Genome characterization of parsley severe stunt-associated virus in Iran.

Parsley severe stunt-associated virus (PSSaV) is a recently identified nanovirus first reported in Germany. During a survey for identification of nanoviruses infecting apiaceous plants in south-eastern Iran, PSSaV was identified and characterized using a combination of rolling circle amplification (RCA) and high-throughput sequencing. Parsley plant samples were collected from vegetable production farms in Kerman province. From two symptomatic samples (39Ba and 40Ba), seven PSSaV components (DNA-C, -S, -M, -R, -N, -U1 and -U2) with two phylogenetically distinct variants of DNA-R (R1 and R2) were identified. In common with the German isolate of PSSaV, no DNA-U4 component was identified. In addition, associated alphasatellite molecules were identified in samples 39Ba [n = 6] and 40Ba [n = 5]. Sequence analyses showed that concatenated component sequences of the two Iranian PSSaVs share 97.2% nucleotide identity with each other and 82% to the German isolate. The coat proteins (CPs) of the PSSaV Iranian sequences share 97.2% amino acid identity and ~ 84% identity with that of the German isolate. Sequence and phylogenetic analyses of a total of 11 recovered alphasatellites from the two samples can be classified into the genera Fabenesatellite [n = 2], Milvetsatellite [n = 1], Mivedwarsatellite [n = 2], Subclovsatellite [n = 2], Sophoyesatellite [n = 4] in the family Alphasatellitidae. Identification of PSSaV and other nanoviruses in wild and cultivated plants in Iran reveals that nanoviruses could be causing yield reduction in crops plants in this country.

A Population Genetics Perspective on Geminivirus Infection.

UNLABELLED: The selective accumulation of both DNA components of a bipartite geminivirus, Abutilon mosaic virus, was recorded during early systemic infection of Nicotiana benthamiana plants. Purified nuclei were diagnosed for viral DNA using hybridization specific for DNA A or DNA B to detect these individual genome components either alone or both simultaneously by dual-color staining. Although this virus needs both components for symptomatic infection, DNA A alone was transported to upper leaves, where it was imported into phloem nuclei and replicated autonomously. The coinfection with DNA A and DNA B revealed an independent spread of both molecules, which resulted in a stochastic distribution of DNA A- and DNA A/B-infected nuclei. A population genetics evaluation of the respective frequencies was compared to a model computation. This elucidated a surprisingly simple relationship between the initial frequencies of the viral DNA components and the number of susceptible cells during the course of early systemic infection. IMPORTANCE: For bipartite begomoviruses, DNA B-independent long-distance spread of DNA A has been described before, but it has never been shown whether viral DNA A alone invades nuclei of systemic tissues and replicates therein. This is demonstrated now for the first time. During infection with DNA A and DNA B, a similar solitary spread of DNA A can be recognized at early stages. We describe a population genetics model of how the hit probabilities of DNA A and DNA B for susceptible cells determine the relative frequencies of either genome component during the course of infection.

Early function of the Abutilon mosaic virus AC2 gene as a replication brake.

UNLABELLED: The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors. IMPORTANCE: The (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology.

Rad54 is not essential for any geminiviral replication mode in planta.

The circular single-stranded DNA of phytopathogenic geminiviruses is propagated by three modes: complementary strand replication (CSR), rolling circle replication (RCR) and recombination-dependent replication (RDR), which need host plant factors to be carried out. In addition to necessary host polymerases, proteins of the homologous recombination repair pathway may be considered essential, since geminiviruses are particularly prone to recombination. Among several others, Rad54 was suggested to be necessary for the RCR of Mungbean yellow mosaic India virus. This enzyme is a double-stranded DNA-dependent ATPase and chromatin remodeller and was found to bind and modulate the viral replication-initiator protein in vitro and in Saccharomyces cerevisiae. In contrast to the previous report, we scrutinized the requirement of Rad54 in planta for two distinct fully infectious geminiviruses with respect to the three replication modes. Euphorbia yellow mosaic virus and Cleome leaf crumple virus were inoculated into Rad54-deficient and wildtype Arabidopsis thaliana plant lines to compare the occurrence of viral DNA forms. Replication intermediates were displayed in the time course of infection by one and two-dimensional agarose gel electrophoresis and Southern hybridization. The experiments showed that Rad54 was neither essential for CSR, RCR nor RDR, and it had no significant influence on virus titers during systemic infection.

KU80, a key factor for non-homologous end-joining, retards geminivirus multiplication.

KU80 is well-known as a key component of the non-homologous end-joining pathway used to repair DNA double-strand breaks. In addition, the KU80-containing DNA-dependent protein kinase complex in mammals can act as a cytoplasmic sensor for viral DNA to activate innate immune response. We have now, to our knowledge for the first time, demonstrated that the speed of a systemic infection with a plant DNA geminivirus in Arabidopsis thaliana is KU80-dependent. The early emergence of Euphorbia yellow mosaic virus DNA was significantly increased in ku80 knockout mutants compared with wild-type sibling controls. The possible impact of KU80 on geminivirus multiplication by generating non-productive viral DNAs or its role as a pattern-recognition receptor against DNA virus infection is discussed.

