RNA interference-based resistance against a legume mastrevirus
© Nahid et al; licensee BioMed Central Ltd. 2011
Received: 15 March 2011
Accepted: 2 November 2011
Published: 2 November 2011
RNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan.
Here a hairpin (hp)RNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the Cauliflower mosaic virus 35S promoter has been produced and stably transformed into Nicotiana benthamiana. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic N. benthamiana plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants.
The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.
RNA interference (RNAi) is a homology-dependent mechanism that involves the specific degradation of cellular RNA by a complex of enzymes. The phenomenon was first discovered in plants and was called "post-transcriptional gene silencing" (PTGS) . RNAi is involved in controlling developmental processes and also as a defense against viruses, transposons and foreign nucleic acids. The key role in RNAi is played by small RNAs [known as short interfering RNA (siRNA) and micro RNA (miRNA)], which act as effectors of silencing . Plants recognize dsRNA as foreign/aberrant and this is cleaved into 21-26 nt siRNAs by a ribonuclease III-like enzyme called Dicer . One strand of the siRNA is incorporated into a ribonuclease complex known as the RNA-induced silencing complex (RISC) and serves as the guide for sequence-specific degradation of homologous mRNAs . siRNAs homologous to promoter regions of target genes induce transcriptional gene silencing (TGS) which results in promoter methylation and consequent inhibition of transcription. siRNAs homologous to coding regions induce PTGS, which results in sequence-specific RNA degradation. PTGS and TGS are mechanistically related, as both involve the production of siRNA .
RNAi can be used to engineer resistance in plants against viruses. Plants expressing a copy of a viral gene in sense and/or antisense orientation can show resistance upon infection with the virus (or other virus containing identical sequences) through RNAi. One of the first studies investigating DNA-directed RNAi in plants compared the ability of constructs expressing transcripts of sense, antisense and both polarities to yield resistance to an RNA virus in tobacco and silence an endogenous GUS reporter gene in rice . In both cases it was shown that duplex RNA (expression of both polarities simultaneously) was more effective than expression of either sense or antisense RNA alone. Further studies showed that RNAi can be more efficiently induced using transgenes that express self-complementary "hairpin" (hp)RNA . The hpRNA transgene is simply composed of a plant promoter and terminator between which an inverted repeat sequence of the target gene (sense and anti-sense) is inserted with a spacer region or intron between the repeats. The RNA transcribed from such a transgene hybridizes with itself to form a hairpin structure comprising a single-stranded loop region, encoded by the spacer region/intron, and a base-paired stem encoded by the inverted repeats, which mimics the dsRNA structure that induces RNAi. The whole length of the stem acts as a substrate for the generation of siRNAs, whereas the spacer region/intron is not involved in siRNA production but is required for the stability of the construct and appears to enhance the efficacy of silencing when directed against viruses .
Geminiviruses are single-stranded (ss)DNA viruses that affect a wide range of economically important crops throughout the warmer regions of the world [9–11]. Viruses of the family Geminiviridae are divided between the four genera (Mastrevirus, Curtovirus, Topocuvirus and Begomovirus) based on host range, genome organization and insect vector . Although they are ssDNA viruses that replicate in the nucleus and have no dsRNA phase within their replication cycle, geminiviruses are known to trigger PTGS in plants with the production of virus-specific siRNA [13, 14]. In contrast to RNA viruses, which can only be affected by PTGS, geminiviruses may be targeted by both PTGS and TGS. TGS is implicated when siRNAs corresponding to the promoter regions are produced that lead to methylation of the promoter and thus inhibition of transcription . TGS was shown to be effective against the begomovirus Mungbean yellow mosaic virus (MYMV) in a transient assay .
The genus Mastrevirus encompasses viruses with monopartite genomes that are transmitted by leafhoppers. Although the majority of mastreviruses infect monocotyledonous plants, a small number infect dicots [17–21]. A group of closely related mastreviruses, that includes Chickpea chlorotic dwarf Pakistan virus (CpCDPKV), cause chickpea stunt disease (CSD) and also infect a number of other legume and non-leguminous crops. CpCDPKV causes significant losses to chickpea cultivation in Pakistan.
