Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

Cotton leaf curl disease (CLCuD) is a major constraint to cotton production in Pakistan. Infectious clones of the monopartite begomovirus cotton leaf curl virus (CLCuV), associated with diseased cotton, are unable to induce typical symptoms in host plants. We have identified and isolated a single-stranded DNA molecule approximately 1350 nucleotides in length which, when coinoculated with the begomovirus to cotton, induces symptoms typical of CLCuD, including vein swelling, vein darkening, leaf curling, and enations. This molecule (termed DNA beta) requires the begomovirus for replication and encapsidation. The CLCuV/DNA 1/DNA beta complex, together with a similar complex previously identified in Ageratum conyzoides, represent members of an entirely new type of infectious, disease-causing agents. The implications of this finding to our understanding of the evolution of new disease-causing agents are discussed.

Ageratum conyzoides L., a weed species widely distributed throughout southeast Asia, frequently exhibits striking yellow vein symptoms associated with infection by Ageratum yellow vein virus (AYVV), a member of the Geminiviridae (genus Begomovirus). Most begomoviruses have bipartite genomes (DNAs A and B), but only a DNA A has been identified for AYVV. We demonstrate that yellow vein disease of A. conyzoides results from co-infection by AYVV DNA A (2,741 nt) and a circular DNA that is approximately half its size (1,347 nt) that we designate DNA beta. Apart from the sequence TAATATTAC, common to all geminiviruses and containing the initiation site of rolling circle replication, DNA beta shows negligible sequence homology either to AYVV DNA A or to DNA B associated with bipartite begomoviruses. DNA beta depends on DNA A for replication and is encapsidated by DNA A-encoded coat protein and so has characteristics of a DNA satellite. However, systemic infection of A. conyzoides by DNA A alone is sporadic and asymptomatic, and DNA A accumulation is reduced to 5% or less of its accumulation in the presence of DNA beta. Therefore, DNA A and DNA beta together form a previously unrecognized disease-inducing complex. Our data also demonstrate that the nanovirus-like DNA 1 component associated with infected A. conyzoides plays no essential role in the disease and represents a satellite-like DNA. Furthermore, the satellite DNA previously found associated with tomato leaf curl virus is probably a defective DNA beta homologue.

Two bipartite begomoviruses, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), have been isolated from mosaic-diseased cassava originating from central India and Sri Lanka, respectively. ICMV was transmitted with low efficiency from cassava to Nicotiana benthamiana by sap inoculation to give leaf curl symptoms. SLCMV was much more virulent in this host, producing severe stunting, leaf curl, and chlorosis. These symptoms were reproduced when their cloned genomic components (DNAs A and B) were introduced into N. benthamiana by either mechanical or Agrobacterium-mediated inoculation (agroinoculation). SLCMV is more closely related to ICMV (DNA A, 84%; DNA B, 94% nucleotide identity) than African cassava mosaic virus (ACMV) (DNA A, 74%; DNA B, 47% nucleotide identity). Sequence comparisons suggest that SLCMV DNA B originated from ICMV DNA B by a recombination event involving the SLCMV DNA A intergenic region. Pseudorecombinants produced by reassortment of the cloned components of ICMV and ACMV were not infectious in N. benthamiana, emphasising their status as distinct virus species. In contrast, a pseudorecombinant between ACMV DNA A and SLCMV DNA B was infectious. Consistent with these observations, iteron motifs located within the intergenic region that may be involved in the initiation of viral DNA replication are conserved between SLCMV and ACMV but not ICMV. When introduced into N. benthamiana by agroinoculation, SLCMV DNA A alone produced a severe upward leaf roll symptom, reminiscent of the phenotype associated with some monopartite begomoviruses. Furthermore, coinoculation of SLCMV DNA A and the satellite DNA beta associated with ageratum yellow vein virus (AYVV) produced severe downward leaf curl in N. glutinosa and yellow vein symptoms in Ageratum conyzoides, resembling the phenotypes associated with AYVV DNA A and DNA beta infection in these hosts. Thus, SLCMV DNA A has biological characteristics of a monopartite begomovirus, and the virus probably evolved by acquisition of a DNA B component from ICMV.

