- Open Access
Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus
© Hu et al; licensee BioMed Central Ltd. 2011
- Received: 15 November 2010
- Accepted: 27 January 2011
- Published: 27 January 2011
An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses.
Inverted repeats of the partial Tobacco mosaic virus (TMV) movement protein (MP) gene and the partial Cucumber mosaic virus (CMV) replication protein (Rep) gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58) immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32) immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃) did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA.
We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.
- Transgenic Plant
- Tobacco Mosaic Virus
- Cucumber Mosaic Virus
- Transgenic Tobacco Plant
- Movement Protein
The plant disease caused by Tobacco mosaic virus (TMV) or Cucumber mosaic virus (CMV) is found worldwide. The two viruses are known to infect more than 150 species of herbaceous, dicotyledonous plants including many vegetables, flowers, and weeds. TMV and CMV cause serious losses on several crops including tobacco, tomato, cucumber, pepper and many ornamentals. During the last decade, several laboratories have tried to introduce resistance to TMV or CMV by genetic engineering. Virus resistance in plants containing virus-derived transgene, usually by the expression of functional or dysfunctional coat protein, movement protein or polymerase gene, has been widely reported. The TMV coat protein gene was used in the first demonstration of virus-derived, protein-mediated resistance in transgenic plants . Pathogen-derived resistance for CMV often showed only partial resistance or very narrow spectrum of resistance to the virus .
RNA silencing or post-transcriptional gene silencing (PTGS), developed during plant evolution, functions as a defense mechanism against foreign nucleic acid invasions (viruses, transponsons, transgenes) . Since the phenomenon of RNA silencing was first observed by Napoli , research has been carried out to elucidate its mechanism. PTGS is a mechanism closely related to RNA interference, which is involved in plant defense against virus infection [5, 6]. It was found that when a inverted repeated sequences of partial cDNA from a plant virus are introduced into host plants for expression of dsRNA and induction of RNA silencing, the transgenic plants can silence virus corresponding gene and are resistant to virus infection [7, 8]. More than 90% of transgenic Nicotiana benthamiana lines were resistant to the virus when engineered with hairpin constructs using Plum pox virus P1 and Hc-Pro genes sequences under the 35S-cauliflower mosaic virus promoter . For the current study, we expressed the partial TMV movement protein (MP) gene and the partial CMV replication protein (Rep) gene in the form of an intermolecular intron-hairpin RNA in transgenic tobacco. We analyzed the resistance of T0 to T4 transgenic plants. We found that the two T4 transgenic lines with single copy were completely resistant to the corresponding virus, and the viral resistance of transgenic plants did not be affected by the low temperature (15℃).
Transformation and analysis of T0 plants
Testing of T0 and T1 transgenic plants for TMV or CMV resistance.
T0 line number
T0 reaction to TMVa
T0 reaction to CMV
T0 copy number of transgene (Southern)
T1 segregation immune:susceptible
3 ~ 4
Resistant response of T0 to T4 transgenic progenies to infection of TMV or CMV
The successive generation seeds were obtained by self-pollination from inoculated plants and the progenies of different lines were gained simultaneously for further analyses. Seedlings per each line were randomly taken from the resultant regenerates for virus inoculation tests. The T1 progenies of T0 parental lines, MP16, MP31, MP39, MP53, Rep15, Rep17, Rep25 and Rep53 contained some plants that were immune and others that were susceptible, whereas the T0 parental line MP36 or Rep727 which was susceptible to the virus yielded only susceptible progenies in successive generations (Table 1). The progeny of T0 lines MP16 and Rep15 was confirmed to a have a segregation ratio of 3:1 (immune: susceptible), suggesting the presence of a single dominant transgene locus in each line, and Southern blot analysis revealed that the loci each appear to have a single transgene (Table 1).
Segregation of TMV or CMV resistance over the T2, T3 and T4 generations of Nicotiana tabacum transformed with inverted repeats of the partial TMV movement protein (MP) gene or the partial CMV Replication protein (Rep) gene.
