ساخت همسانه عفونت‌زای ویروئید‌های مرکبات و اثبات بیماری‌زایی آنها

نوع مقاله: مقاله کامل پژوهشی

نویسندگان

گروه گیاه‌پزشکی، بخش بیماری شناسی گیاهی، دانشکده کشاورزی، دانشگاه فردوسی مشهد، مشهد، ایران.

چکیده

ویروئیدها آر ان ا های بیماری‌زا در گیاهان هستند که توانایی رمز کردن پروتئین را ندارند. تاکنون هفت ویروئید از چهار جنس خانواده Pospiviroidaeاز مرکبات گزارش شده‌اند.با ساخت همسانه عفونت­زای ویروئید­های مرکبات امکان سنجش بیماری­زایی آنها فراهم شد. از آر ان ا کل استخراج‌شده از نهال پرتقال خونی مورو ژنوم کامل ویروئیدهای CEVd، HSVd، CBCVd، CDVd، CVdV و CBLVd در واکنش RT-PCR تکثیر و توالی‌یابی شد. سپس ژنوم کامل هر ویروئید بطور جداگانه در ناقل دوتایی pBin62SK تحت کنترل پیشبر s35 ویروس موزائیک گل‌کلم (Cauliflower mosaic virus, CaMV) قرار گرفت. بیماری­زایی ویروئیدها در گیاهان خیار(Cucumis sativus)، گوجه‌فرنگی (Solanum lycopersicum) و نارنج سه برگ (Poncirus trifoliata) از طریق مایه‌زنی اگروباکتریوم حاوی سازه‌های عفونت‌زای آنها انجام شد. سه هفته پس از مایه‌زنی حضور ویروئیدها در برگ­های جدید گیاهان مایه‌زنی‌شده با آزمون RT-PCR با استفاده از آغازگرهای اختصاصی و هیبریداسیون نقطه ای با کاوشگر اختصاصی هر ویروئید تائید شد. پنج هفته پس از مایه‌زنی، علائم ریزبرگی، پیسک و روخمشی توسط CVdV و CBCVd، پیسک توسط CDVd و علایم روخمشی و بدشکلی برگ بوسیله CEVd، HSVd و CBLVd در برگ­های گوجه فرنگی ظاهر شدند. مایه‌زنی مکانیکی عصاره آلوده به گیاهان سالم نیز علائم مشابهی را تولید کرد. ردیابی ویروئید در برگ­های جدید گیاهان مایه‌زنی‌شده بیانگر عفونت‌زایی سازه ساخته شده است. همسانه‌های عفونت‌زای CEVd، HSVd و CBCVd پیش­تر ساخته شده است ولی این اولین گزارش از ساخت همسانه عفونت‌زای ویروئیدهای CDVd، CBLVd وCVdV است.

کلیدواژه‌ها


عنوان مقاله [English]

Construction of infectious clones and demonstration of pathogenicity of citrus viroids

نویسندگان [English]

  • L. Ebrahimi-moghadam
  • M. Zakiaghl
  • B. Jafarpour
  • M. Mehrvar
چکیده [English]

Viroids are pathogenic RNAs in plants without protein encoding capacity. Seven viroids belonging to four genera in the Pospiviroidae family have been reported from citrus species. By constructing the infectious clones of citrus viroids, it was possible to carried out their pathogenicity test. Full-length genome of CEVd, HSVd, CBCVd, CDVd, CVdV and CBLVd was separately amplified in RT-PCR using total RNA extracted from a Moro blood orange plant and resulting PCR products were sequenced. The full-length genome of each viroid was inserted under the control of cauliflower mosaic virus 35S promoter in a pBin62SK binary vector. Pathogenicity of viroids was tested by agroinoculation of their infectious clones to cucumber (Cucumis sativus), tomato (Solanum lycopersicum) and trifoliate orange (Poncirus trifoliata). Three weeks after inoculation, viroids were detected in newly grown leaves of inoculated plants by RT-PCR using specific primers and by dot blot hybridization using viroid specific probes. Five weeks after inoculation, symptoms including little leaf, mottling and epinasty by CVdV and CBCVd, mottling by CDVd, and epinasty and leaf deformation by CEVd, HSVd and CBLVd in tomato leaves were observed. Mechanical inoculation of infected sap of infected tomato plants induced similar symptoms in healthy tomato plants. Identification of the viroids in newly grown leaves of inoculated plants indicated the infectiousness of the generated constructs. Infectious clones of CEVd, HSVd and CBCVd were previously made, but it is the first report of the construction of the infectious clone for CDVd, CBLVd and CVdV.

