Amplification and Cloning of Large cDNA Fragments of the Citrus tristeza virus Genome.

Citrus tristeza virus (CTV) is probably the most destructive viral pathogen of citrus. It causes chronic losses to commercial citrus production in all citrus-growing areas. The complete sequences of at least 42 genomes of different CTV strains have been obtained using different technologies including sequencing of multiple overlapped RT-PCR-amplified fragments with sizes of less than 4 kb, or from small viral RNA (svRNA), through next-generation high-throughput sequencing (NGS) technologies. The large size of CTV genome (>19.2 kb) makes it impractical to obtain and amplify full-length cDNA in a single step. The strategy of ligation of multiple cDNA fragments to assemble a full-length cDNA clone involves several serial cloning steps and sometimes subcloning phases using enzymatic digestion with restriction nucleases and ligation reactions. In this protocol, we describe a strategy to clone the entire genome of CTV obtained from two RT-PCR amplified products. These 5'- and 3'-genomic halves, which were designed to be overlapped in 15 nt in their 3'- and 5'-ends, respectively, were used as templates for further overlapped PCR to amplify the entire ~20 kb CTV genome. The resultant full cDNA PCR product was then inserted into pCAMBIA-binary vector.

Transmission of a New Polerovirus Infecting Pepper by the Whitefly Bemisia tabaci.

Many animal and plant viruses depend on arthropods for their transmission. Virus-vector interactions are highly specific, and only one vector or one of a group of vectors from the same family is able to transmit a given virus. Poleroviruses (Luteoviridae) are phloem-restricted RNA plant viruses that are exclusively transmitted by aphids. Multiple aphid-transmitted polerovirus species commonly infect pepper, causing vein yellowing, leaf rolling, and fruit discoloration. Despite low aphid populations, a recent outbreak with such severe symptoms in many bell pepper farms in Israel led to reinvestigation of the disease and its insect vector. Here we report that this outbreak was caused by a new whitefly (Bemisia tabaci)-transmitted polerovirus, which we named Pepper whitefly-borne vein yellows virus (PeWBVYV). PeWBVYV is highly (>95%) homologous to Pepper vein yellows virus (PeVYV) from Israel and Greece on its 5' end half, while it is homologous to African eggplant yellows virus (AeYV) on its 3' half. Koch's postulates were proven by constructing a PeWBVYV infectious clone causing the pepper disease, which was in turn transmitted to test pepper plants by B. tabaci but not by aphids. PeWBVYV represents the first report of a whitefly-transmitted polerovirus.IMPORTANCE The high specificity of virus-vector interactions limits the possibility of a given virus changing vectors. Our report describes a new virus from a family of viruses strictly transmitted by aphids which is now transmitted by whiteflies (Bemisia tabaci) and not by aphids. This report presents the first description of polerovirus transmission by whiteflies. Whiteflies are highly resistant to insecticides and disperse over long distances, carrying virus inoculum. Thus, the report of such unusual polerovirus transmission by a supervector has extensive implications for the epidemiology of the virus disease, with ramifications concerning the international trade of agricultural commodities.

Differential expression of cucumber RNA-dependent RNA polymerase 1 genes during antiviral defence and resistance.

RNA-dependent RNA polymerase 1 (RDR1) plays a crucial role in plant defence against viruses. In this study, it was observed that cucumber, Cucumis sativus, uniquely encodes a small gene family of four RDR1 genes. The cucumber RDR1 genes (CsRDR1a, CsRDR1b and duplicated CsRDR1c1/c2) shared 55%-60% homology in their encoded amino acid sequences. In healthy cucumber plants, RDR1a and RDR1b transcripts were expressed at higher levels than transcripts of RDR1c1/c2, which were barely detectable. The expression of all four CsRDR1 genes was induced by virus infection, after which the expression level of CsRDR1b increased 10-20-fold in several virus-resistant cucumber cultivars and in a broad virus-resistant transgenic cucumber line expressing a high level of transgene small RNAs, all without alteration in salicylic acid (SA) levels. By comparison, CsRDR1c1/c2 genes were highly induced (25-1300-fold) in susceptible cucumber cultivars infected with RNA or DNA viruses. Inhibition of RDR1c1/c2 expression led to increased virus accumulation. Ectopic application of SA induced the expression of cucumber RDR1a, RDR1b and RDRc1/c2 genes. A constitutive high level of RDR1b gene expression independent of SA was found to be associated with broad virus resistance. These findings show that multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles.

Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

The ORF3-encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infection and localize to the plasmodesmata.

The genomic RNA of vitiviruses contains 5 open reading frames (ORF). ORF3 encodes a protein to which the function of a movement protein (MP) was assigned, based on sequence homology with other viral proteins. The aim of the research described in this paper was to gain further insight in distribution profile of the ORF3 product encoded by the vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). Expression of the GVA MP-GFP fusion protein via the virus genome in Nicotiana benthamiana leaves resulted in the formation of irregular spots and fibrous network structures on the outermost periphery of epidermal cells. Expression of GVA MP-GFP and GVB MP-GFP was involved in the formation of the tubule-like and punctate structures on the periphery of N. benthamiana and Vitis vinifera protoplasts. Co-expression of the GVA MP-GFP and GVA MP-RFP in protoplasts resulted in co-localization of these proteins into the same punctate structures, indicating that the MP is not accumulated randomly onto the cell surface, but targeted to particular sites at the cell periphery, where punctate and tubule-like structures are likely formed. With the use of cytoskeleton and secretory pathway inhibitors, we showed that the cytoskeletal elements are not likely to be involved in targeting of the MP-GFP to the punctate cellular structures. In addition to MP, a functional coat protein was found to be essential for virus spread within inoculated leaves.

Grapevine virusA-mediated gene silencing in Nicotiana benthamiana and Vitis vinifera.

Virus-induced gene silencing (VIGS) is an attractive approach for studying gene function. Although the number of virus vectors available for use in VIGS experiments has increased in recent years, most of these vectors are applied in annual or herbaceous plants. The aim of this work was to develop a VIGS vector based on the Grapevine virus A (GVA), which is a member of the genus Vitivirus, family Flexiviridae. The GVA vector was used to silence the endogenous phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. In addition, an Agrobacterium-mediated method for inoculating micropropagated Vitis vinifera cv. Prime plantlets via their roots was developed. Using this method, it was possible to silence the endogenous PDS gene in V. vinifera plantlets. The GVA-derived VIGS vector may constitute an important tool for improving functional genomics in V. vinifera.

Post-transcriptional gene silencing and virus resistance in Nicotiana benthamiana expressing a Grapevine virus A minireplicon.

Grapevine virus A (GVA) is closely associated with the economically important rugose-wood disease of grapevine. In an attempt to develop GVA resistance, we made a GFP-tagged GVA-minireplicon and utilized it as a tool to consistently activate RNA silencing. Launching the GVA-minireplicon by agroinfiltration delivery resulted in a strong RNA silencing response. In light of this finding, we produced transgenic Nicotiana benthamiana plants expressing the GVA-minireplicon, which displayed phenotypes that could be attributed to reproducibly and consistently activate post-transcriptional gene silencing (PTGS). These included: (i) low accumulation of the minireplicon-derived transgene; (ii) low GFP expression that was increased upon agroinfiltration delivery of viral suppressors of silencing; and (iii) resistance against GVA infection, which was found in 60%, and in 90-95%, of T1 and T2 progenies, respectively. A grafting assay revealed that non-silenced scions exhibited GVA resistance when they were grafted onto silenced rootstocks, suggesting transmission of RNA silencing from silenced rootstocks to non-silenced scions. Despite being extremely resistant to GVA infection, the transgenic plants were susceptible to the closely related vitivirus, GVB. Furthermore, infection of the silenced plants with GVB or Potato virus Y (PVY) resulted in suppression of the GVA-specific defense. From these data we conclude that GVA-minireplicon-mediated RNA silencing provides an important and efficient approach for consistent activation of PTGS that can be used for controlling grapevine viruses. However, application of this strategy for virus resistance necessitates consideration of possible infection by other viruses.

Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants.

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a approximately 7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.