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Wheat dwarf virus (WDV) adversely affects cereal production in Asia, Europe, and North Africa. In this study, sequences of several WDV isolates from Iran which is located in the Fertile Crescent were analyzed. Analysis revealed a new geographic cluster for WDV-Wheat from Iran. Recombination analysis demonstrated the existence of several breakpoints in different regions of the viral genome. Data analysis demonstrated that WDV-Barley has an older history and lower diversity than WDV-Wheat. Sequence analysis identified a rare occasion of a co-infection of wheat with WDV-Wheat and WDV-Barley.

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

Betasatellites are single-stranded circular DNAs associated with a number of monopartite begomoviruses. Betasatellites rely on the helper begomoviruses for replication and movement in plant tissues and plant-to-plant transmission by vectors. Their genomes are approximately half the size of the helper viruses and consist of three main regions including the βC1 gene, an adenine-rich (A-rich) region, and the satellite conserved region (SCR). In this study, we investigated the minimal sequences required for Cotton leaf curl Multan betasatellite (CLCuMB) replication and movement. Mutational analysis of CLCuMB DNA genome indicated that βC1 gene and A-rich region were not required for trans-replication and movement of CLCuMB in host plants by a helper virus. Deletion of βC1 gene and a fragment (135 nt in length) upstream of this gene impaired CLCuMB replication. However, CLCuMB mutant with deletion of βC1 gene and a further 163 nucleotides replicated at a lower level as compared to the wild-type betasatellite. This suggests that there are essential elements in the fragment upstream of βC1 gene, which are required for the replication of CLCuMB rather than the size limitation of CLCuMB DNA.

Geminiviruses cause curly top disease, in dicotyledonous plants which constrains host crop production. Beet curly top Iran virus (BCTIV) is a widespread Becurtovirus (family Geminiviridae ) in numerous areas within Iran. In this study, we isolated and analyzed a full-length genomic DNA of a new variant of BCTIV from pepper crops in the Kaftark region, east of Shiraz (proposed acronym: BCTIV-Kaf [IR: Kaf:2016:Pepper]). Infected pepper plants showed shortening of internodes, severe interveinal chlorosis, upward leaf rolling and leaf curling. Sequence and phylogenetic analysis showed this BCTIV variant grouped with sugar beet isolates of BCTIV and has the highest similarity to a sugar beet BCTIV isolate from Negar town in Kerman province, Iran. It was more distantly related to a bean isolate of BCTIV from northeast region of Iran. A tandem repeat partial dimmer of BCTIV was constructed and found to be infectious in pepper, tomato and Nicotiana benthamiana plants. Results of this study indicated that BCTIV-Kaf is a new variant of BCTIV infecting pepper plants in Shiraz and that geographic location rather than the type of host plant has more effect on genetic diversity of BCTIV in Iran.

The betasatellite DNA associated with Cotton leaf curl Multan virus (CLCuMB) contains a single complementary-sense ORF, βC1, which is a pathogenicity determinant. CLCuMB was able to replicate in plants in the presence of diverse helper geminiviruses, including Tomato leaf curl virus-Australia (TLCV-Au), Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]), and Beet curly top virus (BCTV-Svr), and can be used as a plant gene delivery vector. To test the hypothesis that CLCuMB has the potential to act as an animal gene delivery vector, a specific insertion construct was produced by the introduction of a human B-cell lymphoma 2 (Bcl-2) cDNA into a mutant DNA of CLCuMB in which the βC1 was deleted (β∆C1). The recombinant βΔC1-Bcl-2 construct was successfully replicated in tomato and tobacco plants in the presence of TLCV-Au, BCTV-Svr and TYLCV-[Ab]. Real-time PCR and Western blot analyses of plants containing the replicative forms of recombinant βΔC1-Bcl-2 DNA showed that Bcl-2 gene was expressed in an acceptable level in these plants, indicating that β∆C1 can be used as a tool to deliver and express animal genes in plants. This CLCuMB-based system, having its own promoter activity, offers the possibility of production of animal recombinant proteins in plants.

