MYMIV replication initiator protein (Rep): roles at the initiation and elongation steps of MYMIV DNA replication

In order to explore the mechanism of geminivirus DNA replication, we show that the Replication initiator (Rep) protein encoded by Mungbean yellow mosaic India virus (MYMIV), a member of the family Geminiviridae, binds specifically to the iterons present in the viral DNA replication origin (CR-A) in a highly ordered manner that might be a prerequisite for the initiation of replication. MYMIV Rep also acts as a helicase during the post-initiation stage and is upregulated in presence of the RPA32 subunit of Replication Protein A. The implication of these findings on the initiation and elongation stages of MYMIV DNA replication has been discussed.     

Sequencing and computational analysis of complete genome sequences of <Emphasis Type="Italic">Citrus yellow mosaic badna virus</Emphasis> from acid lime and pummelo

Citrus yellow mosaic badna virus (CMBV), a member of the Family Caulimoviridae, Genus Badnavirus, is the causative agent of Citrus mosaic disease in India. Although the virus has been detected in several citrus species, only two full-length genomes, one each from Sweet orange and Rangpur lime, are available in publicly accessible databases. In order to obtain a better understanding of the genetic variability of the virus in other citrus mosaic-affected citrus species, we performed the cloning and sequence analysis of complete genomes of CMBV from two additional citrus species, Acid lime and Pummelo. We show that CMBV genomes from the two hosts share high homology with previously reported CMBV sequences and hence conclude that the new isolates represent variants of the virus present in these species. Based on in silico sequence analysis, we predict the possible function of the protein encoded by one of the five ORFs. The nucleotide sequences reported in this paper have been submitted to the NCBI GenBank nucleotide sequence database and have been assigned the accession numbers EU489744 and EU489745.     

RNA viruses and their silencing suppressors boost Abutilon mosaic virus, but not the Old World Tomato yellow leaf curl Sardinia virus

Mixed viral infections can induce different changes in symptom development, genome accumulation and tissue tropism. These issues were investigated for two phloem-limited begomoviruses, Abutilon mosaic virus (AbMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in Nicotiana benthamiana plants doubly infected by either the potyvirus Cowpea aphid-borne mosaic virus (CABMV) or the tombusvirus Artichoke mottled crinkle virus (AMCV). Both RNA viruses induced an increase of the amount of AbMV, led to its occasional egress from the phloem and induced symptom aggravation, while the amount and tissue tropism of TYLCSV were almost unaffected. In transgenic plants expressing the silencing suppressors of CABMV (HC-Pro) or AMCV (P19), AbMV was supported to a much lesser extent than in the mixed infections, with the effect of CABMV HC-Pro being superior to that of AMCV P19. Neither of the silencing suppressors influenced TYLCSV accumulation. These results demonstrate that begomoviruses differentially respond to the invasion of other viruses and to silencing suppression.     

Transreplication of a Tomato yellow leaf curl Thailand virus DNA-B and replication of a DNAbeta component by Tomato leaf curl Vietnam virus and Tomato yellow leaf curl Vietnam virus

The genomes of two tomato-infecting begomoviruses from Vietnam were cloned and sequenced. A new variant of Tomato leaf curl Vietnam virus (ToLCVV) consisting of a DNA-A component and associated with a DNAbeta molecule as well as an additional begomovirus tentatively named Tomato yellow leaf curl Vietnam virus (TYLCVV) consisting also of a DNA-A component were identified. To verify if monopartite viruses occurring in Vietnam and Thailand are able to transreplicate the DNA-B component of Tomato yellow leaf curl Thailand virus-[Asian Institute of Technology] (TYLCTHV-[AIT]) infectivity assays were performed via agroinoculation and mechanically. As result, the DNA-B component of TYLCTHV-[AIT] was transreplicated by different DNA-A components of viruses from Vietnam and Thailand in Nicotiana benthamiana and Solanum lycopersicum. Moreover, the TYLCTHV-[AIT] DNA-B component facilitated the mechanical transmission of monopartite viruses by rub-inoculation as well as by particle bombardment in N. benthamiana and tomato plants. Finally, defective DNAs ranging from 735 to 1457 nucleotides were generated in N. benthamiana from those combinations containing TYLCTHV-[AIT] DNA-B component.     

Immunochemical studies of turnip yellow mosaic virus--II. Localization of a viral epitope in the N-terminal residues of the coat protein

The N-terminal region of the coat protein of turnip yellow mosaic virus has been shown by immunochemical means to be present on the external surface of the virion. Seven synthetic peptides corresponding to overlapping regions of residues 1–12 of the coat protein have been prepared. The inhibitory activity of these peptides was measured by complement fixation tests, RIA and ELISA, using either antibodies directed to the depolymerized viral subunits or antibodies to the capsid. The activity of peptides varied markedly in different assays, illustrating the advantage of using several different immunochemical methods for the analysis of viral antigenic determinants.     

