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Highlights • EVEs are widespread in eukaryotic genomes. Particular types are NIRVS and EPRVS. • The genome of most vascular plants contains a EPRV homologous to caulimovirus' MP. • These endogenous proteins may still retain the ability to move RNAs or ribonucleoproteins from cell-to-cell. • Arabidopsis endogenous MP-like gene is expressed in different plant tissues along development, particularly in flowers. • AtMP may play a role in plant development, sensitivity to salt during germination and play a role in virus infection. Abstract Genes orthologous to the 30K-superfamily of movement proteins (MP) from plant viruses have been recently discovered by bioinformatics analyses as integrated elements in the genome of most vascular plants. However, their functional relevance for plants is still unclear. Here, we undertake some preliminary steps into the functional characterization of one of these putative MP genes found in Arabidopsis thaliana. We found that the AtMP gene is expressed at different stages of the plant development, with accumulation being highest in flowers but lowest in mature siliques. We also found down-regulation of the gene may result in a small delay in plant development and in an exacerbation of the negative effect of salinity in germination efficiency. We have also explored whether changes in expression of the endogenous AtMP have any effect on susceptibility to infection with several viruses, and found that the infectivity of tobacco rattle tobravirus was strongly dependent on the expression of the endogenous AtMP. Finally, we have cloned the endogenous MP from four different plant species into an expression vector that allows for specifically assessing their activity as cell-to-cell movement proteins and have shown that though some may still retain the ancestral activity, they do so in a quite inefficient manner, thus suggesting they have acquired a novel function during adaptation to the host genome. [ABSTRACT FROM AUTHOR]

Over the past decades, several studies have examined the subcellular localization of the cauliflower mosaic virus (CaMV) P6 protein by tagging it with GFP (P6-GFP). These investigations have been essential in the development of models for inclusion body formation, nuclear transport, and microfilament-associated intracellular movement of P6 inclusion bodies for delivery of virions to plasmodesmata. Although it was shown early on that the translational transactivation function of P6-GFP was comparable to wild type P6, it has not been possible to incorporate a P6-GFP gene into an infectious clone of CaMV. Consequently, it has not been possible to formally prove that a P6-GFP fusion is comparable in function to the unmodified P6 protein. Here we show that transient expression of P6-GFP can complement a defective CaMV replicon through gene expression, replication and encapsidation, which validates the relevance of P6-GFP subcellular localization studies for understanding the development of CaMV infections. • CaMV P6-GFP fusions have been integral for elucidating the subcellular location of the multifunctional P6 protein. • Separate delivery of either P6 or P6-GFP can complement a defective CaMV isolate through replication and virion formation. • This work validates the previously published subcellular localization studies conducted with P6-GFP. [ABSTRACT FROM AUTHOR]

A virus isolate designated Angelica bushy stunt virus (AnBSV), provisionally representing a new species in the genus Caulimovirus, was discovered in the medicinal plant Angelica dahurica. The complete 8,300-nt genomic DNA of AnBSV had seven putative open reading frames containing conserved domains/motifs, which are typical features of caulimoviruses, and showed the greatest nucleotide sequence identity (74% identity and 27% query coverage) to a lamium leaf distortion virus isolate. Interestingly, the new caulimovirus exists as endogenous pararetroviral sequences in the host plant and is considered to have multiple defective plant genome-integrated copies that may lead to the generation of subgenomic DNA species. [ABSTRACT FROM AUTHOR]

A total of nine contigs related to caulimovirus-like sequences were detected using high-throughput paired-end RNA sequencing. An attempt to find the plant sample infected with this type of virus identified the medicinal plant Atractylodes macrocephala Koidzumi showing mild mottle symptoms. Subsequently, the complete DNA genome sequence of the Atractylodes virus was determined. The 8,105-nt genome of the virus was composed of six open reading frames and displayed the highest nucleotide sequence identity (70 %) with soybean Putnam virus. Based upon the symptoms observed on the source plant, we propose to refer to this new member of the genus Caulimovirus as atractylodes mild mottle virus. [ABSTRACT FROM AUTHOR]

