Transgenic Research

, Volume 20, Issue 6, pp 1367–1377 | Cite as

Production of transgenic rice new germplasm with strong resistance against two isolations of Rice stripe virus by RNA interference

Original Paper

Abstract

Rice stripe disease, with the pathogen Rice stripe virus (RSV), is one of the most widespread and severe virus diseases. Cultivating a resistant breed is an essential and efficient method in preventing rice stripe disease. Following RNA interference (RNAi) theory, we constructed three RNAi binary vectors based on coat protein (CP), special-disease protein (SP) and chimeric CP/SP gene sequence. Transgenic lines of rice cv. Yujing6 were generated through Agrobacterium-mediated transformation. We inoculated T1 generation plants from each line derived from CP/SP, CP, and SP transgenic rice plants with two RSV isolates from Shandong Province and Jiangsu Province using viruliferous vector insects. In these resistance assays, chimeric CP/SP RNAi lines showed stronger resistance against two isolates than CP or SP single RNAi lines. Stable integration and expression of RNAi transgenes were confirmed by Southern and northern blot analysis of independent transgenic lines. In the resistant transgenic lines, lower levels of transgene transcripts and specific short interference RNAs were observed relative to the susceptible transgenic plant, which showed that virus resistance was increased by RNAi. Genetic analysis demonstrated that transgene and virus resistance was stably inherited in the T2 progeny plants.

Keywords

Rice stripe virus CP gene SP gene RNA interference Transgenic rice 

Abbreviations

Bar

Bialaphos resistance gene

CP

Coat protein

dsRNA

Double-strand RNA

hpRNA

Hairpin RNA

nt

Nucleotide

ORF

Open reading frame

PPT

Phosphinothricin

PCR

Polymerase chain reaction

PTGS

Posttranscriptional gene silencing

RMVR

RNA-mediated virus resistance

RNAi

RNA interference

siRNA

Short interfering RNA

SP

Special-disease protein

vRNA

Viral RNA

vcRNA

Viral complementary RNA

Supplementary material

11248_2011_9502_MOESM1_ESM.pdf (8.9 mb)
Supplementary material 1 (PDF 9069 kb)

