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Systemic Agrobacterium tumefaciens–mediated transfection of viral replicons for efficient transient expression in plantsSylvestre Marillonnet 1,2,Carola Thoeringer 1,2,Romy Kandzia 1,Victor Klimyuk 1&Yuri Gleba 1Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants:stable genetictransformation and transient expression using viral vectors.Although much faster,the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes.We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium–mediated delivery of these constructs results in gene amplification in all mature leaves of a plant simultaneously (systemic transfection).This process,called ‘magnifection’,can be performed on a large scale and with different plant species.This technology combines advantages of three biological systems (the transfection efficiency of A.tumefaciens ,the high expression yield obtained with viral vectors,and the post-translational capabilities of a plant),does not require genetic modification of plants and is faster than other existing methods.Viral vectors designed for expression of recombinant proteins in plants hold great promise because of high absolute and relative yields,and because of the speed provided by transient expression.Most of the results of practical interest achieved so far have been obtained with vectors built on the backbones of plus-sense RNA viruses such as tobacco mosaic virus (TMV)or potato virus X 1–4.We have recently shown that TMV-based vectors can be delivered to plant tissues using A.tumefaciens 5(agroinfection).However,one step of this process,namely the formation of active replicons from the primary nuclear transcript,is inefficient.In a standard leaf transfec-tion experiment,this inefficiency is masked by the subsequent ability of the replicons to move to neighboring cells by cell-to-cell movement.Here we show that this bottleneck can be fully remedied by incorpora-tion of silent nucleotide substitutions into the vector and by addition of multiple introns.We demonstrate that such modifications provide for efficient processing of the DNA information into active replicons in almost all cells (as high as 94%)of Nicotiana benthamiana ,an up to 1,000-fold improvement over nonoptimized TMV-based vectors,and an even higher improvement (4106-fold)in Nicotiana tabacum (tobacco).Finally,we show that the resulting vectors allow the development of a fully scalable and versatile whole-plant transfection protocol,that we term magnifection,for production of heterologous proteins in plants.RESULTSViral replication following agroinfiltration of TMV-based vectors Agroinfiltration of a TMV-based viral vector containing the gene encoding green fluorescent protein (GFP)(pICH16707,Fig.1a )into N.benthamiana leaves leads to the formation of foci of GFPfluorescence 3d post-infiltration (d.p.i.)(shown in ref.5and in Supplementary Fig.1online).T o quantify the proportion of cells initiating viral replication,a 489-bp deletion was made within the movement protein (MP)coding sequence,resulting in construct pICH14833(Fig.1a ).Replicons derived from this construct cannot move from cell-to-cell but are able to replicate autonomously within each infected cell.Three days after agroinfiltration of pICH14833in N.benthamiana leaf (OD 600of the A.tumefaciens in infiltration solution was 0.7),a small number of cells expressing GFP appeared (see Supplementary Fig.1online),and the same pattern was still visible 2weeks after infiltration.By counting protoplasts prepared from the infiltrated area (Figs.1and 2),we found that 0.6–1.6%of cells initiated viral replication.There are several reasons why RNA viral vectors might have difficulties starting the replication cycle.First,RNA viruses,such as TMV ,replicate in the cytoplasm and never enter the nucleus,and have therefore evolved in an environment where they are not exposed to the nuclear pre-mRNA processing machinery.As a result,pre-mRNA transcripts made in the nucleus from viral constructs may not be re-cognized and processed properly.Second,viral vector constructs encode very large transcripts (B 7.6kb for the primary transcript of a viral vector containing a GFP gene),a size much larger than the average size (1–2kb)of plant genes.Moreover,in nature,large eukaryotic genes often contain numerous introns that facilitate processing and export of the pre-mRNAs from the nucleus 6.We therefore hypothesized that modifications of the constructs that would increase the efficiency of processing and export of primary transcripts from the nucleus to the cytoplasm could lead to an increase in the number of cells that would initiate viral replication.Two types of modifications were made:Published online 8May 2005;doi:10.1038/nbt10941IconGenetics,Biozentrum Halle,Weinbergweg 22,D-06120Halle (Saale),Germany.2These authors contributed equally to this work.Correspondence and requests for materials should be addressed to Y.G.