No. of T2plants showing R/Sa
No. of T3plants showing R/S
Responses of T4progenies
Comparative analysis of the T2 or T4 transgene and the mode of expression in terms of resistance
The virus-specific siRNA was detected by Northern blot analysis of low weight RNAs prepared from the leaves of T4 transgenic and non-transgenic tobacco plants using [α-32P]dCTP-labelled partial MP or Rep gene as a probe and the result showed distinct hybridization signal bands of expected size for siRNA (approximately 21-24 nts, homologous to the MP or Rep transcripts) only existed in immune transgenic plants whether virus was inoculated or not. No siRNA could be detected in healthy wild-type control plants (Figure 4C and 4D).
T4 transgenic plant height and seed weight comparing with wild plant
Reaction to virus
Height per plant (m)
Seed weight per plant (g)
Mean ± SEf
Mean ± SEf
1.298 ± 0.101 a
3.251 ± 0.392 a
1.237 ± 0.094 a
3.067 ± 0.586 a
0.573 ± 0.101 b
0.665 ± 0.108 b
1.251 ± 0.074 a
3.472 ± 0.454 a
1.076 ± 0.083 a
2.879 ± 0.363 a
1.195 ± 0.065 a
2.794 ± 0.331 a
0.795 ± 0.069 b
1.257 ± 0.255 b
1.254 ± 0.089 a
2.974 ± 0.327 a
RNA silencing-based virus resistance phenotypes were kept at low temperature
CMV infection did not break resistance to TMV in transgenic tobacco plants expressing TMV hairpin MP RNA
TAS-ELISA detection of T4 transgenic and wild type plants inoculated with TMV/CMV, CMV/TMV or TMV+CMV.
Numerous examples of pathogen-derived resistance have been reported for a wide range of plant viruses. Transgenic plants expressing viral coat proteins have been successfully conferred the resistance to the corresponding viruses [1, 11, 12]. Expression of sequences corresponding to other viral genes have also become a successful tool for inducing pathogen-derived resistance, such as replicase gene [13–16], protease gene [17, 18] and movement protein gene [19–21]. Transgenic pants expressing dsRNA by introduction of an inverted repeat sequence, spaced by an intron, into plants could reach 90% efficiency of gene silencing [22, 23]. An effective method for obtaining resistant transgenic plants is therefore to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many viruses [7, 9, 24–30]. We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene or CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection. Due to the dsRNA nature, engineered specific RNA molecules were targeted for degradation, so only small steady-state amounts of the actual hairpin transcripts could be expected in the transgenic lines [28, 31, 32]. Our results also showed only very little transcript of the transgene could be detected after or before virus inoculation. Occurrence of siRNA is one of the most important characteristics of RNA silencing and can be a reliable molecular marker that is closely associated with viral resistance in transgenic plants expressing viral genes [31, 33, 34]. We also found siRNAs characteristic to RNA silencing were detected to accumulate in high levels in resistant transgenic plants whether virus was inoculated or not. These results indicated that TMV or CMV resistance observed in the resistant transgenic tobacco plants is attributed to RNA silencing.
Multiple complex patterns of transgene integration have been detected in many species such as tomato , cereal [7, 35] and wood perennial tree (Prunus domestica) . No general conclusions can be made as to whether a second copy of the transgene would increase the likelihood of virus resistance , so it is suggested no correlation between the copy number of insertions and types of RNA silencing [36, 37]. We also found no correlation between the resistance and the copy number of the transgene.
Kalantidis K et al.  reported the concentration of siRNA reached a plateau at 30 days post-germination (one month) and then remained stable in the course of further development (two months). But Missiou et al.  reported that the accumulation and composition of transgene-specific siRNA was changed when plants were grown. Our results showed that there was little change of accumulation and composition of siRNAs at both one and three months after virus inoculation.
