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First published online September 28, 2007; 10.1104/pp.107.108217 Plant Physiology 145:1251-1263 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Universal Expression/Silencing Vector in Plants[C],[OA]Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Institute for Plant Sciences and Genetics, Rehovot 76100, Israel
A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.
A common technology for the introduction and expression of foreign genes in plants is Agrobacterium-mediated transformation (Horsch et al., 1985  ). Another general approach for introducing and expressing foreign genes involves physical means, such as bombardment of DNA-coated inert beads into the plant (Klein et al., 1987; Sanford et al., 1987). Viral vectors have also been engineered for the delivery of genetic material and the expression of recombinant proteins in plants (Chapman et al., 1992; Scholthof et al., 1996; Shivprasad et al., 1999; Yusibov et al., 1999; Mallory et al., 2002; Pogue et al., 2002; Porta and Lomonossoff, 2002; Gleba et al., 2004; Istomina et al., 2004). With DNA plant viruses as vectors, the size of the insert is very limited, and when a viral gene is replaced by a foreign one to accommodate larger inserts, replication and movement are hampered and expression is restricted to the affected cells only (Shepherd, 1989; Ugaki et al., 1991; Stanley, 1993; Tamilselvi et al., 2004; Carrillo-Trip et al., 2006). In the case of RNA viruses, cloning of a foreign sequence downstream of a duplicated viral subgenomic promoter brings about expression of the foreign gene. In many cases, the foreign gene is, however, gradually deleted (Dawson et al., 1989). Viral-mediated delivery and expression of foreign genes are nontransgenic; therefore, they have been designated as "transient" (Scholthof et al., 1996; Marillonnet et al., 2005).
Tomato yellow leaf curl virus (TYLCV) is a monopartite geminivirus (Stanley, 1985
Three viral gene products are implicated in viral movement within the plant: the coat proteins (CPs) V1, V2, and C4 (Rojas et al., 2001
Early on, conflicting reports were already being published with respect to the form of the geminiviral DNA that shuttles between the nucleus and the cytoplasm and across the plasma membrane, suggesting ssDNA (Pascal et al., 1994
Many (but not all) geminiviruses are considered to be phloem restricted, but this tropism is not strict. Closely related viruses may differ in their tissue tropism (Wege et al., 2001
TYLCV infection of tomato (Solanum lycopersicum) is harmful and causes serious agricultural/economic problems (Czosnek and Laterrot, 1997
Geminiviral sequences have been found in the genomes of several host plants (Ashby et al., 1997
One plant reaction to viral infection is the stimulation of viral-specific RNA silencing triggered by the appearance of replicative forms of viral dsRNA (e.g. Baulcombe, 2005
Grapevine virus A (GVA) is a vitivirus affecting grapevines and is transmissible to Nicotiana benthamiana. A GVA infectious clone has been produced, leading to a functional analysis of the viral genome (Galiakparov et al., 1999 We engineered a symptomless deletion clone of TYLCV and, as discussed further on, chose to insert a plasmid within the rep gene. Long (at least 5 kb) inserts can thus be introduced into TYLCV-DNA. The virus-plasmid vector and the foreign gene inserted into it move systemically and are expressed in plants. The engineered construct is easily introduced into the plant and it replicates in both plant and Escherichia coli cells. A target gene could be silenced by expressing dsRNA. In addition, we engineered a series of artificial satellite DNAs activated to express or silence target genes following virus infection. By substituting the native virus with IL-60-BS, expression/silencing could be achieved without causing disease. We also induced viral resistance using a unique silencing approach.
A TYLCV-Based Vector and Its Derivatives Replicate and Spread in Plants without Causing Disease
We produced a full-length clone of the aforementioned Israeli strain of TYLCV (GenBank accession no. X15656). A 1.8-genome-long construct, when agroinoculated into tomato plants, caused severe infection. The viral CP is not involved in geminivirus-DNA replication (Noris et al., 1998
The virus-plasmid vector IL-60-BS, as well as its derivatives carrying genes for GUS or GFP, were first introduced into tomato, a natural host of TYLCV. PCR analyses indicated the presence of vector in all tested leaves, as well as in flowers, fruits, and roots. As shown in Figure 2 , the vector was found in parts remote from the point of injection (at least four leaves above it) as early as 3 d postinjection (p.i.) and persisted throughout the plant's life span (12 months p.i.).