Cauliflower mosaic virus gene VI product (P6) is an essential protein that forms cytoplasmic, inclusion bodies (IBs). P6 contains four regions involved in self-association, termed D1–D4. D3 binds to D1, along with D4 and contains a spacer region (termed D3b) between two RNA-binding domains. Here we show D3b binds full-length P6 along with D1 and D4. Full-length P6s harboring single amino acid substitutions within D3b showed reduced binding to both D1 and D4. Full-length P6s containing D3b mutations and fused with green fluorescent protein formed inclusion-like bodies (IL-Bs) when expressed in Nicotiana benthamiana leaves. However, mutant P6s with reduced binding to D1 and D4, showed smaller IL-Bs, than wild type. Likewise, viruses containing these mutations showed a decrease in inoculated leaf viral DNA levels and reduced efficiency of systemic infection. These data suggest that mutations influencing P6 self-association alter IB formation and reduce virus infection. [ABSTRACT FROM AUTHOR]

Microsatellites are known to exhibit ubiquitous presence across all kingdoms of life including viruses. Members of the Caulimoviridae family severely affect growth of vegetable and fruit plants and reduce economic yield in diverse cropping systems worldwide. Here, we analyzed the nature and distribution of both simple and complex microsatellites present in complete genome of 44 species of Caulimoviridae . Our results showed, in all analyzed genomes, genome size and GC content had a weak influence on number, relative abundance and relative density of microsatellites, respectively. For each genome, mono- and dinucleotide repeats were found to be highly predominant and are overrepresented in genome of majority of caulimoviruses. AT/TA and GAA/AAG/AGA was the most abundant di- and trinucleotide repeat motif, respectively. Repeats larger than trinucleotide were rarely found in these genomes. Comparative study of occurrence, abundance and density of microsatellite among available RNA and DNA viral genomes indicated that simple repeats were least abundant in genomes of caulimoviruses. Polymorphic repeats even though rare were observed in the large intergenic region of the genome, indicating strand slippage and/or unequal recombination processes do occur in caulimoviruses. To our knowledge, this is the first analysis of microsatellites occurring in any dsDNA viral genome. Characterization of such variations in repeat sequences would be important in deciphering the origin, mutational processes, and role of repeat sequences in viral genomes. [ABSTRACT FROM AUTHOR]

The Panel on Plant Health performed a pest categorisation of Strawberry vein banding virus (SVBV) for the European Union (EU) territory. SVBV is a well-defined virus species of the genus Caulimovirus for which the entire genome sequence is known and molecular detection assays are available. SVBV is transmitted by vegetative multiplication of infected hosts and through the activity of aphid vectors, the most efficient being Chaetosiphon spp. The virus is reported from all continents and is present in three EU Member States: the Czech Republic, Italy and Slovakia. The host range of SVBV is restricted to cultivated and wild strawberries. It is listed in Annex IAI of Directive 2000/29/EC. SVBV is not expected to be affected by ecoclimatic conditions wherever its hosts are present and has the potential to establish in large parts of the EU territory, and to subsequently spread through the action of its Chaetosiphon fragaefolii vector, which is present in many Member States. SVBV does not cause severe symptoms, and modern cultivars are mostly symptomless if infected with SVBV alone. SVBV can, however, contribute to more severe symptoms when it occurs in mixed infections with other strawberry viruses. Despite this, SVBV is considered a minor problem in strawberry production as a consequence of modern practices including the systematic use of certified planting materials and the use of short crop cycles, which have greatly reduced the impact of strawberry viruses. Overall, SVBV does not have the potential to be a quarantine pest as, given current agricultural practices, it does not fulfil the pest categorisation criteria defined in the International Standards for Phytosanitary Measures No 11 of having a severe impact. However, SVBV has the potential to be a regulated non-quarantine pest because it fulfils all pest categorisation criteria defined in the International Standards for Phytosanitary Measures No 21. [ABSTRACT FROM AUTHOR]

