| Abstract Detail
Pepper - Afternoon Perez, Kari [1], Yeam, Inhwa [1], Jahn, Molly [2], Kang, Byoung-Cheorl [3]. Megaprimer-Mediated Domain Swapping for Construction of Chimeric Viruses. CLONES that encode viral genomes constructed from two viruses with contrasting biological properties have been widely used in studies of viral-host interactions, particularly when the objective is to determine the identity of the viral component recognized by the host in a resistant response, known as the avirulence factor. This work presents an efficient method based on megaprimer-mediated domain swapping for the construction of clones encoding chimeric viral genomes as a versatile and widely applicable alternative to conventional restriction enzyme digestion and ligation methods. Potato Virus X (PVX)-derived vectors expressing genes encoding fluorescent proteins were used to demonstrate this concept. The cyan fluorescent protein (CFP) gene was cloned into a binary PVX vector and subsequently replaced with the yellow fluorescent protein (YFP) gene using the megaprimer amplification reaction. DNA fragments up to 1480 bp could be replaced efficiently and quickly. Most viral clones showed the expected change in phenotype without altered infectivity. Sequence analysis revealed mutations were not introduced into the four domain-swapped plasmids. This approach will provide a valuable tool for determining which domains of a viral genome are essential for infectivity, avirulence, or otherwise determine biologically significant properties of plant viruses.
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1 - Cornell University, Plant Breeding and Genetics, 306 Bradfield Hall, Ithaca, NY, 14853, USA
2 - Cornell University, Department of Plant Breeding and Genetics, Ithaca, NY, 14853, USA
3 - Seoul National University, Plant Science, San 56-1, Shillim 9-dong, Gwanak-gu, Seoul, 151-742, Republic of Korea
Keywords:
avirulence
potyvirus
PVX.
Session: Poster-59
Location: Ballroom CD/Monona Terrace
Date: Tuesday, July 25th, 2006
Time: 8:00 AM
Abstract ID:303 |