To elucidate the mechanism of formation of cowpea mosaic virus (CPMV) particles, RNA-2-encoded precursor proteins were expressed in Spodoptera frugiperda cells. Processing of the 105K and 95K polyproteins in trans to give the mature Large (L) and Small (S) coat proteins required both the 32K proteinase cofactor and the 24K proteinase itself, while processing of VP60, consisting of the fused L-S protein, required only the 24K proteinase. Release of the L and S proteins resulted in the formation of virus-like particles (VLPs), showing that VP60 can act as a precursor of virus capsids. Processing of VP60 expressed in plants also led to efficient production of VLPs. Analysis of the VLPs produced by the action of the 24K proteinase on precursors showed that they were empty (RNA-free). This has important implications for the use of CPMV VLPs in biotechnology and nanotechnology as it will permit the use of noninfectious particles.

Ageratum yellow vein disease (AYVD) is caused by the geminivirus ageratum yellow vein virus (AYVV) and an associated DNA beta satellite. We have mapped a DNA beta transcript to a highly conserved open reading frame (betaC1 ORF). The most abundant transcript 5'-terminus is located 8 bases upstream of the betaC1 ORF putative initiation codon while the transcript terminates at multiple sites downstream from the putative termination codon. Disruption of betaC1 protein expression by the introduction of an internal nonsense codon prevented infection of the AYVV-satellite complex in ageratum and altered the phenotype in Nicotiana benthamiana to that produced by AYVV alone although the mutant was maintained in systemically infected tissues. Modification of the putative initiation codon to a nonsense codon produced an intermediate phenotype in N. benthamiana and a mild yellow vein phenotype in ageratum, suggesting that betaC1 protein expression could be initiated from an alternative site. N. benthamiana plants containing a dimeric DNA beta transgene produced severe developmental abnormalities, vein-greening, and cell proliferation in the vascular bundles. Expression of betaC1 protein from a potato virus X (PVX) vector also induced abnormal plant growth. Our results demonstrate that the satellite encodes at least one protein that plays a major role in symptom development and is essential for disease progression in ageratum, the natural host of the AYVD complex.

Yellow vein disease of Ageratum conyzoides, a weed species that is widely distributed throughout Asia, has been attributed to infection by the geminivirus Ageratum yellow vein virus (AYVV). In addition to a single AYVV genomic component (DNA A), we have previously demonstrated that infected plants contain chimeric defective viral components, comprising DNA A and nongeminiviral sequences, that act as defective interfering DNAs. A database search has revealed that the nongeminiviral sequences of one such defective component (def19) show significant homology with sequences of nanovirus components that encode replication-associated proteins (Reps). Primers designed to hybridise to the nongeminiviral DNA were used to PCR-amplify a full-length nanovirus-like component, referred to as DNA 1, from an extract of infected A. conyzoides. DNA 1 is unrelated to AYVV DNA A but resembles nanovirus components that encode Reps and is most closely related (73% identity) to a nanovirus-like DNA recently isolated from geminivirus-infected cotton. DNA 1 is dependent on AYVV DNA A for systemic infection of A. conyzoides and Nicotiana benthamiana and can systemically infect N. benthamiana in the presence of the bipartite geminivirus African cassava mosaic virus. A. conyzoides plants coinfected with AYVV DNA A and DNA 1 remain asymptomatic, indicating that additional factors are required to elicit yellow vein disease. Our results provide direct evidence for recombination between distinct families of plant single-stranded DNA viruses and suggest that coinfection by geminivirus and nanovirus-like pathogens may be a widespread phenomenon. The ability of plant DNA viruses to recombine in this way may greatly increase their scope for diversification.

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).