(gleba@icongenetics.de).A R T I C L E S©2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g y(i)removal of sequence features that might be improperly recognized by the RNA processing machinery (such as cryptic splice sites and thymine-rich intron-like sequences),and (ii)addition of introns.Removal of putative intron-like featuresT o identify sequence features that might induce abnormal RNA processing events,we analyzed the sequence of pICH14833using the NetgeneII program (http://www.cbs.dtu.dk/services/NetGene2/(ref.7))with parameters set for Arabidopsis thaliana sequences.We noticed several intron-like sequence features consisting of putative cryptic splice sites and several thymine-rich sequences (see Supple-mentary Fig.2online).We first removed some of the putative cryptic splice sites by using PCR with primers designed to introduce silent nucleotide substitu-tions.However,the resulting constructs (pICH15011and pICH17266)were not significantly more efficient than the initial construct,pICH14833(Fig.1and Supplementary Table 1online).Then,we mutagenized a 0.6-kb thymine-rich region located at the beginning of the RdRp coding sequence by introducing 54silent nucleotide sub-stitutions (two substitutions being the two splice site mutations also present in pICH15011)to increase its GC content.The resulting clone,pICH15466,worked substantially better than the unmodified clone,with 13%of cells in the infiltrated area initiating viral replication (Fig.1)compared to 1.6%for pICH14833.Another potentially problematic region corresponds to a 220-bp thymine-rich sequence at the 3¢end of the RdRp coding sequence,a region that contains the MP subgenomic promoter (see Supplementary Fig.2online).Forty-three silent nucleotide substitutions were introduced in this area (leaving all other regions unmodified).With the resulting construct,pICH15900,53%of cells from the infiltrated area expressed GFP (Fig.2b ,Fig.2e infiltration 4).In a separate assay,we measured the amount of GFP fluorescence in the infiltrated area,and found an increase from 3.3fluorescence units (for pICH14833)to 53.3,a relative increase consistent with the increase in the number of GFP-expressing protoplasts.A construct similar to pICH15900but with no deletion in the MP ,pICH16989,gave similar rates of initiation of replication,indicating that the improvement was due to modification of the codon usage and not to the deletion in the MP (Fig.1).T o test whether these modifications had an effect on viral replica-tion,pICH15466and pICH15900were infiltrated into the leaves of transgenic N.benthamiana plants expressing MP (plants transformed with pICH10745).The size of the GFP-expressing foci and the intensity of GFP fluorescence appeared similar for both constructs and pICH14833(Fig.2f ),showing that modification of the RNA sequence did not significantly affect the replication or cell-to-cell movement abilities of the viral vector.pICH15025 pICH15034pICH16877pICH15488pICH15755pICH15477 pICH17200 pICH15922 pICH15499pICH16433 pICH14030 pICH15041pICH16100pICH16191pICH16200 pICH15860 pICH16141pICH17494 (pICH16707)pICH18535(pICH18000)pICH18722(pICH18711)pICH17466 (pICH16424)pICH17474(pICH17272)pICH18523(pICH17282) pICH15466 pICH15900pICH17266 pICH15011 pICH14833 pICH16989pICH5661(pICH15662)pICH17144pICH18000pICH17272abFigure 1Constructs maps and quantificationof the efficiency of initiation of replication.(a )Schematic representation of the constructs.The MP is shown as a gray box between the TVCV RNA-dependent RNA polymerase (RdRp)and the GFP coding sequence.Deletions of 489and 575nt are labeled d1and d2,respectively.Introns are shown as narrow white boxes,and the designation of the insertion sites (numbered 1–23,position given in the methods section)are indicated by a number above the introns.1a and 1b refer to the insertion of two different introns at the sameposition (position 1).Vertical black lines show the position of mutated putative splice sites.Two mutagenized regions containing 54and 43silent nucleotide substitutions are shown as a gray box underlined with a black line,and as a dotted box,respectively.A frameshift in the MP is shown as an X at the beginning of the MP .Construct numbers written in italics under construct names correspond to versions of the constructs not containing a frameshift in the MP .Act2,A.thaliana ACT2promoter;N,cr-TMV 3¢untranslated region;T,Nos terminator.(b )Quantification of the efficiency of initiation of replication of viral constructs measured by counting the proportion of protoplasts expressing GFP in infiltrated areas (gray columns,expressed as a percentage of all protoplasts)or bymeasuring GFP fluorescence with a luminescence spectrometer (black columns,expressed influorescence units,all values were multiplied by 0.7in order to be visualized on the same scale as the protoplast counts).Error bars indicate standard deviation.For protoplasts counts,two samples of 400–500protoplasts were counted from each protoplast preparation.For GFPfluorescence,values were determined from three samples taken from different infiltrated leaves.A R T I C L E S©2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g yAddition of intronsNext,introns were added at up to 19positions within the RdRp and the MP sequences.When introns were inserted into the MP coding sequence,the MP was made nonfunctional by a frameshift rather than by a deletion.The presence of most introns increased the efficiency of initiation of viral replication,although some introns were not as effi-cient as others (Fig.1b ).Insertion of additional introns usually further increased the efficiency of initiation of viral replication.For example,with pICH18535,which contains 12introns,92%(90–94%)of protoplasts from the infiltrated area expressed GFP .With the same construct (but with a functional MP),pICH18000,93%(90–96%)of protoplasts expressed GFP .This suggests that with constructs with more than 12introns,initiation of viral replication starts in virtually every N.benthamiana cell,at least when infiltration solution containing agrobacteria at an OD 600of 0.7is used.Also,at the protein level,expression of GFP was similar in 16-intron constructs with or without a functional MP (pICH18711-described below,and pICH18722,Fig.2g ).We also tested the effect of the presence of introns within the gene of interest.Four introns were inserted within the GFP coding sequence,resulting in construct pICH17144.This construct worked better than the same construct without introns,with 9.5%of cells in the infiltrated area expressing GFP .Viral replication of fully-optimized viral vectorsWe then tested the performance of pICH18711,a fully optimized construct containing most of the modifications described above (the first mutagenized region at the beginning of the RdRp and 16introns in the RdRp and MP coding sequences),but also containing a functional MP (Fig.1a ).A dilution series of A.tumefaciens in infiltration solution was infiltrated into N.benthamiana and tobacco leaves (Fig.3a ,b ).Whereas no GFP-expressing sector could be detected with the original construct in N.benthamiana at dilutions higher than 10À3,GFP-expressing foci were obtained until the 10À6dilution with pICH18711.Infiltration of a 10À4dilution for pICH18711gave a similar number of GFP-expressing foci as the 10À1dilution for the nonoptimized construct,pICH16707,indicatinga 1,000-fold increase in the efficiency of initiation of viral replication.The number of bacteria present in each dilution was estimated by plating an aliquot of the infiltration solution and counting the number of colonies for the 10À5and 10À6dilutions.This showed that approximately eight A.tumefaciens cells are required perGFP-expressing event in N.benthamiana ,whereas 5,700agrobacteria are required for the unmodified control construct.This represents a 712-fold improvement,in accordance with the 1,000-fold increase cited above.For tobacco,23agrobacteria were required per GFP-expressing foci.In contrast,a nonoptimized construct did not work properly in tobacco,even at high bacterial concentration:at the 10À1and 10À2dilutions,only a few individual cells in the infiltrated area expressed GFP ,but replicons were unable to move outside of these few initial cells.Optimized viral vectors were also tested in N.excelsior,and the same level of improvement was seen as in N.benthamiana .Characterization of the optimized viral vectorsWe replaced the relatively small GFP coding sequence (0.7kb)in pICH16707and pICH18711by the larger coding sequence (1.8kb)of the b -glucuronidase gene (GUS),resulting in constructs pICH18841(0introns)and pICH18851(16introns),respectively.With pICH18841,fewer replication foci were observed in infiltrated areas than when using a construct expressing GFP (not shown),suggesting that longer genes may negatively affect the frequency of initiation of viral replication.By using the intron-optimized viral vector pICH18851,the entire infiltrated area was expressing GUS.A time-course experiment was carried out to measure the time required to obtain maximal gene expression after infiltration with either GFP -or GUS -containing constructs (Fig.3c ,d ).A 10À1dilutionea b c d1234681112fu gFigure 2Performance of different synthetic vectors.(a –d )Protoplastsprepared from N.benthamiana leaves with pICH14833(a ),pICH15900(b ),pICH18722(c )and pICH18711(d ),photographed under blue light.Scale bar,100m m.(e )N.benthamiana leaves were infiltrated with pICH14833(1),pICH15011(2),pICH15466(3),pICH15900(4),pICH15477(5),pICH15034(6),pICH16433(7),pICH16141(8),pICH17466(9),pICH17144(10),pICH18722(11),pICH18711(12)and photographed under UV light at 7d.p.i.Infiltrations 13–15were similar to infiltrations 10–12except that pICH10745was coinfiltrated to provide transient MP expression.Infiltrations in a –e were performed with A.tumefaciens in infiltration solution at an OD 600of 0.7.(f )Leaf of a transgenic N.benthamiana plant (pICH10745)infiltrated with pICH14833(1),pICH15466(2)and pICH15900(3).A.tumefaciens infiltration solutions were diluted to between 10À3to 10À4relative to the overnight-grownA.tumefaciens culture in order to obtain separate GFP foci.(g )Coomassie-stained SDS protein gel loaded with crude extracts prepared fromN.benthamiana noninfiltrated leaf tissue (u)or from the infiltrated areas shown in e (numbering is as in panel e ).Molecular weights (kDa)are shown on the left.The arrow indicates GFP .A R T I C L E S©2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g yof the A.tumefaciens infiltration solution (OD 600¼0.35)was used for this experiment.For both GFP and GUS,levels of expression increased faster and reached higher levels with intron-containing constructs.As a control for transient expression using nonreplicating constructs,a time course was performed for GFP expressed from the CaMV 35S promoter (pICH5290)in the absence or presence of the suppressor of silencing p19to enhance expression 8(Fig.3c ).In both cases,GFP expression levels were lower than with viral vectors.Transfection of whole plants:‘magnifection’Since TMV-based vectors that lack a coat protein gene cannot move systemically,production in entire plants requires inoculating all leaves of a plant.However,this process is inefficient with unimproved viral vectors owing to low infectivity of the vectors.Having improved viral vectors in hand,we attempted to inoculate entire plants using a variety of treatments,including immersing or spraying entire plants,applying (or not)a vacuum treatment,infiltrating whole plants or detached leaves,wilting plants/leaves before treatments,using detergents and solubilizers.The best and most reliable results were obtained by simply immersing all aerial parts of an entire plant into a bacterial suspensionand applying a weak vacuum (0.5–1bar)for 1–2min,followed by a gentle (o 1min)gradual return to atmospheric pressure.This simple procedure,which is similar to a protocol commonly used for transientexpression in detached leaves 9,leads to infiltration of A.tumefaciens suspension into the intercellular space of all mature leaves of tobacco or N.benthamiana plants.The treated plants are then simply returned to the greenhouse (under standard conditions)where they fully recover.This procedure leads to GFP expression in all leaves,with the exception of the young nonexpanded leaves of the apex (Fig.4).The infiltration procedure was tested on plants and seedlings of different ages,ranging from 2weeks old to flowering age (6–8weeks depending on growth conditions).High levels of GFP expression were obtained in the mature leaves of all plants,but a higher ratio of expressing to nonexpressing tissue was obtained for larger plants (43weeks old).High levels of GFP expression were also obtained with plants infiltrated at flowering stage,although at later stages,older leaves showed reduced expression.We also tested bacterial suspensions that were diluted 10À1to 10À6relative to a saturated overnight bacterial culture (OD 600of the 10À1dilution was 0.35or B 1.8Â108colony forming units per ml).Infiltrations were performed with N.benthamiana and N.tabacum plants.For both species,the 10À3dilution provided the highest yield,indicating that the 10À1and 10À2dilutions are somewhat inhibitory or toxic to plant cells (Fig.5).For the 10À3or lower dilutions,development of the infection was delayed,indicating that the primary infection by agrobacteria occurred in a minority of cells only,and thatc d.p.i.d.p.i.Figure 3Efficiency of ‘agrodelivery’and of gene expression of the final,fully optimized construct.(a )Tobacco leaf infiltrated with dilution series ofA.tumefaciens in infiltration solution for constructs pICH16707(lower half of the leaf)and pICH18711(upper half).Dilutions labeled –1to –6correspond to 10À1to 10À6dilutions of the A.tumefaciens relative to the starting overnight culture (OD 600of the 10À1dilution was 0.35).The picture was taken under UV light at 9d.p.i.(b )Same as in a but in N.benthamiana.The picture was taken at 5d.p.i.(c )Time course showing the level of GFP fluorescence in N.benthamiana leaf from 2or 3to 10d.p.i.All samples from individual curves were harvested from the same leaf.Leaves A and B were infiltrated with pICH18711,leaves C and D with pICH16707,leaf E with pICH5290(35S-GFP )and leaf F with pICH5290+pICH6692(35S-p19);A.tumefaciens concentration was at 0.35under OD 600for all infiltrations.f.u.,fluorescence units.(d )As in c but with GUS fluorescence by infiltration of pICH18851(16introns)in leaves A and B and pICH18841(0intron)in leaves C and D.Figure 4Expression of GFP in N.benthamiana plants and Beta vulgaris .Whole plants were vacuum-infiltrated with pICH18711and viewed under UV light.(a –d )N.benthamiana plants 4d after infiltration with infiltration solution containing agrobacteria diluted 10À1(d ),10À2(c ),10À3(b )or 10À4(a )relative to the overnight saturated A.tumefaciens culture (OD 600¼0.35of the 10À1dilution).(e )N.benthamiana plants of various ages (17–35d after sowing)wereinfiltrated with a 10À1diluted infiltration solution and photographed 4d later.(f )same plant as shown in e )(infiltrated 28d after sowing),but pictured 6d.p.i.(g ,h )Beta vulgaris var.conditiva vacuum-infiltrated with pICH18711(A.tumefaciens OD 600¼0.35),photographed under normal (g )or UV light (h )10d.p.i.A R T I C L E S©2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g y(virally controlled)cell-to-cell spread of the replicons was then required to complete transfection of all leaf tissue.The absolute protein yield seen in our experiments was as high as 4g of recombinant protein per kg of fresh leaf biomass in N.benthamiana and up to 2.5g/kg in tobacco (N.tabacum),and the relative yield as high as 25%and 40%of total soluble protein in both species,respectively (Fig.5).Assuming such protein yield,and based on realistic yields of 100tons of plant leaf biomass per hectare of a greenhouse per year,a 1ha facility should be capable of producing 280–400kg of recombinant protein per year.Our measurements show that infiltration consumes 1–1.5liters of bacterial suspension per kg of plant leaf biomass,indicating that,at the optimal 10À3dilution,1liter of overnight A.tumefaciens culture is sufficient to treat 700–1,000kg of N.benthamiana biomass and to produce 2.5–4.0kg of recombinant protein.Analysis of magnifection in different plant speciesWhole-plant infiltration was tested on over 50dicotyledonous plant species belonging to eight plant families.Good expression was found in seven species (petunia,cucumber,sunflower,red beets,spinach,Chenopodium capitatum and Tetragonia expansa ),representing five plant families (expression in red beets shown in Figure 4).Some expression was also detected in six other species,including A.thaliana ,Brassica spp.and Lepidium sativum .Since the leaves of some species supporting the transfection,including red beets,spinach,Chenopo-dium or Tetragonia,can be used as uncooked food,the technology proposed here can in principle be used for manufacturing edible/topical vaccines or for production of minimally processed functional foods/feeds.DISCUSSIONThe first demonstration of A.tumefaciens –mediated infection of a plant with a TMV-based vector was reported in 1993(ref.10).The authors quantified the effectiveness of the process and concluded that for a wild-type TMV virus (the U1strain),transfection is very inefficient,requiring B 108bacteria for one successful infection event in a tobacco plant.Since agroinfection by DNA viruses is generally much more effective (103–105bacteria per event),the authors concluded that the low efficiency for RNA viruses is a result of either viral RNA degradation in the nucleus,premature termination of transcription,or low in vivo rates of transcription or poor transcript transport to the cytoplasm.The results of our study both support those conclusions and provide effective remedies,resulting in a process that requires 20bacteria to generate one transfection eventin a tobacco plant,an up to 107-fold improvement over the original process.Several investigators had previously modified the cDNA of RNA viruses by introduction of introns,but mainly with the goal of eliminating the toxicity caused by viral sequences in bacteria 11–13.The general principle of modifying a DNA copy of an RNA virus-derived replicon for increased infectivity is most likely also applicable to other cytoplasmic plant and animal RNA viruses other than the crucifer-specific TMV strain used in our experi-ments 14,15(see also ref.16).Having a more infectious viral vector allowed us to develop magnifection,an efficient whole-plant infiltration protocol.This straightforward protocol requires,in addition to well-established industrial upstream (plant cultivation)and downstream (protein extraction and purification)components,a simple technology block that contains an apparatus for vacuum-infiltration of batches of plants and a chamber/greenhouse for subsequent short-term incubation,as well as a small bacterial fermenter 17.Such a block would of course require certain safety locks so as to prevent the release of agrobacteria into the open environment and to protect the operating personnel.The magnifection process relies on vectors that do not express a coat protein.Although such vectors cannot move systemically,the combination with magnifection provides a solution that has many advantages over existing systems:(i)the vectors are less affected by transgene size since they do not have to move systemically;(ii)simultaneous infection of multiple cells in multiple leaves means that expression is more synchronous and faster;(iii)a larger propor-tion of the host plant is infected by the virus than with traditional vectors (which move systemically primarily to young emerging host tissue);(iv)all metabolic resources of the cell are directed toward synthesis of the protein and not wasted to make large amounts of coat protein;(v)replicon RNAs are not packaged into viral particles and therefore cannot be transferred to secondary untransfected plants or generate wild-type virus.The magnifection process described here is safe,since,in addition to the lack of of viral particle formation,the high yield achievable allows for full containment of the process,thus avoiding open field cultiva-tion.We have also shown that vectors lacking MP are fully comple-mented by host plants engineered to express this viral protein,and therefore using such vectors would lead to an even safer process (unpublished data).Finally,A.tumefaciens is a ubiquitous soil bacter-ium that,for industrial-scale applications,can be genetically rendered unable to survive in the natural environment 18or to transfer Ti plasmid via conjugation to other bacteria.Undesired DNA transfer can also be limited by deleting from the bacterial genome some10–110–3G F P (m g /g f r e s h w e i g h t )adm u 2456789101112s u3dpiFigure 5Time course of GFP expression in N.benthamiana and tobacco.(a )GFP in N.benthamiana plants infiltrated with pICH18711with infiltration solution diluted 10À1to 10À5relative to the overnight saturated A.tumefaciens culture,2–12d.p.i.(b)Coomassie-stained SDS gels loaded with crude protein extracts from N.benthamiana plants infiltrated with the 10À1and 10À3diluted infiltration solutions.m,molecular weight markers (94,67,43,30,20and 14kDa);u,uninfiltrated plant;s,GFP standard.(c )As in a ,but with tobacco.(d )As in b ,but with tobacco.A R T I C L E S©2005 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e b i o t e c h n o l o g yessential functions necessary for T-DNA transfer (from bacteria to plant host)and engineering the plant host to provide those in trans 19,20,or by mixing two agrobacteria that require intercellular complementa-tion for the transfer to occur (ref.21and unpublished data).METHODSConstructs.pICH16707is a GFP-expressing TMV-based viral vector derived from pICH4351(ref.5),but differs from this construct by lack of a LoxP recombination site and by a different vector backbone.pICH15011and pICH17266were made by mutating two putative splice sites (CG/G T GA to CG/G A GA,position 829relative to GenBank accession no.BRU03387,and GCA G /GA to GCA A /GA,position 1,459)or four putative sites (AA/G TAC to AA/A TAC,position 4,201;GC A G/CC to GC C G/CC,position 4411;AA/G TAT to AA/A TAT,position 4,570;AT A G/TC to AT C G/TC position 4,884),respec-tively.Several derivatives were also made from pICH16707by introducing 54and 43silent nucleotide substitutions in areas extending from nt 827to 1,462and 4,655to 4,871,respectively (numbering relative to GenBank accession no.BRU03387,Fig.1a ).The mutated areas were synthesized by PCR using overlapping oligonucleotides containing the desired modifications.Introns ranging in size from 91to 443nt were amplified from A.thaliana genomic DNA by PCR.Sites for intron insertion in the viral sequence were selected that either matched the consensus AG/GT or that could be mutated with silent nucleotide substitutions to match the consensus.Nineteen different positions (shown in Fig.1a )were selected (position given relative to turnip vein-clearing virus (TVCV)sequence,GenBank accession no.BRU03387):1,nt 209;2,nt 423;3,nt 828;4,nt 1,169;5,nt 1,378;6,nt 1,622;7,nt 1,844;8,nt 2,228;9,nt 2,589;10,nt 2,944;11,nt 3,143;12,nt 3,381;13,nt 3,672;14,nt 3,850;15,nt 4,299;16,nt 4,497;17,nt 5,099;18,nt 5,287;19,nt 5,444.Four insertion sites were also selected in GFP (positions given relative to the start of the ORF:20,nt 155;21,nt 275;22,nt 383;23,nt 490).The TVCV MP coding sequence was amplified by PCR from cloned TVCV cDNA (GenBank accession no.BRU03387,bp 4,802–5,628)and subcloned in pICBV10(a pBIN19-derived binary vector)under control of the 35S promoter,resulting in plasmid pICH10745.pICH6692contains the suppressor of silenc-ing p19amplified from tomato bushy stunt virus (TBSV)cDNA using primers 5¢-TTCCATGGAACGAGCTATACAAGGAAACG-3¢and 5¢-CGGGATCCTTAC TCGCTTTCTTCTTCGAAGGT-3¢and cloned under control of a 1.3kb 35S promoter fragment in pICBV10.pICH5290contains the gene encoding GFP 22under control of a 1.3-kb 35S promoter fragment in pICBV1(a pBIN19-derived binary vector).Infiltration of plants and protoplast isolation.Infiltrations of individual leaf sectors were performed as described 5.Agrobacteria were resuspended in infiltration solution at various dilutions relative to the overnight saturated A.tumefaciens culture,from a 5-fold dilution (OD 600¼0.7)to a 10À6dilution (see main text and figure legends).For inoculation of entire plants,Agrobacteria were inoculated to 300ml of Luria-Bertani medium containing 50m g/ml rifampicin and 50m g/ml kanamycin (selection for the binary vector)and grown at saturation.The bacteria were pelleted at 4,800g for 10min and resuspended in 3liters of infiltration buffer (10mM 2-[N -morpholino]ethanesulfonic acid (MES)pH 5.5,10mM MgSO 4)in order to get a 10À1dilution relative to the saturated A.tumefaciens culture or in a larger volume for higher dilutions.A beaker containing the infiltration solution was placed in a vacuum chamber (30-cm diameter),with the aerial parts of a plant dipped into the solution.A vacuum was applied for 2min using a Type PM 16763-860.3pump from KNF Neuberger,with pressure ranging from 0.5to 0.9bar.The plants were returned to the greenhouse under standard conditions.The protoplast isolation procedure is also as previously described 5.Quantification of GFP and GUS.GFP quantification was performed by spectrofluorometry as previously described 5.Absolute GFP proteinconcentration was determined by comparing values of the protein extracts to a standard curve made with recombinant GFP (rGFP from Roche,concentra-tion 1mg/ml).The GUS assay were performed using 50-mg leaf tissue samples according to the protocol described 23.Note:Supplementary information is available on the Nature Biotechnology website.ACKNOWLEDGMENTSWe thank Robert Erwin and Yuri Dorokhov for useful discussions.COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnology website for details).Received 14December 2004;accepted 15March 2005Published online at /naturebiotechnology/1.Porta,C.&Lomonossoff,G.P .Viruses as vectors for the expression of foreign sequencesin plants.Biotechnol.Genet.Eng.Rev.19,245–291(2002).2.Pogue,G.P .,Lindbo,J.A.,Garger,S.J.&Fitzmaurice,W.P .Making an ally from anenemy:plant virology and the new agriculture.Annu.Rev.Phytopathol.40,45–74(2002).3.Gleba,Y.,Marillonnet,S.&Klimyuk,V.Engineering viral 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