Genetically improved monolayer-forming tobacco mosaic viruses to generate nanostructured semiconducting bio/inorganic hybrids.

The genetically determined design of structured functional bio/inorganic materials was investigated by applying a convective assembly approach. Wildtype tobacco mosaic virus (wt TMV) as well as several TMV mutants were organized on substrates over macroscopic-length scales. Depending on the virus type, the self-organization behavior showed pronounced differences in the surface arrangement under the same convective assembly conditions. Additionally, under varying assembly parameters, the virus particles generated structures encompassing morphologies emerging from single micrometer long fibers aligned parallel to the triple-contact line through disordered but dense films to smooth and uniform monolayers. Monolayers with diverse packing densities were used as templates to form TMV/ZnO hybrid materials. The semiconducting properties can be directly designed and tuned by the variation of the template architecture which are reflected in the transistor performance.

Evolutionary liberties of the Abutilon mosaic virus cluster.

Two new strains of Abutilon mosaic virus (AbMV; Geminiviridae) from Germany (Stuttgart) and France (Paris) have been characterized by circomics, direct pyrosequencing of rolling circle amplification (RCA) products, as well as conventional cloning and Sanger sequencing. RCA combined with an analysis of restriction fragment length polymorphisms confirmed the completeness of the sequence determination and a close relationship of both isolates for DNA A with 99 % nucleotide sequence identity. Phylogenetic tree reconstruction supported their clustering with other AbMV strains in a clade with Middle American begomoviruses, whereas South American begomoviruses that infect Abutilon or Sida micrantha are less closely related. Comparing the coat protein (CP) genes of the AbMV cluster, with those of related Middle and South American begomoviruses revealed a remarkable overrepresentation for non-synonymous nucleotide exchanges for certain amino acid positions in the AbMV cluster. Projection of these positions to a structural model of the African cassava mosaic virus CP yielded a non-random distribution at the periphery and, most importantly, highlighted those amino acids that had been identified in whitefly-transmission experiments before. These results establish the basis for an analysis of the evolutionary liberty of certain amino acid positions of the CP, and their impact on the deciphering of insect transmission determinants is discussed.

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.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.

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.

Dynamic subcellular distribution of begomoviral nuclear shuttle and movement proteins.

The Abutilon mosaic virus (AbMV) encodes a nuclear shuttle protein (NSP), and a movement protein (MP) which cooperatively accomplish viral DNA transport through the plant. Subcellular distribution patterns of fluorescent protein-tagged NSP and MP were tracked in Nicotiana benthamiana leaves in presence or absence of an AbMV infection using light microscopy. NSP was located within the nucleus and associated with early endosomes in the presence of MP. MP appeared at the plasma membrane, plasmodesmata and in motile vesicles, trafficking along the endoplasmic reticulum in an actin-dependent manner. MP and NSP did not co-localize and employed separate cellular pathways. Correspondingly, Förster resonance energy transfer analysis did not support physical interaction between NSP and MP. Time lapse movies illustrate the cellular dynamics of both proteins on their way around the nucleus and to the cell periphery and provide a first hint for the nuclear egress of NSP complexes.

Replicative intermediates of maize streak virus found during leaf development.

Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombination-dependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR.

Plant geminivirus rep protein induces rereplication in fission yeast.

The replication-associated protein (Rep) of geminiviruses, single-stranded DNA viruses of higher plants, is essential for virus replication. Since these viruses do not encode their own polymerases, Rep induces differentiated plant cells to reenter the cell cycle by interacting with the plant homologues of retinoblastoma proteins in order to activate the host DNA synthesis machinery. We have used fission yeast (Schizosaccharomyces pombe) as a model organism to analyze the impact of ectopically expressed African cassava mosaic virus Rep protein on the cell division cycle in closer detail. Upon expression, Rep showed its characteristic DNA cleavage activity, and about 10% of the cells exhibited morphological changes. They were elongated threefold, on average, and possessed a single but enlarged and less compact nucleus in comparison to noninduced or vector-only control cells. Flow cytometry of Rep-expressing cultures revealed a distinct subpopulation of Rep protein-containing cells with aberrant morphology. The other 90% of the cells were indistinguishable from control cells, and no Rep was detectable. Rep-expressing cells exhibited DNA contents beyond 2C, indicating ongoing replication without intervening mitosis. Because a second open reading frame (ORF), AC4, is present within the Rep gene, the role of AC4 was examined by destroying its start codon within the AC1 ORF. The results confirmed that Rep is necessary and sufficient to induce rereplication in fission yeast. The unique potential of this well-investigated model for dissecting the cell cycle control by geminiviral proteins is discussed.

Isolation and characterization of a novel geminivirus from parsley.