The presence of vector sequences in remote tissues for long periods and their detection by Southern-blot analysis (Fig. 3 ) indicated its replication and spread in almost all of the injected plants (>90%). As shown below, the vector also replicated and spread in various other plants belonging to several different families, including monocots and woody trees.
IL-60 Does Not Integrate into the Plant Genome and Does Not Produce ssDNA Total DNA was extracted from tomato leaves remote from the point of IL-60-BS injection and was subjected to Southern-blot analysis without cleaving or shearing. The membranes were reacted with a digoxigenin-labeled probe corresponding to part of the TYLCV-CP gene (Fig. 3). Two major bands were detected, as with TYLCV (presented as a positive control). In samples from leaves of vector-administered plants, the two corresponding bands were of a larger size than those of TYLCV alone, due to the presence of the plasmid. Southern analysis is far less sensitive than PCR, and, therefore, positive reactions in remote tissues after long periods cannot be attributed to residual, dilute samples of the DNA originally administered to the plant. The absence of a ssDNA band in the IL-60-BS lanes is indicative of the lack of ssDNA viral progeny, due to inactivation of rep and the consequent inability to initiate the rolling-circle phase.
BglII does not cleave within IL-60-BS. If the vector had been integrated into the plant genome, then cleavage with BglII would have resulted in bands longer than those in the uncleaved samples. The bands obtained from cleaved and uncleaved samples were of the same size (Fig. 3), indicating that the vector had not been integrated into the plant genome. These bands were insensitive to digestion with S1-nuclease, confirming their dsDNA nature (Fig. 3C). Northern and western analyses indicated that viral genes are expressed in plants treated with IL-60-BS (Fig. 4
). The transcription termination site of the RNA transcribed from ORFs V1 and V2 has not been determined. In fact, the transcription of long transcripts in both orientations has been implicated in the formation of the dsRNA required for gene silencing of geminiviruses (Bisaro, 2006
Expression of Foreign Genes Inserted in IL-60-BS
IL-60-BS-GUS and IL-60-BS-GFP (see "Materials and Methods") were introduced into tomato plants. Replication of the constructs was monitored by PCR using primers of the reporter genes (167/408 for GUS and 345/895 for GFP; Table I
). Positive reactions with DNA templates from leaves further up from the point of injection were observed as early as 3 d p.i. (an example is shown in Fig. 5A
). GUS activity was detected by staining (Jefferson et al., 1987
IL-60-BS-Derived Constructs Are Not Heritable and Are Not Transmitted by the Whitefly B. tabaci Tomato plants carrying IL-60-BS-GUS and expressing GUS produced fruits. Seeds were collected from those fruits, and progeny (F1) plants were grown alongside their parents. We self-pollinated three GUS-expressing primary plants and checked 108 F1 progeny plants for the presence of GUS and of IL-60-BS by amplifying the junction between TYLCV and the plasmid (Table I). In addition, we checked 60 F2 progeny plants and 10 F3 plants. Whereas the parent plants reacted positively in all of the PCR tests and still carried and expressed GUS, only one F1 plant faintly expressed GUS. The F2 progeny plants of the GUS-expressing F1 plant did not express GUS. Overall, only one out of 178 progeny plants was found to carry IL-60-BS-GUS, and this was not carried on to further generations. An example of PCR analysis with primers for GUS is presented in Figure 6A . The weak GUS expression found in the single progeny plant (Fig. 6A, lane 2) may have been the result of "mechanical" contamination with the vector.