Transgenic N. benthamiana plants are resistant to CpCDPKV infection
Determination of virus titer in inoculated plants by quantitative PCR
Quantitative real-time PCR estimation of viral DNA levels for CpCDPKV-inoculated transgenic and non-transgenic plants
Mean viral DNA concentration@ (ng/10 ng genomic DNA)
Standard deviation of CT
Standard deviation viral DNA concentration
PCR reactions with DNA extracted from healthy, non-inoculated N. benthamiana plants and from a reaction with no input (template) DNA had Ct values equivalent to the total number of cycles used in the experiment, indicating that the threshold level was not achieved and thus that these reactions contained no detectable viral DNA. In contrast, four CpCDPKV-infected control (non-transgenic) N. benthamiana plants were shown to contain relatively large amounts of viral DNA (between 0.46 and 2.3 ng per 10 μg of genomic DNA). This relatively large variation (~5 fold) in viral DNA levels between different infected plants likely results from the plants being at different infection stages at the time of sampling (meaning that differing numbers of cells were infected).
Although Southern blot analysis was unable to detect viral DNA in the inoculated transgenic plants, the quantitative PCR analysis showed the presence of viral DNA at between 0.0813 and 0.172 pg per 10 μg of genomic DNA. This is only an ~2 fold variation, meaning that the transgenic plants showed less variability in viral DNA levels, even though more plants were examined. However, the difference in viral DNA levels between transgenic and non-transgenic plants was striking - between 2,600 and 28,000 fold.
To assess the specificity of the PCR, a melt curve analysis of the resulting PCR products was conducted (Additional file 1). The analysis showed only a single peak, indicative of the melting of the PCR product at a single temperature, showing that only a single product was amplified.
The RNAi approach has been investigated extensive as a means of delivering resistance to begomoviruses in plants [23, 24]. In at least one case this has led to the first successful field tests of RNAi-based resistant plant lines [25, 26]. However, the lack of success of other studies has led to some ask whether this approach can, ultimately, succeed; with virus diversity and virus-encoded suppressors of RNAi as possible negative indicators of the ultimate success of the RNAi-based approach [27, 28]. For mastreviruses, specifically the monocot-infecting Maize streak virus, a protein-mediated resistance approach has proven successful  and recently, using a transient cell culture assay, the potential usefulness of the RNAi approach to MSV resistance was shown . Here we have shown that stable integration of a hpRNAi construct provides N. benthamiana with resistance to a dicot-infecting mastrevirus.
Although all transgenic plants remained symptomless and Southern blot analysis did not show the presence of viral DNA, quantitative PCR analysis was able to show the presence of very low viral DNA titers; a 2,600 to 28,000 fold difference between transgenic and non-transgenic plants. These results are consistent since PCR is a far more sensitive technique than Southern blotting, indicating that virus DNA levels in transgenic plants are below the detection threshold of Southern blotting. This finding is very similar to the results obtained for infections of the DNA A components of many bipartite begomoviruses in the absence of the DNA B. For bipartite begomoviruses both the DNA A and DNA B components are required for symptomatic infections of plants [31, 32]. Nevertheless, for most bipartite begomoviruses examined, the DNA A component can spread systemically in plants in the absence of the DNA B [33–35]. Plants infected with only DNA A exhibit no symptoms and reduced viral DNA levels. These results suggested that, following Agrobacterium-mediated inoculation, the DNA A is able to gain access to the phloem and spread throughout the plant (either as virions or a nucleoprotein complex involving the CP), but is unable to reestablish infection in the upper (younger, actively growing) parts of the plant in the absence of the genes products encoded by DNA B (which are involved in virus intra- and intercellular movement . The RNAi construct used here, by virtue of containing sequences of both the MP gene and its promoter, should induce both PTGS and TGS of the viral MP gene. It is thus possible, in a manner analogous to the situation with DNA A only infections, that RNAi-mediated down-regulation of MP expression prevented efficient spread of the virus from the site of inoculation to young, actively growing tissues of the plant.
An alternative, and possibly more plausible, explanation of the phenomenon seen in the hpRNAi construct containing plants is that virus replication (and possibly also movement) is severely impaired. However, possibly minute amounts of virus can move into the phloem and spread throughout the plant, but at levels that are insufficient to reestablish infection of cells in the upper (younger, actively growing) parts of the plant - cells which also harbor the hpRNAi construct. The lack of variability in DNA levels in the younger tissues of inoculated transgenic plants (~2 fold in comparison to ~5 fold in non-transgenic infected plants) might suggest that viral DNA replication is not occurring, or occurring at very low levels, in these tissues.