Fresh leaf vegetables are a significant part of the Persian food. Following a survey for identification of nanoviruses and geminivirus infecting leaf vegetables, a novel geminivirus was identified in a diseased parsley sample showing upward marginal leaf curling, marginal leaf yellowing, dwarfing and reduced leaf size in south-eastern Iran. The genome was identified through combination of rolling circle amplification (RCA) and high throughput sequencing (HTS) approaches. The full-length genome (2779 nts) of the cloned geminivirus, parsley yellow leaf curl virus (PYLCV), shares <66 % genome-wide pairwise identity with all other known geminiviruses. The PYLCV genome has six open reading frames (ORFs) and appears to be a hybrid with the virion sense encoded proteins being most similar to those of becurtoviruses and curtoviruses, whereas the complementary sense encoded proteins are most similar to those of begomoviruses. In comparison with other geminivirus encoded capsid proteins (CPs) and replication associated proteins (Reps), the CP of PYLCV shares <56 % amino acid pairwise identity whereas the Rep shares <73 % amino acid pairwise identity. To demonstrate the pathogenicity of the geminivirus, a partial dimer infectious clone was constructed and used to agro-infect parsley as well as Nicotiana benthamiana, turnip, radish and tomato. The agro-inoculation resulted in infection with symptoms in 83.7 % (82/98) of the tested plant. Based on the similarity of the CP encoded by PYLCV to those of becurtoviruses and curtoviruses, it is likely that leafhoppers may be the primary transmission vector.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus Begomovirus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer. For Abutilon mosaic virus (AbMV), phosphorylation of MP expressed in bacteria, yeast, and Nicotiana benthamiana plants, respectively, has been demonstrated in previous studies. Three phosphorylation sites (T221, S223, and S250) were identified in its C-terminal oligomerization domain by mass spectrometry, suggesting a regulation of MP by posttranslational modification. To examine the influence of the three sites on the self-interaction in more detail, MP mutants were tested for their interaction in yeast by two-hybrid assays, or by Förster resonance energy transfer (FRET) techniques in planta. Expression constructs with point mutations leading to simultaneous (triple) exchange of T221, S223, and S250 to either uncharged alanine (MPAAA), or phosphorylation charge-mimicking aspartate residues (MPDDD) were compared. MPDDD interfered with MP-MP binding in contrast to MPAAA. The roles of the phosphorylation sites for the viral life cycle were studied further, using plant-infectious AbMV DNA-B variants with the same triple mutants each. When co-inoculated with wild-type DNA-A, both mutants infected N. benthamiana plants systemically, but were unable to do so for some other plant species of the families Solanaceae or Malvaceae. Systemically infected plants developed symptoms and viral DNA levels different from those of wild-type AbMV for most virus-plant combinations. The results indicate a regulation of diverse MP functions by posttranslational modifications and underscore their biological relevance for a complex host plant-geminivirus interaction.

Different forms of African cassava mosaic virus capsid protein within plants and virions.

One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~ 30 kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~ 30, 32 kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway.

Mitochondrial plasmids of sugar beet amplified via rolling circle method detected during curtovirus screening.

Crops of sugar beet have been considerably impaired by infection with Beet curly top virus (BCTV) during the past decades. Quick and reliable diagnostic techniques are therefore desirable to detect this circular single-stranded DNA-containing geminivirus. Techniques combining either tissue printing or blot hybridization, or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP) were compared. Although they easily detected BCTV with certainty, both exhibited apparent false positive results which have been scrutinized in closer detail. Uninfected control plants revealed unspecific signals due to probe attachment on tissue blots, and dominant fragment patterns upon RCA/RFLP which did not hybridize with BCTV-specific probes. Cloning and sequencing of these DNA fragments showed that they were amplified from mitochondrial plasmids. Examination of their genome structure revealed no relationship with geminiviruses or their satellites.

Post-translational modifications of Abutilon mosaic virus movement protein (BC1) in fission yeast.

The movement protein (MP) of Abutilon mosaic virus (AbMV, Geminiviridae) exhibited a complex band pattern upon gel electrophoresis indicating its post-translational modification when expressed in AbMV-infected plants or, ectopically, in fission yeasts. High-resolution separation according to charge and molecular weight in acetic acid/urea polyacrylamide or sodium dodecyl sulphate polyacrylamide gels followed by western blot analysis revealed a pattern of AbMV MP from infected plants more related to that from fission yeast than from bacteria. For this reason, expression in fission yeast was established as an experimental system to study post-translational modifications of AbMV MP. Metabolic labelling with 32P-orthophosphate confirmed a phosphorylation of all MP variants suggesting multiple phosphorylation sites. Treatment with calf intestinal alkaline phosphatase removed this label completely, but was unable to eliminate all protein bands with lower electrophoretic mobility. Thus, multiple phosphorylations contribute to the complex migration behaviour of MP, but additional post-translational modifications may occur as well.

Barcoding of Plant Viruses with Circular Single-Stranded DNA Based on Rolling Circle Amplification.

The experience with a diagnostic technology based on rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP) analyses, and direct or deep sequencing (Circomics) over the past 15 years is surveyed for the plant infecting geminiviruses, nanoviruses and associated satellite DNAs, which have had increasing impact on agricultural and horticultural losses due to global transportation and recombination-aided diversification. Current state methods for quarantine measures are described to identify individual DNA components with great accuracy and to recognize the crucial role of the molecular viral population structure as an important factor for sustainable plant protection.