Plant-virus interactions are strongly modified by environmental factors, especially by temperature. High temperature is frequently associated with attenuated symptoms and with low virus content . But rapid spread of virus disease and development of severe symptoms are frequently associated with low temperature . Studied have shown that low temperature inhibited the accumulation of siRNAs in insect, plant and mammalian cells [10, 40, 41]. At low temperature, RNA silencing induced by virus or transgene was inhibited, which leads to enhancing virus susceptibility, to loss of silencing-mediated transgenic phenotypes and to dramatically reducing the level of siRNA, but the accumulation level of miRNA was not influenced by temperature . So RNA silencing-based transgenic phenotypes were reported to be lost at low temperature (15°C). We found that RNA silencing-based transgenic phenotypes were not lost at low temperature (15°C). The virus siRNAs level was stable at both 24°C and 15°C and no obvious decrease of virus siRNAs accumulation was found at 15°C as compared with that at 24°C. Bonfim et al.  reported that the amount of siRNA at 25 °C showed a slight decrease as compared with that at 15 °C compared, but they did not test whether the resistance of transgenic bean plants with an intron-hairpin construction was influent. The differences of low temperature on RNA silencing-based transgenic phenotypes were unknown.
The PTGS pathway can be inhibited by suppressors encode by plant viruses [42, 43]. The 2b protein of CMV suppresses PTGS by directly interfering with the activity of the mobile silencing signal [44, 45]. Guerini and Murphy  reported that Capsicum annum cv. Avelar plants resisted systemic infection by the Florida isolate of Pepper mottle potyvirus (PepMoV-FL). However, co-infection of Avelar plants with CMV alleviated this restricted movement, allowing PepMoV-FL to invade young tissues systemically. Our results showed that the TMV-resistant transgenic tobacco plants were clearly not impacted by the suppressor, the 2b protein of CMV.
It's clear that regardless of the mechanistic details, the expression of viral dsRNA seems to be a highly efficient way to engineer virus-resistant plants, and the resistance induced by the hairpin RNA can be stably inherited through self-pollination for the fourth generations. Through this strategy, we can select for the most promising lines that are immune to viruses. Besides the high efficiency for generating transgenic plants resistant to a viral pathogen, the RNA-mediated resistance is good for environmental biosafety over the different protein mediated resistance as potential risks of heterologous encapsidation and recombination of virus are diminished.
We expressed the partial TMV movement protein (MP) gene and the partial CMV replication protein (Rep) gene in the form of an intermolecular intron-hairpin RNA in transgenic tobacco. We analyzed the resistance of T0 to T4 transgenic plants. We found that T4 transgenic lines with single copy were completely resistant to the corresponding virus, and viral resistance of transgenic plants did not be affected by the low temperature (15℃). No significant correlation between the resistance and the copy number of the transgene was found. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA.
Plant material and viruses
Nicotiana tabacum cv. Yunyan 87 was provided by Dr. Liu Yong (Yunnan Institute for Tobacco Science, China). TMV and CMV were isolated by the author's laboratory and maintained on Nicotiana tabacum cv. Xanthi nn in greenhouse.
Construction of plant expression plasmids
Plant expression vector pBIN-TMVΔMP(i/r), which contains inverted repeats of partial TMV MP gene (ΔMP) separated by the soybean intron was constructed previously . For plant expression plasmid containing inverted repeats of CMV partial Rep gene (ΔRep) (Figure 1), specific primersΔRep-F (CGGTCGAC GATAACTAAGTGGTGG, underline was Sal I site) and ΔRep-R (CGATCGAT CCAGACTTCTTGTATTTC, underline was Cla I site) designed according to the published CMV Rep gene (D00355) were used for PCR amplification using the plasmid pFny209 containing CMV Rep gene (kindly provided by professor Jialin Yu, China Agriculture University) and the amplified fragments were inserted into pUCm-T (Shanghai Sango, Shanghai, China) to produce recombinant plasmids pUCm-ΔRep(as) (antisense) and pUCm-ΔRep(s) (sense), respectively. The plasmid pSK-In-ΔRep containing soybean intron and antisense ΔRep fragment was obtained by digesting pUCm-ΔRep(as) with Pst I and Bam HI and inserted into the vector pSK-In (kindly provided by professor Johansen, Danish Plant and Soil Graduate School) between the Pst I and Bam HI site. The plasmid pSK-In-ΔRep was digested by Sal I and Bam HI, and inserted into the Sal I and Bam HI site of the plant expression vector pBIN438 to produce recombinant expression vector pBIN-In-CMVΔRep. The sense ΔRep fragment was obtained by digesting pUCm-ΔRep(s) with Sal I, and then inserted into the Sal I site of pBIN-In-CMVΔRep to produce recombinant plant expression vector pBIN-CMVΔRep(i/r) (Figure 1), containing inverted repeats sequence of CMV ΔRep separated by the soybean intron.
