- Short report
- Open Access
Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus
© Mubin et al; licensee BioMed Central Ltd. 2007
- Received: 18 October 2006
- Accepted: 19 January 2007
- Published: 19 January 2007
Whitefly-transmitted geminiviruses (genus Begomovirus) are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2) to develop resistance against Tomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.
- Transgenic Plant
- Monopartite Begomoviruses
- Tabacum Plant
- Nuclear Shuttle Protein
- Bipartite Begomovirus
The geminiviruses (family Geminiviridae) are a group of phytopathogenic viruses having circular, single-stranded (ss) DNA genomes packed within twinned quasi-icosahedral (geminate) particles. These viruses replicate in the nucleus and are transmitted by insect vectors. Vector specificity for transmission is determined by the coat protein . Geminiviruses have been classified into four genera based on insect vectors and genome organization . The whitefly-transmitted geminiviruses are numerous and form the genus Begomovirus. These viruses have either two genomic components (DNA A and DNA B) or a single genomic component equivalent to DNA A. Some geminivirus diseases in the Old World are caused by monopartite begomoviruses which require a ssDNA satellite, known as DNA β, to infect and cause symptoms in the hosts from which they were isolated [3, 4]. More than 100 species of begomoviruses have been completely sequenced and phylogenetic studies have shown that begomoviruses can be broadly divided into two groups, those originating from the Old World begomoviruses (Europe, Africa, Asia and Australia) and those originating from the New World begomoviruses (the Americas) . However, modern agriculture and trade is disseminating these viruses to new locations. All begomoviruses from the New World are bipartite and lack a virion-sense open reading frame (ORF) designated AV2. This is involved in the movement of monopartite viruses but its function for bipartite begomoviruses is not fully understood . Begomoviruses infecting tomato are the most diverse group and include monopartite, bipartite as well as monopartite begomoviruses that require DNA β.
Begomoviruses are amongst the most devastating phytopathogenic viruses and cause great yield losses throughout the world, particularly in warmer climates that are more suitable for their whitefly vector, Bemisia tabaci Genn. Genetically-engineered resistance has the potential to control these viruses and at least some success has been reported . Most of the efforts based on the concept of "pathogen-derived resistance" have been focused on targeting complementary-sense genes involved in virus replication. However, for begomoviruses, these sequences are more diverse than virion-sense genes and thus are less likely to result in a broad-spectrum resistance. Here, we have targeted a virion-sense gene (AV2), using anti-sense RNA technology, and show that transgenic plants are resistant to Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus that occurs widely across the Indian subcontinent in a number of hosts .
Transformed and non-transformed plants were inoculated with the cloned components of ToLCNDV by agroinfiltration, as described previously . Replication of ToLCNDV inoculated plants was assessed by Southern hybridization using the radioactively-labelled full-length clone of ToLCNDV DNA A as a probe. For Southern hybridization total DNA from transgenic lines as well as control plants was isolated and quantified by spectrophotometry. Equal amounts of total DNA was resolved in agarose gels, transferred to nylon membrane and probed with α32PdCTP-labelled probe of ToLCNDV DNA A. A semi-quantitative PCR was also done to assess the relative level of viral DNA for plants in which viral DNA could not be detected by Southern hybridization. The primers used, NSPf (5'-GTCCCGGGTATGTCACGTATCCATCA-3') and NSPr (5'-GTATCGATTTATCCAATGTAATTAAGAAT-3'), were designed to amplify the DNA B-encoded nuclear shuttle protein gene and amplify an approximately 550 bp fragment .
Ten N. tabacum plants, primary transformants resulting from independent transformation events, were selected randomly for further evaluation. All tested plants were positive for the presence of the transgene as well as the CaMV 35S promoter. The presence of the transgene had no apparent adverse effect on the growth of plants, which were morphologically normal and indistinguishable from non-transformed plants. 10 T0 transgenic and 5 non-transgenic N. tabacum plants were agroinfiltrated with ToLCNDV components and maintained in an insect-proof glasshouse. All 5 non-transgenic plants developed symptoms typical of ToLCNDV, consisting of leaf curling and stunting, within three weeks of inoculation (Fig 1B, left panel). In contrast, no such symptoms were observed on the antisense AV2 transgenic plants (Figure 1B, right panel). Southern blot hybridisation of nucleic acids extracted from systemic (non-inoculated) leaves 25 days after agroinfiltration showed the presence of viral DNA in all 5 non-transgenic plants but not in transgenic plants (Fig 1C).
