 1993
; Mueller et al., 1995
; Pang et al., 1996
, 1997
; Prins et al., 1996
; Sijen et al., 1996
; Smith et al., 1994
; Tanzer et al, 1997
). RNA-mediated virus resistance is also known as homology-dependent resistance (Mueller et al., 1995
). Several models have been postulated to account for the mechanism of PTGS and the resulting virus resistance (Baulcombe, 1996b
): (i) the direct production of antisense RNA model (Grierson et al., 1991
); (ii) the expression threshold model (Dougherty & Parks, 1995
; Smith et al., 1994
); (iii) the aberrant RNA (ectopic pairing) model (English et al., 1996
; Baulcombe & English, 1996
) and (iv) the double-stranded RNA-induced model (Metzlaff et al., 1997
; Montgomery & Fire, 1998
; Prins & Goldbach, 1996
; Waterhouse et al., 1998
).
Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus. The virus genome consists of three single-stranded RNAs that are designated as L (~8900 nucleotides), M (~5000 nucleotides) and S (~2900 nucleotides). The S and M RNAs contain two open reading frames (ORFs) of an ambisense gene arrangement (de Haan et al., 1990 ; Kormelink et al., 1992b
; Law et al., 1991
, 1992
; Maiss et al., 1991
; Pang et al., 1993a
), which are expressed via the synthesis of subgenomic mRNAs (Kormelink et al., 1992a
). The S RNA encodes a 52 kDa nonstructural protein (NSS) in the viral RNA strand and the 29 kDa nucleocapsid (N) protein in the viral complementary RNA strand. The M RNA encodes the precursor to the envelope glycoproteins G2 (58 kDa) and G1 (78 kDa) in the viral complementary RNA strand and a 34 kDa nonstructural protein (NSM), which possibly functions as a virus cell-to-cell movement protein, in the viral RNA strand (Kormelink et al., 1994
). The L RNA is of negative polarity and encodes a 330 kDa putative viral polymerase (de Haan et al., 1991
).
We previously reported that transgenic plants expressing a large segment (longer than 387 bp) of the N gene of TSWV conferred virus resistance through PTGS while N transgene segments smaller than 235 bp did not (Pang et al., 1997 ). However, resistance to TSWV was obtained when small N gene segments were fused to the non-target green fluorescent protein (GFP) gene. These data suggested that GFP was triggering PTGS of the chimeric gene while the TSWV N gene segment of the chimeric gene conferred resistance to the attacking TSWV. This system should thus allow us to determine the minimum unit of viral transgene that can confer resistance via the PTGS mechanism and to test whether a chimeric gene made of short virus segments could provide a simple way to develop transgenic plants that are resistant to multiple viruses. Here we demonstrate that minimum lengths of 59110 bp were required, fused with a silencer DNA, for RNA-mediated tospovirus resistance. In addition, the same lines expressing the hybrid transgene are protected against both TSWV and tobacco mosaic virus (TMV)GFP, demonstrating the feasibility of this simple strategy for engineering multiple virus resistance in transgenic plants.
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Methods |
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Cloning and transformation.
Maps of the transgenes used in this study are shown in Fig. 1(b). The 9/16N gene segment was amplified by PCR by using oligomer primers 96-5 (5' TCTTGAGGATCCATGGAATAAGAGGTAAGCTACCT), which is identical to the S RNA of TSWV-BL at positions 23232341, and 92-54 (5' TACAGTTCTAGAACCATGGATGATGCAAAGTCTGTGAGG), which is complementary to the S RNA at positions 23592378. The 17/32N gene segment was generated by annealing oligomer primers 96-6 (5' CTAGACCATGGATGATGCAAAGTCTGTGAGGCTTG) and 96-4 (5' GATCCAAGCCTCACAGACTTTGCATCATCCATGGT). The 17/32N gene segment was located at positions 23552378 of S RNA with NcoI site and XbaI overhang sequences in the 5' end and a BamHI overhang sequence in the 3' end. The 9/16N and 17/32N segments were cloned in the sense orientation into the XbaI/BamHI sites of a plant expression vector pBI525 (Pang et al., 1992
). For construction of N gene segment fusions with GFP, the translatable GFP ORF was amplified with GFP primers (Pang et al., 1997
) from the plasmid pGFP (Clontech) and cloned alone or as a transcriptional fusion into the NcoI site upstream of the N gene segments 9/16N and 17/32N in pBI525. The resulting plant expression vectors were digested with HindIII and EcoRI and the HindIIIEcoRI segments containing the corresponding gene cassettes were isolated and introduced into the same sites of pBIN19 (Clontech). The resulting binary vectors were transferred into Agrobacterium tumefaciens LBA4404 and cultures of A. tumefaciens containing the vectors were used to inoculate leaf discs of N. benthamiana plants, essentially as described by Horsch et al. (1985)
.
