Base Sequence; DNA Primers; DNA, Viral/genetics; Manihot/virology; Molecular Sequence Data; Mosaic Viruses/genetics; Plant Leaves/genetics/virology; Plants, Genetically Modified/virology; Promoter Regions, Genetic; RNA, Small Interfering/genetics; RNA, Viral/genetics
Abstract :
[en] Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21-24 nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common region.
Akbergenov R, Si-Ammour A, Blevins T, Amin I, Kutter C, Vanderschuren H, Zhang P, Gruissem W, Meins F Jr, Hohn T, Pooggin MM (2006) Molecular characterization of geminivirus-derived small RNAs in different plant species. Nucleic Acids Res 34:462-471
Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, Vazquez F, Robertson D, Meins F Jr, Hohn T, Pooggin MM (2006) Four plant dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res 34:6233-6246
Cecchini E, Al-Kaff NS, Bannister A, Giannakou ME, McCallum DG, Maule AJ, Milner JJ, Covey SN (1998) Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana. J Exp Bot 49:731-737
Chellappan P, Masona MV, Vanitharani R, Taylor NJ, Fauquet CM (2004a) Broad spectrum resistance to ssDNA viruses associated with transgene-induced gene silencing in Cassava. Plant Mol Biol 56:601-611
Chellappan P, Vanitharani R, Fauquet CM (2004b) Short interfering RNA accumulation correlates with host recovery in DNA virus-infected hosts, and gene silencing targets specific viral sequences. J Virol 78:7465-7477
Chellappan P, Vanitharani R, Ogbe F, Fauquet CM (2005) Effect of temperature on geminivirus-induced RNA silencing in plants. Plant Physiol 138:1828-1841
Chen YK, Lohuis D, Goldbach R, Prins M (2004) High frequency induction of RNA-mediated resistance against cucumber mosaic virus using inverted repeat constructs. Mol Breed 14:215-226
Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313:68-71
Fojtova M, Van Houdt H, Depicker A, Kovarik A (2003) Epigenetic switch from posttranscriptional to transcriptional silencing is correlated with promoter hypermethylation. Plant Physiol 133:1240-1250
Goodall GJ, Filipowicz W (1989) The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58:473-483
Jørgensen K, Bak S, Busk PK, Sørensen C, Olsen CE, Puonti-Kaerlas J, Møller BL (2005) Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosides, their site of synthesis and tansport, and blockage of the biosynthesis by RNA interference technology. Plant Physiol 139:363-374
Kalantidis K, Psaradakis S, Tabler M, Tsagris M (2002) 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 15:826-833
Lee JM, Hartman GL, Domier LL, Bent AF (1996) Identification and map location of TTR1, a single locus in Arabidopsis thaliana that confers tolerance to tobacco ringspot nepovirus. Mol Plant Microbe Interact 9:729-735
Legg JP, Thresh JM (2003) Cassava virus diseases in Africa. In: Proceedings of the first international conference on plant virology in Sub-Saharan Africa. 4-8 June, Ibadan, Nigeria, pp 517-552
Liu SJ, Bedford ID, Briddon RW, Markham PG (1997) Efficient whitefly transmission of African cassava mosaic geminivirus requires sequences from both genomic components. J Gen Virol 78:1791-1794
Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. Embo J 19:5194-5201
Moissiard G, Voinnet O (2006) RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci U S A 103:19593-19598
Pandolfini T, Molesini B, Avesani L, Spena A, Polverari A (2003) 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 3:7-15
Pooggin MM, Shivaprasad PV, Veluthambi K, Hohn T (2003) RNAi targeting of DNA virus in plants. Nat Biotechnol 21:131-132
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Shivaprasad PV, Akbergenov R, Trinks D, Rajeswaran R, Veluthambi K, Hohn T, Pooggin MM (2005) Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus. J Virol 79:8149-8163
Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression-Total silencing by intron-spliced hairpin RNAs. Nature 407:319-320
Soni R, Murray JAH (1994) Isolation of intact DNA and RNA from plant tissues. Analyt Biochem 218:474-476
Van Houdt H, Bleys A, Depicker A (2003) RNA target sequences promote spreading of RNA silencing. Plant Physiol 131:245-253
Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet 17:29-35
Wang MB, Abbott DC, Waterhouse PM (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 1:347-356
Waterhouse PM, Fusaro AF (2006) Viruses face a double defense by plant small RNAs. Science 313:54-55
Xie Z, Fan B, Chen C, Chen Z (2001) An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proc Natl Acad Sci USA 98:6516-6521
Zhang P, Gruissem W (2003) Efficient replication of cloned African cassava mosaic virus in cassava leaf disks. Virus Res 92:47-54
Zhang P, Potrykus I, Puonti-Kaerlas J (2000a) Efficient production of transgenic cassava using negative and positive selection. Transgenic Res 9:405-415
Zhang P, Legris G, Coulin P, Puonti-Kaerlas J (2000b) Production of stably transformed cassava plants via particle bombardment. Plant Cell Rep 19:939-945
Zhang P, Vanderschuren H, Futterer J, Gruissem W (2005) Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes. Plant Biotechnol J 3:385-397