Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Genomic Analysis of Satellite RNA of Cucumber mosaic virus-Paf Related with Mild Symptoms

Cucumber mosaic virus Paf 계통의 약독 병징과 관련된 satellite RNA의 유전자 해석

  • Sung, Mi-Young (Division of Biological Environment, Kangwon National University) ;
  • Jung, Min-Young (Division of Biological Environment, Kangwon National University) ;
  • Lee, Sang-Yong (Division of Forest Resources, Kangwon National University) ;
  • Ryu, Ki-Hyun (Division of Environmental and Life Sciences, Seoul Women's University) ;
  • Choi, Jang-Kyung (Division of Biological Environment, Kangwon National University)
  • 성미영 (강원대학교 생물환경학부) ;
  • 정민영 (강원대학교 생물환경학부) ;
  • 이상용 (강원대학교 산림자원학부) ;
  • 류기현 (서울여자대학교 환경생명과학부) ;
  • 최장경 (강원대학교 생물환경학부)
  • Published : 2004.12.01
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Abstract

Recently, we reported a satellite RNA (Paf-satRNA) which is encapsidated in a pepper isolate of Cucumber mosaic virus (CMV-Paf) regulated symptom attenuation of the helper virus. To characterize mild symptom domain of Paf-satRNA, a series of chimeric cDNAs of satRNAs were created by using full-length cDNA clones of Paf-satRNA and a Pep-satRNA, chlorosis-inducing satRNA in pepper plants, and analyzed for determinants of symptom attenuation. When compared the nucleotide sequences, the 3' and 5' terminal sequences of the two wild-type (wt) satRNAs contained relatively conserved sequences which are the typical to CMV satRNA. Ten bases insertions were found in PepY-satRNA, and two variable regions, 81st to 113th and 183rd to 265th from the 5'-end, were located in the middle parts of the satRNAs. To delineate the attenuated symptom-related domain for the Paf-satRNA, in vitro transcripts RNAs transcribed from the wt cDNAs and constructed chimeric cDNAs were combined with genomic RNAs, RNA1, RNA2 and RNA3, of CMV-Fny and inoculated onto Nicotiana benthamiana plants. These transcripts were fully infectious onto the N. benthamiana and infectivity was confirmed by the RT-PCR. Chimeric Paf(H/N)-satRNA and PepY(N/A)-satRNA as well as Paf-satRNA induced very mild mosaic or symptomless infection on N. benthamiana. By contrast, typical mosaic symptom and stunting of infected plants were induced when PepY-satRNA, PepY(H/N)-satRNA and Paf(N/A)-satRNA were infected to N. benthamiana. Paf-satRNA coinfected with CMV-Fny RNAs induced very mild to sympomless on pepper plants whereas PepY-satRNA-infected pepper expressed typical chlorosis mosaic symptom. Two kinds of chimeric mutants, Paf(H/N)-satRNA and PepY(N/A)-satRNA, induced mild mosaic or symptomless infection onto pepper plants, while PepY(H/N)-satRNA and Paf(N/A)-satRNA showed typical chlorosis and mosaic symptom with stunting. This results suggest that mild symptom-related domain for the Paf-satRNA was located on HpaI-NarI region.