Further studies will address the precise mechanism of the resistance by, for example looking for small RNAs derived from the transgene and whether TGS, in addition to PTGS, is involved. Additionally the ability of the hpRNAi construct, derived from CpCDPKV, will be assessed for its ability to provide protection to other related viruses; such as Chickpea chlorotic dwarf Sudan virus and the recently identified Chickpea chlorotic dwarf Syria virus. Ultimately the aim is to produce chickpea lines harboring an hpRNAi construct which can provide a broad spectrum resistance to these legume-infecting mastreviruses.
Production of a hairpin RNAi construct against CpCDPKV
Oligonucleotide primers used in the production of the RNAi construct
5'CCAACTCGAG TTATCAAGCTGGACAAGAGC 3'
5'CCTCCCATGG TTCGCCTTAAACAGAAGG 3'
5'CCTCTCTAGA TTCGCCTTAAACAGAAGG 3'
5'CCAATCTAGA TTATCAAGCTGGACAAGAGC 3'
N. benthamiana was transformed with the RNAi construct (ChP6pFGC5941) by the leaf disc method using Agrobacterium tumefaciens strain LBA4404 [37, 38]. Integration of the construct in the transgenic line was confirmed by PCR using the primer sets used to produce the construct (Table 2). The transgene showed no adverse effect on the growth of plants with all transgenic plants showing a normal phenotype and producing viable seed (results not shown).
Inoculation of plants with CpCDPKV
Twenty-five T1 transgenic N. benthamiana plants harboring the RNAi construct, obtained from the self-pollination of To plants, were agro-infiltrated with a construct for the infectivity of CpCDPKV as described previously . Ten non-transgenic plants of the same age (4 weeks old) were infiltrated as controls. All agro-infiltrated plants were observed periodically for the appearance of symptoms. The presence of virus in transgenic and non-transgenic plants was detected by Southern hybridization and real-time PCR.
Southern hybridization for the detection of viral DNA
Total DNA was isolated from the inoculated plants  and 500 ng of DNA was resolved on 1% agarose gel in 0.5 × TAE buffer. Blotting was performed as described earlier . The blot was hybridized with a 1000 bp biotin-labeled DNA probe (prepared using a Biotin DecaLabel™DNA labeling kit [Fermentas]) containing portions of genes encoding movement protein (MP) and coat protein (CP). The hybridization and DNA detection was performed according to the manufacturer's recommendations.
Real-time PCR quantification of viral DNA in plants
Quantitative PCR (qPCR) was performed for the quantification of viral DNA in transgenic plants (showing resistance against CpCDPKV) and non-transgenic plants (showing infection for CpCDPKV) by using SYBER green dye (IQ SYBER Green supermix, BIO-RAD USA) as described previously . Based on the nucleotide sequence of CpCDPKV (AM849097), a primer pair QF (5'TAAAAGGCGCACTAATGGGTAGACCGTAGA3' - spanning nucleotide coordinates 102-131) and QR (5'GGCGATAACCACCTTCCCG3' - spanning nucleotide coordinates 251-233) was designed to amplify a product of 150 bp specific to CpCDPKV. Young leaf samples were collected from the inoculated plants at 17 dpi and qPCR was performed in three replications along with the standards and controls.
NN was supported by a fellowship from Higher Education Commission (HEC), Government of Pakistan. RWB is employed by HEC under "Foreign Faculty Hiring" program. This work was supported in part by a research grant from International Center for Biotechnology and Genetic Engineering (ICGEB), Trieste, Italy.