Plant transformation, PCR and Southern blot analysis
The recombinant vector pBIN-TMVΔMP(i/r) or pBIN-CMVΔRep(i/r) was transformed into Agrobacterium tumerfaciens EHA105, respectively, by the tri-parental mating method  and transgenic Nicotiana tabacum cv. Yunyan 87 plants were obtained using a leaf disc method as described . Rooted plants were subsequently transferred to soil and grown to maturity in a greenhouse. Following self-fertilization of T0, T1, T2, T3, T4 progenies were tested for antibiotic sensitivity by rooting the seedlings on 50 mg/L of kanamycin. The presence and copy number of integrated intron-hairpin construction in selected tobacco transgenic lines were assessed by PCR and Southern blot. Tobacco genomic DNA was extracted from both the transgenic and non-transgenic leaf tissues (3 g) by the CTAB method , and analyzed for the presence of MP or Rep gene by PCR with primers TMV MP-F1 and TMV MP-R1 specific for TMV MP and primers ΔRep-F andΔRep-R specific for CMV Rep. Genomic DNA extracted from the PCR-positive plants (20-30 μg) was completely digested with Dra I or Eco RI or Eco RV, fractionated in 0.8% agarose gels and transferred onto Hybond N+ nylon membranes (Amersham Biosciences, Bucks, UK). DNA was cross-linked to the membrane using an UL-1000 ultraviolet crosslinker (UVP, Upland, CA, USA). Hybridization was conducted as described  using the [α-32P]dCTP-labelled TMV MP or CMV Rep gene as probe prepared by random primer procedure according to the Prime-a-Gene Labeling System (Promega, Madison, WI, USA).
Virus resistance assays
Transgenic plants and wild plants were grown in greenhouse condition for 5 weeks before virus inoculation. Plants were mock-inoculated with phosphate buffer or inoculated with leaves sap extracts [diluted in 0.02 M phosphate buffer (pH 7.2)] from plants infected with TMV, CMV or both TMV and CMV (TMV+CMV). The inoculated plants were observed for virus symptoms after virus inoculation.
Leaf tissues (0.1 g) from new emergent leaves of each plant infected with TMV, CMV, TMV+CMV or inoculated with buffer were collected at 15, 25, 45 dpi. The virus concentration in the inoculated plants was detected by triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) as described . The absorbance values were measured in a Model 680 Microplate Reader (BIO-RAD, Hercules, CA, USA) at 405 nm.
RNA isolation and analysis
Plants tissues were ground to a fine powder in liquid nitrogen and RNAs were extracted with TRIzol (Invitrogen, Grand Island, N.Y., USA), according to the manufacturer's instructions. The same RNA extract was separated to high- and low-molecular-mass RNAs using 30% PEG (molecular weight 8000, Sigma, Santa Clara, CA, USA) and 3 M NaCl as described . The high-molecular-mass RNAs (20 μg) from transgenic plant tissues were separated on a 1% formaldehyde agarose gel and transferred to Hybond N+ nylon membranes (Amersham Biosciences) for Northern blot analysis. The low-molecular-mass RNAs (15 μg) were separated on a 15% sodium dodecyl sulfate (SDS) polyacrylamide gel with 7M urea and transferred to Hybond-N+ nylon membranes (Amersham Biosciences) by electrophoresis transfer at 400 mA for 45 min using a Bio-Rad semidry Trans-Blot apparatus. To verify equal amounts of siRNAs in each lane, gels also were stained with SYBR® Gold nucleic acid gel stain (Invitrogen). Membranes were hybridized as described  with [α-32P]dCTP-labelled MP or Rep gene as probe prepared by random primer procedure according to the Prime-a-Gene Labeling System (Promega) overnight at 40℃ in 50% formamide buffer. 10-min three time post-hybridization washes were performed sequentially at 40℃ with 1× sodium chloride-sodium citrate buffer (SSC) supplemented with 0.1% SDS. Hybridization signals were detected by phosphorimaging using a Typhoon 9200 imager (GE Healthcare, Piscataway, NJ, USA).
