Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus Article First Online: 18 January 2007 Received: 10 August 2006 Accepted: 30 November 2006 DOI:
10.1007/s00705-006-0914-9 Cite this article as: Knierim, D. & Maiss, E. Arch Virol (2007) 152: 941. doi:10.1007/s00705-006-0914-9 Summary.
Tomato plants grown in greenhouses in Thailand developed typical symptoms of a tomato yellow leaf curl Thailand virus (TYLCTHV) infection. After confirmation by ELISA, a Phi29 DNA polymerase approach was chosen for further molecular analysis of TYLCTHV. Total DNA purified from infected tomato leaves was subjected to rolling-circle amplification (RCA) of DNA-A and DNA-B of TYLCVTHV. In addition, a new monopartite geminivirus with a putative recombinant background was identified by RCA and tentatively named tobacco leaf curl Thailand virus (TbLCTHV). To confirm the composition of both geminiviruses, full-length clones were established and used for inoculation of
Nicotiana benthamiana by particle bombardment or agroinfection. When TYLCTHV DNA-A and DNA-B were applied together by particle bombardment or agroinfection, severe stunting, yellowing, and leaf curling were observed. Whereas TYLCTHV DNA-A and TbLCTHV revealed no infection after'particle bombardment, similar symptoms in N. benthamiana, like leaf upward curling and yellowing were observed following agroinfection.
DNA components of TYLCTHV DNA-A and DNA-B were excised from their respective plasmids, ligated, and amplified by Phi29 DNA polymerase. The ability of viral concatamere inoculation was evaluated in particle co-bombardment experiments on
N. benthamiana. Thus, particle bombardment of RCA-derived multimeric products proved to be at least as effective as inoculation with a partial repeat construct and tenfold as effective as inoculation with excised unit-lengths of DNA-A and DNA-B of TYLCVTHV when using each DNA component in an amount of 5 ng. References Accotto, GP, Navas-Castillo, J, Noris, E, Moriones, E, Louro, D 2000 Typing of tomato yellow leaf curl viruses in Europe Eur J Plant Pathol 106 179 186 CrossRef Google Scholar Andrade, EC, Manhani, GG, Alfenas, PF, Calegario, RF, Fontes, EPB, Zerbini, FM 2006 Tomato yellow spot virus, a tomato-infecting begomovirus from Brazil with a closer relationship to viruses from Sida sp., forms pseudorecombinants with begomoviruses from tomato but not from Sida J Gen Virol 87 3687 3696 CrossRef PubMed Google Scholar Anonymus 2005 Diagnostics. 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