Transmission assays with B. tabaci were carried out as described in "Materials and Methods." Briefly, the insects were fed on tomato plants carrying IL-60-BS and were later transferred to untreated plants. PCR assays were carried out with DNA extracted from the donor and recipient plants. Similar assays were carried out with TYLCV-infected plants (positive controls for the validity and efficiency of the assay). As shown in Figure 6B, the recipient plants did not carry IL-60-BS. As a general rule, 15 insects per plant are sufficient for TYLCV transmission. Suspecting that IL-60-BS may still be transmissible, albeit at a lower efficiency, in one experiment we colonized approximately 150 Bemisia individuals per plant and still did not find the construct in the recipient plants.
pIR-X are a series of constructs in which the IR of TYLCV is placed in the plasmid pDrive, enabling the insertion of foreign genes downstream of it (Fig. 1B; "Materials and Methods"). The satellites pIR-GUS and pIR-GFP did not replicate or express the reporter gene in protoplasts. Therefore, satellites of the structure pIR-X could not, by themselves, replicate and express in plants but could be propagated in E. coli. However, co-administration of IL-60-BS and pIR-GUS or pIR-GFP resulted in the replication of both constructs and strong GUS and GFP expression in plants (Figs. 7 and 8 , respectively). As already mentioned, plants injected with only pIR-GUS did not express GUS. After 14 d, the plants were challenged with a wild-type TYLCV. Extensive GUS expression was noted within a few days, but the plants developed severe disease symptoms. Challenge inoculation with IL-60-BS, however, induced expression without causing any symptoms. As shown in Figure 7, expression of a gene placed under the control of IR becomes visible following challenge infection with a helper-disarmed virus. Because IR-derived expression requires only host factors, it is conceivable that the foreign gene is expressed by itself in the few cells it has entered and that the helper virus enables its movement throughout the plant, rendering its expression readily detectable.
The expression and movement of a gene placed under the control of TYLCV-IR can thus be transactivated at a later time by challenge inoculation with a helper geminivirus. The disarmed IL-60-BS is able to transactivate in a harmless manner. In fact, these constructs are TYLCV satellites, as they depend on TYLCV or IL-60-BS as helper viruses for replication, movement, and expression.
The host range of TYLCV is quite broad, infecting plants belonging to several botanical families (Mansour and Al-Musa, 1992 In many plants, expression of the reported gene was initially restricted to the vascular system; however, eventually the expressing construct moved to non-phloem tissues. In some plants (wheat, onion, dill, and others; Fig. 7, frames 3, 5, and 7), expression was already found outside the vascular system immediately following injection.
The co-administration of IL-60-BS and pIR-GUS (or pIR-GFP) appeared, to the naked eye, to be a better expressing system than IL-60-BS-GUS (or IL-60-BS-GFP) alone. The levels of GUS and GFP expression, when co-administered with IL-60-BS, were tested quantitatively (see "Materials and Methods") relative to their expression in transgenic plants driven by the strong plant promoter 35S (Fig. 8). In both cases, IL-60-BS-derived expression was comparable to that of 35S-derived expression. Expression levels of GUS in the different tested cases were approximately 0.25- to 2-fold those in the control transgenic plants. The actual rates for GUS, obtained by the 4-methylumbelliferyl glucuronide assay (fluorescence units per microgram protein per hour) were 3.7 for 35S-derived expression in tobacco, 2.9 for IL-60-BS-derived expression in N. benthamiana, 0.9 in petunia, and 6.6 in onion. The rates for GFP (fluorescence units per cell) were 1.67 for 35S-GFP and 1.81 for pIR-GFP co-injected with Il-60-BS (both in tobacco). While in transgenic plants, GFP expression was localized in the cytoplasm, expression in the IL-60-BS-driven system was localized in the vacuole. This level of expression is comparable to other viral vectors; the level of expression from the currently best-known plant virus vector is about 2-fold higher than that reported here (discussed further on).
