Identification of microRNAs regulated by tobacco curly shoot virus co-infection with its betasatellite in Nicotiana benthamiana.

BACKGROUND: MicroRNAs (miRNAs) are a class of 21-24 nucleotide endogenous non-coding small RNAs that play important roles in plant development and defense responses to biotic and abiotic stresses. Tobacco curly shoot virus (TbCSV) is a monopartite begomovirus, cause leaf curling and plant stunting symptoms in many Solanaceae plants. The betasatellite of TbCSV (TbCSB) induces more severe symptoms and enhances virus accumulation when co-infect the plants with TbCSV. METHODS: In this study, miRNAs regulated by TbCSV and TbCSB co-infection in Nicotiana benthamiana were characterized using high-throughput sequencing technology. RESULTS: Small RNA sequencing analysis revealed that a total of 13 known miRNAs and 42 novel miRNAs were differentially expressed in TbCSV and TbCSB co-infected N. benthamiana plants. Several potential miRNA-targeted genes were identified through data mining and were involved in both catalytic and metabolic processes, in addition to plant defense mechanisms against virus infections according to Gene Ontology (GO) analyses. In addition, the expressions of several differentially expressed miRNAs and their miRNA-targeted gene were validated through quantitative real time polymerase chain reaction (qRT-PCR) approach. CONCLUSIONS: A large number of miRNAs are identified, and their target genes, functional annotations also have been explored. Our results provide the information on N. benthamiana miRNAs and would be useful to further understand miRNA regulatory mechanisms after TbCSV and TbCSB co-infection.

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the ß-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels. RESULTS: A hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants. CONCLUSIONS: These results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

Association of satellites with a mastrevirus in natural infection: complexity of Wheat dwarf India virus disease.

UNLABELLED: In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. IMPORTANCE: Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat Dwarf India Virus (WDIV), isolated from two distant locations in India. This study reports the first identification of the satellites in a monocot plant. The satellites enhanced accumulation of WDIV and severity of disease symptoms. The satellites lowered the concentration of virus-specific small RNAs in wheat plants, indicating their silencing suppressor activity. The involvement of the satellites in symptom severity of the mastrevirus can have implications in the form of economic impact of the virus on crop yield. Understanding the role of the satellites in disease severity is important for developing disease management strategies.

Zamilon, a novel virophage with Mimiviridae host specificity.

Virophages, which are potentially important ecological regulators, have been discovered in association with members of the order Megavirales. Sputnik virophages target the Mimiviridae, Mavirus was identified with the Cafeteria roenbergensis virus, and virophage genomes reconstructed by metagenomic analyses may be associated with the Phycodnaviridae. Despite the fact that the Sputnik virophages were isolated with viruses belonging to group A of the Mimiviridae, they can grow in amoebae infected by Mimiviridae from groups A, B or C. In this study we describe Zamilon, the first virophage isolated with a member of group C of the Mimiviridae family. By co-culturing amoebae with purified Zamilon, we found that the virophage is able to multiply with members of groups B and C of the Mimiviridae family but not with viruses from group A. Zamilon has a 17,276 bp DNA genome that potentially encodes 20 genes. Most of these genes are closely related to genes from the Sputnik virophage, yet two are more related to Megavirus chiliensis genes, a group B Mimiviridae, and one to Moumouvirus monve transpoviron.

Cotton leaf curl Multan betasatellite as a plant gene delivery vector trans-activated by taxonomically diverse geminiviruses.

Cotton leaf curl Multan betasatellite (CLCuMB) replicates in tobacco, tomato and datura plants in the presence of the helper viruses tomato leaf curl virus-Australia, Iranian isolates of tomato yellow leaf curl virus, tomato leaf curl Karnataka virus, and beet severe curly top virus (BSCTV). Infectious recombinant CLCuMB constructs were made in which segments of either the CaMV 35S or the petunia ChsA promoter replaced the CLCuMB ßC1 ORF, and these were designated pBinßΔC1-35S and pBinßΔC1-ChsA, respectively. Inoculation of tobacco plants containing a functional 35S-GUS transgene with pBinßΔC1-35S, and normal petunia plants with pBinßΔC1-ChsA, in the presence of helper viruses resulted in silencing of GUS and ChsA activities in transgenic tobacco and non-transgenic petunia plants, respectively. Replication of CLCuMB with different geminiviruses, especially BSCTV, a curtovirus with a broad host range, makes it a valuable gene delivery vector to the large number of host plant species of geminiviruses that support CLCuMB.

Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a betasatellite for induction of disease symptoms.

Tomato leaf curl disease (ToLCD) has emerged as a major constraint on tomato production in some parts of West Africa. In this study, begomoviruses associated with ToLCD in Togo and Nigeria were characterized, as well as a betasatellite associated with the disease in Togo. The genome organization of both viruses is typical of Old World monopartite begomoviruses. Sequence analysis revealed that the begomovirus from Togo is a variant of tomato leaf curl Kumasi virus (ToLCKuV) from Ghana, and it is designated ToLCKuV-[Togo:Pagouda:2006] (ToLCKuV-[TG:Pag:06]). The begomovirus from Nigeria has a recombinant genome, composed of sequences of ToLCKuV (major parent) and a cotton leaf curl Gezira virus (CLCuGV)-like virus, and possesses an unusual non-reiterated replication-associated protein (Rep) binding site. Moreover, because the sequence has <89% identity with those of previously characterized begomoviruses, it is a new species and is designated tomato leaf curl Nigeria virus-[Nigeria:Odogbo:2006] (ToLCNGV-[NG:Odo:06]). The cloned DNAs of ToLCKuV-TG and ToLCNGV were infectious and induced leaf curl symptoms in tomato plants, but ToLCNGV was comparatively more virulent. Both viruses also induced stunted growth and leaf curl symptoms in other solanaceous species (various Nicotiana spp. and Datura stramonium), whereas ToLCNGV but not ToLCKuV-TG induced symptoms in common bean plants. The betasatellite associated with ToLCD in Togo is genetically distinct (i.e., <78% nucleotide sequence identity with previously identified betasatellites) and is designated tomato leaf curl Togo betasatellite-[Togo:Pagouda:2006] (ToLCTGB-[TG:Pag:06]). Replication and systemic spread of ToLCTGB in tomato was mediated by ToLCKuV-TG and ToLCNGV; however, the betasatellite had no effect on disease symptoms induced by either begomovirus. In contrast, ToLCTGB increased symptom severity induced by both viruses in Nicotiana spp. and D. stramonium. Thus, although ToLCTGB increased symptom severity in a host-dependent manner, it does not appear to play a role in ToLCD and may have been present with ToLCKuV-TG as a reassortant.

A new strategy for generating geminivirus resistant plants using a DNA betasatellite/split barnase construct.

The betasatellite DNA associated with cotton leaf curl disease contains a single ORF, ßC1, which is a pathogenicity determinant. Deletion of the ßC1 ORF showed that it was not required for betasatellite replication in the presence of Tomato leaf curl virus-Australia (TLCV-Au). A series of betasatellite/split mutant barnase gene constructs, in which a direct repeat of the Bacillus amyloliquefaciens barnase gene flanked the betasatellite, were shown to replicate in tobacco in the presence of TLCV-Au. A betasatellite/split intact barnase gene construct, with the optimal direct repeat unit of the barnase gene, was introduced into Nicotiana tabacum plants. Approximately one third of the transgenic lines containing the betasatellite/split barnase gene constructs were shown to be completely resistant to the TLCV-Au infection. The betasatellite/split intact barnase gene cassette ensures that there is no expression of the barnase in the absence of TLCV-Au, but upon infection of the cell with the virus, release of the betasatellite/split barnase cassette as a replicating molecule resulting in the reconstitution and expression of an active barnase gene and the destruction of the infected cell. This system offers the potential to provide resistance in a variety of plant species against geminiviruses that support the replication of betasatellite.

Satellite panicum mosaic virus coat protein enhances the performance of plant virus gene vectors.

The coat protein of satellite panicum mosaic virus (SPCP) is known to effectively protect its cognate RNA from deleterious events, and here, we tested its stabilizing potential for heterologous virus-based gene vectors in planta. In support of this, a Potato virus X (PVX) vector carrying the SPMV capsid protein (PVX-SPCP) gene was stable for at least three serial systemic passages through Nicotiana benthamiana. To test the effect of SPCP in trans, PVX-SPCP was co-inoculated onto N. benthamiana together with a Tomato bushy stunt virus (TBSV) vector carrying a green fluorescent protein (GFP) gene that normally does not support systemic GFP expression. In contrast, co-inoculation of TBSV-GFP plus PVX-SPCP resulted in GFP accumulation and concomitant green fluorescent spots in upper, non-inoculated leaves in a temperature-responsive manner. These results suggest that the multifaceted SPMV CP has intriguing effects on virus-host interactions that surface in heterologous systems.