This work was financially supported by the Important National Science & Technology Specific Projects of China (2009ZX08009-026B) and the Grant from Yunnan Tobacco Company (07A03).
- Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN: Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 1986, 232: 738-743. 10.1126/science.3457472View ArticlePubMedGoogle Scholar
- Beachy RN: Mechanisms and applications of pathogen-derived resistance in transgenic plants. Curr Opin Biotechnol 1997, 8: 215-220. 10.1016/S0958-1669(97)80105-XView ArticlePubMedGoogle Scholar
- Voinnet O: RNA silencing: small RNAs as ubiquitous regulators of gene expression. Curr Opin Plant Biol 2002, 5: 444-451. 10.1016/S1369-5266(02)00291-1View ArticlePubMedGoogle Scholar
- Napoli C, Lemieux C, Jorgensen R: Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in Trans. Plant Cell 1990, 2: 279-289. 10.1105/tpc.2.4.279PubMed CentralView ArticlePubMedGoogle Scholar
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature 1998, 391: 806-811. 10.1038/35888View ArticlePubMedGoogle Scholar
- Waterhouse PM, Wang MB, Lough T: Gene silencing as an adaptive defence against viruses. Nature 2001, 411: 834-842. 10.1038/35081168View ArticlePubMedGoogle Scholar
- Wang MB, Abbott DC, Waterhouse PM: A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 2000, 1: 347-356. 10.1046/j.1364-3703.2000.00038.xView ArticlePubMedGoogle Scholar
- Frizzi A, Huang S: Tapping RNA silencing pathways for plant biotechnology. Plant Biotechnol J 2010, 8: 655-677. 10.1111/j.1467-7652.2010.00505.xView ArticlePubMedGoogle Scholar
- Di Nicola-Negri E, Brunetti A, Tavazza M, Ilardi V: Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transgenic Res 2005, 14: 989-994. 10.1007/s11248-005-1773-yView ArticlePubMedGoogle Scholar
- Szittya G, Silhavy D, Molnar A, Havelda Z, Lovas A, Lakatos L, Banfalvi Z, Burgyan J: Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation. EMBO J 2003, 22: 633-640. 10.1093/emboj/cdg74PubMed CentralView ArticlePubMedGoogle Scholar
- Palukaitis P, Zaitlin M: Replicase-mediated resistance to plant virus disease. Adv Virus Res 1997, 48: 349-377. full_textView ArticlePubMedGoogle Scholar
- Tepfer M: Risk assessment of virus-resistant transgenic plants. Annu Rev Phytopathol 2002, 40: 467-491. 10.1146/annurev.phyto.40.120301.093728View ArticlePubMedGoogle Scholar
- Golemboski DB, Lomonossoff GP, Zaitlin M: Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci USA 1990, 87: 6311-6315. 10.1073/pnas.87.16.6311PubMed CentralView ArticlePubMedGoogle Scholar
- Longstaff M, Brigneti G, Boccard F, Chapman S, Baulcombe D: Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase. EMBO J 1993, 12: 379-386.PubMed CentralPubMedGoogle Scholar
- Audy P, Palukaitis P, Slack SA, Zaitlin M: Replicase-mediated resistance to potato virus Y in transgenic tobacco plants. Mol Plant Microbe Interact 1994, 7: 15-22. 10.1094/MPMI-7-0015View ArticlePubMedGoogle Scholar
- Brederode FT, Taschner PE, Posthumus E, Bol JF: Replicase-mediated resistance to alfalfa mosaic virus. Virology 1995, 207: 467-474. 10.1006/viro.1995.1106View ArticlePubMedGoogle Scholar
- Maiti IB, Murphy JF, Shaw JG, Hunt AG: Plants that express a potyvirus proteinase gene are resistant to virus infection. Proc Natl Acad Sci USA 1993, 90: 6110-6114. 10.1073/pnas.90.13.6110PubMed CentralView ArticlePubMedGoogle Scholar
- Vardi E, Sela I, Edelbaum O, Livneh O, Kuznetsova L, Stram Y: Plants transformed with a cistron of a potato virus Y protease (NIa) are resistant to virus infection. Proc Natl Acad Sci USA 1993, 90: 7513-7517. 10.1073/pnas.90.16.7513PubMed CentralView ArticlePubMedGoogle Scholar