The dahlia ( Dahlia variabilis) genome contains an endogenous pararetrovirus sequence (EPRS) tentatively designated as DvEPRS. The DvEPRS shares genome structure and organization that is typical of members of the Caulimovirus genus. Studies were carried out to better understand the nature of this integration and to determine the gene expression of this DvEPRS. Genomic Southern hybridization showed multiple and random integration events of the DvEPRS in the dahlia genome. To investigate the presence of DvEPRS transcripts, RT-PCR was done on DNase-treated total RNA from DvEPRS-infected dahlia plants. Results showed the expression of open reading frames I, V, and VI. Direct PCR from sap extracts produced more intense DNA amplicons of Dahlia mosaic virus and Dahlia common mosaic virus which are believed to exist as typical episomal caulimoviruses, whereas significantly less intense amplicon was seen in case of DvEPRS in comparison with internal transcribed spacer region of dahlias amplicon. The DvEPRS in wild and cultivated species of Dahlia offer a model system to study the molecular events underlying the ecology, evolution and spread of DvEPRS within natural and managed ecosystems and the factors affecting integration of these EPRS in the plant genome. [ABSTRACT FROM AUTHOR]

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus, and an endogenous plant pararetroviral sequence (DvEPRS) were reported in Dahlia spp. DvEPRS, previously referred to as DMV-D10, was originally identified in the US from the cultivated Dahlia variabilis, and has also been found in New Zealand, Lithuania and Egypt, as well as in wild dahlia species growing in their natural habitats in Mexico. Sequence analysis of three new EPRSs from cultivated dahlias from Lithuania [D10-LT; 7,159 nucleotide level (nt)], New Zealand (D10-NZ, 7,156 nt), and the wild species, Dahlia rupicola, from Mexico (D10-DR, 7,133 nt) is reported in this study. The three EPRSs have the structure and organization typical of a caulimovirus species and showed identities among various open reading frames (ORFs) ranging between 71 and 97 % at the nt when compared to those or the known DvEPRS from the US. Examination of a dataset of seven full-length EPRSs obtained to date from cultivated and wild Dahlia spp. provided clues into genetic diversity of these EPRSs from diverse sources of dahlia. Phylogenetic analyses, mutation frequencies, potential recombination events, selection, and fitness were evaluated as evolutionary evidences for genetic variation. Assessment of all ORFs using phylogenomic and population genetics approaches suggests a wide genetic diversity of EPRSs occurring in dahlias. Phylogenetic analyses show that the EPRSs from various sources form one clade indicating a lack of clustering by geographical origin. Grouping of various EPRSs into two host taxa (cultivated vs. wild) shows little divergence with respect to their origin. Population genetic parameters demonstrate negative selection for all ORFs, with the reverse transcriptase region more variable than other ORFs. Recombination events were found which provide evolutionary evidence for genetic diversity among dahlia-associated EPRSs. This study contributes to an increased understanding of molecular population genetics and evolutionary pathways of these reverse transcribing viral elements. [ABSTRACT FROM AUTHOR]

In this study a new real-time PCR assay for the detection of figwort mosaic virus (FMV) DNA is described. This assay targets a 113-bp-long sequence of the FMV open reading frame Vll, a non-conserved coding region among the caulimoviruses. Detection of FMV DNA is useful to complement screening for the FMV 34S promoter (P-FMV), a genetic element present in several genetically modified (GM) plants. The specificity of the assay was assessed against closely related plant viruses, plant species of agronomic importance or susceptible to infection by FMV, and various GM plants containing the P-FMV sequence. No cross-reactivity of the assay against the tested nontarget organisms was observed. The limit of detection of the FMV real-time PCR assay was determined at approximately 5 copies per reaction. The robustness of the method was tested using different real-time PCR instruments and PCR master mixes with no negative effects on the PCR efficiency and linearity being observed. When amplifying the FMV target DNA at a concentration level close to the detection limit, no negative influence on the PCR performance was observed in the presence of background genomic DNA from various plant species. The precision data of the in-house validation were in line with generally accepted performance requirements. In summary, the method appears fit for the purpose of a specific identification test for the presence of genomic FMV DNA, while preventing false-positive results during P-FMV screening. [ABSTRACT FROM AUTHOR]

