BACKGROUND
using plant viruses as vehicles for foreign protein production in plants offers many advantages over more traditional prokaryotic-based, and even over transgenic plant-based protein production systems.
for example, plants are relatively easy and inexpensive to grow, plants are able to perform post-translational protein modifications  not possible with prokaryotes, and plant cells can secrete appropriately engineered proteins allowing for simplified product purification  <cit> .
transgenic plants engineered to produce desirable proteins offer some of these advantages, but engineered plants require substantial time, effort and cost to develop and do not offer flexibility for rapid change if modifications to the protein product are desired.
by contrast, using plant viruses to produce desired proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields.
cucumber mosaic virus  is the one of the viruses that has been used for protein production in plants  <cit> .
cmv has an extremely wide plant host range  <cit>  which opens the door for using plants other than only nicotiana spp.
for producing proteins, and thus an optimized cmv-based protein production system would be very desirable.
but cmv also has some potential drawbacks for foreign protein production.
cmv has a tripartite single-stranded rna genome and each genomic rna is packaged separately within icosahedral capsids  <cit> .
cmv genomic rnas  <dig> and  <dig> encode the 1a and 2a proteins, respectively, which are involved in viral rna replication  <cit> .
rna  <dig> also encodes a small protein called 2b, which affects virulence and is known to suppress the initiation of the plant defense, rna silencing, and to play a role in promoting cell-to-cell movement  <cit> .
rna  <dig> also is bicistronic, encoding the cell-to-cell movement protein  and the virion capsid protein .
all three cmv genomic rnas are essential for the systemic plant infection and all five cmv-encoded proteins directly or indirectly affect the movement of cmv within the plant host  <cit> .
still, cmv genome segments  <dig> and  <dig> have been modified in some cases for insertion of specific sequences which can give foreign protein production in plants  <cit> .
in our previous work we engineered a binary plasmid to contain modified complementary dnas  representing the complete cmv tripartite genome, in which the cmv coat protein gene was replaced by the gene encoding α-1-antitrypsin   <cit> .
we deleted a region of the cmv rna  <dig> leader sequence to ensure that the viral replicase was not able to replicate the truncated rna  <dig> and since coat protein was lacking, infectious cmv was not generated thereby eliminating possible unwanted spread of the recombinant cmv.
furthermore, because one of the key cmv-encoded protein components of the viral replicase  is under the control of a relatively tightly regulated chemically inducible promoter , recombinant viral amplicons were produced intracellularly only after addition of the inducer .
the high efficiency and specificity are among the major advantages of the xve system, and thus it provides a potent tool for research in plant biotechnology.
despite the advantage of having all cmv components on a single plasmid   <cit> , the cmviva plasmid proved to not be easy for subsequent manipulation.
its size alone  made subsequent cloning manipulations difficult.
therefore, here we explored development of new cmv-based smaller-sized variants by separating genome components onto different plasmids to give a bipartite inducible  and tripartite, autonomously replicating forms of cmv .
we also assessed the effects of mrna <dig> leader sequence variants and compared two cmv genotypes for their abilities to give in planta production of two proteins, the green fluorescent protein  and the acidothermus cellulolyticus endo- <dig>  4-β-glucanase , a cellulose degrading enzyme.
this heat-stable,  <dig>  mw well-studied endoglucanase has been produced previously in different species of transgenic plants  <cit> , and is believed to have potential application for cellulose biomass conversion to sugars and use in biofuel production.
here we show that active e <dig> can be rapidly produced in non-transgenic plants by using agroinfiltration with the cmv-based systems.
this reduces the time and energy required to generate transgenic plants and still gives the comparable yields of active e <dig> to those obtained previously by others.
methods
plants and photography
three-week-old nicotiana benthamiana plants and nine day old zucchini squash  plants were used for virus inoculations or agroinfiltration.
plants were photographed with a cannon g <dig> digital camera equipped with a tiffen deep yellow  <dig> filter.
for photographing gfp expression, plants were illuminated with a hand-held long-wave uv lamp.
cloning and plasmid construction
in order to develop cmvin  and cmvar , the gene-of-interest was inserted into the coat protein coding region of cmv-q rna  <dig>   <cit>  to give pcmvar rna  <dig>  we modified the cmv rna  <dig> intergenic region, which also gives rise to the mrna  <dig> leader sequence, by pcr primer tagging to introduce additional restriction enzyme sites for easier cloning.
