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.

The defective RNAs of Closteroviridae.

The family Closteroviridae consists of two genera, Closterovirus and Ampelovirus with monopartite genomes transmitted respectively by aphids and mealybugs and the Crinivirus with bipartite genomes transmitted by whiteflies. The Closteroviridae consists of more than 30 virus species, which differ considerably in their phytopathological significance. Some, like beet yellows virus and citrus tristeza virus (CTV) were associated for many decades with their respective hosts, sugar beets and citrus. Others, like the grapevine leafroll-associated ampeloviruses 1, and 3 were also associated with their grapevine hosts for long periods; however, difficulties in virus isolation hampered their molecular characterization. The majority of the recently identified Closteroviridae were probably associated with their vegetative propagated host plants for long periods and only detected through the considerable advances in dsRNA isolation and sequencing of PCR amplified replicons. Molecular characterization of CTV and several other Closteroviridae revealed that, in addition to genomic and subgenomic RNAs, infected plants contain several different subviral defective RNAs (dRNAs). The roles and biological functions of dRNAs associated with Closteroviridae remain terra incognita.

Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus.

Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

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.

The grapevine-infecting vitiviruses, with particular reference to grapevine virus A.

A number of vitiviruses infect grapevine, arguably the most important fruit crop, and the host from which they derive their genus name. In contrast to most grapevine viruses, the etiological role of these viruses is unclear, albeit that they are associated with several well-known--and a number of emerging--diseases of grapevine. Here, we review the genus Vitivirus, with special reference to its most omnipresent member, grapevine virus A. We discuss the latest taxonomic status of the genus, as well as the genome and genomic organisation, replication mechanism, and genetic variability of GVA, and we also present the latest research progress with vitivirus-based vectors; the identification of a new vitivirus, GVE, the discovery of VIGG, a unique GVA-induced host protein, the molecular characterisation of hitherto unknown or puzzling genetic elements in the GVA genome, and the latest developments in vitivirus diagnostics.

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.

Construction of a multiprobe for the simultaneous detection of viroids infecting citrus trees.

Infections with different viroid species are common among cultivated fruit trees and grapevines, and many old-clone citrus varieties contain up to five citrus viroids (CVds) within a single tree. This paper describes the construction of a CVd-Multiprobe consisting of full-length clones of Hop stunt viroid, Citrus exocortis viroid, Citrus bent leaf viroid and CVd-III. The CVd-Multiprobe was tested against RNA transcripts of the four viroids and RNA extracts from plants singly infected with CEVd or HSVd or multiply infected with different CVds. The viroids were effectively diagnosed with the DIG labeled CVd-Multiprobe when tested by Northern hybridization or dot blot analyses. The CVd-Multiprobe does not provide information on the specific viroid resulting in a positive signal. However, this should not be considered as a problem, since most citrus certification programs will discard budwood source trees infected with any of the known CVds.

Transgenes consisting of a dsRNA of an RNAi suppressor plus the 3' UTR provide resistance to Citrus tristeza virus sequences in Nicotiana benthamiana but not in citrus.

In an attempt to utilize post-transcriptional gene silencing (PTGS) as a means to impart resistance against Citrus tristeza virus (CTV) into citrus plants, the p23 + 3'UTR sequence (p23U) of the VT strain of CTV was engineered to fold into a double-stranded (ds) RNA structure. The resulting construct (p23UI) was introduced into Nicotiana benthamiana and Alemow (Citrus macrophylla) plants by Agrobacterium-mediated transformation. Transgenic p23UI- N. benthamiana were resistant to infection with a viral vector made of Grapevine virus A (GVA) + p23U (GVA-p23U), as indicated by the absence of the chimeric virus from inoculated plants. Inoculation of transgenic p23UI Alemow plants with CTV resulted in delayed appearance of symptoms in 9 out of the 70 transgenic plants. However, none of the plants showed durable resistance, as indicated by the obtaining of similar Northern hybridization signals from both transgenic and non-transgenic citrus plants. The possible causes for the failure of transgenic citrus plants to confer durable resistance to CTV are discussed.

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.

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon.

Launching the Beet yellows virus (BYV) minireplicon by agrobacterial delivery resulted in an unexpectedly low number of infected cells per inoculated leaf. This effect was due to a strong RNA silencing response in the agroinfiltrated leaves. Strikingly, ectopic co-expression of p21, a BYV RNA silencing suppressor, increased minireplicon infectivity by three orders of magnitude. Mutational analysis demonstrated that this effect correlates with suppressor activity of p21. Five diverse, heterologous viral suppressors were also active in this system, providing a useful approach for a dramatic, up to 10,000-fold, increase of the efficiency of agroinfection. The minireplicon agroinfection assay was also used to identify a new suppressor, a homolog of BYV p21, derived from Grapevine leafroll-associated virus-2. In addition, we report preliminary data on the suppressor activity of the p10 protein of Grapevine virus A and show that this protein belongs to a family of Zn-ribbon-containing proteins encoded by filamentous plant RNA viruses from three genera. The members of this family are predicted to have RNA silencing suppressor activity.