Dahlia mosaic disease of the ornamental flowering plant Dahlia is caused by two caulimoviruses, dahlia mosaic virus (DMV) and dahlia common mosaic virus (DCMV). We used a rolling-circle amplification method to amplify, clone and determine for the first time the full genome sequence of a DCMV isolate from New Zealand (DCMV-NZ). Within the 7949-bp circular double-stranded retro-transcribing DCMV-NZ DNA, we identified six putative open reading frames, typical of all genomes in the family Caulimoviridae. The availability of the complete DCMV sequence provides a reference genome against which all others can be compared. [ABSTRACT FROM AUTHOR]

The genome structure and organization of endogenous caulimovirus sequences from dahlia ( Dahlia spp), dahlia mosaic virus (DMV)-D10 from three wild species, D. coccinea (D10-DC), D. sherffii (D10-DS) and D. tenuicaulis (D10-DT), were determined and compared to those from cultivated species of dahlia, D. variabilis (DvEPRS). The complete ca. 7-kb dsDNA genomes of D10-DC, D10-DS, and D10-DT had a structure and organization typical of a caulimovirus and shared 89.3 to 96.6% amino acid sequence identity in various open reading frames (ORF) when compared to DvEPRS . The absence of the aphid transmission factor and the truncated coat protein fused with the reverse transcriptase ORF were common among these DMV-D10 isolates from wild Dahlia species. [ABSTRACT FROM AUTHOR]

Abstract: The genome of the floriculture pathogen Dahlia mosaic caulimovirus (DMV) encodes six open reading frames. Generally, caulimovirus gene VI products (P6s) are thought to be multifunctional proteins required for viral infection and it is likely that self-association is required for some of these functions. In this study, yeast two-hybrid and maltose binding protein (MBP) pull-down assays indicated that full-length DMV P6 specifically self-associates. Further analyses indicated that only the DMV P6 N-terminal region, consisting of 115 amino acids, interacts with full-length P6 and with itself. This distinguishes the DMV P6 from its Cauliflower mosaic virus counterpart, which contains four regions involved in self-association. Thus, our results suggest that each caulimovirus P6 may possess a unique pattern of protein–protein interactions. Bioinformatic tools identified a putative nuclear exclusion signal located between amino acid residues 10–20, suggesting another possible function for the P6 N-terminal region. [Copyright &y& Elsevier]

Selected wild Dahlia species in their natural habitats from west-central Mexico were tested for the presence of three caulimoviruses known to be associated with cultivated dahlia ( Dahlia variabilis), viz. Dahlia mosaic virus (DMV), DMV-D10 and Dahlia common mosaic virus. Virus species-specific primers and PCR were used followed by cloning and sequencing of the amplicons. Results showed that the wild dahlia species in their natural habitat contained DMV-D10, which is an endogenous plant pararetrovirus. Viral sequences were found in 91% of the samples ( n = 56) representing four different wild species. The gene coding for the movement protein of DMV-D10 from Dahlia coccinea and all other species was cloned and sequenced. Sequence comparisons showed divergence of this gene when compared to that of DMV-D10 from cultivated dahlias. The discovery of plant pararetroviruses in wild dahlia species in their natural habitats suggests a possible emergence, co-existence and co-evolution of pararetroviruses and their host plants. [ABSTRACT FROM AUTHOR]