this was done by pcr amplifying the cp coding region using tagged forward primers  and the reverse primer , and goflexi taq dna polymerase .
amplified fragments were transferred to pgem-t easy  and sequences were verified.
plasmids were then digested by pst i and tth <dig> i, and the desired fragment was transferred to pqa <dig>  <cit>  .
then sequences containing the camv 35s  promoter, rna  <dig> and 35s terminator were pcr amplified using primers 35spfwd and 35strev  and pfu dna polymerase , and the resulting fragments were ligated into the sma i site of the mini binary vector, pcb <dig>  <cit>  .
these were then used as the rna  <dig> source for cmvar and cmvin variants.
the higher producing constructs  were selected for further experiments.
the e <dig> sequence  used here was first codon-optimized for dicots and constructed to contain the rice alpha amylase  signal peptide at its n’-terminus, and a 6-his tag at its c’ terminus  .
the green fluorescent protein  and e <dig> coding sequences were pcr amplified and cloned into the cp coding region of pcmvar rna  <dig>
first, primer sets downstreamfwd and the rna4wtrev, rna42rev, rna46rev, rna48rev , were used for reverse pcr to remove the cp coding region .
the e <dig> gene was amplified by pcr using specific primers set  and ligated into coat protein gene-deleted pcmvar rna  <dig> by blunt end ligation , yielding pcmvar e. the gfp coding sequence was pcr amplified from pcmviva gfp  <cit>  using the specific primer sets , and cloned into the coat protein region of pcmvar rna  <dig> using the same methods as for e <dig>  resulting in pcmvar g .
to generate the cmvar replicating constructs, cmv rna  <dig> and rna  <dig> segments  were pcr amplified using the specific forward and reverse primer sets .
the rna  <dig> region of pcmvar rna  <dig> was removed and replaced by the rna  <dig> or  <dig> genome segments and gave i and ii pcmvar rna  <dig> and  <dig> .
the subgroup i rna  <dig> and rna  <dig> were originally from a california cmv  <cit> , and the subgroup ii rna  <dig> and rna  <dig> were from cmv-q .
for the cmvin system, rna  <dig> and  <dig> segments came from pduxlr1r <dig> .
pcr products were eluted from an agarose gel and self ligated to make pcmvar 33 g  <dig>   <dig>   <dig>  wt and pcmvar 33e  <dig>   <dig>   <dig>  wt variants, respectively .
table  <dig> shows the names, activities and genotypes for the expression system variants used in this paper.
1all rna <dig> constructs are from subgroup ii cmv-q.
2rna  <dig> and  <dig> sources are subgroup i or subgroup ii as indicated.
cells containing required plasmids were mixed and infiltrated into non-transgenic n. benthamiana plants as described.
agroinfiltration
binary plasmids purified from e. coli cultures were transformed into agrobacterium tumefaciens gv <dig> or eha <dig> cells using electroporation.
transformed a. tumefaciens cells were plated on luria-bertani plates containing rifampicin  and gentamycin  for gv <dig> and kanamycin  for specific constructs, and rifampicin  and tetracycline  for eha <dig> and gentamycin  for specific constructs, respectively.
for agroinfiltration, a single colony was inoculated into 5 ml l-mesa media ,  <dig> μl  <dig>  m acetosyringone) and grown to an od <dig> of  <dig> .
cells were harvested by centrifuging for 10 min at  <dig>  g and resuspended in induction media ,  <dig> μl  <dig>  m acetosyringone), and allowed to sit at room temperature for 3 hrs before infiltration.
when mixtures of a. tumefaciens cells were infiltrated into plants, cultures were prepared separately in induction medium and combined immediately before infiltration.
for inoculating small sugar pumpkin plants, a. tumefaciens cells containing the constructs were infiltrated into n. benthamiana plants.
leaves were harvested 6 days after infiltration, and used for standard rub inoculation.
rna extraction and realtime rt-pcr
samples for rna and protein extraction were harvested from infiltrated and non-infiltrated leaves at 6 days after infiltration.
total rna was extracted using the rneasy kit  following the manufacturer’s instructions.
complementary dna  synthesized from dnase-digested total rna was used for reverse transcription using the rna 3end primer as listed in table  <dig> and superscript ii reverse transcriptase, as described by the manufacturer .