Cotton leaf curl Multan betasatellite (CLCuMB) is responsible for symptom expression of a devastating disease of cotton in the Indian subcontinent. CLCuMB depends on helper virus replication-associated protein for its replication and on viral coat protein (CP) for its encapsidation. However, no direct evidence of encapsidation of CLCuMB in viral CP has been available. In the present study, non-viruliferous whiteflies were placed on tomato plants that had been agroinoculated with infectious clones of an Iranian isolate of tomato yellow leaf curl virus (TYLCV-[Ab]) and CLCuMB for an acquisition access period of 72 h and then transferred to healthy tomato seedlings at the 3- to 4-leaf stage. Typical symptoms of TYLCV-[Ab] appeared on inoculated seedlings 30-45 days post-inoculation. The presence of TYLCV-[Ab] and CLCuMB DNAs in symptomatic test plants and viruliferous whiteflies was confirmed by PCR analysis using specific primers and DIG Southern blotting. Furthermore, the possibility of CLCuMB DNA encapsidation in TYLCV-[Ab] CP within infected plants was examined by immunocapture PCR. The results showed that CLCuMB DNA was encapsidated in TYLCV-[Ab] CP. Whitefly-mediated transmission of CLCuMB in the presence of helper virus is additional evidence for encapsidation of CLCuMB by TYLCV-[Ab] CP.

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 βC1 ORF, and these were designated pBinβΔC1-35S and pBinβΔC1-ChsA, respectively. Inoculation of tobacco plants containing a functional 35S-GUS transgene with pBinβΔC1-35S, and normal petunia plants with pBinβΔC1-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.

The betasatellite DNA associated with cotton leaf curl disease contains a single ORF, βC1, which is a pathogenicity determinant. Deletion of the βC1 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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

DNA beta is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA beta contains a single gene, betaC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the beta-glucuronidase reporter gene driven by a betaC1 promoter-deletion series of the DNA beta associated with cotton leaf curl Multan virus identified a 68 nt region (between -139 and -207) which is important for betaC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the betaC1 start codon. Mutation of the G-box resulted in a significant reduction in betaC1 promoter activity and DNA beta replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA beta.

Tomato leaf curl virus (TLCV) satellite DNA (sat-DNA) constructs containing functional segments of the cauliflower mosaic virus (CaMV) 35S promoter, replicate in tobacco in the presence of helper TLCV and silence GUS activity in transgenic tobacco plants containing a CaMV 35S-GUS expression cassette. We have analysed these plants for evidence of the hallmarks of silencing. The GUS transcript was not detectable in the leaves of GUS-silenced tobacco plants. These plants contained siRNAs of approximately 23 nt in length homologous to both the 35S promoter region and the GUS ORF. The absence of GUS expression and the existence of siRNAs in transgenic plants show that the silencing induced by TLCV sat-DNA is due to RNA silencing. To test the utility of this silencing system, a 341 nucleotide promoter sequence of the petunia chalcone synthase A (ChsA) was inserted into the sat-DNA and inoculated into petunia plants, together with the helper TLCV, and found to markedly reduce pigmentation of flowers and the level of ChsA transcript. This DNA-based silencing system has the potential to introduce epigenetic traits via short DNA inserts to a variety of plants that are hosts to different geminiviruses supporting the sat-DNA.

Distinct subgenomic DNA species known as defective (df) DNA molecules were found in plants infected with tomato leaf curl virus (TLCV). Four df DNAs derived from TLCV Type and Darwin 1 strains were found to contain large deletions that disrupt all of the viral genes required for viral replication, encapsidation and spread. However, the viral origin of replication (ori), including the replication-associated protein (Rep) binding domains, was present in all four df DNAs. Co-agroinfection of leaf strips with tandem repeat constructs of the viral and df DNAs resulted in their replication in the presence of the respective TLCV strain. However, the df DNAs failed to move in whole plants when co-inoculated with TLCV. The df DNAs were shown to be associated with TLCV coat protein, which may indicate encapsidation. Mutational analysis showed the minimum sequence requirements for df DNA replication by TLCV to be the intergenic region containing the Rep-binding domains.

Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.

The 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.