Geminiviruses package single-stranded circular DNA and replicate via double-stranded DNA intermediates. During the past decade, increasing evidence has led to the general acceptance that their replication follows a rolling-circle replication mechanism like bacteriophages with single-stranded DNA. In a recent study, we showed that this is also true for Abutilon mosaic geminivirus (AbMV), but that this particular virus may also use a recombination-dependent replication (RDR) route in analogy to T4 phages. Because AbMV is a special case, since it has been propagated on ornamental plants for more than a hundred years, it was interesting to determine whether RDR is common among other geminiviruses. We analyzed geminiviruses from different genera and geographic origins by using BND cellulose chromatography in combination with an improved high resolution two-dimensional gel electrophoresis, and we conclude that multitasking in replication is widespread, at least for African cassava mosaic, Beet curly top, Tomato golden mosaic, and Tomato yellow leaf curl virus.Geminiviruses have spread worldwide during the past three decades, causing severe diseases in important crop plants (23,35). Several factors have promoted the epidemics: transportation of plant material, increase and diversification of insect vector populations, and recombination between geminiviruses that coinfect host plants. Geminiviruses may contain monopartite or bipartite genomes (45), and consequently recombination may occur in two different ways: by exchange of viral chromosomes (interchromosomal recombination, or "pseudorecombination" as plant virologists call this type of reassortment) (54,55,61,62) or by crossover of chromosomes (intrachromosomal recombination) (5,6,17,19,38,46,68,69). Moreover, geminiviruses are able to adopt satellite-like DNA circles which have an additional impact on pathogenesis (7,12,47). In comparison to RNA-containing plant viruses, geminiviruses are relatively prone to recombination and harbor frequent footprints of recombination events within their genomes (38). This phenomenon is conceivable on the basis of a recombination-dependent replication mode of geminiviruses as was proposed recently (26).Geminiviruses package single-stranded circular DNA in twin-shaped capsids (67) and complement their DNA by using RNA-primed DNA polymerization (49). The resulting doublestranded circular DNA is packed into minichromosomes (1, 42). New rounds of replication were originally identified by two-dimensional gel electrophoresis (48), and these results have founded a rolling-circle replication (RCR) model for geminivirus multiplication (reviewed in reference 22) in analogy to the replication of bacteriophages with single-stranded circular DNA (32). A single viral protein ("AC1," also known as "AL1" or "C1" for different virus species or "Rep" for replication-associated protein to indicate its function) is necessary and sufficient for initiation by nicking the viral origin of replication (22). The RCR mode was confirmed by direct electron-mic...

The Abutilon mosaic virus (AbMV, Geminiviridae) DNA B component encodes a movement protein (MP), which facilitates viral transport within plants and affects pathogenicity. The presence of phosphorylated serine and threonine residues was confirmed for MP expressed in yeast and Nicotiana benthamiana by comparative Western blot analysis using phospho-amino acid- and MP-specific immunodetection. Mass spectrometry of yeast-derived MP identified three phosphorylation sites located in the C-terminal domain (Thr-221, Ser-223 and Ser-250). To assess their functional relevance in plants, several point mutations were generated in the MP gene of DNA B, which replace Thr-221, Ser-223 and Ser-250, either singly or in combinations, with either an uncharged alanine or a phosphorylation-mimicking aspartate residue. When co-inoculated with DNA A, all mutants were infectious. In systemically infected plants the symptoms and/or viral DNA accumulation were significantly altered for several of the mutants.

A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.Geminiviruses belong to a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (9). The Geminiviridae family (58) is divided into four genera according to their genome organization and biological properties. The genus Begomovirus includes members that are transmitted by whiteflies, infect only dicotyledonous plants, and may have bipartite (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV) or Tomato yellow leaf curl virus (TYLCV).Although among plant virus a minority, geminiviruses have a great economical impact, affecting many different crops worldwide (42). TYLCSV and TYLCV are two distinct species causing tomato yellow leaf curl disease, one of the most important threats to tomato crops in many tropical and subtropical regions of the world (45). Both viruses possess genomes of about 2.7 kb in size, which encode at least six proteins and contain an intergenic region (IR) that comprises the origin of replication and viral promoters. The open reading frames (ORFs) in the complementary sense orientation encoding a replication-associated protein (Rep, also known as AC1, AL1, C1, or L1), a transcription activator protein (TrAP, also known as AC2, AL2, C2, or L2), and a replication enhancer protein (REn) partially overlap; a small ORF, C4, is located within the Rep ORF but in a different reading frame (25). Rep and REn are required for efficient viral DNA replication, although...

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