Rolling circle replication is a mechanism for copying singlestranded genomes by means of double-stranded intermediates. A multifunctional replication inititiator protein (Rep) is indispensable for the precise initiation and termination of this process. Despite the ubiquitous presence and fundamental importance of rolling circle replication elements, structural information on their respective replication initiators is still missing. Here we present the solution NMR structure of the catalytic domain of Rep, the initiator protein of tomato yellow leaf curl virus. It is composed of a central five-stranded anti-parallel ␤-sheet, flanked by a small twostranded ␤-sheet, a ␤-hairpin and two ␣-helices. Surprisingly, the structure reveals that the catalytic Rep domain is related to a large group of proteins that bind RNA or DNA. Identification of Rep as resembling the family of ribonucleoprotein͞RNA-recognition motif fold proteins establishes a structure-based evolutionary link between RNA binding proteins, splicing factors, and replication initiators of prokaryotic and eukaryotic single-stranded DNA elements and mammalian DNA tumor viruses.

The Rep protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a single-stranded DNA virus of plants, is the replication initiator essential for virus replication. TYLCSV Rep has been classified among ATPases associated with various cellular activities (AAA؉ ATPases), in superfamily 3 of small DNA and RNA virus replication initiators whose paradigmatic member is simian virus 40 large T antigen. Members of this family are DNA-or RNA-dependent ATPases with helicase activity necessary for viral replication. Another distinctive feature of AAA؉ ATPases is their quaternary structure, often composed of hexameric rings. TYLCSV Rep has ATPase activity, but the helicase activity, which is instrumental in further characterization of the mechanism of rolling-circle replication used by geminiviruses, has been a longstanding question. We present results showing that TYLCSV Rep lacking the 121 N-terminal amino acids has helicase activity comparable to that of the other helicases: requirements for a 3 overhang and 3-to-5 polarity of unwinding, with some distinct features and with a minimal AAA؉ ATPase domain. We also show that the helicase activity is dependent on the oligomeric state of the protein.Initiation of DNA replication is a highly specific and controlled event which depends on the coordinated assembly of large protein complexes at the origin of replication. This process, which is universal, is activated by recognition and binding of the initiator protein to its cognate replication origin on the chromosome. Other steps follow, such as melting at the origin of replication and further unwinding carried out by replicative helicases recruited at the site of replication (31). In the case of extrachromosomal elements, such as viruses and plasmids, the initiator protein is often multifunctional and performs several of these steps, as well as interacting with host factors necessary for replication (42).The Rep protein is the initiator protein of tomato yellow leaf curl Sardinia virus (TYLCSV), a begomovirus of the Geminiviridae family of plant single-stranded DNA (ssDNA) viruses. The Rep proteins of geminiviruses are closely related and show substantial sequence conservation. Rep, also named C1 and AL1, is a multifunctional protein and the only viral protein absolutely required for virus replication. Four functional domains have been delineated for begomovirus Rep: the N-terminal domain (amino acids [aa] 1 to 120), which is involved in initiation of the rolling-circle replication (RCR) utilized by geminiviruses (4,34,35); the oligomerization domain (aa 121 to 180), leading to interactions with itself (34) and with host factors (17); the ATPase domain (aa 181 to 330), which is characterized by the presence of a P loop (discussed in more detail below); and a carboxyl-terminal domain (aa 331 to 359) of unknown function but shown to be required for viral replication in the case of tomato golden mosaic virus (35) (Fig. 1).The ATPase domain of geminivirus Rep proteins was identified as a common element among proteins encoded by...

Nicotiana tabacum cv. Xanthi transgenic plants expressing ORF13 of Agrobacterium rhizogenes 8196 T‐DNA under the 35S RNA promoter from the cauliflower mosaic virus displayed developmental abnormalities. They were small, with short and variable internodal lengths, their root systems were poorly developed; leaves were small, asymmetric, rounded, wrinkled and dark green; flowers were short, and irregularly shaped. They exhibited reduced apical dominance and regularly produced offshoots at the base of the plant. This phenotype was also exhibited by offshoots of normal N. tabacum cv. Xanthi stock grafted with a transgenic scion indicating that expression of ORF13 influences plant development via diffusible factor(s).

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA--which we refer to as rol alpha, beta and gamma--were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5' non-coding region of rol alpha and rol beta was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol beta in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol alpha-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.

Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene. ORF13 exhibited high activity in roots but with different patterns of expression. The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem. The ORF13 promoter is wound inducible in a limited area adjacent to the wound site. The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses. A series of 5' deletions of the ORF13 promoter fused to the beta-glucuronidase gene was examined for expression in roots and leaves of transgenic plants. Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected.

SummaryThe multipartite genome of the nanovirus Faba bean necrotic yellows virus, which consists of one gene on each DNA component, was exploited to construct a series of virus-based episomal vectors designed for transient replication and gene expression in plants. This nanovirus based expression system yields high levels of protein which allows isolation of recombinant protein and protein complexes from plant tissues. As examples, we demonstrated in planta interaction between the nanovirus F-box protein Clink and SKP1, a constituent of the ubiquitin-dependent protein turnover pathway. Thus, replicative nanovirus vectors provide a simple and ef®cient means for in planta characterization of protein-protein interaction.

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