Circomics of Cuban geminiviruses reveals the first alpha-satellite DNA in the Caribbean.

Circomics (circular DNA genomics), the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analysis and pyro-sequencing, has been used recently to identify geminiviruses with high efficiency and low costs. Circular DNAs associated with Cuban geminiviruses were characterised by RCA/RFLP analysis and 454 sequencing of two batches of DNA amplified from selected plant samples as well as individual cloning and Sanger sequencing of DNA components and compared to other geminiviral DNAs by phylogenetic analysis. Cuban geminiviruses that were closely related to each other challenged the circomics approach. Ten geminiviral components and one alpha-satellite DNA were determined and compared to three geminiviral components obtained by conventional cloning. New strains of Sida yellow mottle virus (SiYMoV), tomato yellow distortion leaf virus (ToYDLV), Sida golden mosaic Florida virus (SiGMFV) and Sida golden mosaic Liguanea virus (SiGMLV) are described with host plant species being classified by molecular PCR-based bar coding. A new virus species is named Peristrophe mosaic virus. The first alpha-satellite found in Middle America establishes the New World branch of these elements which are related to nanoviruses and were previously thought to be restricted to the Old World. In conclusion, circomics is efficient for complex infections and closely related viruses to detected unexpected viral DNAs, but may need some scrutinisation by direct sequencing and cloning of individual components for certain cases.

Cloned tomato golden mosaic virus back in tomatoes.

Clones of tomato golden mosaic virus (TGMV), a key model for geminivirus research, have been transmitted back to their original host tomato for the first time. In contrast to the high pathogenicity in other solanaceous species, TGMV induced only very mild symptoms: a few chlorotic spots on the leaf lamina for the common variant (formerly strain cs), and limited vein yellowing for the yellow vein variant (yv). Symptoms disappeared over time, though viral DNA remained detectable in newly developed leaves. Both TGMV variants invaded phloem and, occasionally, also mesophyll parenchyma cells in tomato. Complete direct sequencing of rolling circle amplification products of the viral progeny in tomato plants revealed the consensus of the DNA populations for the two genome components (DNA-A, DNA-B) of both TGMV variants. The DNA-A components showed 98.5% and 99.9% nucleotide sequence identity, respectively, with the independently cloned TGMV molecule sequenced initially in 1984, confirming the classification of csTGMV and yvTGMV as variants. The results are discussed with reference to the history of the Brazilian "mosaico dourado" disease in tomato, and the odyssey of TGMV passaging through Nicotiana benthamiana plants and bacteria of numerous laboratories worldwide.

Circular DNA genomics (circomics) exemplified for geminiviruses in bean crops and weeds of northeastern Brazil.

Circomics was coined to describe the combination of rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) and pyro-sequencing to investigate the genome structures of small circular DNAs. A batch procedure is described using 61 plant samples from Asia, South America and Central America which revealed 83 contig sequences of geminiviral DNA components and 4 contig sequences of DNA satellites. The usefulness of this approach is validated for the Brazilian begomoviruses, and the sequence fidelity is determined by comparing the results with those of conventional cloning and sequencing of Bolivian begomoviruses reported recently. Therefore, circomics has been proven to be a major step forward to economize costs and labor and to characterize reliably geminiviral genomes in their population structure of the quasispecies.

The genomes of four novel begomoviruses and a new Sida micrantha mosaic virus strain from Bolivian weeds.

Begomovirus is the largest genus within the family Geminiviridae and includes economically important plant DNA viruses infecting a broad range of plant species and causing devastating crop diseases, mainly in subtropical and tropical countries. Besides cultivated plants, many weeds act as virus reservoirs. Eight begomovirus isolates from Bolivian weeds were examined using rolling-circle amplification (RCA) and restriction fragment length polymorphism (RFLP). An efficient, novel cloning strategy using limited Sau3A digestion to obtain tandem-repeat inserts allowed the sequencing of the complete genomes. The viruses were classified by phylogenetic analysis as typical bipartite New World begomoviruses. Four of them represented distinct new virus species, for which the names Solanum mosaic Bolivia virus, Sida mosaic Bolivia virus 1, Sida mosaic Bolivia virus 2, and Abutilon mosaic Bolivia virus are proposed. Three were variants of a new strain of Sida micrantha mosaic virus (SimMV), SimMV-rho[BoVi07], SimMV-rho[Bo:CF1:07] and SimMV-rho[Bo:CF2:07], and one was a new variant of a previously described SimMV, SimMV-MGS2:07-Bo.

Characterization of DNA intermediates of an arising geminivirus.

Weeds of the genus Sida collected in Brazil have harbored several geminiviruses persistently over decades of vegetative propagation. They serve as cradles for new geminiviruses originating from pseudorecombination (reassortment) or molecular recombination, as has been exemplified by Sida micrantha mosaic-associated viruses (SimMV). One of such viruses has developed recently and naturally by recombination between a DNA A and a DNA B of different ancestors. We used two-dimensional gel electrophoresis and hybridization to visualize viral DNA intermediates in mixed infections as well as after transfer of single viruses into test plants. DNA intermediates which indicate multitasking in replication (rolling circle and recombination-dependent replication) were readily detected in all cases. A conspicuous increase in multimerization of circular single-stranded (ss) DNA could be attributed to the recently recombined geminivirus, suggesting poor adaptation to the host and/or inefficient gene regulation. Consequences of the accumulation of multimeric ssDNA were analyzed using nucleoprotein particle purification and electron microscopy. SimMV nucleoprotein exhibited pleomorphic structures in addition to the typical twin particles. This report provides the first analysis of DNA intermediates of an arising geminivirus.