realtime pcr was performed using gene specific primers for each cmv rna segment .
real-time pcr was performed using sybr green pcr master mix  in an abi prism  <dig> sequence detection system  under standard amplification conditions .
the threshold cycle  is defined as the fractional cycle number at which the fluorescence exceeded the fixed threshold.
statistical analyses were performed using the bonferroni  t test using the sas  <dig>  program.
protein extraction and immunoblotting
proteins were extracted from leaves using protein extraction buffer , 10 mm edta, 5 mm dtt, 150 mm nacl,  <dig> % triton x- <dig>  1x protease inhibitor  and tissue maceration using a bead-beater.
samples were centrifuged at  <dig>  g for 20 min to remove cell debris, and protein concentrations were determined by bradford assay using coomassie plus  with bovine serum albumin as the standard.
proteins were analyzed by sds-page in 12% polyacrylamide gels and transferred to hybond-c extra membranes .
membranes were incubated with rabbit cmv anti-cp polyclonal antibody at 1: <dig>  dilution, followed by goat anti-rabbit igg-alkaline phosphatase conjugate  at 1: <dig>  dilution.
for e <dig> detection, membranes were incubated with specific mouse monoclonal igg anti-e <dig> antibody  at 1: <dig>  dilution, followed by goat anti-mouse igg alkaline phosphatase conjugate  at 1: <dig>  dilution.
after washing with tris-buffered saline  with  <dig> % tween- <dig> for three times, the membrane was developed to a purple color using colorimetric ap conjugate substrate reagent kit  including premixed bcip  and nbt  substrate solutions .
endoglucanase  activity assays
endoglucanase  activity assays were done as described  <cit> .
a 60 μl aliquot of diluted supernatant containing e <dig> was added to 540 μl of acetate buffer  and 200 μl of substrate ).
then, 200 μl of the reaction was sampled at time zero and after 30 min, and added to 800 μl of stop buffer .
the change in fluorescence as released methylumbelliferine  over time was measured with a versafluor fluorometer .
fluorescence was converted to activity, and specific activity was determined as described  <cit> .
RESULTS
modified rna <dig> leader sequences affect mrna and coat protein levels
to facilitate cloning desired genes into the cmv cp region, we first created a separate cmv rna 3-based plasmid and modified the intergenic nucleotide sequence upstream of the cp orf start codon to contain desired restriction endonuclease sites .
this also resulted in changes to the mrna  <dig> nucleotide leader sequence immediately preceding the aug start codon and therefore could affect mrna translation efficiency  <cit> .
therefore, we first compared the respective cmvin and cmvar variants for their ability to replicate and to express cmv cp within infiltrated leaves.
we used real-time pcr to quantify levels of progeny rnas  <dig>   <dig>   <dig>  and  <dig> plus  <dig> .
when we compared the cmvinii variants with cmviva, all showed accumulation of genomic and subgenomic rnas, and of cmv cp .
because we are most interested in protein production from rna  <dig>  comparison of the rna  <dig> &  <dig> data show that the modified variants  all showed slightly more rnas  <dig> and  <dig> than did the wildtype cmvinii, but none were higher than cmviva .
although the levels of rnas  <dig> and  <dig> were not statistically different, comparison of cmv cp accumulation showed more cmv cp was detected for cmvinii variants  <dig>   <dig>   <dig> and cmviva, than was for the wildtype cmvinii.
cmviva and cmvinii variants both have  <dig> nucleotide deletions in the inducible cmv rna  <dig> such that although it is transcribed and the resulting rna  <dig> serves for translation to yield the 1a protein, the rna  <dig> genome segment is not replicated as are genome segments rnas  <dig> and  <dig> .
because the 1a component of the replicase complex is under the control of a relatively tightly regulated chemically inducible promoter, the recombinant viral amplicons are only produced under induction conditions.
when we compared the cmvarii variants, all showed accumulation of all cmv rnas, and so long as inocula contained all three cmv genomic rnas, accumulation of rnas  <dig>   <dig> and  <dig> was not significantly different for the different variants.
however, realtime pcr analysis showed lower accumulation of rnas 3 +  <dig> for cmvarii variant <dig> when compared to the monopartite pq <dig> .
all cmvarii variants showed much less cp accumulation than was seen for pq <dig>  and cmvarii  <dig>   <dig> and  <dig> showed less cp than did cmvarii wt .