Two types of constructs were developed for silencing purposes. A construct of tandem sense:antisense repeats of a segment of the gene for phytoene desaturase (PDS) was inserted into pIR-GUS, replacing GUS with the PDS repeats (see "Materials and Methods"). The resultant construct (IR-PDS repeats) was induced to transcribe the PDS repeats by injecting it, along with IL-60-BS, into tomato, petunia, and N. benthamiana plants. The resultant RNA was expected to fold back on itself to make dsRNA and thus induce PDS silencing. Within 1 to 4 weeks (depending on the plant species), some leaves started to turn yellow (in tomato) or white (in N. benthamiana and petunia) due to chlorophyll bleaching resulting from PDS silencing. Quantitative reverse transcription (RT)-PCR indicated approximately 64-fold inhibition of expression (Fig. 9 ). Another silencing method consisted of inserting a target sequence between two opposing IR promoters (IR-X-RI), leading to the production of dsRNA upon transactivation with virus or IL-60-BS. PDS silencing was also achieved in this manner (data not shown).
Creating GVA Resistance/Tolerance in N. benthamiana Posttranscriptional gene silencing has been reported to play a role in the plant's reaction to infection, and equilibrium between silencing and silencing suppression determines pathogenecity. We therefore attempted to produce nontransgenic resistance/tolerance by silencing the GVA-silencing suppressor p10, thereby arresting the virus's ability to exercise counter-silencing measures. The entire ORF 5 of GVA (coding for p10) was placed between two opposing IRs, as described in "Materials and Methods," producing IR-p10-RI. This construct was injected into N. benthamiana plants along with IL-60-BS. Five days later, the plants were inoculated with GVA. Symptom appearance was monitored daily. While the control (noninjected) plants developed symptoms within a week, plants treated with IR-p10-RI + IL-60-BS were divided into two groups: asymptomatic plants and plants with very mild symptoms (Fig. 10 ). Semiquantitative RT-PCR indicated an approximately 60-fold reduction of p10 expression in symptomless treated plants, as compared to untreated plants (Fig. 10B), and an approximately 30-fold reduction in plants exhibiting mild symptoms (data not shown). Thus, suppression of the viral silencing suppressor gave rise to plants that are GVA resistant or tolerant.
This article describes a platform for expression or silencing in plants that is fast responding and applicable to all plants tested to date. Thus, studies in hard-to-manipulate plants, such as wheat, pepper, and grapevine, can now be performed within days (2–3 weeks in woody plants). The IR-carrying segments of TYLCV fused to a foreign gene and introduced into plants (pIR-X) are stable, even though they do not replicate or spread in the plant. Their stability may be attributed to the inherent IR sequence. A helper geminivirus may be introduced at a later time, contributing transactivating factors and inducing the spread of, and expression from, the IR-carrying segment. In fact, pIR-X and IR-X-RI constructs are satellites, depending on a helper virus for replication, expression, and movement (see also Morilla et al., 2006 ). If the challenging entity is IL-60-BS, the outcome is harmless. This provides an inducible expression system. It also appears that double-injection of the aforementioned IR-carrying segment with IL-60-BS brings about stronger expression of the foreign gene, apparently due to its proximity to the IR. The level of expression from the strong plant promoter 35S ranges from 1% to 3% of the plant's soluble proteins (Liu et al., 1994; De Jaeger et al., 2002; Krishnan et al., 2002; Kang et al., 2006). We determined an up to 2-fold increase of expression in the IL-60-BS-derived system compared to that of 35S-derived expression, yielding roughly up to 6% of the soluble protein. In comparison, expression rate in plants from the best known viral (Tobacco mosaic virus) vector is approximately 10% of the soluble protein (Shivprasad et al., 1999). The IL-60-BS-driven system, therefore, expresses at about one-half the level of the Tobacco mosaic virus-derived vector. By expressing constructs leading to the formation of dsRNA, the system can also induce silencing of a target gene. Insertion of short inverted repeats of PDS downstream of IR resulted in PDS silencing. GVA-resistant/tolerant plants were created by placing ORF 5 (p10) of GVA between two opposing IRs.
