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) has been reported to be one of the major mechanisms of RNA-mediated resistance in transgenic plants (Dougherty & Parks, 1995 ; Baulcombe, 1996
; Baulcombe & English, 1996
; Dawson, 1996
; Prins & Goldbach, 1996
; Beachy, 1997
; van den Boogaart et al., 1998
). Recently, we showed that transgenes consisting of ~400 bp segments of the N gene of TSWV conferred resistance to TSWV in transgenic plants through PTGS (Pang et al., 1997
), but N gene segments of 92235 bp did not. However, transgenic plants expressing transgenes consisting of 218 or 110 bp N gene segments linked to the 720 bp green fluorescent protein (GFP) gene were resistant to TSWV (Pang et al., 1997
). Further work showed that transgenic plants with transgenes consisting of N gene segments of 59 or 24 bp similarly linked to GFP were not resistant to TSWV, even though the transgene showed PTGS (Jan et al., 2000
). Collectively, these data suggested that segments of the N gene of 110 bp or more could confer resistance when linked to a transcribed DNA (designated silencer DNA) that induces PTGS. This observation opened the possibility of developing multiple virus resistance in transgenic plants by using viral DNA as a silencer and linking it to segments of other viral DNA.
In order to test this strategy for multiple virus resistance, we linked the 867 bp TuMV CP gene (Jan et al., 1999 ) to a 218 bp N gene segment of TSWV and transferred the construct into Nicotiana benthamiana. The CP gene of TuMV was used as a silencer DNA because TuMV is considered to be the most important virus of cultivated cruciferous cash crops (Green & Deng, 1985
) and it is known that the CP gene of TuMV induces PTGS and confers resistance to TuMV (Jan et al., 1999
). Here, we report that transgenic plants expressing a transgene consisting of TuMV CP fused with a 218 bp N gene segment of TSWV are resistant to TSWV and TuMV via the PTGS mechanism.
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Methods |
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Inoculation of transgenic plants.
TuMV-ESC8 and TSWV-BL were propagated in turnip cultivar Presto and N. benthamiana, respectively. For inoculations with single viruses, inocula (1:30 dilutions of crude sap) were prepared by grinding TuMV- or TSWV-infected leaves (1 g) in 30 ml 10 mM phosphate buffer (pH 7·0) or buffer 4 (0·033 M KH2PO4, 0·067 M K2HPO4 and 0·01M Na2SO3), respectively. Inocula consisting of both TuMV and TSWV were prepared by mixing equal aliquots of 1:15 dilutions of crude sap from plants infected with each of these viruses, using buffer 4 to prepare extracts. Test plants were inoculated at the 57 leaf stage, except where indicated, by rubbing leaf extracts onto carborundum-dusted leaves and subsequently rinsing the leaves with water. To monitor for the possibility of escapes, control non-transformed plants were inoculated in each experiment and each batch of inoculum extract was applied first to transgenic plants and then to control plants. Inoculated plants were grown in the greenhouse and observed daily for at least 45 days.
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Results |
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A total of 23 R0 transgenic plants with TuMVCP+3/4N and 21 with TuMVCP+5/8N were initially confirmed to be transgenic by PCR analysis and nptII ELISA. Northern analysis of some R0 plants showed that the level of TuMV CP transcript was tightly correlated with that of the N gene transcript, indicating that they are transcribed as a single transcription unit as designed (data not shown). All of the R0 plants were self-pollinated to produce R1 seeds for inoculation tests.
A TSWV N gene segment fused to the TuMV CP gene confers resistance to both viruses
Prior to testing for resistance, R1 seedlings from the transgenic lines were screened by nptII ELISA to identify transgenic and non-transgenic plants in the populations. Unlike negative-control plants, which were all susceptible to the virus, a proportion of R1 plants expressing the TuMVCP+3/4N and TuMVCP+5/8N transgenes were resistant to TuMV and TSWV. Table 1 summarizes the reactions of the inoculated R1 plants to TSWV-BL and TuMV-ESC8. The reactions could be grouped into three different categories: (i) resistant to both TuMV and TSWV, (ii) resistant to TuMV but susceptible to TSWV and (iii) susceptible to both TuMV and TSWV. No resistance to TSWV and TuMV was observed in a total of 262 plants examined from nine of the 18 TuMVCP+3/4N transgene lines (Table 1
). Some plants from four of the other nine lines (55-4, 55-6, 55-9, 55-17) displayed resistance to TSWV and TuMV while, interestingly, the five remaining lines (55-8, 55-11, 55-16, 55-20, 55-21) had plants that were resistant to TuMV but susceptible to TSWV (Table 1
). In contrast, plants resistant to TSWV and TuMV were found in only one of the 14 tested lines with the TuMVCP+5/8N transgene (Table 1
).
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Discussion |
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Transgenic plants with multiple resistance have also been generated previously by combining the entire CP genes of more than one virus, with each gene being driven by a promoter and a terminator. Transgenic potato expressing the CP genes of potato virus X and potato virus Y were resistant to both viruses (Lawson et al., 1990 ; Kaniewski et al., 1990
). Similarly, transgenic tobacco with resistance to three tospoviruses (TSWV, tomato chlorotic spot virus and groundnut ringspot virus) (Prins et al., 1995
), transgenic squash with resistance to two or three viruses [cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV2) and zucchini yellow mosaic virus (ZYMV)] (Fuchs & Gonsalves, 1995
; Tricoli et al., 1995
; Fuchs et al., 1998
), transgenic cantaloupe with resistance to CMV, ZYMV and WMV2 (Fuchs et al., 1997
) and transgenic tomato with resistance to CMV isolates of subgroups I and II (Kaniewski et al., 1999
) have been developed. Our approach differs from these in that we need only one transgene with a promoter and a terminator and the transgene may contain only a segment of one of the viral genes.