however, all cmvarii variants were able to initiate systemic infections in n. benthamiana and zucchini squash plants .
rt-pcr and nucleotide sequence analysis of the cmv progeny showed that the modified leader sequences were retained in rnas extracted from systemically-infected leaves .
cmvinii and cmvarii variants yield high gfp fluorescence
to assess foreign protein production we first cloned the gene for gfp into the cp coding region for the cmvinii and cmvarii variants.
non-transgenic n. benthamiana plants were infiltrated and leaves were examined for fluorescence at  <dig> and 10 days post-infiltration.
at 6 days post-infiltration bright gfp fluorescence was seen in the infiltrated regions .
in general, the regions of the leaves infiltrated with cmvinii and cmvarii variants showed very bright fluorescence.
by 10 days post-infiltration, bright gfp fluorescence was observed for the cmvinii and cmvarii variants, regardless of leader sequence.
despite the fact that cmvarii variants producing the cmv cp spread within plants giving systemic infections, cmvarii variants producing gfp did not, and fluorescence was localized to the infiltrated areas.
this is most likely because these variants do not produce cmv cp, which is known to be a determinant of cmv systemic spread in plants  <cit> , and thus these infections were localized to the infiltrated regions of the treated leaves.
we also tested another reporter protein, the red fluorescent protein , for expression using cmvinii and cmvarii and obtained essentially identical results to those shown for the variants expressing gfp .
e <dig> was produced in plants using both cmvinii and cmvarii variants
our intent is to develop easy-to-use cmv variants that give efficient production of desirable proteins in non-transgenic plants, including proteins with potential biofuel applications.
therefore, we next assessed e <dig> accumulation in leaves of infiltrated n. benthamiana plants.
we first used immunoblot analysis to detect total e <dig> accumulation in leaves infiltrated with the cmvinii and cmvarii variants.
e <dig> was detected in the infiltrated leaves for both cmvinii and cmvarii wild type variants .
the leader sequence cmvinii variants  <dig> and  <dig> showed higher e <dig> compared to the wildtype cmvinii.
for cmvarii variants, the wild type showed higher e <dig> accumulation, but the  <dig> and  <dig> variants also gave good e <dig> accumulation .
interestingly, the intact e <dig> migrated as a ca.
<dig> kda protein as shown in figure  <dig> and additional file 7: figure s <dig>  even though the calculated mw of e <dig> is  <dig>  kda, including the histidine tag and rice amylase signal peptide .
similar reports for anomalous e <dig> migration in sds-page have been previously reported  <cit> .
the immunoblot experiments showed e <dig> protein accumulation but our interest is in production of enzymatically active e <dig>  therefore we used activity assays on plant extracts to estimate yields of active e <dig>  in repeated experiments, the cmvinii e <dig> and e <dig> consistently yielded  <dig> to almost  <dig> fold higher relative e <dig> accumulation than wildtype cmvinii e, while the yield for cmvinii e <dig> was negligible.
by contrast, the wildtype cmvarii e consistently yielded more e <dig> than the other cmvarii variants .
cmvarii e <dig> gave negligible e <dig> accumulation while cmvarii e <dig> and e <dig> showed low yields, but much less than wildtype cmvarii e. we had anticipated that cmvarii would give higher protein accumulation in plants because of rna  <dig> replication, which is lacking in cmvinii , but this proved not to be the case, the highest overall yields were obtained with cmvinii e  <dig> and  <dig>  the cmvarii is easier to use since there is no requirement for adding the rna  <dig> inducer, estradiol, and if we could achieve higher protein accumulation with cmvarii this would be our choice.
therefore we next attempted to increase cmvar-driven protein accumulation by two additional approaches: to increase cmv rna replication and to increase cmv spread within plants.