We modified the native TYLCV genome to achieve the desired functional alterations, i.e. abolishing symptoms but maintaining spreading capability. The CP of geminiviruses plays no role in viral DNA replication but is involved in viral movement, systemic spread, and symptom development in the plant (Liu et al., 1994
The previously suggested size limitation of inserts in geminivirus expression vectors (Stanley, 1993 As already mentioned, the IR-carrying segment is stable, and the introduction of a helper geminivirus will allow its spread and expression, thereby providing an inducible expression system. By expressing constructs leading to the formation of dsRNA, the system can also silence a target gene.
The agricultural use of genomically modified plants is a matter of public debate, and in many countries it is prohibited by law or regulation. The main concerns voiced against the use of transgenic plants are the fear of inappropriate selection of the transgenic lineage (due to masked deleterious positional effects), possible cross-fertilization with weeds and other crops, further genome alterations due to recombination (especially when copies of endogenous genes are added), and possible transduction of the foreign sequences to plant and soil microorganisms. Introduction of antibiotic-resistant genes to food and the environment is also a major concern. The IL-60-derived systems remain active after removal of the bacterial origin of replication and the ampicillin-resistance gene (data not shown), these alterations making them potentially biosafe. Geminiviruses are not seed transmissible (Kashina et al., 2003 In addition to the use of the IL-60 system for expression, it can be employed for silencing. We demonstrated a case of inducible silencing of an endogenous gene (PDS). We proceeded by building a system in which virus infection stimulates the silencing of one of its own genes, bringing about resistance/tolerance. The treated plants become resistant/tolerant within a few days of injection, as compared to conventional breeding which, after many years of development, has been only partially successful.
Clones and Constructs
For reasons discussed earlier, we deleted 60 bp (positions 552–612) of a full-length clone of the Israeli strain of TYLCV (Navot et al., 1991 The plasmid Bluescript II KS+ was SacI-linearized and ligated to the SacI site of IL-60. The resultant 5,682-bp-long virus-plasmid construct was termed IL-60-BS. This construct replicates in Escherichia coli cells, and milligram quantities of it can be obtained by a simple midi-prep procedure for plasmid isolation. The plasmid was introduced into position 2,443 of IL-60, thus interrupting the latter's rep gene by placing a 2,960-bp insert near its 5' end. The inserted spacer also caused a frameshift, so that the two parts of rep were in different reading frames. Surprisingly, a construct bearing a single length of viral genome can thus be administered directly into plants without Agrobacterium mediation, and the construct replicates and spreads in the affected plants. Reporter genes were introduced into IL-60-BS as follows. IL-60-BS was cleaved with EcoRV (which cleaves solely within the multiple cloning site of BS). The coding region of the reporter gene GUS (bases 1,466–3,274; GenBank accession no. M14641) was PCR-amplified adding a SacI restriction site to one end and a SalI site to the other. All of the restriction sites were blunt-ended with T4-DNA polymerase, and the GUS was inserted into the EcoRV site of the vector. A construct containing the coding sequence of the GFP (bases 1–797; GenBank accession no. U87974) was inserted into IL-60-BS in exactly the same way. The resultant constructs were termed IL-60-BS-GUS and IL-60-BS-GFP. In addition, constructs carrying the plasmid pDrive instead of Bluescript were prepared and behaved similarly to the aforementioned constructs. The IR serves as the origin of replication as well as a strong bi-directional promoter for the expression of all viral genes. A satellite construct consisting of the IR, pre-coat ORF, and a short part of the 5' untranslated region of CP was fused to the aforementioned coding sequence of GUS. This construct was termed IR-GUS and was force-cloned into pDRIVE with SacI and SalI. The resultant construct was termed pIR-GUS. Various other sequences were also inserted by replacing GUS to produce pIR-X. These constructs cannot replicate in plants, but can be induced to replicate, move, and