ORF 5 of grapevine virus A encodes a nucleic acid-binding protein and affects pathogenesis.

A previous functional analysis of the genome of grapevine virus A (GVA) was not conclusive as to the role of open reading frame 5 (ORF 5). This ORF encodes a 10-kDa protein (p10) carrying two distinct domains: a basic, arginine-rich domain and a zinc-finger domain. P10 was cloned and expressed in Escherichia coli, and was shown by northwestern assays to interact with nucleic acids. In-frame deletion of the basic region abolished P10's nucleic acid-binding capability, whereas substitution of cysteine residues by serine in the zinc-finger domain did not affect binding. These mutations were inserted into the full-length infectious clone. It has been shown that ORF 5 mutations do not affect replication of GVA-RNA. However, plants inoculated with the aforementioned mutations did not develop symptoms, and Western blot analysis revealed markedly reduced expression of the movement protein (the product of ORF 3).

Effect of benzyladenine on recovery of cryopreserved shoot tips of grapevine and citrus cultured in vitro.

The effect of N6-benzyladenine (BA) on the recovery of cryopreserved shoot tips of the LN33 hybrid (Vitis L.) and Troyer citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis [L.] Osbeck.] cultured in vitro was examined. For the LN33 hybrid, the presence of BA in the recovery medium was essential for survival of control and cryopreserved shoot tips, although the BA concentration did not influence the survival percentage. BA at 5, 2, and 5 microM or higher induced callus formation in control, and shoot tips cryopreserved by vitrification, and by encapsulation-dehydration, respectively. While a BA concentration of 4 microM was found optimal for recovery of control shoot tips, 1 and 2-4 microM produced the best recovery of shoot tips cryopreserved by vitrification and encapsulation-dehydration, respectively. A similar pattern of effect of BA on recovery was found for 'Troyer' citrange. Low survival of control and cryopreserved shoot tips was observed with a BA-free recovery medium. The addition of BA to the recovery medium significantly increased survival. The BA concentration that induced callus formation in shoot tips cryopreserved by encapsulation-vitrification was higher than that which induced it in those cryopreserved by encapsulation-dehydration. Recovery of control shoot tips was best with an addition of 6-10 microM BA to the medium. Optimal recovery of shoot tips cryopreserved by encapsulation-vitrification and encapsulation-dehydration was achieved with 3-4 and 2 microM BA, respectively. Results from the present study suggest that an optimal BA concentration for recovery of control shoot tips may be different from that for cryopreserved shoot tips; furthermore, the optimal BA concentration for recovery of cryopreserved shoot tips may also differ among different cryogenic procedures.

Effects of modification of the transcription initiation site context on citrus tristeza virus subgenomic RNA synthesis.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3'-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5' termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5' or 3' of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements.

Grapevine virus A (GVA), a species of the recently established genus Vitivirus, consists of an approximately 7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). The virus, which is closely associated with the grapevine rugose wood disease complex, has been poorly investigated genetically. We explored the production of viral RNAs in a GVA-infected Nicotiana benthamiana herbaceous host and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Neither 3'- nor 5'-terminal sgRNAs, which would correspond to ORF5, was detected, suggesting that expression of this ORF occurs via a bi- or polycistronic mRNA. The 5'-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 3' end of 5.5-kb 5'-terminal sgRNA and the 5' end of the 1.8-kb 3'-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Apparently, the production of the 5'- and 3'-terminal sgRNAs was controlled by sequences upstream of the 5'-terminus of each of ORFs 2-4. Detection of both plus and minus strands of the 5'- and 3'-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3'-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3'-terminal minus-strand RNA, a 5'-terminal plus-strand sgRNA, and the corresponding 5'-terminal minus-strand RNA.

A novel class of large and infectious defective RNAs of Citrus tristeza virus.

Citrus tristeza virus (CTV)-infected plants contain one or more populations of defective RNAs (dRNAs), mostly with a size range of ca. 2.0 to 5.0 kb. Several CTV dRNAs have been characterized and found to consist mainly of the two termini of the genomic RNA, with extensive internal deletions. The present paper describes a new class of large ( approximately 12.0 kb) dRNAs from three different CTV isolates with two unusual features. First is their composition with intact replicase genes. These dRNAs contained a large 5' portion of the genomic RNA terminus, which apparently corresponded to the recently described 5' large single-stranded subgenomic RNA (sgRNA) of ORF1a+1b (Che et al., 2001). The 3' portion of the large dRNAs varied among the 10 different cDNA clones examined in this work. In 2 dRNAs this portion consisted of truncated ORF10 (p20), and in 5 dRNAs it contained truncated ORF11 (p23). Two dRNA molecules were found with a 3' portion that started in the exact 5' position of the intergenic region between the p20 and p23 ORFs. In one dRNA, this portion coincided with the full-length sgRNA corresponding to ORF10. The second unusual feature was their ability to be readily transmitted mechanically to citrus plants by stem slashing and also to Nicotiana benthamiana protoplasts. The possibility that these dRNAs may be encapsidated and be capable of self-replication is discussed.