کلیدواژه‌ها [English]

  • Viroid
  • citrus
  • infectious clone
  • pathogenicity
 
Ambros S., Hernandez C. and Flores R. 1999. Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host. Journal of General Virology 80: 2239–52.
Ashulin L., Lachman O., Hadas R. and Bar-Joseph M. 1991. Nucleotide sequence of a new viroid species, Citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Research 19:4767.
Astruc N., Marcos J. F., Macquaire G., Candresse T., Pallas V. 1996. Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European Journal Plant Pathology 102: 837-846.
Barbosa C. J., Pina J. A., Navarro L. and Duran-Vila N. 2002. Replication/accumulation and symptom expression of citrus viroids on some species of citrus and related genera. Fifteenth IOCV Conference 15: 264–271.
Barbosa C. J., Serra P., Pina J. A., Navarro L., Daros J. A., Flores R. and Duran-Vila N. 2005. Identification and preliminary characterization of a viroid-like RNA in Atalantia citroides. Riverside CA, pp. 264-271. In: N. Duran-Vila, M.E. Hilf, M. Pena-Rocha (Eds). Proceedings, XVI International Conference Citrus Virologists.
Bevan M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12: 8711- 8721. 
Bove J. M. 1995. Virus and Virus- like Diseases of Citrus in The Near East Region.FAO publication. 518 pp.
Borkhardt B., Vongsasitorn D. and Albrechtsen S. E. 1994. Chemiluminescent detection of potato spindle tuber viroid in true potato seed using a digoxigenin labelled DNA probe. Potato Research  37: 249–255
Daros J. A. and Flores R. 2004.  Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proceeding of the national academy of Sciences 101: 6792–6797.
Ding B. 2009. The Biology of Viroid-Host Interactions. Annual Review of Phytopathology 47: 105-131.
Eiras M., Silva S. R., Stuchi E. S., Carvalho S. A. and Garcez, R. M. 2013. Identification and characterization of viroids in “Navelina ISA 315” sweet orange, Tropical Plant Pathology 38: 58-62.
Eisel D., Seth O., Grunewald-Janho S., Kruchen B., Ruger B. (Eds). 2008.  DIG Application Manual for Filter Hybridization, Roche Diagnostics GmbH 207 pp.
Fawcett H. S. and Klotz L. J. 1948. Exocortis on trifoliate orange. Citrus Leaves 28:8.
Flores R. Gas M. E., Molina-Serrano D., Nohales M. A., Carbonell A., Gago S., DelaPena M. and Daros J. A. 2009. Viroid replication: rolling-circles, enzymes and ribozymes. Viruses 1: 317-334.
Fagoaga C. and Duran-Vila N. 1996. Naturally occurring variants of citrus exocortis viroid in vegetable crops. Plant Pathology 45: 45-53.
Gandia M. and Duran-Vila N. 2004. Variability of the progeny of a sequence variant of Citrus bent leaf viroid (CBLVd). Archives of Virology 149: 407–16.
Gandia M., Rubio L., Palacio A. and Duran-Vila N. 2005. Genetic variation and population structure of an isolate of Citrus exocortis viroid (CEVd) and of the progenies of two infectious sequence variants. Archives of Virology 150: 1945–1957.
Gomez G. and Pallas V. 2006. Hop stunt viroid is processed and translocated in transgenic Nicotiana benthamiana plants. Molecular Plant Pathology 7: 511-517.
Gora A., Candresse T. and Zagorski W. 1994. Analysis of the population structure of three phenotypically different PSTVd isolates. Archives of Virology 138: 233.
Gora-sochacka A., Kierzek A. and Candresse T. 1997. The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants. RNA 3: 68-74.
Hadidi A., Flores R., Randles J. W. and Semancik J. S. (Eds). 2003. Viroids. CSRO Publishing, Collingwood, Australia. 370 pp.
Hataya T., Nakahara K., Ohara T., Ieki H. and Kano T. 1998. Citrus viroid Ia is a derivative of citrus bent leaf viroid (CVd-Ib) by partial sequence duplications in the right terminal region. Archives of Virology 143: 971–980.
Hellens R. P., Allan A.C., Friel E.N., Bolitho K., Grafton K., Templeton M.D., Karunairetnam S., Gleave A.P. and Laing W.A. 2005. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1: 13-27.
Holsters M., De Waele D., Depicker A., Messens E., van Montagu M. and Schell J. 1978. Transfection and transformation of Agrobacterium tumefaciens.  Molecular Gene Genetic 163, 181-187.
Ito T., Ieki H. and Ozaki, K. 2000. A population of variants of a viroid closely related to citrus viroid-I in citrus plants. Archives of Virology 145: 2105–14.
Jakse J., Radisek S., Pokorn T., Matousek J. and Javornik B. 2015. Deep-sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop. Plant Pathology 64: 831-842.