be expressed by TYLCV infection or IL-60-BS injection. Two sets of constructs were used for silencing. For PDS silencing, the GUS part of IR-GUS was replaced by an inverted-repeat segment of part of the tomato gene for PDS, prepared by amplifying tomato DNA with PDS primers (Table I). The resultant 199-bp product was TA cloned into the plasmid pDrive. The plasmid was then cleaved with BamHI and XbaI, and the resultant fragments were self-ligated, resulting in tandem inverted repeats of various lengths (multiplications of 199 bp). Following electrophoresis, a fragment of approximately 400 bp was extracted from the gel. This fragment is a tandem repeat of the PCR product, one repeat in a sense orientation and the other in an antisense orientation. The PDS fragment was inserted into the IR-carrying pDRIVE (see preparation of IR-GUS) that had been digested with BamHI and XbaI. Another construct for silencing consisted of two IR segments of TYLCV placed in opposite orientations at both ends of the multiple cloning site of the plasmid pDrive, and the construct was termed IR-X-RI. To silence GVA-p10, the viral ORF 5 (bases 7,011–7,287; GenBank accession no. AF007415) was PCR-amplified and TA cloned into pDrive. IRs were then inserted at both ends of p10 (between the KpnI and PstI sites upstream of p10 and between the HindIII and SalI sites downstream of it).
E. coli cells were transformed with the pertinent virus-plasmid construct, propagated under ampicillin selection, and the construct was extracted by standard procedures (Sambrook and Russel, 2001
Southern, northern, western, PCR, and quantitative RT-PCR analyses were carried out according to standard procedures (Sambrook and Russel, 2001 Probes for Southern and northern analyses were labeled by the PCR-digoxigenin procedure (Roche Molecular Biochemicals). Chemiluminescent probes for western blots were prepared with the SuperSignal West Pico kit (Pierce). All probes were prepared according to the respective manufacturer's protocol.
Quantitative PCR was carried out by removing aliquots from an ongoing PCR of a target gene (or cDNA) at different cycles and determining the threshold of band appearance. A similar assay, with the same templates, was carried out with primers for a constitutive gene, and the threshold of its band appearance was determined. Each treatment threshold was given an arbitrary quantitative value according to the formula
GUS staining and GFP fluorescence were monitored as described earlier. GFP images were photographed without a filter to detect any native fluorescence derived from leaf damage and then with a filter (Leica MZ FL III, GFP2). Levels of GFP expression in various treated plants were compared by measuring GFP fluorescence intensity per cell. These determinations were calculated from the microscopic images by the Image Pro 3 program of Media Cybernetics. Levels of GUS expression were determined by 4-methylumbelliferyl glucuronide assay (Jefferson et al., 1987
Whiteflies (Bemisia tabaci; 15–100 per plant) were fed on tomatoes carrying IL-60-BS for 48 h. The insects were then placed in one chamber of a dual-chamber container (the two chambers were separated by stretched Parafilm, the other chamber containing 12.5% Suc). Following 48 h feeding on Suc (through the membrane), the insects were transferred to nontreated plants. Similarly, as a positive control, insects were fed on TYLCV-infected plants, then on Suc and then on the recipient plant. The presence of TYLCV or IL-60-BS was followed by PCR.
The study was carried out in the framework of Minerva's Otto Warburg Center for Plant Biotechnology. We thank the Wolfson Foundation for the use of facilities contributed to the Plant Science Institute. Received August 29, 2007; accepted September 17, 2007; published September 28, 2007.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ilan Sela (sela{at}agri.huji.ac.il).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.108217 * Corresponding author; e-mail sela{at}agri.huji.ac.il.
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ABSTRACT
RESULTS
-helix (positions 99–106), including the catalytic Tyr (Y103; Y101 in the reported isolate of TYLCV), which is required for nicking (Campos-Olivas et al., 2002
ct = 2–(ct target gene – ct constitutive gene), Ct being the cycle threshold. The relative quantitative increase/decrease of templates between control and treated plants was estimated from the ratio of their respective 