- Baulcombe DC: Unwinding RNA Silencing. Science 2000, 290: 1108. 10.1126/science.290.5494.1108View ArticlePubMedGoogle Scholar
- Brodersen P, Voinnet O: The diversity of RNA silencing pathways in plants. Trends Genet 2006, 22: 268-280. 10.1016/j.tig.2006.03.003View ArticlePubMedGoogle Scholar
- Bernstein E, Caudy AA, Hammond SM, Hannon GJ: Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001, 409: 363-366. 10.1038/35053110View ArticlePubMedGoogle Scholar
- Hammond SM, Bernstein E, Beach D, Hannon GJ: An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000, 404: 293-296. 10.1038/35005107View ArticlePubMedGoogle Scholar
- Vaucheret H, Fagard M: Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 2001, 17: 29-35. 10.1016/S0168-9525(00)02166-1View ArticlePubMedGoogle Scholar
- Waterhouse P, Graham MW, Wang M-B: Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 1998, 95: 13959-13964. 10.1073/pnas.95.23.13959PubMed CentralView ArticlePubMedGoogle Scholar
- Smith NA, SP S, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM: Total silencing by intron-spliced hairpin RNAs. Nature 2000, 407: 319-320. 10.1038/35030305View ArticlePubMedGoogle Scholar
- Wesley SV, Helliwell C, Smith NA, Wang M-B, Rouse D, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, et al.: Constructs 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
- Moffat AS: Geminiviruses emerge as serious crop threat. Science 1999, 286: 1835. 10.1126/science.286.5446.1835View ArticleGoogle Scholar
- Boulton MI: Geminiviruses: major threats to world agriculture. Ann Appl Biol 2003, 142: 143. 10.1111/j.1744-7348.2003.tb00239.xView ArticleGoogle Scholar
- Mansoor S, Briddon RW, Zafar Y, Stanley J: Geminivirus disease complexes: an emerging threat. Trends Plant Sci 2003, 8: 128-134. 10.1016/S1360-1385(03)00007-4View ArticlePubMedGoogle Scholar
- Stanley J, Bisaro DM, Briddon RW, Brown JK, Fauquet CM, Harrison BD, Rybicki EP, Stenger DC: Geminiviridae. In Virus Taxonomy, VIIIth Report of the ICTV. Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. London: Elsevier/Academic Press; 2005:301-326.Google Scholar
- Lucioli A, Noris E, Brunetti A, Tavazza R, Ruzza V, Castillo AG, Bejarano ER, Accotto GP, Tavazza M: Tomato yellow leaf curl Sardinia virus Rep-derived resistance to homologous and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-mediated transgene silencing is activated. J Virol 2003, 77: 6785-6798. 10.1128/JVI.77.12.6785-6798.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Chellappan P, Vanitharani R, Fauquet CM: Short interfering RNA accumulation correlates with host recovery in DNA virus-infected hosts, and gene silencing targets specific viral sequences. J Virol 2004, 78: 7465-7477. 10.1128/JVI.78.14.7465-7477.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJM: Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 2000, 19: 5194-5201. 10.1093/emboj/19.19.5194PubMed CentralView ArticlePubMedGoogle Scholar
- Pooggin M, Shivaprasad PV, Veluthambi K, Hohn T: RNAi targeting of DNA virus in plants. Nat Biotechnol 2003, 21: 131-132.View ArticlePubMedGoogle Scholar
- Morris BAM, Richardson KA, Haley A, Zhan X, Thomas JE: The nucleotide sequence of the infectious cloned DNA component of tobacco yellow dwarf virus reveals features of geminiviruses infecting monocotyledonous plants. Virology 1992, 187: 633-642. 10.1016/0042-6822(92)90466-3View ArticlePubMedGoogle Scholar
- Liu L, van Tonder T, Pietersen G, Davies JW, Stanley J: Molecular characterization of a subgroup I geminivirus from a legume in South Africa. J Gen Virol 1997, 78: 2113-2117.View ArticlePubMedGoogle Scholar
- Nahid N, Amin I, Mansoor S, Rybicki EP, van der Walt E, Briddon RW: Two dicot-infecting mastreviruses (family Geminiviridae ) occur in Pakistan. Arch Virol 2008, 153: 1441-1451. 10.1007/s00705-008-0133-7View ArticlePubMedGoogle Scholar
- Thomas J, Parry J, Schwinghamer M, Dann E: Two novel mastreviruses from chickpea ( Cicer arietinum ) in Australia. Arch Virol 2010, 155: 1777-1788. 10.1007/s00705-010-0763-4View ArticlePubMedGoogle Scholar
- Mumtaz H, Kumari SG, Mansoor S, Martin DP, Briddon RW: Analysis of the sequence of a dicot-infecting mastrevirus (family Geminiviridae ) originating from Syria. Virus Genes 2011, 42: 422-428. 10.1007/s11262-011-0586-8View ArticlePubMedGoogle Scholar