By the techniques of DNA shuffling, PCR, and restriction-ligation, chimeric forms of cauliflower ( Brassica oleracea) mosaic virus (CaMV), dahlia ( Dahlia pinnata) mosaic virus (DMV), and carnation ( Dianthus caryophillus) etching ring virus (CERV) promoters were obtained at various combinations. Twelve chimeric promoters were cloned into pCambia binary vectors comprising the reporter GUS gene, and their activities in transgenic tobacco ( Nicotiana tabacum) plants were determined fluorimetrically. 35S promoter and those of DMV (442 bp) and CERV (371 and 501 bp) were used as controls. Seven of analyzed promoters displayed higher and seven promoters lower activity in transgenic tobacco plants than 35S promoter. The highest activity was characteristic of natural DMV promoter, and the least one — natural CERV promoter 501 bp in size. The CERV promoter 371 bp in size was approximately similar in strength to 35S promoter. [ABSTRACT FROM AUTHOR]

Abstract: Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was investigated for the screening of genetically modified organisms (GMOs). Attention was focused on the choice of the coating chemistry that could be used for the immobilization of probe sequences on the gold surface of the quartz crystal. Two immobilization procedures were tested and compared considering the amount of the immobilized P35S probe and the extent of the hybridization reaction with the target oligonucleotide. In wet chemistry procedure, the interaction between the thiol and gold for the immobilization of a thiolated probe was employed. Direct surface functionalization of piezoelectric quartz crystals were achieved in 13.56MHz plasma polymerization reactor utilising ethylenediamine (EDA) precursors for the immobilization of amined probes. Results indicated that immobilization of a thiolated probe provides better immobilization characteristics and higher sensitivity for the detection of the hybridization reaction. The thiolated probe was used for the detection of P35S sequence in PCR-amplified DNAs and in real samples of pflp (ferrodoxin like protein)-gene inserted tobacco plants. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and ultrasonication. The results obtained from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM-based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of GMOs. [Copyright &y& Elsevier]

Promoters of carnation etched ring virus (CERV) and dahlia mosaic virus (DMV) were cloned into binary vectors pCambia 1304, pCambia 1281Z, and pCambia 1291Z with reporter GFP and GUS genes. Activities of these promoters in tobacco protoplasts and transgenic plants were determined using these constructs. Histochemical GUS analysis demonstrated the absence of tissue-specificity in transgenic plants transformed with these promoters. The quantitative analysis of these promoter activities in transgenic tobacco plants, using 4-methylumbelliferone as a substrate, showed that 35S CaMV, CERV, and DMV promoters displayed approximately similar activities in transgenic tobacco plants. [ABSTRACT FROM AUTHOR]

The genome structure and organization of a new and distinct caulimovirus that is widespread in dahlia ( Dahlia variabilis) was determined. The double-stranded DNA genome was ca. 7.0 kb in size and shared many of the features of the members of the genus Caulimovirus, such as the presence of genes potentially coding for the movement protein, the inclusion body protein, and the reverse transcriptase (RT), and an intergenic region consisting of a potential 35S promoter. However, the virus differed from the previously described dahlia mosaic caulimovirus and other known caulimoviruses in that the aphid transmission factor (ATF) was absent and the putative coat protein contained a C-terminal deletion and was fused in-frame with the RT. Sequence identity at the amino acid level with known caulimoviruses including a previously reported caulimovirus from dahlia was low and ranged from 32 to 72%. The absence of an ATF and the highly divergent nature of the genomic sequence are characteristics of this new caulimovirus that is widely associated with dahlia. [ABSTRACT FROM AUTHOR]

Carnation etched ring virus (CERV), is the most widespread virus in carnation cultivars after Carnation mottle virus. It's incidences has been reported worldwide. It has double stranded DNA genome with the length of ∼8 kbp. Primers were designed for CERV coat protein gene (1482 bp) amplification and directional and inframe cloning in expression vector, pET-28a(+) (Novagen, USA), using Escherichia coli strain BL 21 strain competent cells. Expression conditions for maximum recovery of soluble recombinant protein was standardized. The in vitro expressed protein was purified and was used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera production and the antisera was purified. Some of the antisera was used for enzyme conjugate preparation. This antiserum and conjugate were then used for formulation of an ELISA-based diagnostic kit for CERV detection. Its properties were compared with the commercially available kit. In all cases, with both glasshouse and field material, the antibodies had good detectability and specificity. These antibodies combine specificity to the target protein and versatility with regard to all the more important serological techniques. [ABSTRACT FROM AUTHOR]