Cucumber mosaic virus(CMV)-Paf 계통에 포함된 satellite RNA(Paf-satRNA)는 CMV의 병징을 완화시키는 약독병징 관련 유전자로 작용하였다(Choi 등, 2001). 이 연구는 Paf-satRNA의 약독병징 관련 유전자의 도메인을 확인하기 위하여, 고추에서 chlorosis 병징을 발현하는 PepY-satRNA와 키메라 satRNA를 구축하여 분석하였다. 두 종의 satRNA의 염기서열을 비교한 결과, 분자크기가 큰 PepY-satRNA에서 10염기의 삽입이 발견되기는 하였지만, 양 말단영역의 염기는 비교적 안정된 conserved sequence를 보였다. 그러나 이들 satRNA의 중간영역에 존재하는 염기서열, 즉 5' 말단의 81번째 염기로부터 113번째, 그리고 183번째 염기부터 265번째 염기까지의 영역에서는 많은 변화를 나타냈다. 약독병징과 관련된 도메인을 확인하기 위하여 구축한 각 satRNA 및 키메라 satRNA의 cDNA로부터 transcript RNA를 전사시키고, 전사된 각 satRNA transcript를 CMV-Fny의 게놈RNA1, RNA2 및 RNA3의 transcript와 혼합한 후 N. benthamiana에 접종하였다. 그 결과 RT-PCR에 의해서 모든 satRNA-cDNA로부터 전사된 transcript의 감염성이 확인되었으며, Paf-satRNA 및 키메라 Paf(H/N)-satRNA와 PepY(N/A)-satRNA를 접종한 N. benthamiana에서는 모두 약한 모자이크 또는 무병징 감염의 특성을 보였다. 이와는 대조적으로 PepY-satRNA 및 키메라 PepY(H/N)-satRNA와 Paf(N/A)-satRNA를 접종한 식물에서는 전형적인 모자이크 증상과 식물체의 위축을 동반하였다. 이들 각 키메라 satRNA에 감염된 N. benthamiana를 접종원으로 고추에 접종한 결과, Paf-satRNA와 혼합한 CMV-Fny를 접종한 고추에서는 무병징에 가까운 약한 모자이크 증상이 발현되었고, PepY-satRNA를 접종한 고추는 뚜렷한 chlorosis의 모자이크 증상이 발현되었다. 한편 이들 두 종 satRNA의 키메라, Paf(H/N)-satRNA와 PepY(N/A)-satRNA를 접종한 고추에서는 모두 약한 모자이크 또는 무병징 감염의 특성을 보였고, PepY(H/N)-satRNA와 Paf(N/A)-satRNA를 접종한 식물에서는 전형적인 chlorosis의 모자이크 증상과 식물체의 위축을 동반하였다. 이와 같은 결과를 종합해 보았을 때, N. benthamiana에서와 마찬가지로 Paf-satRNA의 약독병징과 관련된 유전자의 도메인은 HpaI-NarI 영역에 존재한다는 것을 나타냈다.

