Virus Research 161 (2011) 194–197 Contents lists available at ScienceDirect Virus Research journa l h o me pag e: www.elsevier.com/locate/virusres Short communication Self-interaction of Abutilon mosaic virus replication initiator protein (Rep) in plant cell nuclei Björn Krenz a,1 , Felix Neugart b , Tatjana Kleinow a , Holger Jeske a,* a Institute of Biology, Dpt. of Molecular Biology and Plant Virology, Pfaffenwaldring 57, University of Stuttgart, D-70550 Stuttgart, Germany b Institute of Cell Biology, Allmandring 30, University of Stuttgart, D-70550 Stuttgart, Germany a r t i c l e i n f o Article history: Received 9 March 2011 Received in revised form 27 July 2011 Accepted 27 July 2011 Available online 5 August 2011 Keywords: Geminivirus Begomovirus Replication a b s t r a c t Geminiviruses replicate their circular single-stranded DNA genome in nuclei of infected plant cells. Their replication initiator proteins (Reps) possess interaction domains for homo- and hetero-oligomerization as shown previously by in vitro studies and yeast two hybrid assays. Here, homo-oligomerization and cellular localization of the Abutilon mosaic virus (AbMV) Rep was analysed with bimolecular fluorescence complementation (BiFC) in epidermal tissues of Nicotiana benthamiana. BiFC revealed that Rep oligomers accumulated within the nucleoplasm, but were excluded from nucleoli as indicated by a nucleoli/cajal body marker. A similar subcellular distribution was observed for Rep fused to full-length cyan fluorescent protein. To examine whether tagged Reps were functionally active, N. benthamiana plants transgenic for a dimeric AbMV DNA B were inoculated with the BiFC expression constructs and nucleic acids were analysed by rolling circle amplification/restriction fragment length polymorphism as well as Southern blot hybridization. The results confirmed that the modified AbMV Rep was able to transreplicate DNA B. © 2011 Elsevier B.V. All rights reserved. Geminiviruses (reviewed by Jeske, 2009) may divide their cir- cular single-stranded DNA (ssDNA) genomes into two components (DNA A and DNA B), which are both required for infectivity in plants. Proteins encoded by DNA A are important for viral repli- cation, transcription and encapsidation, whereas the two DNA B-encoded proteins enable systemic spread within the host plant and affect pathogenicity. The multifunctional replication initia- tor protein (Rep; synonymous AC1, AL1 or C1) is essential for viral ssDNA replication within nuclei of host cells, which utilizes three modes: complementary strand (CSR), rolling circle (RCR) and recombination-dependent (RDR) replication (reviewed by Hanley- Bowdoin et al., 1999, 2004; Jeske, 2007). Since geminiviruses do not encode its own polymerase, they rely on the respective host enzymes, the genes of which may be activated by Rep through cell cycle regulation of differentiated cells (Jeske, 2009). For this purpose, an oligomerization platform in the central portion of the protein serves for self-interactions, binding of other viral proteins such as the viral replication enhancer protein (REn; synonymous AC3 or C3) (Settlage et al., 1996, 2001, 2005) or the coat protein (Malik et al., 2005), and host proteins like the plant retinoblastoma- related protein (pRBR) (Ach et al., 1997; Collin et al., 1996; Horvath et al., 1998; Kong et al., 2000; Settlage et al., 2001; Xie et al., * Corresponding author. Tel.: +49 711 685 65070; fax: +49 711 685 65096. E-mail address: holger.jeske@bio.uni-stuttgart.de (H. Jeske). 1 Present address: Dpt. of Plant Pathology and Plant-Microbe Biology, Cornell University, 334 Plant Science Bldg., Ithaca, NY 14853-5904, United States. 1995), the proliferative cell nuclear antigen (PCNA) (Bagewadi et al., 2004; Castillo et al., 2003), the replication factor C (RFC) (Luque et al., 2002) and a SUMO-conjugating enzyme (Castillo et al., 2004). Homo-oligomerization of geminiviral Reps has been shown previ- ously, either using yeast two-hybrid assays or biochemical analyses of recombinant proteins, mostly derived from ectopic expression in bacteria or insect cells (Choudhury et al., 2006; Clerot and Bernardi, 2006; Missich et al., 2000; Orozco et al., 1997, 2000; Settlage et al., 1996). Some dissent exists in the literature about the oligomer sizes: octamers of full-length Rep were found for tomato golden mosaic virus using gel filtration and of wheat dwarf virus using chemical cross-linking. Clerot and Bernardi (2006) described dode- camers of the truncated tomato yellow leaf curl Sardinia virus Rep which possessed helicase activity, but missed the nicking/closing domain. A similar construct of mungbean yellow mosaic India virus Rep formed 24mers (Choudhury et al., 2006). However, an investi- gation of Rep oligomer generation and the subcellular localization of protein interaction within living plant cells are still pending. In this study, bimolecular fluorescence complementation (BiFC) (Kerppola, 2008; Schütze et al., 2009; Walter et al., 2004) was applied to monitor Abutilon mosaic virus (AbMV) Rep–Rep com- plex formation and the cellular site of this self-interaction in epider- mal tissues of Nicotiana benthamiana. For BiFC analyses, binary con- structs were generated to over-express AbMV Rep fused with either the epitope-tagged N- or C-terminal non-fluorescent half of the yellow fluorescent protein (YFP) (Fig. 1a, supplementary material S1, pSPYNE-35S GATEWAY with cMyc:YFP N or pSPYCE-35S GATEWAY with hemagglutinin [HA]:YFP C , Walter et al., 2004). For com- 0168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2011.07.020