Dahlia mosaic, caused by Dahlia mosaic virus (DMV), is one of the most important viral diseases of dahlia. Molecular characterization of DMV showed the association of two distinct caulimoviruses (DMV-D10, DMV-Portland) and a D10-like sequence variant (DMV-Holland) with the disease. Using primers specific to these two viruses and the sequence variant, a polymerase chain reaction-based assay was used to determine their relative incidence in several dahlia samples from the United States and the Netherlands. Testing was done on samples collected in 2005 and 2006 in the United States and in 2006 in the Netherlands. Results indicated the predominance of DMV-D10 over DMV-Portland and DMV-Holland in both the United States and the Netherlands. Using conserved regions of the viral genome, primers were designed and used to detect all three sequences. Results suggested that DMV-D10 is predominantly associated with dahlia mosaic, but diagnostics should also include testing for DMV-Portland and DMV-Holland. [ABSTRACT FROM AUTHOR]

Dahlia mosaic is a serious disease affecting dahlias. In addition to the Dahlia mosaic virus (DMV) reported previously, we characterized two putative new caulimoviruses, tentatively designated as DMV-D10 and Dahlia common mosaic virus (DCMV), from dahlia. To better understand their relative incidence in dahlia, a total of 213 samples were collected during 2007 and 2008 from several varieties of cultivated dahlia (D. variabilis) in the United States. Samples were tested for the three caulimoviruses using virus-specific primers in a polymerase chain reaction. Amplicons were cloned and sequenced to confirm the infection of dahlia with these viruses. Results showed that DMV-D10 was the most prevalent (94%) followed by DCMV (48.5%) and DMV (23%). Mixed infections were common and viruses were detected irrespective of symptom expression at the time of sampling. Two percent of the samples were not infected by any of the three tested caulimoviruses. Results suggest that caulimovirus infectious are widespread in dahlia and highlight the need for testing and production of virus-free material to reduce their spread. [ABSTRACT FROM AUTHOR]

Abstract: The genus Caulimovirus consists of several distinct virus species with a double-stranded DNA genome that infect diverse plant species. A comparative analysis of the sequences of known Caulimovirus species revealed two regions that are conserved in all Caulimovirus species with the exception of Strawberry vein banding virus. Degenerate primers based on these two regions were designed and tested in a polymerase chain reaction-based assay for broad spectrum detection of members of this genus. Cauliflower mosaic virus, Figwort mosaic virus and three distinct caulimoviruses associated with dahlia (Dahlia variabilis) were used to show the utility of this test in detecting diverse caulimoviruses. The primer pair gave an amplicon of expected size (840bp). Amplicons from each virus were cloned and sequenced to verify their identity. The primer pair and the PCR assay provide approach for the broad spectrum detection of several members of the genus Caulimovirus. [Copyright &y& Elsevier]

The strawberry vein banding virus (SVBV) open reading frame (ORF) VI encodes a P6 protein known as the RNA silencing suppressor. This protein is known to form inclusion like granules of various sizes and accumulate in both the nuclei and the cytoplasm of SVBV-infected plant cells. In this study, we have determined that the P6 protein is the only trans-activator (TAV) encoded by SVBV, and can efficiently trans-activate the translation of downstream gfp mRNA in a bicistron derived from the SVBV. Furthermore, the P6 protein can trans-activate the expression of different bicistrons expressed by different caulimovirus promoters. The P6 protein encoded by SVBV from an infectious clone can also trans-activate the expression of bicistron. Through protein-protein interaction assays, we determined that the P6 protein could interact with the cell translation initiation factor FveIF3g of Fragaria vesca and co-localize with it in the nuclei of Nicotiana benthamiana cells. This interaction reduced the formation of P6 granules in cells and its trans-activation activity on translation. [ABSTRACT FROM AUTHOR]