- Lapidot M, Gafny R, Ding B, Wolf S, Lucas WJ, Beachy RN: A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant J 1993, 4: 959-970. 10.1046/j.1365-313X.1993.04060959.xView ArticleGoogle Scholar
- Malyshenko SI, Kondakova OA, Nazarova Ju V, Kaplan IB, Taliansky ME, Atabekov JG: Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins. J Gen Virol 1993, 74: 1149-1156. 10.1099/0022-1317-74-6-1149View ArticlePubMedGoogle Scholar
- Cooper B, Lapidot M, Heick JA, Dodds JA, Beachy RN: A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility. Virology 1995, 206: 307-313. 10.1016/S0042-6822(95)80046-8View ArticlePubMedGoogle Scholar
- Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM: Gene expression - total silencing by intron-spliced hairpin RNAs. Nature 2000, 407: 319-320. 10.1038/35036500View ArticlePubMedGoogle Scholar
- Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, et al.: Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 2001, 27: 581-590. 10.1046/j.1365-313X.2001.01105.xView ArticlePubMedGoogle Scholar
- Kalantidis K, Psaradakis S, Tabler M, Tsagris M: The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact 2002, 15: 826-833. 10.1094/MPMI.2002.15.8.826View ArticlePubMedGoogle Scholar
- Pandolfini T, Molesini B, Avesani L, Spena A, Polverari A: Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection. BMC Biotechnol 2003, 3: 7. 10.1186/1472-6750-3-7PubMed CentralView ArticlePubMedGoogle Scholar
- Bonfim K, Faria JC, Nogueira EO, Mendes EA, Aragao FJ: RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean ( Phaseolus vulgaris ). Mol Plant Microbe Interact 2007, 20: 717-726. 10.1094/MPMI-20-6-0717View ArticlePubMedGoogle Scholar
- Bucher E, Lohuis D, van Poppel PM, Geerts-Dimitriadou C, Goldbach R, Prins M: Multiple virus resistance at a high frequency using a single transgene construct. J Gen Virol 2006, 87: 3697-3701. 10.1099/vir.0.82276-0View ArticlePubMedGoogle Scholar
- Fuentes A, Ramos PL, Fiallo E, Callard D, Sanchez Y, Peral R, Rodriguez R, Pujol M: Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to tomato yellow leaf curl virus infection in transgenic tomato plants. Transgenic Res 2006, 15: 291-304. 10.1007/s11248-005-5238-0View ArticlePubMedGoogle Scholar
- Krubphachaya P, Juricek M, Kertbundit S: Induction of RNA-mediated resistance to papaya ringspot virus type W. J Biochem Mol Biol 2007, 40: 404-411.View ArticlePubMedGoogle Scholar
- Tyagi H, Rajasubramaniam S, Rajam MV, Dasgupta I: RNA-interference in rice against Rice tungro bacilliform virus results in its decreased accumulation in inoculated rice plants. Transgenic Res 2008, 17: 897-904. 10.1007/s11248-008-9174-7PubMed CentralView ArticlePubMedGoogle Scholar
- Missiou A, Kalantidis K, Boutla A, Tzortzakaki S, Tabler M, Tsagris M: Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Mol Breeding 2004, 14: 185-197. 10.1023/B:MOLB.0000038006.32812.52View ArticleGoogle Scholar
- Nomura K, Ohshima K, Anai T, Uekusa H, Kita N: RNA Silencing of the introduced coat protein gene of Turnip mosaic virus confers broad-spectrum resistance in transgenic Arabidopsis . Phytopathol 2004, 94: 730-736. 10.1094/PHYTO.2004.94.7.730View ArticleGoogle Scholar
- Hamilton A, Voinnet O, Chappell L, Baulcombe D: Two classes of short interfering RNA in RNA silencing. EMBO J 2002, 21: 4671-4679. 10.1093/emboj/cdf464PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang SC, Tian LM, Svircev A, Brown DCW, Sibbald S, Schneider KE, Barszcz ES, Malutan T, Wen R, Sanfacon H: Engineering resistance to Plum pox virus (PPV) through the expression of PPV-specific hairpin RNAs in transgenic plants. Can J Plant Pathol 2006, 28: 263-270. 10.1080/07060660609507295View ArticleGoogle Scholar