PTGS is homology dependent and is the apparent mechanism for RNA-mediated virus resistance. It is, thus, reasonable to think that transgenic plants with resistance to three or more viruses can be obtained simply by transforming plants with a transgene consisting of fused gene segments of different viruses linked to a silencer DNA (e.g. GFP, TuMV CP). Recent data from our laboratory provide support for this approach (unpublished results). N. benthamiana plants with a chimeric transgene consisting of three small N gene segments of three different tospoviruses linked to GFP DNA were resistant to all three tospoviruses. This novel approach has several advantages for controlling viruses. Firstly, multiple resistance could be generated to desired viruses. Secondly, the small non-translatable segments minimize the risks of recombination, transcapsidation, synergism or complementation, which have been raised as disadvantages of some strategies that use full-length viral genes. Thirdly, a chimeric transgene requires only limited genetic elements for expression in transgenic plants, thus reducing the demand for genetic elements to engineer multiple traits into a single biotech product. Finally, the CP gene has been shown to be very effective in conferring resistance and many viral CP genes have been identified and are readily available. In addition, it is likely that this strategy could be used in functional genomics to down-regulate multiple genes coordinately in plants, to identify genes responsible for biochemical pathways or to develop new biotech products with multiple traits.
Similar co-suppression of two genes in a chimeric transgene has been described in a few reports. Introduction in tomato of a chimeric gene construct containing 244 bp of the 5' end of the polygalacturonase gene linked to the 5' end of a 1320 bp pectinesterase gene was able to trigger co-suppression of both genes (Seymour et al., 1993 ). Gene constructs consisting of the
-glucuronidase gene (uidA) linked to the full-length chalcone synthase (chsA) cDNA or the 5' half or the 3' half triggered PTGS of both genes in transgenic petunia (Van Blokland et al., 1994
; Stam et al., 1997
). These results, along with our own, show that silencer sequences are effective when they are located either at the 5'-end (Fig. 1
; Van Blokland et al., 1994
; Stam et al., 1997
; Jan et al., 2000
) or the 3'-end (Seymour et al., 1993
) of chimeric transgenes. Moreover, our results are consistent with observations from other groups, who have shown that there are multiple target sites for PTGS along the transgene. Marano & Baulcombe (1998)
showed that transgenic tobacco plants containing the TMV-U1 54 kDa replicase gene were resistant to potato virus X vectors containing the first half, the middle half or the second half of the replicase gene. Jacobs et al. (1999)
reported that sequences throughout the basic
-1,3-glucanase mRNA coding region are targets for PTGS in transgenic tobacco.
We observed that the 110 bp N gene segment was much less efficient than the 218 bp segment in conferring resistance to TSWV (Table 2), confirming our previous observations (Pang et al., 1997
; Jan et al., 2000
). This also suggests that the minimum N gene length for conferring resistance is between 59 and 110 bp, since the former length does not confer resistance (Jan et al., 2000
). However, the minimum gene segment length for conferring resistance to other viruses or for inactivating other homologous mRNAs probably differs from our observations with the N gene. Recently, Crété & Vaucheret (1999)
showed that transformation of chimeric nii1uidA, uidAnii1 and nii1uidAnii1 transgenes carrying 186 bp of the 5' end and/or 241 bp of the 3' end of the tobacco nitrite reductase nii1 cDNA fused with the uidA gene did not trigger co-suppression of endogenous nii genes in transgenic tobacco. A possible reason for these results might be that the minimum length of nii gene required to trans-inactivate the nii genes is larger than 241 bp. Alternatively, the 5'- and 3'-terminal regions of the nii1 gene may be relatively inefficient targets for PTGS, as was recently reported by Jacobs et al. (1999)
for transgenic tobacco with the gn1
-1,3-glucanase gene.
Interestingly, we also obtained plants that were resistant to TuMV (e.g. line 55-21 in Tables 1 and 2
) and susceptible to TSWV, but not vice versa. These results suggest that there are differences between regions of a transgene in the effectiveness at conferring resistance within the same plant. Logically, one would assume that the chances of obtaining resistance to a particular virus via PTGS get higher as the length of the homologous DNA segment, and thus the target area for degradation, increases. Further analyses of these plants should shed light on the reasons for the occurrence of plants with resistance only to TuMV. Such studies will provide insights into the most effective gene segment lengths to use in developing multiple virus resistance by the above approach.
In summary, we have shown that transgenic plants with resistance to both a potyvirus and a tospovirus can be obtained by fusing a segment of the tospovirus N gene to the potyvirus CP gene. This provides a simple approach to develop plants with resistance to two viruses. More importantly, this work provides strong evidence that transgenic plants with resistance to three or more viruses can be obtained by transforming plants with a transgene consisting of viral segments linked to a DNA silencer, which could also be a viral gene. The practical value of multiple virus resistance is obvious, since crops are frequently infected with more than one virus.
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Acknowledgments |
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Footnotes |
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c Present address: BB5F, Monsanto Company, 700 Chesterfield Village Pkwy N., St Louis, MO 63198, USA.
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References |
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Received 20 January 2000;
accepted 10 April 2000.
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