We further examined the inoculated transgenic plants for the presence of virus by PCR using primers designed to target DNA B (outside the region of the transgene) and serial dilutions of the template DNA (Ten fold dilutions). These analyses detected the presence of virus in four of the ten transgenic plants, but only at low dilutions (high DNA concentrations); whereas in non-transgenic infected plants viral DNA was detected at all dilutions (Figure 1D). These results confirm that antisense AV2 transgenic plants were able to efficiently prevent virus replication and/or movement; with resistance approaching immunity in 6 out of 10 plants.
The aim of this study was to explore the possibility of obtaining resistance to ToLCNDV by silencing the virion-sense AV2 gene. ToLCNDV encodes two genes in the virion-sense, of which only AV2 is essential for the movement of the virus in plants . A recent report has similarly shown that AV2 of Indian cassava mosaic virus (ICMV), another bipartite begomovirus from the Indian subcontinent, is involved in virus spread to neighbouring cells , whereas the V2 (a homolog of AV2) of a monopartite begomovirus is involved in overcoming host defences mediated by post-transcriptional gene silencing as well as in movement [13, 14].
The AV2 sequence is well conserved among begomoviruses from the Old World . This suggests that silencing of AV2 may be a useful strategy for developing broad-spectrum resistance, a highly desirable character in the case of begomoviruses where numerous distinct virus species/strains may cause disease in a particular crop. Our current studies are investigating the spectrum of resistance provided by the antisense construct of ToLCNDV AV2.
MM is supported by a Ph.D. fellowship from Higher Education Commission (HEC), Government of Pakistan. This research has been supported by projects from the Ministry of Science and Technology (MoST), Government of Pakistan, and the International Center for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy. We thank Dr. Richard Jorgenson for providing pFGC5941 vector and Dr. Rob Briddon for critical reading of manuscript and useful comments.
- Briddon RW, Pinner MS, Stanley J, Markham PG: Geminivirus coat protein replacement alters insect specificity. Virology 1990, 177: 85-94. 10.1016/0042-6822(90)90462-ZPubMedView ArticleGoogle Scholar
- Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D: Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit Rev Plant Sci 1999, 18: 71-106. 10.1016/S0735-2689(99)00383-4View 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)00007-4PubMedView ArticleGoogle Scholar
- Mansoor S, Zafar Y, Briddon RW: Geminivirus disease complexes: the threat is spreading. Trends Plant Sci 2006, 11: 209-212. 10.1016/j.tplants.2006.03.003PubMedView ArticleGoogle Scholar
- Bull SE, Briddon RW, Sserubombwe WS, Ngugi K, Markham PG, Stanley J: Genetic diversity and phylogeography of cassava mosaic viruses in Kenya. J Gen Virol 2006, 87: 3053-3065. 10.1099/vir.0.82013-0PubMedView ArticleGoogle Scholar
- Rothenstein D, Krenz B, Selchow O, Jeske H: Tissue and cell tropism of Indian cassava mosaic virus (ICMV) and its AV2 (precoat) gene product. Virology 2006, in press.Google Scholar
- Bisaro DM: Silencing suppression by geminivirus proteins. Virology 2006, 344: 158-168. 10.1016/j.virol.2005.09.041PubMedView ArticleGoogle Scholar
- Padidam M, Beachy RN, Fauquet CM: Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. J Gen Virol 1995, 76: 25-35.PubMedView ArticleGoogle Scholar
- Kerschen C, Napoli R, Jorgensen A, Muller AE: Effectiveness of RNA interference in transgenic plants. FEBS Lett 566: 223-228. 10.1016/j.febslet.2004.04.043Google 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. 10.1126/science.227.4691.1229View ArticleGoogle Scholar
- Hussain M, Mansoor S, Iram S, Fatima AN, Zafar Y: The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant. J Virol 2005, 79: 4434-4439. 10.1128/JVI.79.7.4434-4439.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Padidam M, Beachy RN, Fauquet CM: The role of AV2 ("precoat") and coat protein in viral replication and movement in tomato leaf curl geminivirus. Virology 1996, 224: 390-404. 10.1006/viro.1996.0546PubMedView ArticleGoogle Scholar
- Zrachya A, Glick E, Levy Y, Arazi T, Citovsky V, Gafni Y: Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus -Israel. Virology 2006, 358: 159-165. 10.1016/j.virol.2006.08.016PubMedView ArticleGoogle Scholar
- Rojas MR, Jiang H, Salati R, Xoconostle-Cazares B, Sudarshana MR, Lucas WJ, Gilbertson RL: Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus . Virology 2001, 291: 110-125. 10.1006/viro.2001.1194PubMedView ArticleGoogle Scholar
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