- Boulton MI: Functions and interactions of mastrevirus gene products. Physiological and Molecular Plant Pathology 2002, 60: 243-255. 10.1006/pmpp.2002.0403View ArticleGoogle Scholar
- Vanderschuren H, Stupak M, Fütterer J, Gruissem W, Zhang P: Engineering resistance to geminiviruses - review and perspectives. Plant Biotechnol J 2007, 5: 207-220. 10.1111/j.1467-7652.2006.00217.xView ArticlePubMedGoogle Scholar
- Shepherd DN, Martin DP, Thomson JA: Transgenic strategies for developing crops resistant to geminiviruses. Plant Sci 2009, 176: 1-11. 10.1016/j.plantsci.2008.08.011View ArticleGoogle Scholar
- Aragão F, Faria J: First transgenic geminivirus-resistant plant in the field: The development of a RNAi-based agriculture technology. In Vitro Cell Dev Biol Anim 2010, 46: S5.Google Scholar
- Aragão FJL, Faria JC: First transgenic geminivirus-resistant plant in the field. Nat Biotech 2009, 27: 1086-1088. 10.1038/nbt1209-1086View ArticleGoogle Scholar
- Lucioli A, Sallustio DE, Barboni D, Berardi A, Papacchioli V, Tavazza R, Tavazza M: A cautionary note on pathogen-derived sequences. Nat Biotechnol 2008, 26: 617-619. 10.1038/nbt0608-617View ArticlePubMedGoogle Scholar
- Noris E, Lucioli A, Tavazza R, Caciagli P, Accotto GP, Tavazza M: Tomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes. J Gen Virol 2004, 85: 1745-1749. 10.1099/vir.0.79944-0View ArticlePubMedGoogle Scholar
- Shepherd DN, Mangwende T, Martin DP, Bezuidenhout M, Kloppers FJ, Carolissen CH, Monjane AL, Rybicki EP, Thomson JA: Maize streak virus-resistant transgenic maize: a first for Africa. Plant Biotechnol J 2007, 5: 759-767. 10.1111/j.1467-7652.2007.00279.xView ArticlePubMedGoogle Scholar
- Owor BE, Martin DP, Rybicki EP, Thomson JA, Bezuidenhout M, Lakay F, Shepherd DN: A rep-based hairpin inhibits replication of diverse Maize streak virus isolates in a transient assay. J Gen Virol 2011.Google Scholar
- Stanley J: Infectivity of the cloned geminivirus genome requires sequences from both DNAs. Nature 1983, 305: 643-645. 10.1038/305643a0View ArticleGoogle Scholar
- Hamilton WDO, Bisaro DM, Coutts RHA, Buck KW: Demonstration of the bipartite nature of the genome of a single-stranded DNA plant virus by infection with the cloned DNA components. Nucleic Acids Res 1983, 11: 7387-7396. 10.1093/nar/11.21.7387PubMed CentralView ArticlePubMedGoogle Scholar
- Klinkenberg FA, Stanley J: Encapsidation and spread of African cassava mosaic virus DNA A in the absence of DNA B when agroinoculated to Nicotiana benthamiana . J Gen Virol 1990, 71: 1409-1412. 10.1099/0022-1317-71-6-1409View ArticleGoogle Scholar
- Evans D, Jeske H: DNA B facilitates, but is not essential for, the spread of Abutilon mosaic virus in agroinoculated Nicotiana benthamiana . Virology 1993, 194: 752-757. 10.1006/viro.1993.1316View ArticlePubMedGoogle Scholar
- Briddon RW, Markham PG: Complementation of bipartite begomovirus movement functions by topocuviruses and curtoviruses. Arch Virol 2001, 146: 1811-1819. 10.1007/s007050170067View ArticlePubMedGoogle Scholar
- Sanderfoot AA, Lazarowitz SG: Getting it together in plant virus movement: cooperative interactions between bipartite geminivirus movement proteins. Trends Cell Biol 1996, 6: 353-358. 10.1016/0962-8924(96)10031-3View ArticlePubMedGoogle Scholar
- Horsch RB, Klee HJ: Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process. Proc Natl Acad Sci USA 1986, 83: 4428-4432. 10.1073/pnas.83.12.4428PubMed CentralView ArticlePubMedGoogle Scholar
- Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 1985, 227: 1229-1231.View ArticleGoogle Scholar
- Doyle JJ, Doyle JL: A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 1987, 19: 11-15.Google Scholar
- Sambrook J, Frisch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Azhar MT, Amin I, Anjum ZI, Arshad M, Briddon RW, Mansoor S: Both malvaceous and non-malvaceous betasatellites are associated with two wild cotton species grown under field conditions in Pakistan. Virus Genes 2010, 41: 417-424. 10.1007/s11262-010-0521-4View ArticlePubMedGoogle Scholar
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