- Yang G, Lee YH, Jiang Y, Kumpatla SP, Hall TC: Organization, not duplication, triggers silencing in a complex transgene locus in rice. Plant Mol Biol 2005, 58: 351-366. 10.1007/s11103-005-5101-yView ArticlePubMedGoogle Scholar
- Hily JM, Ravelonandro M, Damsteegt V, Bassett C, Petri C, Liu Z, Scorza R: Plum pox virus coat protein gene Intron-hairpin-RNA (ihpRNA) constructs provide resistance to plum pox virus in Nicotiana benthamiana and Prunus domestica . J Amer Soc for Hort Sci 2007, 132: 850-858.Google Scholar
- Chuang CF, Meyerowitz EM: Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana . Proc Natl Acad Sci USA 2000, 97: 4985-4990. 10.1073/pnas.060034297PubMed CentralView ArticlePubMedGoogle Scholar
- Hull R: Matthews' Plant Virology. 4th edition. Academic Press: London; 2002.Google Scholar
- Harrison BD: Studies on the effect of temperature on virus multiplication in inoculated leaves. Annu Appl Biol 1956, 44: 215-226. 10.1111/j.1744-7348.1956.tb02117.xView ArticleGoogle Scholar
- Fortier E, Belote JM: Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila . Genesis 2000, 26: 240-244. 10.1002/(SICI)1526-968X(200004)26:4<240::AID-GENE40>3.0.CO;2-PView ArticlePubMedGoogle Scholar
- Kameda T, Ikegami K, Liu Y, Terada K, Sugiyama T: A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi. Biochem Biophys Res Commun 2004, 315: 599-602. 10.1016/j.bbrc.2004.01.097View ArticlePubMedGoogle Scholar
- Carrington JC, Kasschau KD, Johansen LK: Activation and suppression of RNA silencing by plant viruses. Virology 2001, 281: 1-5. 10.1006/viro.2000.0812View ArticlePubMedGoogle Scholar
- Vance V, Vaucheret H: RNA silencing in plants--defense and counterdefense. Science 2001, 292: 2277-2280. 10.1126/science.1061334View ArticlePubMedGoogle Scholar
- Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC: Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana . EMBO J 1998, 17: 6739-6746. 10.1093/emboj/17.22.6739PubMed CentralView ArticlePubMedGoogle Scholar
- Guo HS, Ding SW: A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J 2002, 21: 398-407. 10.1093/emboj/21.3.398PubMed CentralView ArticlePubMedGoogle Scholar
- Guerini MN, Murphy JF: Resistance of Capsicum annuum 'Avelar' to pepper mottle potyvirus and alleviation of this resistance by co-infection with cucumber mosaic cucumovirus are associated with virus movement. J Gen Virol 1999, 80: 2785-2792.View ArticlePubMedGoogle Scholar
- Zhang K, Niu YB, Zhou XP: Transgenic tobacco plants expressed dsRNA can prevent tobacco mosaic virus infection. J Agri Biotechnol 2005, 13: 226-229.Google Scholar
- Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 1980, 77: 7347-7351. 10.1073/pnas.77.12.7347PubMed CentralView ArticlePubMedGoogle Scholar
- Huang C, Xie Y, Zhou X: Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component. Plant Biotechnol J 2009, 7: 254-265. 10.1111/j.1467-7652.2008.00395.xView ArticlePubMedGoogle Scholar
- Xiong R, Wu J, Zhou Y, Zhou X: Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus . Virology 2009, 387: 29-40. 10.1016/j.virol.2009.01.045View ArticlePubMedGoogle Scholar
- Wu J, Yu L, Li L, Hu J, Zhou J, Zhou X: Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnol J 2007, 5: 570-578. 10.1111/j.1467-7652.2007.00270.xView ArticlePubMedGoogle Scholar
- Hamilton AJ, Baulcombe DC: A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999, 286: 950-952. 10.1126/science.286.5441.950View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.