Department of Plant Pathology, Cornell University, NYSAES, Geneva, NY 14456, USA1
Seminis Vegetable Seeds, 37437 State Highway 16, Woodland, CA 95695, USA2
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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Squash mosaic virus (SqMV) is a seed-borne, beetle-transmitted comovirus with isometric virus particles about 30 nm in diameter (Campbell, 1971 ). The bipartite viral genome of comoviruses consists of two single-stranded, positive-sense RNA molecules, designated as bottom-component RNA (B-RNA) and middle-component RNA (M-RNA), of about 6000 and 4200 nucleotides, respectively (Goldbach & Wellink, 1996
). Each RNA contains a genome-linked protein (VPg) at the 5' end and has a polyadenylated tract at the 3' end (Goldbach & Wellink, 1996
). Both RNAs are translated into polyproteins from which the functional proteins are derived by proteolytic cleavages. The M-RNA encodes the coat proteins (CP) (42 and 22 kDa) and cell-to-cell movement proteins (Franssen et al., 1982
; Wellink & van Kammen, 1989
) while the B-RNA encodes the VPg, replicase and protease proteins (Lomonossoff & Shanks, 1983
). The sequence for the SqMV CP genes has been determined and is located in the 3' region of the M-RNA (Hu et al., 1993
; Haudenshield & Palukaitis, 1998
).
Recently, we reported that transgenic squash lines expressing both CP genes of the melon strain of SqMV displayed varying reactions to SqMV (Pang et al., 2000 ). The susceptible line SqMV-22 had a high steady-state transgene transcript level but low transcription rate compared with the resistant line SqMV-127, which showed PTGS. Line SqMV-3, designated as a recovery line, initially showed systemic infection but newly developing leaves were free of symptoms 2040 days after inoculation and these leaves were resistant to SqMV infection (Pang et al., 2000
).
This communication reports the further characterization of the recovery phenotype of SqMV-3 and the phenotypes of plants that contain combinations of transgene inserts from lines SqMV-127, -3 and -22. The recovery phenotype of SqMV-3 was observed under greenhouse and field conditions and is due to PTGS being activated at later developmental stages rather than being induced by virus infection. We also show that resistant progeny can be obtained by combining transgenes from the susceptible SqMV-22 and recovery SqMV-3 lines.
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Methods |
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Inoculation of transgenic plants.
The virus isolate, SqMV m-88, was obtained originally from infected melon seeds (Hu et al., 1993 ) and typed as the melon strain by biological comparisons with the type strain (Nelson & Knuhtsen, 1973
). Inocula were prepared by propagating the virus in zucchini squash (Cucurbita pepo L.) and grinding infected leaves in 0·01 M phosphate buffer (pH 7·0). Fifteenfold-diluted leaf extracts were immediately rubbed onto the three upper carborundum-dusted leaves of squash plants and the inoculated leaves were subsequently rinsed with water. Plants were observed for symptoms every other day for at least 60 days. The reaction of transgenic squash to SqMV was confirmed by a double antibody sandwich (DAS)-ELISA (Clark & Adams, 1977
) with a polyclonal antibody against the virion of SqMV (Hu et al., 1993
).
ELISA, Northern and Southern blot analysis of transgenic plants.