References

  1. Chen Z, Xu ZF, Ye JJ, Yao HP, Zheng S, Ding JY (2005) Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression. J Zhejiang Univ Sci 6:236–241CrossRefGoogle Scholar
  2. Chen XM, Liu J, Li X, Jiang F, Xie XY, Zhu CX, Wen FJ (2010) Inhibiting virus infection by RNA interference of the eight functional genes of the potato virus Y genome. J Phytopathol 158:776–784. doi:10.1111/j.1439-0434.2010.01701.x CrossRefGoogle Scholar
  3. Cheng ZB, Ren CM, Zhou YJ, Fan YJ, Xie LH (2008) Pathogenicity of Rice stripe tenuivious isolates from different areas. Acta Phytopathol Sinica 38(2):129–131Google Scholar
  4. Cui GR, Wu H, Liu YG (2004) Selecting herbicide-resistant calli of rice and appraising to seedling with PPT and Basta. Seed 23:7–10Google Scholar
  5. Ding SW, Li H, Lu R, Li F, Li WX (2004) RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res 102:109–115. doi:10.1016/j.virusres.2004.01.021 PubMedCrossRefGoogle Scholar
  6. Duan FP, Liang CY, Li YQ (2001) Research advances of Bar gene and its transgenic crops. Guihaia 21:166–172Google Scholar
  7. Dykxhoorn DM, Lieberman J (2006) Silencing viral infection. PLoS Med 3:1000–1004. doi:10.1371/journal.pmed.0030242 CrossRefGoogle Scholar
  8. Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994. doi:10.1007/BF00014672 PubMedCrossRefGoogle Scholar
  9. Hannon GJ (2002) RNA interference. Nature 418:244–251. doi:10.1038/418244a PubMedCrossRefGoogle Scholar
  10. Hayakawa T, Zhu YF, Itoh K, Kimura Y, Izawa T, Shmamoto K, Toriyama S (1992) Genetieally engineered rice resistant to rice stripe virus, an insect-transmitted virus. Proc Natl Acad Sci USA 89:9865–9869PubMedCrossRefGoogle Scholar
  11. Kogenezawa H, Doi Y, Yora K (1996) Purification of rice stripe virus. Annu Phytopathol Soc Jpn 41:148–164CrossRefGoogle Scholar
  12. Lindbo JA, Silva-Rosales L, Proebsting WM, Dougherty WG (1993) Induction of a highly specific antiviral state in transgenic plant: Implications for regulation of gene expression and virus resistance. Plant Cell 5:1749–1759. doi:10.1105/tpc.5.12.1749 PubMedCrossRefGoogle Scholar
  13. Liu XC, Pan CX, Song YZ, Chen HL, Wen FJ (1995) A simple procedure of DNA isolation from monocotyledonous plants and its application. J Shandong Agric Univ (Natural science) 26:491–495Google Scholar
  14. Ma J, Song YZ, Li KD, Zhu CX, Wen FJ (2008) The analysis of molecular variability of rice stripe virus isolate (RSV-SD-JN2) in Jining Shandong. Zhi Wu Bao Hu Xue Bao 35:415–420Google Scholar
  15. Matzke M, Matzke AJ, Kooter JM (2001) RNA: guiding gene silencing. Science 293:1080–1083PubMedCrossRefGoogle Scholar
  16. Nicola-Negri ED, Brunetti A, Tavazza M, Iiardi V (2005) Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transg Res 14:989–994. doi:10.1007/s11248-005-1773-y CrossRefGoogle Scholar
  17. Ramesh SV, Mishra AK, Praveen S (2007) Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants. Oligonucleotides 17:251–257. doi:10.1089/oli.2006.0063 PubMedCrossRefGoogle Scholar
  18. Ramirez BC, Haenni AL (1994) Molecular biology of tenuiviruses, a remarkable group of plant viruses. J Gen Virol 75:467–475. doi:10.1099/0022-1317-75-3-467 PubMedCrossRefGoogle Scholar
  19. Sambrook J, Fristsch EF, Maniatis T (2001) Molecular cloning: a libratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  20. Sanford JC, Johnson SA (1985) The concept of parasite-derived resistance: deriving resistance genes from the parasite own genome. J Theor Biol 115:395–405. doi:10.1016/S0022-5193 CrossRefGoogle Scholar
  21. Shimizu T, Yoshii M, Wei T, Hirochik H, Omura T (2009) Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus. Plant Biotechnol J 7:24–32. doi:10.1111/j.1467-7652.2008.00366.x PubMedCrossRefGoogle Scholar
  22. Smith NA, Singh SP, Wang M, Stoutjesdijk P, Green A, Waterhouse PM (2000) Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407:319–320. doi:10.1038/35030305 PubMedCrossRefGoogle Scholar
  23. Song E, Lee SK, Dykxhoorn DM, Novina C, Zhang D, Crawford K, Cerny J, Sharp PA, Lieberman J, Manjunath N, Shankar P (2003) Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J Virol 77:7174–7181. doi:10.1128/JVI.77.13.7174-7181.2003 PubMedCrossRefGoogle Scholar
  24. Takahashi M, Toriyama S, Hamamatsu C, Ishihama A (1993) Nucleotide sequence and possible ambisense coding strategy of rice stripe virus RNA segment 2. J Gen Virol 74:769–773. doi:10.1099/0022-1317-74-4-769 PubMedCrossRefGoogle Scholar
  25. Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976. doi:10.1111/j.1365-313X.2006.02836.x PubMedCrossRefGoogle Scholar
  26. Toriyama S (1986) Rice stripe virus: prototype of a new group of viruses that replicate in plants and insects. Microbiol Sci 3:347–351PubMedGoogle Scholar
  27. Toriyama S, Takahashi M, Sano Y, Shimizu T, Ishihama A (1994) Nucleotide sequence of RNA1, the largest genomic segment of rice stripe virus, the proto type of the Tenuiviruses. J Gen Virol 75:3569–3579. doi:10.1099/0022-1317-75-12-3569 PubMedCrossRefGoogle Scholar
  28. Vaucheret H, Beclin C, Fagard M (2001) Post transcriptional gene silencing in plants. J Cell Sci 114:3083–3091PubMedGoogle Scholar
  29. 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. doi:10.1046/j.1364-3703.2000.00038.x PubMedCrossRefGoogle Scholar
  30. Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in Plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964. doi:0027-8424/98/9513959-6$2.00/0 PubMedCrossRefGoogle Scholar
  31. Wei TY, Yang JG, Liao FL, Gao FL, Lu LM et al (2009) Genetic diversity and population structure of rice stripe virus in China. J Gen Virol 90:1025–1034. doi:10.1099/vir.0.006858-0 PubMedCrossRefGoogle Scholar
  32. Wu SJ, Zhong H, Zuo H, Gu MH, Liang GH (2006) Research progress in molecular biology of rice stripe virus and gene engineering of virus resistance. Acta Agric Jiangxi 18:72–77Google Scholar
  33. Xie L (1986) Research on rice virus disease in China. Trop Agric Res Ser 19:45–50Google Scholar
  34. Xue DW, Ma LL, Jiang H, Hua ZH, Guo LB, Huang DN, Qian Q (2005) Safety assessment of herbicide-tolerant transgenic rice. J Agric Biotechnol 13:723–727Google Scholar
  35. Zhou T, Wang L, Chen ZB, Fan YJ, Zhou YJ (2009) Mechanism and inheritance of resistance to rice stripe disease in the japonica rice cultivar Zhendao 88. Sci Agric Sin 42:103–109Google Scholar
  36. Zhu Y, Hayakawa T, Toriyama S, Takahashi M (1991) Complete nucleotide sequence of RNA3 of rice stripe virus: an ambisense coding strategy. J Gen Virol 72:763–767PubMedCrossRefGoogle Scholar
  37. Zhu Y, Hayakawa T, Toriyama S (1992) Complete nucleotide sequence of RNA4 of rice stripe virus isolate T and comparison with another isolate and with maize stripe virus. J Gen Virol 73:1309–1312. doi:10.1099/0022-1317-73-5-1309 PubMedCrossRefGoogle Scholar
  38. Zhu JH, Zhu XP, Wen FJ, Bai QR, Zhu CX, Song YZ (2004) Effect of cDNA fragments in different length derived from Potato Virus Y coat protein gene on the induction of RNA-mediated virus resistance. Sci China Ser C Life Sci 47:382–388. doi:10.1360./03yc0066 CrossRefGoogle Scholar
  39. Zhu CX, Song YZ, Yin GH, Wen FJ (2009) Induction of RNA-mediated multiple virus resistance to potato virus Y, tobacco mosaic virus and cucumber mosaic virus. J Phytopathol 157:101–107. doi:10.1111/j.1439-0434.2008.01449.x CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop BiologyShandong Agricultural UniversityTai’anPeople’s Republic of China
  2. 2.Rice Science Research InstituteShandong Academy of Agricultural ScienceJiningPeople’s Republic of China

Personalised recommendations