Murcia N., Bernad L., Serra P., Bani Hashemian S. M. and Duran-Vila N. 2009. Molecular and biological characterization of natural variants of Citrus dwarfing viroid. Archives of virology 154:1329–34.
King A. M. Q., Adams M. J., Carstens E. B. and Lefkowitz E. J. (eds) 2012. Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press
Kofalvi S. A., Marcos J. F., Canizares M. C., Pallas V. and Candresse T. 1997. Hop stunt viroid (HSVd) sequence variants from Prunes species: Evidence for recombination between HSVd isolates. Journal of General Virology 78: 3177–3186.
Mishra M. D., Hammond R.W., Owens R.A., Smith D.R. and Diener T.O. 1991. Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid. Journal of General Virology 72: 1781-1785.
Mumford R. A., Walsh K. and Boonham N. 2000. A Comparison of Molecular Methods for the Routine Detection of Viroids. Bulletin OEPP/EPPO Bulletin 30: 431- 435.
Nakahara, K., Hataya, T., Hayashi, Y., Sugimoto, T., Kimura, I. and Shikata, E. 1998. A Mixture of Synthetic Oligonucleotide Probes Labelled with Biotin for the Sensitive Detection of Potato Spindle Tuber Viroid. Journal of Virology Methods 71: 219– 227.
Owens R. A. and Hammond R.W. 2009. Viroid pathogenicity: one process, many faces. Viruses 1: 298-316.
Palacio-Bielsa A., Romero-Durban J. and Duran-Vila, N. 2004. Characterization of citrus HSVd isolates. Archives of Virology 149: 537–552.
Podstolski W., Gora-sochacka A. and Zagorski W. 2005. Co-inoculation with two non-infectious cDNA copies of potato spindle tuber viroid (PSTVd) leads to the appearance of novel. Acta Biochimica Polonica 52, 87–98.
Puchta H., Ramm K., Luckinger R., Hadas R., Bar-Joseph M. and Sanger H. 1991. Primary and secondary structure of citrus viroid IV (CVd IV), a new chimeric viroid presents in dwarfed grapefruit in Israel. Nucleic Acids Research 19: 6640.
Rakowski A. G., Szychowski J. A., Avena Z.S and Semancik J. S. 1994. Nucleotide sequence and structural features of the group III citrus viroid. Journal of General Virology 75:3581–3584.
Reanwarakorn K. and Semancik J. S. 1998. Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids. Journal of General Virology 79: 3163–3171.
Rio D. C. 2015. Denaturation and electrophoresis of RNA with formaldehyde. Cold Spring Harbor Protocols 2015: 219–222.
Sano T. Candresse T., Hammond R. W., Diener T. O. and Owens R. A. 1992. Identification of multiple structural domains regulating viroid pathogenicity. Proceedings of the National Academy of Sciences of the United States of America 89: 10104–8.
Semancik J. S. and Weathers L.G. 1972. Pathogenic 10s RNA from exocortis disease recovered from tomato bunchy-top plants similar to potato spindle tuber infection. Virology 49: 622-625.
Serra P., Eiras M., Murcia N., Kitajima E. W., Daros J. A. and Flores R. 2008a. Citrus viroid V : Occurrence, Host Range, Diagnosis, and Identification of New Variants. Virology 98: 1199-1204.
Serra P, Barbosa C. J., Daros J., Flores R. and Duran-Vila N. 2008b. Citrus viroid V: molecular characterization and synergistic interactions with other members of the genus Apscaviroid. Virology 370: 102-12.
Serra P., Bani Hashemian S. M., Fagoaga C., Romero J., Ruiz-Ruiz S., Gorris M. T., Bertolini E. and Duran- Vila N. 2014. Virus-viroid interactions: Citrus Tristeza Virus enhances the accumulation of Citrus Dwarfing Viroid in Mexican lime via virus-encoded silencing suppressors. Journal of Virology 88: 1394–7.
Singh R. P. and Boucher A. 1987. Electrophoretic separation of a sever from mild strains of potato spindle tuber viroid.  Phytopathology 77: 1588-1591.
Shikata E. 1990. New viroids from Japan. Seminars in Virology 1: 107-115.
Walia Y., Dhir S., Ram R., Zaidi A. A. and Hallan V. 2014. Identification of the herbaceous host range of Apple scar skin viroid and analysis of its progeny variants. Plant Pathology 63: 684-690.
Weathers L.G. and Calavan E. C. 1961. Additional indicator plants for exocortis and evidence for strain differences in the virus. Phytopathlogy 51: 262-264.
Weathers L.G., Greer F.C. and Harjungs M.K. 1967. Transmission of exocortis virus of citrus to herbaceous plants. Plant Disease Reporter 51: 868-871.
Weathers L.G and Greer F.C. 1968. Additional herbaceous hosts of the exocortis virus of citrus. Phytopathology 58: 1071.
Yoon J., Cho I., Choi G. and Choi S. 2014. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate. Plant Pathology Journal 30: 68–74.