Journal of Virological Methods 135 (2006) 254–262 Megaprimer-mediated domain swapping for construction of chimeric viruses Kari Perez, Inhwa Yeam, Molly M. Jahn, Byoung-Cheorl Kang ∗ Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853, United States Received 11 January 2006; received in revised form 18 March 2006; accepted 21 March 2006 Available online 15 May 2006 Abstract 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 paper 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 1480bp 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Potyvirus; Avirulence determinant; PVX; CFP; YFP 1. Introduction The component(s) of a plant virus responsible for triggering a resistance response is known as the viral avirulence determi- nant. In plant–pathogen interactions, pathogen avirulence genes have long been defined by the fact that the gene or its corre- sponding gene product are essential in eliciting the resistant response in hosts containing a disease resistance (R) gene (Flor, 1971; Keen, 1990). In plant–viral interactions, the avirulence determinant for a specific host/virus combination is typically identified by creating infectious chimeric viral clones derived from two viral genotypes that contrast with respect to viru- lence (Hull, 2002). Once an avirulence domain is identified, subsequent mutational analysis via site-directed methods can be used to identify specific point mutations responsible for vir- ∗ Corresponding author at: Department of Plant Science, Seoul National Uni- versity, San 56-1, Shillim 9-dong, Gwanak-gu, Seoul 151-742, South Korea. E-mail addresses: kwp6@cornell.edu (K. Perez), iy25@cornell.edu (I. Yeam), mmj9@cornell.edu (M.M. Jahn), bk54@snu.ac.kr (B.-C. Kang). ulence (Hull, 2002). Creating chimeric clones, however, can be problematic if the necessary endonuclease restriction sites are unavailable or unsuitable. Despite this constraint, very few studies have employed alternative methods that do not rely on endonuclease and ligase (Charlier et al., 2003; Dekker et al., 2000; Liang et al., 2004). Alternative methods published to date typically are very restricted in their application. An easy, effi- cient and widely applicable strategy to generate chimeric clones of viral genomes is therefore highly desirable. The polymerase chain reaction (PCR)-based site-directed mutagenesis (SDM) technique is widely used to introduce desired mutations into target DNA sequences (Ishii et al., 1998). A variety of PCR-based SDM protocols have been established to achieve efficient mutagenesis. Among them, the ‘megaprimer PCR’ method is particularly attractive because it is simple and relatively inexpensive (Kammann et al., 1989; Landt et al., 1990; Sarkar and Sommer, 1990; Sarkar and Sommer, 1992). This approach relies on the fact that DNA synthesized in vitro is not methylated and therefore is resistant to digestion by the enzyme DpnI. In the megaprimer method, in vitro DNA synthesis takes place in two successive steps (Kirsch and Joly, 1998; Sarkar 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.020