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.
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.
Knierim
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Institute of Plant Diseases and Plant Protection, Leibniz University Hannover, Hannover, Germany.
Maiss
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E
D017103:Bacillus Phages / Q000201:enzymology; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D017791:Geminiviridae / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D016679:Genome, Viral / Q000235:genetics*; D018551:Solanum lycopersicum / Q000821:virology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D013785:Thailand; D014026:Nicotiana; D014764:Viral Proteins; D014774:Virulence
D004279:DNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase