A Versatile Transreplication-Based System To Ident
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病菌学杂志 2006年第7期
Unidad de Genetica, Departamento de Biología Celular, Genetica, y Fisiología, Universidad de Málaga, 29071 Málaga, Spain
Biologisches Institut, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universitt Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany
ABSTRACT
A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.
INTRODUCTION
Geminiviruses belong to a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (9). The Geminiviridae family (58) is divided into four genera according to their genome organization and biological properties. The genus Begomovirus includes members that are transmitted by whiteflies, infect only dicotyledonous plants, and may have bipartite (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV) or Tomato yellow leaf curl virus (TYLCV).
Although among plant virus a minority, geminiviruses have a great economical impact, affecting many different crops worldwide (42). TYLCSV and TYLCV are two distinct species causing tomato yellow leaf curl disease, one of the most important threats to tomato crops in many tropical and subtropical regions of the world (45). Both viruses possess genomes of about 2.7 kb in size, which encode at least six proteins and contain an intergenic region (IR) that comprises the origin of replication and viral promoters. The open reading frames (ORFs) in the complementary sense orientation encoding a replication-associated protein (Rep, also known as AC1, AL1, C1, or L1), a transcription activator protein (TrAP, also known as AC2, AL2, C2, or L2), and a replication enhancer protein (REn) partially overlap; a small ORF, C4, is located within the Rep ORF but in a different reading frame (25). Rep and REn are required for efficient viral DNA replication, although only Rep is essential. Rep is a multifunctional protein that initiates and terminates plus-strand replication and specifically binds during origin recognition to a DNA sequence motive located in the IR (2, 10, 21, 29, 35, 50; for a review, see reference 25). TrAP and the C4 protein (also known as AC4, AL4, or L4), described as pathogenicity factors playing an important role in the infection process, are able to reverse RNA silencing in plants and to suppress local silencing in transient assays (62-64, 66).
Geminiviruses do not encode their own DNA polymerases and rely on the nuclear DNA replication machinery, like many mammalian DNA tumor viruses do. They replicate in nuclei of mature cells, which are inactive in DNA replication. An early step in geminivirus infection is therefore the induction of host DNA replication enzymes (23-25). Correspondingly, proliferating cell nuclear antigen (PCNA) was found to be expressed in differentiated cells of TGMV-infected plants and also in transgenic plants expressing TGMV Rep (46). TGMV Rep binds to a plant homolog of the animal cell cycle regulator, retinoblastoma protein (pRb) (1, 34). By analogy to mammalian DNA viruses, the interaction between Rep and pRb may bypass a pRb phosphorylation requirement for the G1/S transition and S-phase progression during geminivirus infection (41, 57). Several interactions between geminivirus Rep and plant cellular proteins have been identified by biochemical (affinity chromatography) or genetic (yeast two-hybrid system) assays. It has been demonstrated that Rep interacts with a serine/threonine protein kinase; a kinesin; the histone H3 (33); geminivirus Rep A-binding protein 1 (GRAB1) and GRAB2, two members of the NAC family (70); and with the SUMO-conjugating enzyme NbSCE1 (12). Additionally, Rep interactions with some elements of the cellular replication machinery have also been reported, such as the replication factor C complex from wheat (TmRFC-1) (39) and PCNA from tomato and tobacco (6, 11).
Geminiviruses, with their simple genome organization, broad host range, high copy number, and generation of infective clones, have many advantages as recombinant virus-based gene amplification systems in infected transgenic plants (60). Extrachromosomal amplification from geminivirus-based constructs has been exploited for the production of valuable peptides and proteins (28, 43, 51) or to analyze the function of Rep in replication (30).
Here, we present a TYLCSV-based green fluorescent protein (GFP) amplification system to identify the plant organs or tissues where viruses replicate and to follow virus replication within plants in real-time and without destruction.
We also demonstrate that this new tool, in combination with virus-induced gene silencing (VIGS) technology, could be an attractive instrument in functional genomics to easily identify host proteins required for geminivirus infection. We present an example of the system's feasibility using VIGS to suppress the expression of PCNA, a plant protein known to be required for viral DNA replication.
MATERIALS AND METHODS
Microorganisms, plants, and general methods. Virus strains used in this work are tomato yellow leaf curl Sardinia virus strain TYLCSV-ES[2] (GenBank accession no. L27708) and tomato yellow leaf curl virus strain TYLCV-[ES72/97] (GenBank accession no. AF071228).
Escherichia coli strain DH5- was used for subcloning. Southern gel blots from plants were performed as previously described (12). Agrobacterium tumefaciens was grown and selected with the appropriate antibiotics as described previously (17). Recombinant binary plasmids were introduced into A. tumefaciens LBA4404 by electroporation (65). A. tumefaciens LBA4404 was transformed with the constructs pGTYA2, pGTYOMGFP, pBINGFP, and pCACS1; transformants were selected with the appropriate antibiotics. DNA blot analysis was performed to check construct integrity before using constructs in transgenic plants.
Nicotiana benthamiana Domin was grown in an insect-free greenhouse with supplementary lighting. Manipulations of Escherichia coli and nucleic acids were performed by standard methods (56).
Plasmids and cloning. The first step in producing p2IR-GFP was to produce pTYO30, a plasmid that contains a tandem repeat of a TYLCSV IR fragment, positions 2441 thorough 2779, to nucleotide 158 in pBSKII+ (Strategene). A 945-bp DNA fragment corresponding to nucleotides 2441 to 606 of TYLCSV was amplified by PCR from pTYA50 (a plasmid containing an EcoRI monomer of TYLCSV) (J. Reina and E. R. Bejarano, unpublished data) using primers UIB-1 (5'-ATATGGAGATGAGGTTSCCC-3') and LCP-1 (5'-CAGAACCCCTAGTTACATCAC-3'). This PCR product was digested with SacII; the resulting 804-bp fragment, corresponding to a single copy of IR, was blunt-end ligated into EcoRV site pBSKII+, thus generating pTYO11. To obtain a tandem repeat, pTYO11 was digested with BamHI, and the fragment was blunt ended and ligated into the HincII site of pBSKII+ to yield pTYO29. Next, another BamHI fragment from pTYO11 was cloned into the BamHI site of pTYO29, yielding pTYO30.
pSMGFP (16), provided by the Arabidopsis Information Resource, was digested with XbaI-SacI to remove the GFP gene. A synthesized double-stranded DNA (dsDNA) fragment containing an HpaI site (5'-CTAGGTTAACCCCCGGGAGATCTCAGCT-3' and 5'-CAATTGGGGGCCCTCTAGAAG-3) was cloned into the XbaI-SacI sites from the previous plasmid to yield pSN1. pSN1 contained a unique HpaI site between the 35S cauliflower mosaic virus (CaMV) promoter (49) and the nopaline synthase (NOS) terminator. A HindIII-EcoRI fragment from pSN1 (including the 35S promoter-HpaI-NOS terminator fragment) was blunt ended and ligated into EcoRV of pTYO30 to yield pTYOM.
pGTYOM was made in two steps. First, pGA482-H was constructed by digestion of pGA482 with HpaI and BglI, blunt ended, and religated to eliminate the HpaI site. Secondly, a SacI-BglII fragment from pTYOM (which contains the 35S promoter and the NOS terminator between a tandem of TYLCSV IR) was blunt ended and subcloned into the blunt-end SacI site of pGA482-H to yield pGTYOM. The GFP gene was obtained by digestion of pSMGFP with SacI and BamHI, blunt ended, and ligated into the unique HpaI site of pGTYOM to yield p2IR-GFP.
pBINGFP was obtained by cloning a HindIII-EcoRI fragment from pSMGFP into HindIII-EcoRI sites from pBINPLUS (61).
Plasmid pACS1 was derived from the A. tumefaciens binary vector pGA482 (4) carrying the TYLCSV Rep gene in the sense orientation, under the regulation of the 35S CaMV promoter and the octapine synthase terminator. This plasmid was constructed as follows: plasmid pTYA55 (Reina and Bejarano, unpublished), carrying a complete TYLCSV genome, was digested with NcoI and KpnI to release a 1,154-bp DNA fragment including full-length Rep and C4 genes. This fragment was blunt-end ligated into the SmaI site of pBSKII+, thus generating pTYC1. The latter was digested with EcoRI and BamHI, releasing a fragment including the full-length Rep gene, which was then blunt-end ligated into the SmaI site of pJC2ENa (17), thus placing the Rep gene downstream of the 35S CaMV promoter and upstream of the octopine synthase terminator carried by pJC2Ena. As a result, a new plasmid (pJC1) was generated carrying Rep in the sense orientation. pJC1 was then digested with HindIII and XbaI, releasing a 2,746-bp DNA fragment, blunt ended, and ligated into the HpaI site of pGA482 to yield pACS1.
pTV00 and pBINTRA6 were kindly provided by the Sainsbury Laboratory and are described in reference 55. pTV-LePCNA132-255 contains a tomato PCNA partial clone (from amino acids 132 to 255) amplified by PCR from pCNATOM-87 (11) and ligated into the SmaI-ClaI sites of pTV00.
Plant transformation. N. benthamiana plants were grown in Murashige and Skoog (MS) medium (Sigma) supplemented with sucrose (30 g/liter). During plant transformation, MS medium was supplemented with 0.1 mg of -naphthalene acetic acid (Duchefa, N 0903)/liter and 0.95 mg of kinetin (6-furfurylaminopurine) (Sigma, K-0753)/liter. Antibiotic selection of transformed plants was achieved by supplementing the MS medium with 100 μg of kanamycin/ml; this concentration was reduced to 50 μg/ml for seed germination. Plants were grown in a controlled-environment growth chamber at 22 to 24°C with a 16-h photoperiod.
N. benthamiana transgenic plants were generated by transformation of leaf disks with A. tumefaciens carrying the corresponding binary plasmids (pTYOMGFP and pBINGFP), following a previously described protocol (31). For each plasmid, a number of independent transformants originated from separate calli were selected for their resistance to kanamycin. Plants were regenerated from these transformants, and a reduced number of regenerated plants were selected for further work. Seeds obtained by autofecundation of each of these selected transgenic plants were grown in kanamycin-supplemented MS medium to determine the number of independent loci where the 2IRGFP transgene was integrated. Total DNA extracted from transgenic plants (18) was used to analyze the integrity and the copy number of the integrated T-DNA by Southern blotting.
Geminivirus infection assays and detection of replicon DNA. Viral infections of N. benthamiana plants were performed by the agroinoculation technique as previously described (20). Plants were agroinoculated with plasmids pGTYA2 for TYLCSV and pGTYLES for TYLCV (binary vectors that contain a dimer of TYLCSV-ES[2] or TYLCV-Mld[ES72/97], respectively) (44, 47). For control, plants were mock inoculated with A. tumefaciens harboring the empty binary vector pGA482 or pBIN+.
Total plant DNA was extracted from N. benthamiana leaves at different days postinfection. For plant DNA gel blots, total DNA samples (2 μg to detect viral particles and 5 μg to detect replicon molecules) were used and hybridized as described previously.
For two-dimensional agarose gel electrophoresis, 1 g of plant material was processed as described previously (54) to purify total DNA using benzoylated-naphtholyated DEAE (BND)-cellulose chromatography. DNA amounts, each corresponding to 200 mg of the initial plant material, were applied to a single two-dimensional gel, Southern transferred, and hybridized against a GFP-specific probe covering the whole gene and, after the probe was stripped, against a TYLCSV-IR-specific probe.
PCR-amplified DNAs comprising the intergenic regions of TYLCSV-ES or TYLCVMld-[ES7297] were used to generated a virus-discriminating probe as previously described (44) For GFP, an XbaI-SacI fragment from the plasmid pSMGFP was used. For replicon molecules, a DNA fragment (HindIII-XbaI) comprising the 35S CaMV promoter was used.
Primers upperGFP (5'-GTGGCCGAGGATGTTTCCGTCCTCC-3) and lowerGFP (5'-AAGTTGGAATACAACTACAACTCCCAC-3') were used to PCR amplify a fragment of 2.3 kb of the episomal replicons using 1 μg of total DNA from N. benthamiana plants. PCR conditions were as follows: 95°C for 1 min; 25 cycles, each consisting of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 7 min at 72°C.
VIGS. VIGS with tobacco rattle virus (TRV) in N. benthamiana plants were performed as described in reference 55, and independent cultures of Agrobacterium carrying pTV00 (TVR RNA2), pBINTRA6 (TVR RNA1), pTV-LePCNA132-255, or pGTYA2 were grown overnight to saturation in LBroth plus appropriate antibiotics. Cultures were resuspended in VIGS buffer (10 mM morpholineethanesulfonic acid, 10 mM MgCl2, and 150 μM acetosyringone) and incubated at room temperature overnight. For infections, cultures containing pBINTRA6 and pTV00 (or pTV-LePCNA132-255) were mixed at a 1:1 ratio. A volume of this mixed culture was combined with the same volume of a culture carrying pGTYA2, and this mixture was used to infiltrate the underside of two leaves from a 3-week-old 2IRGFP N. benthamiana plant.
Photography. Geminivirus-infected plants were photographed by using a Nikon digital camera (Coolpix 4500). Close-up images were obtained with epifluorescense microscope Leica MZ FLIII. Images were processed using Adobe Photoshop.
RESULTS
Amplification of 2IRGFP trans replicons during TYLCSV infection. Transgenic plants (2IRGFP) were generated by transformation of Nicotiana benthamiana with p2IR-GPF, a construct containing a direct repeat of the IR of TYLCSV flanking the cauliflower mosaic virus 35S promoter, the green fluorescent protein open reading frame, and the NOS terminator sequence (Fig. 1A). For control, transgenic plants (BINGFP) lacking the IR repeats were generated in parallel (Fig. 1B). Six to eight independent plant lines for each construct (2IR-GFP and pBINGFP) were analyzed by Southern blot hybridization to confirm the integrity and the number of copies of the transgenes integrated into the plant genome (data not shown). Lines 2IRGFP-15 and BINGFP-3, containing a single copy of the p2IR-GFP or the pBINGFP construct, respectively, were selected for further characterization. All experiments shown were performed using T2 homozygous plants.
Plants from both transgenic lines developed typical systemic symptoms when infected with TYLCSV or TYLCV (data not shown). Additionally, DNA gel blots confirmed that a similar amount of viral DNA accumulated in leaves of transgenic and nontransgenic lines (Fig. 1C). However, when DNA extracted from infected and noninfected plants was hybridized with a 35S promoter-specific probe, an episomal DNA (hereafter referred to as the mGFP replicon) was only detected in 2IRGFP plants infected with TYLCSV (Fig. 1D). To confirm the presence of the expected circular mGFP replicon in these plants, DNA extracted 21 days postinfection (dpi) was digested with EcoRI, EcoRV, or both and analyzed by Southern blot hybridization using a GFP-specific probe. EcoRI or EcoRV (cutting twice and once within the mGFP replicon) produced a single 2.43-kb and 2.46-kb band, respectively (Fig. 1D, lanes EI and EV), whereas the double digestion lighted up one fragment of approximately 1.65 kb containing the complete GFP ORF, the NOS terminator sequence, a complete IR, and a small fragment of the 35S promoter (Fig. 1D, lanes EI and EV). The mGFP replicon was detected neither in transgenic BINGFP plants infected with TYLCSV nor in 2IRGFP plants infected with the heterologous TYLCV (Fig. 1D).
The restriction analysis indicated that trans replicons accumulated predominantly as open circular (oc) and covalently closed circular (ccc) dsDNA. The two bands observed in the undigested sample vanished when DNA was digested with EcoRI or EcoRV, yielding a single visible band (EcoRI digestion generates two fragments, but the smallest one, 25 bp long, cannot be detected). This was consistent with a linear dsDNA molecule of the mobilized version of the transgene containing the complete GFP expression cassette and a single copy of the IR. This conclusion was further confirmed by digestion of DNA from TYLCSV-infected 2IRGFP plants with other restriction enzymes and probing with GFP-, 35S-, or IR-specific probes (data not shown).
Our data are consistent with a specific generation of circular episomal trans replicons from the transgene during viral infection, as a consequence of the interaction between Rep and one of its cognate origin of replication, followed by nicking and ligation of the displaced nascent ssDNA at the second origin after replication of the sequence (25). A circular episomal replicon was produced that WAs subsequently amplified. Accordingly, an isolate of TYLCV (TYLCV-Mld[ES72/97]) was unable to mobilize the TYLCSV-derived trans replicon when infecting the 2IRGFP plants (Fig. 1C and 1D), probably because TYLCV Rep does not recognize TYLCSV IR-binding motifs.
During the initial experiments, only low levels of dsDNA but no trans replicon-specific ssDNA was detectable. Since it was possible that monomeric circular DNA of the expression cassette may have arisen from recombination rather than replication, it was mandatory to identify the replicative intermediates in closer detail. DNA from 2IRGFP plants which had been infected with TYLCSV was purified by BND-cellulose chromatography, separated on two-dimensional agarose gels (a recently developed technique) (54), and hybridized after Southern blotting, first against a GFP-specific probe and subsequently against an IR-specific probe, which recognized trans replicon and viral replicon with the same specificity (Fig. 2A to D). BND-cellulose chromatography enriches dsDNA preferentially in the wash fraction (Fig. 2A and C) and ssDNA-containing DNAs in the eluate fraction (Fig. 2B and D), the single forms of which have been extensively described previously (54). From this separation, it is obvious that the earlier failure to detect ssDNA was determined by the detection limit of the technique. ssDNA of the trans replicon was present as discrete monomer, dimer, and trimer, as well as in the form of heterogeneous molecules, which are the best indication of ongoing ssDNA replication. Unfortunately, the concentration of the trans replicons was still too low to detect the replicative intermediates of complementary strand replication, rolling-circle replication, or recombination-dependent replication (RDR), which have been found in all geminiviruses analyzed to date (3, 32, 54) and which were obvious if the blots were hybridized against a TYLCSV-IR-specific probe (Fig. 2D). Only a faint arc, classified as RDR in the results shown in Fig. 2B, could be assigned at the original blot but was too low in intensity to be properly reproduced. Nevertheless, the intermediates shown so far strengthen the conclusion that the trans replicons have undergone replication and not only recombination. The small relative amount of trans replicons compared to that of viral replicons may be imagined from the gel shown in Fig. 2C, where viral cccDNA and trans replicon cccDNA were detected side by side with the same probe.
Analysis of GFP expression in TYLCSV-infected 2IRGFP plants. Under long-wavelength UV light, uninfected 2IRGFP plants appeared slightly green, due to expression of GFP from the transgene (Fig. 3B). Green fluorescence was evenly distributed in the leaves, but this pattern changed dramatically when 2IRGFP plants were infected with TYLCSV (Fig. 3C). Three weeks postinfection (wpi), GFP fluorescence was concentrated at the leaf veins, and it was no longer detected on the leaf lamina. An increase in GFP expression was also noticeable in other TYLCSV-infected organs such as stems, roots, or flowers (Fig. 3G, K, and N, right) compared to organs of mock-inoculated plants (Fig. 3F, J, and N, left). GFP signal in roots and stems was also concentrated in transport tissues (Fig. 3I and M for TYLCSV-infected plants and 3H and L for mock-inoculated plants).
The changes observed in the GFP expression pattern of 2IRGFP plants were dependent on TYLCSV-specific infection, since they were not detected when plants were mock inoculated (Fig. 3B) or infected with TYLCV (Fig. 3D). Furthermore, the change in GFP expression produced by TYLCSV infection was dependent on the presence of the IR in the transgene, since GFP expression of transgenic BINGFP plants was not affected by infection (Fig. 3E).
To further characterize the changes in GFP expression in transgenic 2IRGFP plants, we carried out a time course experiment with infected plants. We agroinoculated 120 2IRGFP young plants with TYLCSV at the 3- to 4-leaf stage in four independent experiments and followed the expression of GFP in all leaves up to 6 wpi. Leaves were numbered according to the inoculation site, with the first leaf placed upon the agroinfection point considerd leaf + 1. As previously described (Fig. 3), we observed an increase of GFP expression at 21 dpi in the veins of the most apical leaves, together with a reduction of expression in the leaf lamina. Interestingly, GFP expression on the leaves placed just above the inoculation point had completely disappeared. Figure 4A shows representative photographs of each kind of leaves described, and a diagram that summarizes the standard expression patterns of all T2 plants infected with TYLCSV. GFP expression disappeared from leaf + 1 to leaf + 7, while the vein pattern associated with GFP expression appeared in the most apical leaves (leaf + 8 to leaf + 12). The intensity and spread of the vein expression was stronger in younger leaves (Fig. 4, leaf + 9 versus leaf + 11 or + 12). Six weeks postinfection, plants displayed a similar expression pattern to that detected at 3 wpi. A vein pattern was only noticeable in apical leaves, while GFP expression had entirely disappeared in all other leaves, even those where the GFP expression had been very high 3 weeks before (e.g., leaf + 12). To confirm that the increase of GFP expression is associated with the generation of the episomal replicon, we analyzed the accumulation of viral and replicon molecules in leaves displaying a similar pattern to leaf + 12 from Fig. 4A by Southern blotting. As shown in Fig. 4B, GFP expression correlated with the presence of episomal molecules. Leaves expressing GFP 3 wpi (lanes W3) contained replicons, while 3 weeks later (lanes W6), when the expression of GFP had vanished, the amount of these molecules was considerably reduced.
When viral DNA accumulation was analyzed further (Fig. 4C), we found a significant reduction in the amount of ssDNA and dsDNA accumulated in leaves collected at 3 wpi, compared to those collected 3 weeks later (6 wpi). This reduction was much higher for the dsDNA than for the ssDNA forms of the viral genome. Quantification of viral DNA forms showed that a leaf + 12 at 6 wpi accumulated approximately 70% of the ssDNA form presented at 3 wpi, but only 15% of the dsDNA. Considering that dsDNA represents replicative intermediates of the viral genome, we can conclude that a decrease in GFP expression, produced between 3 and 6 wpi, correlates with a reduction in virus replication.
2IRGFP replicon generation and GFP overexpression are both Rep dependent. To investigate whether the presence of Rep from TYLCSV was sufficient to mobilize and amplify the mGFP replicon in 2IRGFP plants, even in the absence of the other viral protein involved in replication (REn), we agroinfiltrated leaves with a construct expressing Rep, and concomitantly C4, under the control of the 35S CaMV promoter (pACS1). Four days later, a large increase in GFP fluorescence was detected on all infiltrated leaves (Fig. 5A, panel pACS1). Similar results were obtained when leaves were agroinfiltrated with an infectious clone of TYLCSV (Fig. 5A, panel TYLCSV). The large increase in fluorescence detected in TYLCSV-infiltrated leaves was, however, much lower than that detected in pACS1-infiltrated leaves, most likely because the levels of Rep protein are higher when Rep is expressed from the 35S promoter rather than from its own viral promoter. No increase in green fluorescence was detected when leaves were agroinfiltrated with the empty binary plasmid or with an infectious clone of TYLCV (Fig. 5A, panels pGA482 and TYLCV). More than 20 leaves from several 2IRGFP plants were agroinfiltrated with each construct, and similar results were obtained in all the experiments.
The increase of GFP expression in agroinfiltrated leaves correlated with the production of mGFP replicons in transgenic plants. We detected the accumulation of replicons by PCR (Fig. 5B) and by Southern blot analysis (Fig. 5C) in leaves agroinfiltrated with pACS1 or with TYLCSV but not in those inoculated with TYLCV or the binary vector pGA482. Furthermore, replicon levels in TYLCSV-infiltrated leaves were significantly lower than those in pACS1-infiltrated ones. Taken together, these results indicate that TYLCSV Rep protein is necessary to mobilize and amplify the mGFP replicon from the transgene present in 2IRGFP plants.
Gene silencing of PCNA prevents GFP induction and TYLCSV replication. Our data suggested that the distribution of the green fluorescence in infected 2IRGFP transgenic plants highlights the plant organs or tissues where the virus has expressed Rep and is therefore actively replicating its DNA. So, we consider whether these transgenic plants could be used to identify plant genes required for TYLCSV replication. If a cell function necessary for virus replication was silenced in TYLCSV-infected transgenic plants, we could readily detect it by illuminating the plants with UV light, as a green fluorescent vein pattern would not be produced. Silencing of host function could be obtained by dsRNA-mediated suppression of genes through the production of sense or antisense transcripts or even more efficiently by using single-stranded self-complementary (hairpin) RNA containing an intron (13, 36, 68, 69). However, both strategies rely on the generation of transgenic plants, which is a time-consuming task. Alternatively, temporal silencing of the genes could be obtained by infecting plants with viral vectors carrying host-derived sequences. This technology, VIGS, has been widely used to analyze gene functions, particularly for genes involved in defense against pathogens (for a review, see reference 38; see reference 52 for an example).
Hence, we assessed our system by silencing by VIGS a host gene already known to be required for begomovirus replication, the PCNA gene. PCNA is an essential, ubiquitous, and highly conserved protein in eukaryotes that functions as a DNA sliding clamp required for DNA replication and repair (67). Previous studies demonstrated that systemic begomovirus infection was significantly reduced when PCNA was silenced by geminivirus-derived vectors (53). We silenced N. benthamiana PCNA (NbPCNA) using a TRV vector designed to induce VIGS (55). TRV was selected because it offers advantages over other viral systems, as it does not induce symptoms and it is able to target host RNAs in the tissue growth area of plants, where it is believed that geminiviruses replicate. A 369-nucleotide fragment from tomato PCNA (LePCNA132-255) encoding amino acids 132 to 255 was cloned into VIGS vector pTV00. We selected this fragment of tomato PCNA previously isolated in our laboratory (11) because its nucleotide sequence is almost identical to that of cloned NbPCNA (90.3% identity with fragments of up to 35 identical nucleotides in a row) (19). It has been proposed that VIGS can target any gene with just over 20 nucleotides perfectly matched to the insert cloned in the VIGS vector (38). Thus, we expected that expression levels of NbPCNA would be reduced after LePCNA overexpression.
Transgenic 2IRGFP plants were agroinfected with TRV RNA1 and either TRV RNA2 (gene-silencing negative control) or RNA2-LePCNA132-255 (TRV RNA2 with an LePCNA132-255 fragment). Immediately afterward, all plants were also infected with TYLCSV (different time lapses between TRV and TYLCSV infections were assayed previously to select this timing). Ten days after infection, a phenotype, similar to that previously described for PCNA silencing in N. benthamiana (53), was displayed by plants infected with RNA2-LePCNA132-255. Primary growth was interrupted at the apical meristem, and new leaves showed progressively reduced expansion. This PCNA-suppressed phenotype was obvious at 21 dpi (Fig. 6A, left, TVR-PCNA). At that time control plants inoculated with TRV vector and TYLCSV exhibited the typical symptoms produced by begomovirus infection (Fig. 6A, left panel, TRV) and displayed the same GFP expression pattern that 2IRGFP plants infected with TYLCSV alone (Fig. 6A, right, TVR; Fig. 3C). Although transgenic plants infected with RNA2-LePCNA132-255 seemed to develop fewer symptoms of TYLCSV infection than control plants, the PCNA-silenced phenotype displayed by these plants somehow obscured the analysis of this difference. However, a difference was clearly noticed when the plants were illuminated with UV, since GFP vein pattern fluorescence was almost undetectable on most leaves of PCNA-silenced plants located above the inoculation point; only isolated dots of green fluorescence were observed (Fig. 6A, right, TRV-PCNA). The absence of fluorescence in these plants indicated that TYLCSV replication was impaired when the level of PCNA in the cell was reduced. When quantified by Southern blotting, TYLCSV DNA accumulation showed a significant decrease in leaves displaying the PCNA-silenced phenotype compared to leaves from plants infected with the TRV RNA2 vector (Fig. 6B). The reduction of viral DNA accumulation was stronger for dsDNA than for ssDNA molecules. PCNA-silenced leaves accumulated approximately 50% of TYLCSV ssDNA compared to leaves from control plants, but only 25% of dsDNA (average of three Southern blot analyses of different PCNA-silenced leaves). These results confirm that viral TYLCSV infection was reduced although not completely suppressed. This was not an unexpected result, as silencing of an endogenous gene by VIGS is not uniform and it does not occur in the whole plant.
DISCUSSION
We have designed a construct comprising those cis elements essential for TYLCSV replication to allow Rep-dependent release of the replicon and subsequent episomal replication. Although episomes induced by geminivirus infection have already been described for several systems, including mastreviruses (5, 48, 51) and bipartite begomoviruses (26, 27, 30, 40, 43), this is the first time that this strategy has been applied to monitoring monopartite begomovirus replication. Our construct comprises a GFP expression cassette cloned between a tandem repeat of a TYLCSV sequence, which includes the viral origin of replication, the viral promoters, and the 5' region of V2, CP, and Rep genes. Since GFP expression is under the control of the 35S CaMV promoter, constitutive expression of the reporter gene was expected to take place in transgenic plants carrying this construct. This was confirmed by the fact that most of the transgenic 2IRGFP lines obtained displayed green fluorescence in all tissues. However, we noticed that the level of GFP expression in 2IRGFP plants was lower than that displayed by transgenic plants carrying a GFP expression cassette without the viral IRs (BINGFP plants). It is possible that the IR viral sequence cloned upstream of the 35S promoter might interfere with its transcriptional efficiency through the binding of cellular factors to the TYLCV promoters present in the viral sequence.
The results obtained when 2IRGFP transgenic plants were infected with TYLCSV are consistent with a Rep-assisted generation of mGFP replicons from the 2IRGFP transgene, where Rep is provided in trans by the TYLCSV genome. This will lead to the overexpression of the GFP from the many replicon units thus generated, in those cells where Rep is being expressed by the virus.
The replicon was mobilized and amplified when Rep was expressed from either the TYLCSV genome or from a binary vector. As was reported for a similar system based in African cassava mosaic virus (30), the mGFP replicon accumulated at a level much lower than that of the TYLCSV genomes. Double- and single-stranded DNA forms of the replicon were detected, but in contrast to geminivirus genome where the ssDNA form is the major form of viral molecules, the amount of replicon ssDNA molecules was significantly smaller than that of dsDNA forms. This reduction or even absence of ssDNA accumulation has been also observed with other geminivirus replicon-based systems (43, 48, 51) and could mean either that the transition from the synthesis of a replicative form to the synthesis of ssDNA has not taken place in the episomal molecules or that ssDNA episomal molecules are degraded immediately after they are synthesized.
The results obtained when 2IRGFP plants were infected with TYLCSV suggest that GFP overproduction highlights plant tissues where the virus is replicating. In fact, the GFP expression pattern in infected 2IRGFP plants matches that obtained by in situ hybridization of N. benthamiana plants, showing that TYLCSV is restricted to the phloem (44). This hypothesis is also supported by the results obtained by Southern blot analysis of leaves that express GFP and of leaves where GFP expression has vanished (Fig. 5C). Although the loss of GFP expression in the leaves was associated with a reduction in the accumulation of both ssDNA and dsDNA viral forms, the relative decrease of dsDNA was manifestly larger than that of ssDNA. Geminiviruses causing tomato yellow leaf curl disease are important pathogens that have been the subject of numerous investigations. Despite an extensive amount of work, very little is known of the development of the infection and the spread of the virus within infected plants. Hybridization techniques used to study the accumulation of viral DNA in the plant have shown that the virus spreads following the flow of photoassimilates, which is somehow expected, since both move along phloematic tissues. They are first transported to the roots and the shoot apex and then to the neighboring leaves and flowers. In older leaves, the virus concentration is practically imperceptible (8, 15). GFP overexpression patterns from TYLCSV-infected 2IRGFP transgenic plants are consistent with these studies, since we detected GFP overexpression in the most apical leaves, in roots, and in flowers. However, hybridization studies cannot determine whether the viral molecules that accumulate in a particular plant organ or tissue have been produced there or whether they have replicated elsewhere in the plant and later transported there. In a plain step forward, our system allows us to easily identify the tissue where the virus is actively replicating. In contrast with the current notion that viral DNA accumulation in the roots is due to transport of viral DNA rather than to viral replication in situ (8, 15), the results obtained here indicate that active viral replication is indeed occurring at the root tissues, since we detect a conspicuous GFP overproduction in these tissues (Fig. 3). GFP expression in aerial parts of transgenic plants changed along the course of infection. Although the vein pattern was maintained in GFP-expressing leaves, only the most apical leaves displayed green fluorescence. We also observed that the intensity of the fluorescence decreased as the infection developed. These results suggest that TYLCSV is only able to replicate in a limited number of leaves, with the level of replication, reaching a peak at 3 wpi.
The extent of TYLCSV spread and the progress of the infection do not seem to be affected by the presence of the viral sequences integrated into the genome of the 2IRGFP plants, as no significant differences were observed in symptom development in viral DNA accumulation between either 2IRGFP or wild-type infected plants (data not shown).
The C4 ORF completely overlaps with the Rep ORF, so we could not completely rule out from our experiments the possibility that C4 played a role in virus-dependent GFP expression. Although it is unlikely that C4 would be involved in the generation of mGFP replicons and induction of GFP expression, it is possible that this protein and/or TrAP would be responsible for the maintenance of green fluorescence in the veins when GFP expression was suppressed in the surrounding plant tissues. Homologs of both proteins from several begomovirus have been shown to suppress RNA silencing. Hence, although Rep is responsible for generating the mGFP replicon and for raising GFP expression, the presence of TrAP and/or C4 would be required to maintain the green fluorescence blocking the RNA silencing signal that affects other noninfected plant tissues. Thus, the appearance of green fluorescence will mark the tissues where the Rep is expressed and the virus starts its replication, while GFP expression will vanish if Rep or the gene-silencing suppressors C4 and/or TrAP are not present. Future experiments to determine the biological consequences of TrAP and/or C4 inactivation will address this hypothesis.
The information generated by high-throughput techniques for gene discovery and expression analysis could be combined with posttranscriptional gene silencing approaches to determine gene function on a genome-wide scale. Posttranscriptional gene silencing, and particularly VIGS systems, can be used as tools to speed up studies of gene function by reverse genetic analysis. VIGS technology has already been used for function analysis of defense-related genes; however, phenotype evaluation remains one of the major constraints to the use of VIGS to identify host genes involved in viral infections. Traditionally, the level of geminivirus infection has been determined by evaluating symptom development and quantifying viral DNA accumulation by nucleic acid hybridization. Both methods present a substantial inconvenience when used in large-scale VIGS analyses. The use of recombinant viruses containing a reporter gene is a better alternative for evaluating whether gene silencing of a target gene interferes with viral infection. Many RNA viruses have already been labeled with reporter genes such as the GFP gene (7, 14). However, few recombinant geminiviruses tagged with reporter genes have been described, and GFP targeting has only been successful with Bean dwarf mosaic virus (37, 59). There are many limiting features that hinder the tagging of geminiviruses. DNA viruses suffer a stronger genome packaging size limitation than RNA viruses (22, 26). Furthermore, in monopartite begomoviruses such as TYLCSV, all six ORFs are essential for efficient replication and long-distance movement within the plant.
The episomal amplification GFP system described in this paper overcomes most of these problems, as follows. (i) Viral Rep recognition of the transgene IR produces an easily detectable phenotype by GFP overexpression. (ii) The system is not affected by the infection with the VIGS vector. (iii) All leaves from the assayed plants can be evaluated at the same time. (iv) The results are not hindered by the replication of the virus in nonsilenced tissues.
The experiments achieving PCNA silencing demonstrate that our system is suitable for easily determining the effect of the suppression of a certain host gene in viral replication or movement in combination with VIGS, and they open the possibility of performing a wide screen to identify plant proteins required for viral infection with a cDNA-VIGS library.
ACKNOWLEDGMENTS
We are grateful to Javier Ruiz Albert, Enrique Viguera, and Ana Grande for critical reading the manuscript and making many helpful suggestions. We thank L. Cruzado for excellent technical assistance, E. Moriones for providing us with the TYLCV-[ES72/97] clone, and the Sainsbury Laboratory, John Innes Centre, for providing us with the TRV vectors.
This research was supported by a grant from the Spanish Ministerio de Ciencia y Tecnología (AGF98-0439-C05-05). G.M. was awarded a predoctoral fellowship from FIAPA, and A.G.C. was awarded a predoctoral fellowship from the Spanish Ministerio de Educación y Cultura.
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Biologisches Institut, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universitt Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany
ABSTRACT
A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.
INTRODUCTION
Geminiviruses belong to a large family of plant viruses with circular, single-stranded DNA (ssDNA) genomes packaged within geminate particles (9). The Geminiviridae family (58) is divided into four genera according to their genome organization and biological properties. The genus Begomovirus includes members that are transmitted by whiteflies, infect only dicotyledonous plants, and may have bipartite (A and B components), like Tomato golden mosaic virus (TGMV), or monopartite genomes, like Tomato yellow leaf curl Sardinia virus (TYLCSV) or Tomato yellow leaf curl virus (TYLCV).
Although among plant virus a minority, geminiviruses have a great economical impact, affecting many different crops worldwide (42). TYLCSV and TYLCV are two distinct species causing tomato yellow leaf curl disease, one of the most important threats to tomato crops in many tropical and subtropical regions of the world (45). Both viruses possess genomes of about 2.7 kb in size, which encode at least six proteins and contain an intergenic region (IR) that comprises the origin of replication and viral promoters. The open reading frames (ORFs) in the complementary sense orientation encoding a replication-associated protein (Rep, also known as AC1, AL1, C1, or L1), a transcription activator protein (TrAP, also known as AC2, AL2, C2, or L2), and a replication enhancer protein (REn) partially overlap; a small ORF, C4, is located within the Rep ORF but in a different reading frame (25). Rep and REn are required for efficient viral DNA replication, although only Rep is essential. Rep is a multifunctional protein that initiates and terminates plus-strand replication and specifically binds during origin recognition to a DNA sequence motive located in the IR (2, 10, 21, 29, 35, 50; for a review, see reference 25). TrAP and the C4 protein (also known as AC4, AL4, or L4), described as pathogenicity factors playing an important role in the infection process, are able to reverse RNA silencing in plants and to suppress local silencing in transient assays (62-64, 66).
Geminiviruses do not encode their own DNA polymerases and rely on the nuclear DNA replication machinery, like many mammalian DNA tumor viruses do. They replicate in nuclei of mature cells, which are inactive in DNA replication. An early step in geminivirus infection is therefore the induction of host DNA replication enzymes (23-25). Correspondingly, proliferating cell nuclear antigen (PCNA) was found to be expressed in differentiated cells of TGMV-infected plants and also in transgenic plants expressing TGMV Rep (46). TGMV Rep binds to a plant homolog of the animal cell cycle regulator, retinoblastoma protein (pRb) (1, 34). By analogy to mammalian DNA viruses, the interaction between Rep and pRb may bypass a pRb phosphorylation requirement for the G1/S transition and S-phase progression during geminivirus infection (41, 57). Several interactions between geminivirus Rep and plant cellular proteins have been identified by biochemical (affinity chromatography) or genetic (yeast two-hybrid system) assays. It has been demonstrated that Rep interacts with a serine/threonine protein kinase; a kinesin; the histone H3 (33); geminivirus Rep A-binding protein 1 (GRAB1) and GRAB2, two members of the NAC family (70); and with the SUMO-conjugating enzyme NbSCE1 (12). Additionally, Rep interactions with some elements of the cellular replication machinery have also been reported, such as the replication factor C complex from wheat (TmRFC-1) (39) and PCNA from tomato and tobacco (6, 11).
Geminiviruses, with their simple genome organization, broad host range, high copy number, and generation of infective clones, have many advantages as recombinant virus-based gene amplification systems in infected transgenic plants (60). Extrachromosomal amplification from geminivirus-based constructs has been exploited for the production of valuable peptides and proteins (28, 43, 51) or to analyze the function of Rep in replication (30).
Here, we present a TYLCSV-based green fluorescent protein (GFP) amplification system to identify the plant organs or tissues where viruses replicate and to follow virus replication within plants in real-time and without destruction.
We also demonstrate that this new tool, in combination with virus-induced gene silencing (VIGS) technology, could be an attractive instrument in functional genomics to easily identify host proteins required for geminivirus infection. We present an example of the system's feasibility using VIGS to suppress the expression of PCNA, a plant protein known to be required for viral DNA replication.
MATERIALS AND METHODS
Microorganisms, plants, and general methods. Virus strains used in this work are tomato yellow leaf curl Sardinia virus strain TYLCSV-ES[2] (GenBank accession no. L27708) and tomato yellow leaf curl virus strain TYLCV-[ES72/97] (GenBank accession no. AF071228).
Escherichia coli strain DH5- was used for subcloning. Southern gel blots from plants were performed as previously described (12). Agrobacterium tumefaciens was grown and selected with the appropriate antibiotics as described previously (17). Recombinant binary plasmids were introduced into A. tumefaciens LBA4404 by electroporation (65). A. tumefaciens LBA4404 was transformed with the constructs pGTYA2, pGTYOMGFP, pBINGFP, and pCACS1; transformants were selected with the appropriate antibiotics. DNA blot analysis was performed to check construct integrity before using constructs in transgenic plants.
Nicotiana benthamiana Domin was grown in an insect-free greenhouse with supplementary lighting. Manipulations of Escherichia coli and nucleic acids were performed by standard methods (56).
Plasmids and cloning. The first step in producing p2IR-GFP was to produce pTYO30, a plasmid that contains a tandem repeat of a TYLCSV IR fragment, positions 2441 thorough 2779, to nucleotide 158 in pBSKII+ (Strategene). A 945-bp DNA fragment corresponding to nucleotides 2441 to 606 of TYLCSV was amplified by PCR from pTYA50 (a plasmid containing an EcoRI monomer of TYLCSV) (J. Reina and E. R. Bejarano, unpublished data) using primers UIB-1 (5'-ATATGGAGATGAGGTTSCCC-3') and LCP-1 (5'-CAGAACCCCTAGTTACATCAC-3'). This PCR product was digested with SacII; the resulting 804-bp fragment, corresponding to a single copy of IR, was blunt-end ligated into EcoRV site pBSKII+, thus generating pTYO11. To obtain a tandem repeat, pTYO11 was digested with BamHI, and the fragment was blunt ended and ligated into the HincII site of pBSKII+ to yield pTYO29. Next, another BamHI fragment from pTYO11 was cloned into the BamHI site of pTYO29, yielding pTYO30.
pSMGFP (16), provided by the Arabidopsis Information Resource, was digested with XbaI-SacI to remove the GFP gene. A synthesized double-stranded DNA (dsDNA) fragment containing an HpaI site (5'-CTAGGTTAACCCCCGGGAGATCTCAGCT-3' and 5'-CAATTGGGGGCCCTCTAGAAG-3) was cloned into the XbaI-SacI sites from the previous plasmid to yield pSN1. pSN1 contained a unique HpaI site between the 35S cauliflower mosaic virus (CaMV) promoter (49) and the nopaline synthase (NOS) terminator. A HindIII-EcoRI fragment from pSN1 (including the 35S promoter-HpaI-NOS terminator fragment) was blunt ended and ligated into EcoRV of pTYO30 to yield pTYOM.
pGTYOM was made in two steps. First, pGA482-H was constructed by digestion of pGA482 with HpaI and BglI, blunt ended, and religated to eliminate the HpaI site. Secondly, a SacI-BglII fragment from pTYOM (which contains the 35S promoter and the NOS terminator between a tandem of TYLCSV IR) was blunt ended and subcloned into the blunt-end SacI site of pGA482-H to yield pGTYOM. The GFP gene was obtained by digestion of pSMGFP with SacI and BamHI, blunt ended, and ligated into the unique HpaI site of pGTYOM to yield p2IR-GFP.
pBINGFP was obtained by cloning a HindIII-EcoRI fragment from pSMGFP into HindIII-EcoRI sites from pBINPLUS (61).
Plasmid pACS1 was derived from the A. tumefaciens binary vector pGA482 (4) carrying the TYLCSV Rep gene in the sense orientation, under the regulation of the 35S CaMV promoter and the octapine synthase terminator. This plasmid was constructed as follows: plasmid pTYA55 (Reina and Bejarano, unpublished), carrying a complete TYLCSV genome, was digested with NcoI and KpnI to release a 1,154-bp DNA fragment including full-length Rep and C4 genes. This fragment was blunt-end ligated into the SmaI site of pBSKII+, thus generating pTYC1. The latter was digested with EcoRI and BamHI, releasing a fragment including the full-length Rep gene, which was then blunt-end ligated into the SmaI site of pJC2ENa (17), thus placing the Rep gene downstream of the 35S CaMV promoter and upstream of the octopine synthase terminator carried by pJC2Ena. As a result, a new plasmid (pJC1) was generated carrying Rep in the sense orientation. pJC1 was then digested with HindIII and XbaI, releasing a 2,746-bp DNA fragment, blunt ended, and ligated into the HpaI site of pGA482 to yield pACS1.
pTV00 and pBINTRA6 were kindly provided by the Sainsbury Laboratory and are described in reference 55. pTV-LePCNA132-255 contains a tomato PCNA partial clone (from amino acids 132 to 255) amplified by PCR from pCNATOM-87 (11) and ligated into the SmaI-ClaI sites of pTV00.
Plant transformation. N. benthamiana plants were grown in Murashige and Skoog (MS) medium (Sigma) supplemented with sucrose (30 g/liter). During plant transformation, MS medium was supplemented with 0.1 mg of -naphthalene acetic acid (Duchefa, N 0903)/liter and 0.95 mg of kinetin (6-furfurylaminopurine) (Sigma, K-0753)/liter. Antibiotic selection of transformed plants was achieved by supplementing the MS medium with 100 μg of kanamycin/ml; this concentration was reduced to 50 μg/ml for seed germination. Plants were grown in a controlled-environment growth chamber at 22 to 24°C with a 16-h photoperiod.
N. benthamiana transgenic plants were generated by transformation of leaf disks with A. tumefaciens carrying the corresponding binary plasmids (pTYOMGFP and pBINGFP), following a previously described protocol (31). For each plasmid, a number of independent transformants originated from separate calli were selected for their resistance to kanamycin. Plants were regenerated from these transformants, and a reduced number of regenerated plants were selected for further work. Seeds obtained by autofecundation of each of these selected transgenic plants were grown in kanamycin-supplemented MS medium to determine the number of independent loci where the 2IRGFP transgene was integrated. Total DNA extracted from transgenic plants (18) was used to analyze the integrity and the copy number of the integrated T-DNA by Southern blotting.
Geminivirus infection assays and detection of replicon DNA. Viral infections of N. benthamiana plants were performed by the agroinoculation technique as previously described (20). Plants were agroinoculated with plasmids pGTYA2 for TYLCSV and pGTYLES for TYLCV (binary vectors that contain a dimer of TYLCSV-ES[2] or TYLCV-Mld[ES72/97], respectively) (44, 47). For control, plants were mock inoculated with A. tumefaciens harboring the empty binary vector pGA482 or pBIN+.
Total plant DNA was extracted from N. benthamiana leaves at different days postinfection. For plant DNA gel blots, total DNA samples (2 μg to detect viral particles and 5 μg to detect replicon molecules) were used and hybridized as described previously.
For two-dimensional agarose gel electrophoresis, 1 g of plant material was processed as described previously (54) to purify total DNA using benzoylated-naphtholyated DEAE (BND)-cellulose chromatography. DNA amounts, each corresponding to 200 mg of the initial plant material, were applied to a single two-dimensional gel, Southern transferred, and hybridized against a GFP-specific probe covering the whole gene and, after the probe was stripped, against a TYLCSV-IR-specific probe.
PCR-amplified DNAs comprising the intergenic regions of TYLCSV-ES or TYLCVMld-[ES7297] were used to generated a virus-discriminating probe as previously described (44) For GFP, an XbaI-SacI fragment from the plasmid pSMGFP was used. For replicon molecules, a DNA fragment (HindIII-XbaI) comprising the 35S CaMV promoter was used.
Primers upperGFP (5'-GTGGCCGAGGATGTTTCCGTCCTCC-3) and lowerGFP (5'-AAGTTGGAATACAACTACAACTCCCAC-3') were used to PCR amplify a fragment of 2.3 kb of the episomal replicons using 1 μg of total DNA from N. benthamiana plants. PCR conditions were as follows: 95°C for 1 min; 25 cycles, each consisting of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 7 min at 72°C.
VIGS. VIGS with tobacco rattle virus (TRV) in N. benthamiana plants were performed as described in reference 55, and independent cultures of Agrobacterium carrying pTV00 (TVR RNA2), pBINTRA6 (TVR RNA1), pTV-LePCNA132-255, or pGTYA2 were grown overnight to saturation in LBroth plus appropriate antibiotics. Cultures were resuspended in VIGS buffer (10 mM morpholineethanesulfonic acid, 10 mM MgCl2, and 150 μM acetosyringone) and incubated at room temperature overnight. For infections, cultures containing pBINTRA6 and pTV00 (or pTV-LePCNA132-255) were mixed at a 1:1 ratio. A volume of this mixed culture was combined with the same volume of a culture carrying pGTYA2, and this mixture was used to infiltrate the underside of two leaves from a 3-week-old 2IRGFP N. benthamiana plant.
Photography. Geminivirus-infected plants were photographed by using a Nikon digital camera (Coolpix 4500). Close-up images were obtained with epifluorescense microscope Leica MZ FLIII. Images were processed using Adobe Photoshop.
RESULTS
Amplification of 2IRGFP trans replicons during TYLCSV infection. Transgenic plants (2IRGFP) were generated by transformation of Nicotiana benthamiana with p2IR-GPF, a construct containing a direct repeat of the IR of TYLCSV flanking the cauliflower mosaic virus 35S promoter, the green fluorescent protein open reading frame, and the NOS terminator sequence (Fig. 1A). For control, transgenic plants (BINGFP) lacking the IR repeats were generated in parallel (Fig. 1B). Six to eight independent plant lines for each construct (2IR-GFP and pBINGFP) were analyzed by Southern blot hybridization to confirm the integrity and the number of copies of the transgenes integrated into the plant genome (data not shown). Lines 2IRGFP-15 and BINGFP-3, containing a single copy of the p2IR-GFP or the pBINGFP construct, respectively, were selected for further characterization. All experiments shown were performed using T2 homozygous plants.
Plants from both transgenic lines developed typical systemic symptoms when infected with TYLCSV or TYLCV (data not shown). Additionally, DNA gel blots confirmed that a similar amount of viral DNA accumulated in leaves of transgenic and nontransgenic lines (Fig. 1C). However, when DNA extracted from infected and noninfected plants was hybridized with a 35S promoter-specific probe, an episomal DNA (hereafter referred to as the mGFP replicon) was only detected in 2IRGFP plants infected with TYLCSV (Fig. 1D). To confirm the presence of the expected circular mGFP replicon in these plants, DNA extracted 21 days postinfection (dpi) was digested with EcoRI, EcoRV, or both and analyzed by Southern blot hybridization using a GFP-specific probe. EcoRI or EcoRV (cutting twice and once within the mGFP replicon) produced a single 2.43-kb and 2.46-kb band, respectively (Fig. 1D, lanes EI and EV), whereas the double digestion lighted up one fragment of approximately 1.65 kb containing the complete GFP ORF, the NOS terminator sequence, a complete IR, and a small fragment of the 35S promoter (Fig. 1D, lanes EI and EV). The mGFP replicon was detected neither in transgenic BINGFP plants infected with TYLCSV nor in 2IRGFP plants infected with the heterologous TYLCV (Fig. 1D).
The restriction analysis indicated that trans replicons accumulated predominantly as open circular (oc) and covalently closed circular (ccc) dsDNA. The two bands observed in the undigested sample vanished when DNA was digested with EcoRI or EcoRV, yielding a single visible band (EcoRI digestion generates two fragments, but the smallest one, 25 bp long, cannot be detected). This was consistent with a linear dsDNA molecule of the mobilized version of the transgene containing the complete GFP expression cassette and a single copy of the IR. This conclusion was further confirmed by digestion of DNA from TYLCSV-infected 2IRGFP plants with other restriction enzymes and probing with GFP-, 35S-, or IR-specific probes (data not shown).
Our data are consistent with a specific generation of circular episomal trans replicons from the transgene during viral infection, as a consequence of the interaction between Rep and one of its cognate origin of replication, followed by nicking and ligation of the displaced nascent ssDNA at the second origin after replication of the sequence (25). A circular episomal replicon was produced that WAs subsequently amplified. Accordingly, an isolate of TYLCV (TYLCV-Mld[ES72/97]) was unable to mobilize the TYLCSV-derived trans replicon when infecting the 2IRGFP plants (Fig. 1C and 1D), probably because TYLCV Rep does not recognize TYLCSV IR-binding motifs.
During the initial experiments, only low levels of dsDNA but no trans replicon-specific ssDNA was detectable. Since it was possible that monomeric circular DNA of the expression cassette may have arisen from recombination rather than replication, it was mandatory to identify the replicative intermediates in closer detail. DNA from 2IRGFP plants which had been infected with TYLCSV was purified by BND-cellulose chromatography, separated on two-dimensional agarose gels (a recently developed technique) (54), and hybridized after Southern blotting, first against a GFP-specific probe and subsequently against an IR-specific probe, which recognized trans replicon and viral replicon with the same specificity (Fig. 2A to D). BND-cellulose chromatography enriches dsDNA preferentially in the wash fraction (Fig. 2A and C) and ssDNA-containing DNAs in the eluate fraction (Fig. 2B and D), the single forms of which have been extensively described previously (54). From this separation, it is obvious that the earlier failure to detect ssDNA was determined by the detection limit of the technique. ssDNA of the trans replicon was present as discrete monomer, dimer, and trimer, as well as in the form of heterogeneous molecules, which are the best indication of ongoing ssDNA replication. Unfortunately, the concentration of the trans replicons was still too low to detect the replicative intermediates of complementary strand replication, rolling-circle replication, or recombination-dependent replication (RDR), which have been found in all geminiviruses analyzed to date (3, 32, 54) and which were obvious if the blots were hybridized against a TYLCSV-IR-specific probe (Fig. 2D). Only a faint arc, classified as RDR in the results shown in Fig. 2B, could be assigned at the original blot but was too low in intensity to be properly reproduced. Nevertheless, the intermediates shown so far strengthen the conclusion that the trans replicons have undergone replication and not only recombination. The small relative amount of trans replicons compared to that of viral replicons may be imagined from the gel shown in Fig. 2C, where viral cccDNA and trans replicon cccDNA were detected side by side with the same probe.
Analysis of GFP expression in TYLCSV-infected 2IRGFP plants. Under long-wavelength UV light, uninfected 2IRGFP plants appeared slightly green, due to expression of GFP from the transgene (Fig. 3B). Green fluorescence was evenly distributed in the leaves, but this pattern changed dramatically when 2IRGFP plants were infected with TYLCSV (Fig. 3C). Three weeks postinfection (wpi), GFP fluorescence was concentrated at the leaf veins, and it was no longer detected on the leaf lamina. An increase in GFP expression was also noticeable in other TYLCSV-infected organs such as stems, roots, or flowers (Fig. 3G, K, and N, right) compared to organs of mock-inoculated plants (Fig. 3F, J, and N, left). GFP signal in roots and stems was also concentrated in transport tissues (Fig. 3I and M for TYLCSV-infected plants and 3H and L for mock-inoculated plants).
The changes observed in the GFP expression pattern of 2IRGFP plants were dependent on TYLCSV-specific infection, since they were not detected when plants were mock inoculated (Fig. 3B) or infected with TYLCV (Fig. 3D). Furthermore, the change in GFP expression produced by TYLCSV infection was dependent on the presence of the IR in the transgene, since GFP expression of transgenic BINGFP plants was not affected by infection (Fig. 3E).
To further characterize the changes in GFP expression in transgenic 2IRGFP plants, we carried out a time course experiment with infected plants. We agroinoculated 120 2IRGFP young plants with TYLCSV at the 3- to 4-leaf stage in four independent experiments and followed the expression of GFP in all leaves up to 6 wpi. Leaves were numbered according to the inoculation site, with the first leaf placed upon the agroinfection point considerd leaf + 1. As previously described (Fig. 3), we observed an increase of GFP expression at 21 dpi in the veins of the most apical leaves, together with a reduction of expression in the leaf lamina. Interestingly, GFP expression on the leaves placed just above the inoculation point had completely disappeared. Figure 4A shows representative photographs of each kind of leaves described, and a diagram that summarizes the standard expression patterns of all T2 plants infected with TYLCSV. GFP expression disappeared from leaf + 1 to leaf + 7, while the vein pattern associated with GFP expression appeared in the most apical leaves (leaf + 8 to leaf + 12). The intensity and spread of the vein expression was stronger in younger leaves (Fig. 4, leaf + 9 versus leaf + 11 or + 12). Six weeks postinfection, plants displayed a similar expression pattern to that detected at 3 wpi. A vein pattern was only noticeable in apical leaves, while GFP expression had entirely disappeared in all other leaves, even those where the GFP expression had been very high 3 weeks before (e.g., leaf + 12). To confirm that the increase of GFP expression is associated with the generation of the episomal replicon, we analyzed the accumulation of viral and replicon molecules in leaves displaying a similar pattern to leaf + 12 from Fig. 4A by Southern blotting. As shown in Fig. 4B, GFP expression correlated with the presence of episomal molecules. Leaves expressing GFP 3 wpi (lanes W3) contained replicons, while 3 weeks later (lanes W6), when the expression of GFP had vanished, the amount of these molecules was considerably reduced.
When viral DNA accumulation was analyzed further (Fig. 4C), we found a significant reduction in the amount of ssDNA and dsDNA accumulated in leaves collected at 3 wpi, compared to those collected 3 weeks later (6 wpi). This reduction was much higher for the dsDNA than for the ssDNA forms of the viral genome. Quantification of viral DNA forms showed that a leaf + 12 at 6 wpi accumulated approximately 70% of the ssDNA form presented at 3 wpi, but only 15% of the dsDNA. Considering that dsDNA represents replicative intermediates of the viral genome, we can conclude that a decrease in GFP expression, produced between 3 and 6 wpi, correlates with a reduction in virus replication.
2IRGFP replicon generation and GFP overexpression are both Rep dependent. To investigate whether the presence of Rep from TYLCSV was sufficient to mobilize and amplify the mGFP replicon in 2IRGFP plants, even in the absence of the other viral protein involved in replication (REn), we agroinfiltrated leaves with a construct expressing Rep, and concomitantly C4, under the control of the 35S CaMV promoter (pACS1). Four days later, a large increase in GFP fluorescence was detected on all infiltrated leaves (Fig. 5A, panel pACS1). Similar results were obtained when leaves were agroinfiltrated with an infectious clone of TYLCSV (Fig. 5A, panel TYLCSV). The large increase in fluorescence detected in TYLCSV-infiltrated leaves was, however, much lower than that detected in pACS1-infiltrated leaves, most likely because the levels of Rep protein are higher when Rep is expressed from the 35S promoter rather than from its own viral promoter. No increase in green fluorescence was detected when leaves were agroinfiltrated with the empty binary plasmid or with an infectious clone of TYLCV (Fig. 5A, panels pGA482 and TYLCV). More than 20 leaves from several 2IRGFP plants were agroinfiltrated with each construct, and similar results were obtained in all the experiments.
The increase of GFP expression in agroinfiltrated leaves correlated with the production of mGFP replicons in transgenic plants. We detected the accumulation of replicons by PCR (Fig. 5B) and by Southern blot analysis (Fig. 5C) in leaves agroinfiltrated with pACS1 or with TYLCSV but not in those inoculated with TYLCV or the binary vector pGA482. Furthermore, replicon levels in TYLCSV-infiltrated leaves were significantly lower than those in pACS1-infiltrated ones. Taken together, these results indicate that TYLCSV Rep protein is necessary to mobilize and amplify the mGFP replicon from the transgene present in 2IRGFP plants.
Gene silencing of PCNA prevents GFP induction and TYLCSV replication. Our data suggested that the distribution of the green fluorescence in infected 2IRGFP transgenic plants highlights the plant organs or tissues where the virus has expressed Rep and is therefore actively replicating its DNA. So, we consider whether these transgenic plants could be used to identify plant genes required for TYLCSV replication. If a cell function necessary for virus replication was silenced in TYLCSV-infected transgenic plants, we could readily detect it by illuminating the plants with UV light, as a green fluorescent vein pattern would not be produced. Silencing of host function could be obtained by dsRNA-mediated suppression of genes through the production of sense or antisense transcripts or even more efficiently by using single-stranded self-complementary (hairpin) RNA containing an intron (13, 36, 68, 69). However, both strategies rely on the generation of transgenic plants, which is a time-consuming task. Alternatively, temporal silencing of the genes could be obtained by infecting plants with viral vectors carrying host-derived sequences. This technology, VIGS, has been widely used to analyze gene functions, particularly for genes involved in defense against pathogens (for a review, see reference 38; see reference 52 for an example).
Hence, we assessed our system by silencing by VIGS a host gene already known to be required for begomovirus replication, the PCNA gene. PCNA is an essential, ubiquitous, and highly conserved protein in eukaryotes that functions as a DNA sliding clamp required for DNA replication and repair (67). Previous studies demonstrated that systemic begomovirus infection was significantly reduced when PCNA was silenced by geminivirus-derived vectors (53). We silenced N. benthamiana PCNA (NbPCNA) using a TRV vector designed to induce VIGS (55). TRV was selected because it offers advantages over other viral systems, as it does not induce symptoms and it is able to target host RNAs in the tissue growth area of plants, where it is believed that geminiviruses replicate. A 369-nucleotide fragment from tomato PCNA (LePCNA132-255) encoding amino acids 132 to 255 was cloned into VIGS vector pTV00. We selected this fragment of tomato PCNA previously isolated in our laboratory (11) because its nucleotide sequence is almost identical to that of cloned NbPCNA (90.3% identity with fragments of up to 35 identical nucleotides in a row) (19). It has been proposed that VIGS can target any gene with just over 20 nucleotides perfectly matched to the insert cloned in the VIGS vector (38). Thus, we expected that expression levels of NbPCNA would be reduced after LePCNA overexpression.
Transgenic 2IRGFP plants were agroinfected with TRV RNA1 and either TRV RNA2 (gene-silencing negative control) or RNA2-LePCNA132-255 (TRV RNA2 with an LePCNA132-255 fragment). Immediately afterward, all plants were also infected with TYLCSV (different time lapses between TRV and TYLCSV infections were assayed previously to select this timing). Ten days after infection, a phenotype, similar to that previously described for PCNA silencing in N. benthamiana (53), was displayed by plants infected with RNA2-LePCNA132-255. Primary growth was interrupted at the apical meristem, and new leaves showed progressively reduced expansion. This PCNA-suppressed phenotype was obvious at 21 dpi (Fig. 6A, left, TVR-PCNA). At that time control plants inoculated with TRV vector and TYLCSV exhibited the typical symptoms produced by begomovirus infection (Fig. 6A, left panel, TRV) and displayed the same GFP expression pattern that 2IRGFP plants infected with TYLCSV alone (Fig. 6A, right, TVR; Fig. 3C). Although transgenic plants infected with RNA2-LePCNA132-255 seemed to develop fewer symptoms of TYLCSV infection than control plants, the PCNA-silenced phenotype displayed by these plants somehow obscured the analysis of this difference. However, a difference was clearly noticed when the plants were illuminated with UV, since GFP vein pattern fluorescence was almost undetectable on most leaves of PCNA-silenced plants located above the inoculation point; only isolated dots of green fluorescence were observed (Fig. 6A, right, TRV-PCNA). The absence of fluorescence in these plants indicated that TYLCSV replication was impaired when the level of PCNA in the cell was reduced. When quantified by Southern blotting, TYLCSV DNA accumulation showed a significant decrease in leaves displaying the PCNA-silenced phenotype compared to leaves from plants infected with the TRV RNA2 vector (Fig. 6B). The reduction of viral DNA accumulation was stronger for dsDNA than for ssDNA molecules. PCNA-silenced leaves accumulated approximately 50% of TYLCSV ssDNA compared to leaves from control plants, but only 25% of dsDNA (average of three Southern blot analyses of different PCNA-silenced leaves). These results confirm that viral TYLCSV infection was reduced although not completely suppressed. This was not an unexpected result, as silencing of an endogenous gene by VIGS is not uniform and it does not occur in the whole plant.
DISCUSSION
We have designed a construct comprising those cis elements essential for TYLCSV replication to allow Rep-dependent release of the replicon and subsequent episomal replication. Although episomes induced by geminivirus infection have already been described for several systems, including mastreviruses (5, 48, 51) and bipartite begomoviruses (26, 27, 30, 40, 43), this is the first time that this strategy has been applied to monitoring monopartite begomovirus replication. Our construct comprises a GFP expression cassette cloned between a tandem repeat of a TYLCSV sequence, which includes the viral origin of replication, the viral promoters, and the 5' region of V2, CP, and Rep genes. Since GFP expression is under the control of the 35S CaMV promoter, constitutive expression of the reporter gene was expected to take place in transgenic plants carrying this construct. This was confirmed by the fact that most of the transgenic 2IRGFP lines obtained displayed green fluorescence in all tissues. However, we noticed that the level of GFP expression in 2IRGFP plants was lower than that displayed by transgenic plants carrying a GFP expression cassette without the viral IRs (BINGFP plants). It is possible that the IR viral sequence cloned upstream of the 35S promoter might interfere with its transcriptional efficiency through the binding of cellular factors to the TYLCV promoters present in the viral sequence.
The results obtained when 2IRGFP transgenic plants were infected with TYLCSV are consistent with a Rep-assisted generation of mGFP replicons from the 2IRGFP transgene, where Rep is provided in trans by the TYLCSV genome. This will lead to the overexpression of the GFP from the many replicon units thus generated, in those cells where Rep is being expressed by the virus.
The replicon was mobilized and amplified when Rep was expressed from either the TYLCSV genome or from a binary vector. As was reported for a similar system based in African cassava mosaic virus (30), the mGFP replicon accumulated at a level much lower than that of the TYLCSV genomes. Double- and single-stranded DNA forms of the replicon were detected, but in contrast to geminivirus genome where the ssDNA form is the major form of viral molecules, the amount of replicon ssDNA molecules was significantly smaller than that of dsDNA forms. This reduction or even absence of ssDNA accumulation has been also observed with other geminivirus replicon-based systems (43, 48, 51) and could mean either that the transition from the synthesis of a replicative form to the synthesis of ssDNA has not taken place in the episomal molecules or that ssDNA episomal molecules are degraded immediately after they are synthesized.
The results obtained when 2IRGFP plants were infected with TYLCSV suggest that GFP overproduction highlights plant tissues where the virus is replicating. In fact, the GFP expression pattern in infected 2IRGFP plants matches that obtained by in situ hybridization of N. benthamiana plants, showing that TYLCSV is restricted to the phloem (44). This hypothesis is also supported by the results obtained by Southern blot analysis of leaves that express GFP and of leaves where GFP expression has vanished (Fig. 5C). Although the loss of GFP expression in the leaves was associated with a reduction in the accumulation of both ssDNA and dsDNA viral forms, the relative decrease of dsDNA was manifestly larger than that of ssDNA. Geminiviruses causing tomato yellow leaf curl disease are important pathogens that have been the subject of numerous investigations. Despite an extensive amount of work, very little is known of the development of the infection and the spread of the virus within infected plants. Hybridization techniques used to study the accumulation of viral DNA in the plant have shown that the virus spreads following the flow of photoassimilates, which is somehow expected, since both move along phloematic tissues. They are first transported to the roots and the shoot apex and then to the neighboring leaves and flowers. In older leaves, the virus concentration is practically imperceptible (8, 15). GFP overexpression patterns from TYLCSV-infected 2IRGFP transgenic plants are consistent with these studies, since we detected GFP overexpression in the most apical leaves, in roots, and in flowers. However, hybridization studies cannot determine whether the viral molecules that accumulate in a particular plant organ or tissue have been produced there or whether they have replicated elsewhere in the plant and later transported there. In a plain step forward, our system allows us to easily identify the tissue where the virus is actively replicating. In contrast with the current notion that viral DNA accumulation in the roots is due to transport of viral DNA rather than to viral replication in situ (8, 15), the results obtained here indicate that active viral replication is indeed occurring at the root tissues, since we detect a conspicuous GFP overproduction in these tissues (Fig. 3). GFP expression in aerial parts of transgenic plants changed along the course of infection. Although the vein pattern was maintained in GFP-expressing leaves, only the most apical leaves displayed green fluorescence. We also observed that the intensity of the fluorescence decreased as the infection developed. These results suggest that TYLCSV is only able to replicate in a limited number of leaves, with the level of replication, reaching a peak at 3 wpi.
The extent of TYLCSV spread and the progress of the infection do not seem to be affected by the presence of the viral sequences integrated into the genome of the 2IRGFP plants, as no significant differences were observed in symptom development in viral DNA accumulation between either 2IRGFP or wild-type infected plants (data not shown).
The C4 ORF completely overlaps with the Rep ORF, so we could not completely rule out from our experiments the possibility that C4 played a role in virus-dependent GFP expression. Although it is unlikely that C4 would be involved in the generation of mGFP replicons and induction of GFP expression, it is possible that this protein and/or TrAP would be responsible for the maintenance of green fluorescence in the veins when GFP expression was suppressed in the surrounding plant tissues. Homologs of both proteins from several begomovirus have been shown to suppress RNA silencing. Hence, although Rep is responsible for generating the mGFP replicon and for raising GFP expression, the presence of TrAP and/or C4 would be required to maintain the green fluorescence blocking the RNA silencing signal that affects other noninfected plant tissues. Thus, the appearance of green fluorescence will mark the tissues where the Rep is expressed and the virus starts its replication, while GFP expression will vanish if Rep or the gene-silencing suppressors C4 and/or TrAP are not present. Future experiments to determine the biological consequences of TrAP and/or C4 inactivation will address this hypothesis.
The information generated by high-throughput techniques for gene discovery and expression analysis could be combined with posttranscriptional gene silencing approaches to determine gene function on a genome-wide scale. Posttranscriptional gene silencing, and particularly VIGS systems, can be used as tools to speed up studies of gene function by reverse genetic analysis. VIGS technology has already been used for function analysis of defense-related genes; however, phenotype evaluation remains one of the major constraints to the use of VIGS to identify host genes involved in viral infections. Traditionally, the level of geminivirus infection has been determined by evaluating symptom development and quantifying viral DNA accumulation by nucleic acid hybridization. Both methods present a substantial inconvenience when used in large-scale VIGS analyses. The use of recombinant viruses containing a reporter gene is a better alternative for evaluating whether gene silencing of a target gene interferes with viral infection. Many RNA viruses have already been labeled with reporter genes such as the GFP gene (7, 14). However, few recombinant geminiviruses tagged with reporter genes have been described, and GFP targeting has only been successful with Bean dwarf mosaic virus (37, 59). There are many limiting features that hinder the tagging of geminiviruses. DNA viruses suffer a stronger genome packaging size limitation than RNA viruses (22, 26). Furthermore, in monopartite begomoviruses such as TYLCSV, all six ORFs are essential for efficient replication and long-distance movement within the plant.
The episomal amplification GFP system described in this paper overcomes most of these problems, as follows. (i) Viral Rep recognition of the transgene IR produces an easily detectable phenotype by GFP overexpression. (ii) The system is not affected by the infection with the VIGS vector. (iii) All leaves from the assayed plants can be evaluated at the same time. (iv) The results are not hindered by the replication of the virus in nonsilenced tissues.
The experiments achieving PCNA silencing demonstrate that our system is suitable for easily determining the effect of the suppression of a certain host gene in viral replication or movement in combination with VIGS, and they open the possibility of performing a wide screen to identify plant proteins required for viral infection with a cDNA-VIGS library.
ACKNOWLEDGMENTS
We are grateful to Javier Ruiz Albert, Enrique Viguera, and Ana Grande for critical reading the manuscript and making many helpful suggestions. We thank L. Cruzado for excellent technical assistance, E. Moriones for providing us with the TYLCV-[ES72/97] clone, and the Sainsbury Laboratory, John Innes Centre, for providing us with the TRV vectors.
This research was supported by a grant from the Spanish Ministerio de Ciencia y Tecnología (AGF98-0439-C05-05). G.M. was awarded a predoctoral fellowship from FIAPA, and A.G.C. was awarded a predoctoral fellowship from the Spanish Ministerio de Educación y Cultura.
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