Evaluation of the minimal replication time of Cauliflower mosaic virus in different hosts

Though the duration of a single round of replication is an important biological parameter, it has been determined for only few viruses. Here, this parameter was determined for Cauliflower mosaic virus (CaMV) in transfected protoplasts from different hosts: the highly susceptible Arabidopsis and turnip, and Nicotiana benthamiana, where CaMV accumulates only slowly. Four methods of differing sensitivity were employed: labelling of (1) progeny DNA and (2) capsid protein, (3) immunocapture PCR,, and (4) progeny-specific PCR. The first progeny virus was detected about 21 h after transfection. This value was confirmed by all methods, indicating that our estimate was not biased by the sensitivity of the detection method, and approximated the actual time required for one round of CaMV replication. Unexpectedly, the replication kinetics were similar in the three hosts; suggesting that slow accumulation of CaMV in Nicotiana plants is determined by non-optimal interactions in other steps of the infection cycle.     

Phosphorylation of the Movement Protein of Cucumber Mosaic Virus in Transgenic Tobacco Plants

The 3a protein of Cucumber mosaic virus is essential for the cell-to-cell movement of the viral RNA through plasmodesmata. We have introduced an epitope peptide before the stop codon of the 3a protein and cloned the tagged ORF into a binary vector for Agrobacterium-mediated transformation. The established transgenic tobacco lines produced the 3a protein, which was specifically detected with anti-3a and anti-epitope antisera. Metabolic labeling and subsequent immunoprecipitation revealed that [³²P]-orthophosphate was incorporated into the 3a protein. The phosphoamino acid analysis indicated that the 3a protein contained phosphoserine but not phosphothreonine or phosphotyrosine. This is the first demonstration of the 3a protein phosphorylation in planta.     

Identification and Sequence Analysis of Potato yellow vein virusCapsid Protein Minor Gene

Potato yellow vein virus (PYVV) is a whitefly-transmitted (Trialeurodes vaporariorum) closterovirus (WTC) with an as yet unidentified genome composition. PYVV dsRNA preparations consist of three high molecular weight dsRNA species (dsRNAs 1, 2 and 3) 8.0, 5.5 and 4.0kbp in size respectively, as well as two low molecular weight dsRNA species of 2.0 and 1.8kbp (denoted x and y). The PYVV capsid protein minor (CPm) gene was identified on the dsRNA 3 species, and was subsequently cloned and sequenced. The PYVV CPm gene is 2022 nucleotides long and putatively encodes a protein with estimated size 77.5kDa. The PYVV CPm gene product is considerably larger than the equivalent proteins encoded by the bipartite criniviruses, Lettuce infectious yellows virus (LIYV) and Cucurbit yellow stunting disorder virus (CYSDV) (52 and 53kDa, respectively). The PYVV CPm possesses a centralized domain which is absent from both the LIYV and CYSDV CPm counterparts. Pairwise comparisons as well as phylogenetic analysis based on the available amino acid sequences of the CPm of various WTCs, showed that PYVV is closely related to LIYV, CYSDV and also Beet pseudo-yellows virus.     

Abutilon mosaic virus DNA B component supports mechanical virus transmission, but does not counteract begomoviral phloem limitation in transgenic plants

Different Nicotiana benthamiana lines stably transformed with Abutilon mosaic virus (AbMV) dimeric DNA B were capable of systemically spreading complete bipartite AbMV genomes, following agroinoculation of DNA A alone. Constitutively expressed viral movement protein (BC1) did not induce any persistent disease phenotype, but plants developed transient morphological abnormalities such as radially symmetric leaves after kanamycin withdrawal. Systemic AbMV infection produced symptoms and virus titers indistinguishable from those in non-transgenic plants. In systemically invaded leaves, the begomovirus remained phloem-limited, whereas the plants' susceptibility to mechanical transmission of AbMV was enhanced by a factor of three to five, as compared to non-transgenic controls. Hence, DNA B-encoded movement functions can complement local movement to the phloem after mechanical transmission, but fail to support viral invasion of non-phloem cells in systemically infected organs, indicating that the phloem restriction of AbMV does not result predominantly from a lack of transport competence in mesophyll tissues.     

Cytoplasmic inclusion cistron of Soybean mosaic virus serves as a virulence determinant on Rsv3-genotype soybean and a symptom determinant

Soybean mosaic virus (SMV; Potyvirus, Potyviridae) is one of the most widespread viruses of soybean globally. Three dominant resistance genes (Rsv1, Rsv3 and Rsv4) differentially confer resistance against SMV. Rsv1 confers extreme resistance and the resistance mechanism of Rsv4 is associated with late susceptibility. Here, we show that Rsv3 restricts the accumulation of SMV strain G7 to the inoculated leaves, whereas, SMV-N, an isolate of SMV strain G2, establishes systemic infection. This observation suggests that the resistance mechanism of Rsv3 differs phenotypically from those of Rsv1 and Rsv4. To identify virulence determinant(s) of SMV on an Rsv3-genotype soybean, chimeras were constructed by exchanging fragments between avirulent SMV-G7 and the virulent SMV-N. Analyses of the chimeras showed that both the N- and C-terminal regions of the cytoplasmic inclusion (CI) cistron are required for Rsv3-mediated resistance. Interestingly, the N-terminal region of CI is also involved in severe symptom induction in soybean.     