Transgenic plants containing the 2/2N, 3/4N and 5/8N gene segments fused with GFP were described by Pang et al. (1997) .
Nuclear run-on transcription assays, ELISA and Northern blot analyses of transgenic plants.
Isolation of nuclei and nuclear run-on transcription assays were described previously by Pang et al. (1996) . Double-antibody sandwich ELISA was used to detect the neomycin phosphotransferase (npt) II enzyme in transgenic plants by using an nptII ELISA kit (5 Prime to 3 Prime Inc.). For estimation of RNA transcript levels in transgenic plants by Northern blot, total plant RNAs were isolated as described by Napoli et al. (1990)
and Northern blotting was performed as described by Pang et al. (1993b
). Ten µg total RNA per lane was separated on formaldehyde-containing agarose gels (Sambrook et al., 1989
) and the agarose gels were stained with ethidium bromide to monitor the uniformity of total plant RNA in each lane. Since the N gene segments in transgenic plants containing GFP+9/16N and GFP+17/32N were too short (59 and 24 bp, respectively) to be detected when using probes made by random priming (Feinberg & Vogelstein, 1983
), an oligonucleotide probe was used to detect the N gene in transgenic plants containing GFP linked to 17/32N or 9/16N. The oligonucleotide probe was 32P-labelled as described by Sambrook et al. (1989)
. Hybridizations were performed essentially as described in the manufacturers protocol for GeneScreen Plus membrane (Dupont) except that the hybridization was at 42 °C and washing was at 48 °C. Images of some autoradiograms were photographed with a COHU CCD camera model 49152000 (COHU Inc.). Signals were quantified by using the US National Institutes of Health-Image program version 1.59.
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Results |
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Northern blot and nuclear run-on assays were performed to determine whether the protection of GFP-transgenic plants against TMVGFP and TMVGFPNP was due to the PTGS mechanism. Results of hybridization analysis showed a correlation between the resistance phenotype and low levels of GFP RNA transcript accumulation (Fig. 2a). In addition, the resistant plants with low steady-state RNA accumulation had high RNA transcription rates in nuclei (Fig. 2b
). These results suggest collectively that PTGS not only suppressed GFP expression but also trans-inactivated the replication of the chimeric virus containing the homologous GFP sequence.
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To determine more precisely the minimum length of the TSWV N gene that is required to confer resistance in the GFP+N plants that show PTGS, transgenic N. benthamiana plants were engineered to express 11024 bp of the N gene alone or fused with the GFP gene (Fig. 1b). In one set of experiments, R0 plants expressing these fusions were inoculated with infectious transcripts of TMVGFP or TMVGFPNP and observed for symptoms. Control transgenic plants with only the 9/16N or 17/32N gene segments were similarly inoculated. Plants that showed resistance to TMVGFP or TMVGFPNP or a recovery phenotype (symptoms developed initially but new leaves were symptomless at 1430 days p.i.) were then inoculated with TSWV (Table 3
). Five of 12 R0 plants tested that expressed the 110 bp segment of the N gene linked to GFP (GFP+5/8N) showed resistance or recovery following inoculation with TMVGFP or TMVGFPNP. Two of these five lines were also resistant to TSWV (Table 3
). In contrast, six of 12 GFP+9/16N (59 bp) and five of 12 GFP+17/32N (24 bp) lines were resistant to TMVGFP or TMVGFPNP, but none of these 11 resistant lines showed resistance to TSWV. All six plants with 9/16N or 17/32N gene segments alone were susceptible to TMVGFP or TMVGFPNP.