Antibodies to SqMV (Hu et al., 1993 ) were used in DAS-ELISA (Clark & Adams, 1977
) to detect virus infection and an nptII ELISA kit (5 Prime to 3 Prime) was used to detect the nptII enzyme in transgenic plants. Total DNA isolated according to the method of Doyle & Doyle (1990)
was used in Southern blots. The number of transgene inserts was estimated by the number and the size of expected fragments generated by digestion with BglII and EcoRI or BglII and XhoI. A BglII site is located in the 42 kDa CP gene just after its initiation codon and another is located in the polylinker of the pGA482G vector (Pang et al., 2000
). Thus, digestion with BglII generates a fragment with the 22 kDa CP transgene plus plant genomic DNA. The XhoI and EcoRI enzymes were used to reduce the size of the 22 kDa CP transgene plus genomic DNA fragment by cutting within the plant genomic DNA. Restriction enzyme-digested DNA was separated on agarose gels and then blotted onto GeneScreen Plus membrane (Dupont). For Northern blots, total RNA was isolated by using the procedure described by Napoli et al. (1990)
. Ten µg total RNA was applied in each well and separated by electrophoresis on a formaldehyde-containing agarose gel (Sambrook et al., 1989
). The agarose gels were stained with ethidium bromide to compare the relative amounts of total plant RNA in each well. The Northern and Southern blots were probed with only the 22 kDa CP because Pang et al. (2000)
had shown previously that the 22 kDa CP and the 42 kDa CP transgenes were coordinately expressed in the transgenic lines under investigation. Hybridization conditions for Southern and Northern blots were chosen according to the protocol of the GeneScreen Plus membrane (Dupont) and random-priming methods (Feinberg & Vogelstein, 1983
) were used to generate probes specific to the 22 kDa CP. Images of some autoradiograms were photographed with a COHU CCD camera, model 4915-2000. Signals were quantified by using the US National Institutes of Health Image program version 1.59.
Isolation of nuclei and nuclear run-on transcription assays.
Isolation of nuclei and nuclear run-on transcription assays were performed essentially as described by Dehio & Schell (1994) . The same amount of labelled RNA was used for hybridization to replicated Southern blot membranes that contained 0·2 µg restriction enzyme-digested, electrophoretically separated fragments of the 22 kDa CP, 42 kDa CP, actin and nptII genes prepared similarly, as described previously (Pang et al., 1997
).
Field evaluation.
The R1 seeds of three transgenic squash lines as well as those of non-transformed controls were germinated in a plastic house on July 19, 1995 and seedlings were screened by nptII ELISA to identify the non-transgenic segregants from the R1 transgenic populations. Plants (48 per line) from each of the three transgenic squash lines and the non-transformed control were planted in the field on July 28, 3 feet (~1 m) apart within the row and 6 feet (~2 m) between rows, by using a randomized block design. Twelve to fourteen plants for each transgenic lines and non-transformed plants were inoculated at 1, 3 or 5 weeks post-transplantation. SqMV m-88-infected leaf extracts (1 g infected tissue in 15 ml buffer) were applied to the three upper leaves of squash plants. Observations were made twice a week until the fruits became mature. The reaction of transgenic squash to SqMV was confirmed by DAS-ELISA as described above.
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Results |
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As expected, all inoculated SqMV-22 progeny developed systemic symptoms, while nearly all (25/27) SqMV-127 progeny displayed resistant phenotypes regardless of when the plants were inoculated (Table 1). However, the reactions of SqMV-3 progeny ranged from susceptible (4/9) to recovery (5/9) phenotype when small plants (17 DAG) were inoculated, while some SqMV-3 plants were resistant (3/15) when large plants (31 and 45 DAG) were inoculated (Table 1
).
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Discussion |
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Our results demonstrate that the recovery phenotype of transgenic squash expressing the CP genes of SqMV is due to the activation of PTGS at later plant developmental stages (Table 1; Fig. 1
) rather than by virus infection, as reported by Lindbo et al. (1993)
and Guo & García (1997)
. Thus, at least two types of recovery phenotypes can be observed in transgenic plants: virus-induced (tobacco etch virus, Lindbo et al., 1993
; plum pox virus, Guo & García, 1997
) and development-induced recovery (SqMV, this research). The relationship of PTGS and consequent virus resistance to the age of the leaves was observed in transgenic lettuce expressing the nucleocapsid gene of tomato spotted wilt tospovirus (Pang et al., 1996
). In that study, transgenic hemizygotes and, in some cases, homozygotes consisted of unsilenced lower leaves and silenced upper leaves. However, no recovery phenotypes were observed with plants that were inoculated at the earliest stage. In addition, developmental PTGS has already been observed in transgenic plants (Vaucheret et al., 1995
; Kunz et al., 1996
; Balandin & Castresana, 1997
). For example, Balandin & Castresana (1997)
showed that gene silencing occurs a few weeks after seed germination and is maintained throughout vegetative growth and floral development in all leaves of the plant.