The plasmid status of satellite bacteriophage P4.

P4 is a natural phasmid (phage-plasmid) that exploits different modes of propagation in its host Escherichia coli. Extracellularly, P4 is a virion, with a tailed icosahedral head, which encapsidates the 11.6-kb-long double-stranded DNA genome. After infection of the E. coli host, P4 DNA can integrate into the bacterial chromosome and be maintained in a repressed state (lysogeny). Alternatively, P4 can replicate as a free DNA molecule; this leads to either the lytic cycle or the plasmid state, depending on the presence or absence of the genome of a helper phage P2 in the E. coli host. As a phage, P4 is thus a satellite of P2 phage, depending on the helper genes for all the morphogenetic functions, whereas for all its episomal functions (integration and immunity, multicopy plasmid replication) P4 is completely autonomous from the helper. Replication of P4 DNA depends on its alpha protein, a multifunctional polypeptide that exhibits primase and helicase activity and binds specifically the P4 origin. Replication starts from a unique point, ori1, and proceeds bidirectionally in a straight theta-type mode. P4 negatively regulates the plasmid copy number at several levels. An unusual mechanism of copy number control is based on protein-protein interaction: the P4-encoded Cnr protein interacts with the alpha gene product, inhibiting its replication potential. Furthermore, expression of the replication genes cnr and alpha is regulated in a complex way that involves modulation of promoter activity by positive and negative factors and multiple mechanisms of transcription elongation-termination control. Thus, the relatively small P4 genome encodes mostly regulatory functions, required for its propagation both as an episomal element and as a temperate satellite phage. Plasmids that, like P4, propagate horizontally via a specific transduction mechanism have also been found in the Archaea. The presence of P4-like prophages or cryptic prophages often associated with accessory bacterial functions attests to the contribution of satellite phages to bacterial evolution.

The nucleotide sequence of satellite tobacco necrosis virus strain C and helper-assisted replication of wild-type and mutant clones of the virus.

The complete nucleotide sequence of satellite tobacco necrosis virus strain C (STNV-C) was determined. The genome has a similar overall organization to two STNV isolates studied previously but differs significantly from them in the secondary structure of the translated and untranslated regions (UTRs). STNV-C RNA is naturally uncapped and contains 1221 nt: 101 nt in the 5' UTR, 606 nt in the capsid protein (CP) coding region and 514 nt in the 3' UTR. Using the known sequences of STNV-C and tobacco necrosis virus strain D (TNV-D) RNAs, full-length cDNA clones of both RNAs were constructed. Synthetic transcripts derived from STNV-C cDNA clones only replicated in plants and protoplasts when co-inoculated with TNV-D transcripts. A number of mutant clones in both the 3' and the 5' STNV-C RNA UTRs were constructed which disrupted putative cis-acting elements recognized by helper virus polymerase. Deletion analysis revealed an essential requirement of all 3' and 5' proximal sequences in the STNV-C UTRs for replication. However, an internal region in the 3' UTR could be deleted without loss of infectivity. Likewise, the entire STNV-C CP-encoding region could be deleted and replaced with a marker gene of a similar size without loss of transcript accumulation in plants.

Cucumber-mosaic-virus-associated RNA-5. XII. Symptom-modulating effect is codetermined by the helper virus satellite replication support function.

In tomato, the disease-modulating effects of a cucumber mosaic virus (CMV) satellite isolate from Belgium, here designated T-CARNA-5 (CARNA-5 = CMV-associated RNA-5), were found to be different depending on the supporting helper virus strain. With two CMV strains, T-CARNA-5 induced lethal necrosis, but with a third strain from Ixora spp. (CMV-Ix), aggravated stunting was observed. However, the primary structure of the T-CARNA-5 contained within virus isolated from tobacco or tomato infected with each of these three CMV strains, conformed to the conserved sequence profile of CARNA-5 isolates which are necrogenic in tomato. Dilution endpoint bioassay of T-CARNA-5 established a direct cause-effect relationship between it and tomato necrosis or stunting, depending on the helper virus. Total nucleic acid extracts taken at different times from tomato plants infected with the above CMV strains and T- or S-CARNA-5 (used as non-necrogenic control) showed viral RNA, ssCARNA-5 and dsCARNA-5 to be present in significant amounts, but in sometimes dissimilar proportions depending on the combination; except in CMV-Ix/S-CARNA-5 infection where neither ss-nor dsCARNA-5 was found. The experiments established that CARNA-5 biological expression studies in CMV-infected tomato have to take into account the helper virus satellite replication support function, which may be a primary codeterminant of quantitative or qualitative differences in the symptom modulation observed.

Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA.

Circular and multimeric forms of the satellite RNA of tobacco ringspot virus and their autolytic processing reactions are well known. They suggest replication models in which key elements are rolling circle transcription and the processing of the resulting multimeric RNA to generate the unit, 'monomeric' satellite RNA sequence. We prepared plasmids bearing two distinct sequences of the satellite RNA. Each was arranged to allow transcription of an oligoribonucleotide (r-oligo) of the polarity that is complementary to encapsidated satellite RNA. One sequence has the autolytic processing phosphodiester bond, ApG, and the other is located at a distance of about 150 nucleotide (nt) residues. The second r-oligo accomplished cleavage of the first, in a catalytic fashion. Analysis of truncated forms showed that 10 nt of the ApG junction-containing r-oligo and 46 of the endoribonucleolytic r-oligo were sufficient for recognition in the cleavage reaction. These results map the sequences involved in autolytic processing of the complementary polarity satellite RNA to two regions.

Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus.

The satellite RNA of tobacco ringspot virus (sTobRV) undergoes self-catalysed cleavage during replication. A plasmid for in vitro expression of sTobRV has been constructed and used to obtain a library of mutagenized sTobRV sequences. Screening of these mutants has allowed precise definition of the sequences required for (+) and (-) strand cleavage. The sequences and RNA structures associated with cleavage of each strand differ markedly. Cleavage of the (+) strand requires those sequences flanking the site for cleavage to form a 'hammerhead' domain, similar to those found in other satellite and viroid RNA. In contrast, cleavage of the (-) strand requires only a small region of 12 nucleotides (nt) at the site of cleavage, and a sequence of 55 nt positioned elsewhere in the molecule. Comparison with a closely related satellite suggests that a novel RNA structure may be involved in (-) strand cleavage.

The effect of hexadimethrine bromide (polybrene) on the infection of the primate retroviruses SSV 1/SSAV 1 and BaEV.

Polybrene was shown to enhance the adsorption of simian sarcoma virus-simian sarcoma associated virus complex (SSV-1/SSAV-1) and baboon endogenous virus (BaEV) onto cells in culture. A 16- to 18-fold increased adsorption of both viruses occurred at 8 micrograms/ml polybrene within one hour after infection. The polybrene mediated adsorption was found to be inhibited by the addition of tri-sodium citrate to the culture medium, suggesting the involvement of electrostatic forces. This contention was further supported by the demonstration of temperature independence of the polybrene mediated interaction.

Biological and antigenic characteristics of HEL-12 virus.

The biological and antigenic properties of HEL-12 virus have been compared with gibbon ape lymphosarcoma virus (GALV) and simian sarcoma and simian sarcoma-associated viruses, SiSV and SSAV, respectively, HEL-12 virus did not transform human or marmoset fibroblasts but rescued SiSV focus-forming activity from non-productively transformed marmoset cells (HF/SiSV-NP). Like SSAV and GALV, HEL-12 virus induced syncytia with XC cells. In addition, HEL-12 cells which did not produce virus but which contained HEL-12 proviral DNA, rescued SiSV from HF/SiSV-NP cells in co-cultivation experiments. Results of neutralization and serum cytotoxicity tests utilizing SiSV rescued by HEL-12 [SiSV-(HEL-12)] indicated that HEL-12 virus envelope proteins are very closely related to those of SSAV but readily distinguished from those of GALV. Antigenic diversity of SiSV(SSAV), SiSV(GALV) and SiSV(HEL-12) envelope glycoproteins (gp70) was shown in competition radioimmunoassays (RIA) designed to detect minor antigenic differences using antiserum monospecific for SiSV(SSAV) gp70 or Friend murine leukaemia virus gp70. Antigenic differences between these gp70s were demonstrated in RIA using purified SSAV gp70 or HEL-12 gp70. These data indicate that HEL-12 virus has biological properties similar to those of SSAV and GALV, is distinguished from GALV in neutralization tests and has both distinct and SSAV-related gp70 antigenic determinants.