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Discussion |
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The simplest explanation for our results is that the 720 bp GFP DNA acts as a silencer DNA because it is large enough to induce PTGS, in contrast to N gene segments of 200 bp or less, which are not sufficiently large to induce PTGS. The silencer DNA (GFP in this case) may simply stabilize the transgene fusions or the GFP gene may provide RNA sequence elements required for activating PTGS. Resistance to TSWV is obtained in transgenic plants when these N gene segments (200110 bp) are linked to GFP because they are part of a transgene that is post-transcriptionally silenced. Our previous work also showed that N gene segments of more than 400 bp could serve as silencer DNAs. We speculate that the putative antisense molecules that are produced from the region of the mRNAs of N gene segments that are 59 bp or less are either too small or not sufficiently abundant to degrade the incoming virus effectively and induce a resistant phenotype.
Our findings demonstrate the feasibility of, and define some of the important parameters for, developing multiple virus-resistant transgenic plants by using a chimeric transgene with a silencer DNA linked to small segments that originate from target plant viruses. This novel strategy could have significant practical value because most crops are susceptible to more than one virus and a single crop is often exposed to infections by multiple viruses in a growing season. It is also likely that this strategy could be used to down-regulate multiple genes in plants and obtain transgenic plants with virus resistance and other interesting traits, for example, delayed ripening (Gray et al., 1992 ).
A potato virus X (PVX) vector has been used to show that silencing of a non-viral transgene can suppress a virus with inserted sequences homologous to the silencing transgene (English et al., 1996 ). Tobacco plants displaying PTGS of
-glucuronidase (GUS) or npt were resistant to PVX.GUS and PVX.NPT, respectively, and tomato plants with PTGS of polygalacturonase (PG) were resistant to PVX.PG. Sijen et al. (1996)
also observed that N. benthamiana plants with PTGS of the movement protein (MP) of cowpea mosaic comovirus (CPMV) displayed resistance to PVX.MP.
N. benthamiana infected with TMVGFPNP did not show green fluorescence and the appearance of systemic symptoms was delayed by 68 days compared with plants infected with TMVGFP. Casper & Holt (1996) reported that the approximately 200 bp 3' UTR in the GFP cDNA inhibited GFP expression drastically from TMV. Thus, the N gene sequence at the 3' end of the GFP gene might have severely inhibited transcription and/or translation of the virus subgenomic mRNA.
Studies with CPMV and PVX suggest that there are preferential sites on the transgene mRNA molecules that act as signals for sequence-specific degradation (Sijen et al., 1996 ; English et al., 1996
). The 640 bp 3' region of the transcribed MP transgene of CPMV and the ~700 bp 3' region of the GUS transgene might have been responsible for elimination of the incoming chimeric PVX in those studies (PVX.MP and PVX.GUS, respectively). However, our data show that a single transgene confers resistance to both a tospovirus and TMVGFP, suggesting either that there are multiple signals on the mRNA molecule for degradation or that, once the degradation process is triggered by some specific secondary structure or feature of the RNA, the process is capable of degrading any RNA molecule that shares sequence similarity with the transgene. The latter hypothesis does require a minimum length of homologous sequence to be effective, which is consistent with the data presented here. A similar result was reported by Seymour et al. (1993)
, who showed that the PG and pectinesterase (PE) genes were coordinately down-regulated in transgenic tomato plants transformed with a chimeric gene construct containing the 244 bp 5' end of PG fused to the 5' end of a 1320 bp PE. Moreover, our results are consistent with the recent observation of Marano & Baulcombe (1998)
, who showed that transgenic tobacco plants containing the TMV-U1 54 kDa replicase gene were resistant to PVX vectors containing the first half, middle half or second half of the replicase gene.
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Acknowledgments |
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Footnotes |
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c Present address: BB5K, Monsanto Company, 700 Chesterfield Village Pkwy N., St Louis, MO 63198, USA.
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References |
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Received 16 July 1999;
accepted 14 September 1999.