The recovery line SqMV-3 contained two inserts of CP genes and young plants hemizygous for the CP transgene displayed susceptible or recovery phenotypes. However, increasing the gene dosage by self-pollination or by crossing with the susceptible phenotype (SqMV-22) resulted in plants that displayed the resistant phenotype. This resistant phenotype was due to activation of PTGS at an early stage, as observed by nuclear run-on experiments (Fig. 4; Pang et al., 2000
). These results confirm the transgene-dosage effects on PTGS and virus resistance that have been reported in the literature (Smith et al., 1994
; Goodwin et al., 1996
; Mueller et al., 1995
; Pang et al., 1996
). Thus, our data suggest that resistant plants can be obtained by combining transgenes from susceptible and recovery phenotypes. This approach could have practical applications.
We have shown that the presence of the transgene insert of the resistant line SqMV-127 can silence homologous transgenes from recovery and susceptible transgenic lines, as evidenced by lowered SqMV-3/-22 transgene transcript accumulation in progeny from crosses of SqMV-3x127 and SqMV-22x127 (Table 3; Fig. 3
). A similar observation was reported by Mueller et al. (1995)
, who showed that virus resistance in transgenic tobacco expressing the RNA polymerase gene of potato virus X is associated with the ability of the transgene to silence homologous transgenes from susceptible plants.
It has been reported the PTGS can be affected by environmental factors (Pang et al., 1996 ; Vaucheret et al., 1995
; Kunz et al., 1996
). Consideration of these factors could be important, as engineered resistance in crops needs to be effective under field conditions. Our field experiments showed that SqMV resistance is apparently stable under field conditions. These observations suggest that transgenic SqMV resistance under field conditions should have practical applications (this work; Pang et al., 2000
). This development is especially significant since natural resistance to SqMV in squash and melons has not been identified (Provvidenti, 1993
; Campbell, 1971
).
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Acknowledgments |
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Footnotes |
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References |
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Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell 101, 235-238.[Medline]
Baulcombe, D. C. (1996). Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8, 1833-1844.
Baulcombe, D. C. & English, J. J. (1996). Ectopic pairing of homologous DNA and post-transcriptional gene silencing in transgenic plants. Current Opinion in Biotechnology 7, 173-180.
Beachy, R. N. (1997). Mechanisms and applications of pathogen-derived resistance in transgenic plants. Current Opinion in Biotechnology 8, 215-220.[Medline]
Campbell, R. N. (1971). Squash mosaic virus. CMI/AAB Descriptions of Plant Viruses no. 43.
Clark, M. F. & Adams, A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34, 475-483.[Abstract]
Dawson, W. D. (1996). Gene silencing and virus resistance, a common mechanism. Trends in Plant Science 1, 107-108.
Dehio, C. & Schell, J. (1994). Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing. Proceedings of the National Academy of Sciences, USA 91, 5538-5542.[Abstract]
Dougherty, W. G. & Parks, T. D. (1995). Transgenes and gene suppression: telling us something new? Current Opinion in Cell Biology 7, 399-405.[Medline]
Doyle, J. J. & Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus 12, 13-15.
English, J. J., Mueller, E. & Baulcombe, D. C. (1996). Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8, 179-188.
Feinberg, A. P. & Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6-13.[Medline]
Franssen, H., Goldbach, R., Broekhuijsen, M., Moerman, M. & van Kammen, A. (1982). Expression of middle-component RNA of cowpea mosaic virus, in vitro generation of a precursor to both capsid proteins by bottom-component RNA-encoded protease from infected cells. Journal of Virology 41, 8-17.
Goldbach, R. W. & Wellink, J. (1996). Comoviruses, molecular biology and replication. In The Plant Viruses, pp. 35-76. Edited by B. D. Harrison & A. F. Murant. New York: Plenum Press.
Goodwin, J., Chapman, K., Swaney, S., Parks, T. D., Wernsman, E. A. & Dougherty, W. G. (1996). Genetic and biochemical dissection of transgenic RNA-mediated virus resistance. Plant Cell 8, 95-105.
Guo, H. S. & García, J. A. (1997). Delayed resistance to plum pox potyvirus mediated by a mutated RNA replicase gene: involvement of a gene-silencing mechanism. Molecular PlantMicrobe Interactions 10, 160-170.