Keywords

References

  1. Baulcombe, D. C, Saunders, G. R, Bevan, M. W., Mayo, M. A. and Harrison, B. D. 1986. Expression of biologically active viral satellite RNA from the nuclear genome of trandformed plants. Nature 321: 446-449 https://doi.org/10.1038/321446a0
  2. Bernal, J. J. and Garcia-Arenal, F. 1994. Analysis of the determinants of the satellite RNA of cucumber mosaic cucumovirus for high accumulation in squash. Virology 205: 262-268 https://doi.org/10.1006/viro.1994.1642
  3. Choi, G. S., Kim, J. H., Ryu, K. H., Choi, J. K., Chae, S. Y., Kim, H. R, Chung, B. N., Kim, J. S. and Choi, Y. M. 2002. Occurrence of viruses infecting pepper in Korea. Plant Pathol. J. 18: 380 (abstract)
  4. 최장경, 성미영, 정혜진, 홍진성, 이상용. 2001. Satellite RNA 보유 Cucumber mosaic virus(CMV)의 고추 CMV병에 대한 교차방어 효과. 식물병연구 7: 155-163
  5. Choi, J. K., Kim, H. J., Hong, J. S., Kim, D. W. and Lee, S. Y. 1998. Identification and differentiation of cucumber mosaic virus isolates in Korea. Korean J. Plant Pathol. 14: 7-12
  6. Choi, S. K., Choi, J. K., Park, W. M. and Ryu, K. H. 1999. RTPCR detection and identification of three species of cucumoviruses with a single pair of primer. J. Virol. Methods 83: 67-73 https://doi.org/10.1016/S0166-0934(99)00106-8
  7. Davies, C. and Symons, R. H. 1988. Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA3. Virology 165: 216-224 https://doi.org/10.1016/0042-6822(88)90675-7
  8. Gould, A. R and Symons, R. H. 1978. Alfalfa mosaic virus RNA. Determination of the sequence homology between the four RNA species and a comparison with the four RNA species of cucumber mosaic virus. Eur. J. Biochem. 91: 269-278 https://doi.org/10.1111/j.1432-1033.1978.tb20962.x
  9. Francki, R I. B., Mossop, D. W. and Hatta, T. 1979. Cucumber mosaic virus. CMI/AAB Descriptions of Plant Viruses, No. 213
  10. Hong, J. S., Park, J. c, Kim, J. E. and Choi, J. K. 1999. Comparison of nucleotide sequences and biological properties of three satellite RNAs associated with cucumber mosaic viruses. Plant Pathol. J. 15: 374 (abstract)
  11. Jaegle, M., Devic, M., Longstaff, M. and Baulcombe, D. 1990. Cucumber mosaic virus satellite RNA (Y strain): analysis of sequences which affect yellow mosaic symptoms on tobacco. J. Gen. Virol. 71: 1905-1912 https://doi.org/10.1099/0022-1317-71-9-1905
  12. Kurath, G. and Palukaitis, P. 1989. Satellite RNAs of cucumber mosaic virus: recombinants constructed in vitro reveal independent functional domains for chlorosis and necrosis in tomato. Mol. Plant Microbe Interat. 2: 91-96 https://doi.org/10.1094/MPMI-2-091
  13. Kuwata, S., Masuta, C. and Takanami, Y. 1991. Reciprocal phenotype alterations between two satellite RNAs of cucumber mosaic virus. J. Gen. Virol. 72: 2385-2389 https://doi.org/10.1099/0022-1317-72-10-2385
  14. 이상용, 홍은주, 최장경. 1995. 오이 모자이크바이러스 위성 RNA의 cDNA가 도입 된 형칠 전환담배의 육성. 한식병지 11: 80-86
  15. Masuta, C. and Takanami, Y. 1989. Determination of sequence and structural requirements for pathogenicity of a cucumber mosaic virus satellite RNA (Y strain). Ann. Phytopath. Soc. Japan 54: 332-336
  16. Montasser, M. S., Tousignant, M. E. and Kaper, J. M. 1998. Viral satellite RNAs for prevention of cucumber mosaic virus (CMV) disease in field grown pepper and melon plants. Plant Dis. 82: 1298-1303 https://doi.org/10.1094/PDIS.1998.82.12.1298
  17. Morris, T. J. and Dodds, J. A. 1979. Isolation.and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69: 854-858 https://doi.org/10.1094/Phyto-69-854
  18. Nitta, N., Takanami, Y., Kuwata, S. and Kubo, S. 1988. Inoculation with RNAs 1 and 2 of cucumber mosaic virus induces viral RNA replicase activity in tobacco mesophyll protoplasts. J. Gen. Virol. 69: 2695-2700 https://doi.org/10.1099/0022-1317-69-10-2695
  19. Palukaitis, P.,Roossinck, M. J., Dietzgen, R G. and Francki, R. I. B. 1992. Cucumovirus. Adv. Virus Res. 41: 281-348 https://doi.org/10.1016/S0065-3527(08)60039-1
  20. Peden, K. W. C. and Symons, R. H. 1973. Cucumber mosaic virus contains a functionally divided genome. Virology 53: 487-492 https://doi.org/10.1016/0042-6822(73)90232-8
  21. Rizzo, T. M. and Palukaitis, P. 1990. Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2, and 3: generation of infectious RNA transcripts. Mol. Gen. Genet. 222: 249-256 https://doi.org/10.1007/BF00633825
  22. Sleat, D. E., Zhang, L. and Palukaitis, P. 1994. Mapping determinants within cucumber mosaic virus and its satellite RNA for the induction of necrosis in tomato plants. MPMI 7: 189-195 https://doi.org/10.1094/MPMI-7-0189
  23. Takanami, Y. 1981.A striking change in symptoms on cucumber mosaic virus infected tobacco plants induced by a satellite RNA. Virology 109: 120-126 https://doi.org/10.1016/0042-6822(81)90476-1
  24. Yoshida, K., Goto, T. and Iizuka, M. 1985. Attenuated isolates of cucumber mosaic virus produced by satellite RNA and crossprotection between attenuated isolates and virulent ones. Ann. Phytopathol. Soc. Japan 51: 238-242 https://doi.org/10.3186/jjphytopath.51.238