Molecular evidence and sequence analysis of a natural reassortant between <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and II strains

Cucumber mosaic virus (CMV) is a tripartite RNA virus and has been divided into three subgroups, named IA, IB, and II. Some studies have found a few natural reassortants between CMV subgroups, although reassortment between CMV subgroups is infrequent. In our present work, a CMV reassortant, named CMV-Tsh, was obtained from a tomato plant. The complete sequence of CMV-Tsh genomic RNAs has been determined and analyzed. The results of sequence comparisons and phylogenetic analyses revealed that CMV-Tsh RNAs 1 and 3 are derived from one or two CMV subgroup II strain(s), while RNA2 is derived from a CMV subgroup IA strain. A PCR and restriction enzyme analysis-based method was developed to analyze the possibility of mixed infection by CMV strains of different subgroup in the CMV-Tsh-infected tomato plant. The results of the restriction enzyme analysis proved that CMV-Tsh is the unique strain in the tomato plant. Taken together, CMV-Tsh is a natural reassortant having CMV subgroup IA RNA2 and subgroup II RNAs 1 and 3. The GenBank Accession numbers of the sequences reported in this paper are EF202595- EF202597.     

Sequence variability and phylogenetic relationship of betasatellite isolates associated with yellow vein mosaic disease of mesta in India

Six betasatellite isolates associated with the yellow vein mosaic disease in mesta crops grown under three different geographical locations of India have been characterized. These six isolates and the one previously reported from eastern India could be divided into two distinct Types. The first Type, consisted of four betasatellite isolates characterized from northern and southern regions of India, was observed to be the newer isolates of Ludwigia leaf distortion betasatellite. The second Type, comprised three betasatellite isolates obtained from the eastern part of India, showed highest sequence identity with Cotton leaf curl Multan betasatellite and appeared to be the newer isolates of it. These isolates present within each of these two betasatellite species showed limited variability with respect to their individual group. The results thus indicated the association of two different betasatellite species with yellow vein mosaic disease of mesta in India and highlighted the possible adaptation of mesta crops as a newer hosts by these two betasatellite species.     

Transgenic expression of coat protein gene of <Emphasis Type="Italic">Rice tungro bacilliform virus</Emphasis> in rice reduces the accumulation of viral DNA in inoculated plants

Rice tungro, a devastating disease of rice in south and southeast Asia, is caused by the joint infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to obtain transgenic resistance against RTBV, indica rice cultivar Pusa Basmati-1 was transformed to express the coat protein (CP) gene of an Indian isolate of RTBV. Rice plants containing the transgene integrated in low copy numbers were obtained, in which the CP was shown to accumulate in the leaf tissue. The progenies representing three independent transformation events were challenged with Indian isolates of RTBV using viruliferous Green leafhoppers, and the viral titers in the inoculated plants were monitored using DNA dot-blot hybridization. As compared to non-transgenic controls, two independent transgenic lines showed significantly low levels of RTBV DNA, especially towards later stages of infection and a concomitant reduction of tungro symptoms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of a host determination mechanism of bromoviruses in Arabidopsis thaliana

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.     

Spatial analysis for exclusive interactions between subgroups I and II of Cucumber mosaic virus in cowpea

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.     

Contrasting effects of HC-Pro and 2b viral suppressors from Sugarcane mosaic virus and Tomato aspermy cucumovirus on the accumulation of siRNAs

RNA silencing and suppression of silencing are host and virus interactions for defense and counter-defense. Here, we explored the function effect of HC-Pro encoded by Sugarcane mosaic virus (SCMV) on the suppression of RNA silencing. siRNA northern blotting assay indicated that the replication of SCMV was regulated by host RNA silencing machinery. Co-expression assay demonstrated that the HC-Pro encoded by SCMV suppressed the RNA silencing induced by sense RNA and dsRNA. Transitive RNA silencing assay showed that HC-Pro down-regulated the accumulation of 3' secondary siRNA, but not 5' secondary and primary siRNA. Meanwhile, the 2b gene of Tomato aspermy cucumovirus (Tav) evidently down-regulated the accumulation of 5' secondary siRNA. Importantly, we found that HC-Pro and Tav2b down-regulated the accumulation of RDR6 mRNA. Thus, HC-Pro, an RNA silencing suppressor encoded by SCMV, regulates the accumulation of different siRNAs and has more than one target in the RNA silencing pathway.