Hamilton, A. J. & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952.
Haudenshield, J. S. & Palukaitis, P. (1998). Diversity among isolates of squash mosaic virus. Journal of General Virology 79, 2331-2341.[Abstract]
Hu, J. S., Pang, S.-Z., Nagpala, P. G., Siemieniak, D. R., Slightom, J. L. & Gonsalves, D. (1993). The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts. Archives of Virology 130, 17-31.[Medline]
Kunz, C., Schob, H., Stam, M., Kooter, J. M. & Meins, F. J. (1996). Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression. Plant Journal 10, 437-450.
Lindbo, J. A., Silva, R. L., Proebsting, W. M. & Dougherty, W. G. (1993). Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5, 1749-1759.
Lomonossoff, G. P. & Shanks, M. (1983). The nucleotide sequence of cowpea mosaic virus B RNA. EMBO Journal 2, 2253-2258.
Metzlaff, M., ODell, M., Cluster, P. D. & Flavell, R. B. (1997). RNA-mediated RNA degradation and chalcone synthase A silencing in petunia. Cell 88, 845-854.[Medline]
Montgomery, M. K. & Fire, A. (1998). Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends in Genetics 14, 255-258.[Medline]
Mueller, E., Gilbert, J., Davenport, G., Brigneti, G. & Baulcombe, D. C. (1995). Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant Journal 7, 1001-1013.
Napoli, C., Lemieux, C. & Jorgensen, R. (1990). Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279-290.
Nelson, M. R. & Knuhtsen, H. K. (1973). Squash mosaic virus variability: review and serological comparisons of six biotypes. Phytopathology 63, 920-926.
Pang, S.-Z., Jan, F.-J., Carney, K., Stout, J., Tricoli, D. M., Quemada, H. D. & Gonsalves, D. (1996). Post-transcriptional transgene silencing and consequent tospovirus resistance in transgenic lettuce are affected by transgene dosage and plant development. Plant Journal 9, 899-909.
Pang, S.-Z., Jan, F.-J. & Gonsalves, D. (1997). Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proceedings of the National Academy of Sciences, USA 94, 8261-8266.
Pang, S.-Z., Jan, F.-J., Tricoli, D. M., Russell, P. F., Carney, K. J., Hu, J. S., Fuchs, M., Quemada, H. D. & Gonsalves, D. (2000). Resistance to squash mosaic comovirus in transgenic squash plants expressing its coat protein genes. Molecular Breeding 6, 87-93.
Prins, M. & Goldbach, R. (1996). RNA-mediated virus resistance in transgenic plants. Archives of Virology 141, 2259-2276.[Medline]
Provvidenti, R. (1993). Resistance to viral diseases of cucurbits. In Resistance to Viral Diseases of Vegetables, pp. 8-43. Edited by M. M. Kyle. Portland, OR: Timber Press.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual,2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schiebel, W., Pelissier, T., Riedel, L., Thalmeir, S., Schiebel, R., Kempe, D., Lottspeich, F., Sanger, H. L. & Wassenegger, M. (1998). Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato. Plant Cell 10, 2087-2101.
Smith, H. A., Swaney, S. L., Parks, T. D., Wernsman, E. A. & Dougherty, W. G. (1994). Transgenic plant virus resistance mediated by untranslatable sense RNAs: expression, regulation, and fate of nonessential RNAs. Plant Cell 6, 1441-1453.
van den Boogaart, T., Lomonossoff, G. P. & Davies, J. W. (1998). Can we explain RNA-mediated virus resistance by homology-dependent gene silencing? Molecular PlantMicrobe Interactions 11, 717-723.
Vaucheret, H., Palauqui, J. C., Elmayan, T. & Moffatt, B. (1995). Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants. Molecular & General Genetics 248, 311-317.[Medline]
Waterhouse, P. M., Graham, M. W. & Wang, M. B. (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences, USA 95, 13959-13964.
Wellink, J. & van Kammen, A. (1989). Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins. Journal of General Virology 70, 2279-2286.
Received 2 December 1999;
accepted 12 May 2000.