1numbers indicate e <dig> production as μg/g fresh weight.
average ± standard deviations are shown.
samples were collected 6 days post-infiltration, using  <dig> plants,  <dig> leaves from each plant  for each biological trial.
results for three different biological trials are shown for each test.
the identities of variants tested are given in table  <dig>  group superscript letters next to the numbers represent different statistical groups, means with the same letter are not significantly different by the bonferroni  t test using the sas  <dig>  program.
reassortant cmvari variants yield more protein compared to cmvarii variants
cmv is one of the world’s most widespread plant viruses, and has many genetic variants which are primarily divided into the taxonomic subgroups i and ii  <cit> .
in general, subgroup i cmvs show more severe symptoms in plants than do subgroup ii cmv isolates, which can show mild or even symptomless infections.
this is suggested to be associated with the 2b protein  as a silencing suppressor  <cit> , and effects can vary in different plant hosts  <cit> .
therefore, we generated and compared cmv subgroup i and ii genomic reassortants for their abilities to give greater replication and protein production.
all reassortants contained the same cmv subgroup ii wildtype rna  <dig> or variant constructs for gfp or e <dig>  gfp fluorescence was brighter for all cmvari variants compared with the respective cmvarii variants .
we next compared production of active e <dig> among the cmvari and ii e variants by immunoblotting  and found that the reassortant wildtype cmvari e gave more active e <dig> than did cmvarii e in side-by-side experiments .
by contrast, cmvari e variants  <dig>   <dig> and  <dig> gave very low e <dig> accumulation.
however, wildtype cmvari e gave relatively high e <dig> accumulation, similar to that for cmvinii e variants  <dig> and  <dig>
the mp c-terminal  <dig> amino acid deletion constructs showed increased yields compared to the intact mp constructs
cmv requires both the mp and cp for cell-to-cell movement in plants, both of which are encoded by rna  <dig>  <cit> .
thus for both cmvar and cmvin variants, when foreign sequences are cloned into the cp coding region, there is no cell-to-cell movement due to lack of the cmv-encoded cp, and the desired recombinant proteins  accumulate only in the initially-infected cells.
however, it was shown previously that when the cmv mp was mutated so as to lack the c-terminal  <dig> amino acids, cmv infections were able to move cell-to-cell in plants even in the absence of cp  <cit> .
therefore, we deleted the mp c-terminal  <dig> amino acids and compared e <dig> and gfp accumulation in plants using the cmvari and cmvarii variants.
gfp fluorescence was high for all variants with the  <dig> amino acid truncated mp .
however, comparing e <dig> accumulation for all variants, the highest levels of active e <dig> were obtained for the  <dig> amino acid truncated cmvari ewt variant .
our assays were for intact, enzymatically active e <dig>  and we obtained yields up to 21 μg/g of active e <dig>  corresponding to ~ <dig> % of tsp.
furthermore, unlike for the wildtype mp variants, the cmvinii 33e variants  <dig> and  <dig> gave relatively low accumulation of e <dig>
discussion
several different viruses have been used for protein production in plants, and each has advantages as well as disadvantages  <cit> .
many of the “first generation” plant virus vectors  <cit>  utilized whole plant systemic virus infections to give desired proteins.
while many of these have proven to be very useful there are some significant drawbacks.
systemic infections can take several days to fully develop.
protein production is then asynchronous and yields can vary in different tissues  <cit> .
recombinant viruses also show size constraints for the inserted sequence, often coding sequences of only 1 kb or less  can be inserted  <cit> .
then as the infection develops the viruses partially or completely excise the inserted recombinant sequence, leading to loss of the desired intact protein product  <cit> .
furthermore, some viruses  may have even more severe size constraints if rna encapsidation is a requirement for development of the systemic infection.
then if the coding sequence for the desired protein is large, insertion into the viral rna may preclude encapsidation, thereby preventing efficient spread.
recent progress in developing agrobacterium tumefaciens delivered plant virus-based protein production systems has been made by several research groups using different plant viruses  <cit> .
these plant virus-based amplicon systems offer many advantages including the fact that non-transgenic plants can be used, the desired protein production is rapid, the product can accumulate to high levels, and virus-based expression can be temporally regulated to be almost synchronous in all infiltrated areas.
because a majority of the infiltrated cells become simultaneously infected, virus movement to new cells is not necessary, encapsidation of recombinant rnas is not an issue.
these “second generation” virus-based systems also can retain larger foreign coding sequences and thus produce larger proteins in plants  <cit> , here we produced enzymatically active  <dig>  mw e <dig>
in our previous work, we used the estradiol-inducible, cmv-based cmviva to produce α anti-trypsin  in non-transgenic n. benthamiana plants  <cit> .
cmviva has all three cmv genome components in one large  <dig> kbp plasmid, which, due to its large size is difficult to manipulate.
thus, here we took approaches to develop cmv-based inducible  as well as autonomously replicating  systems, both of which are more easily manipulated and might be able to give high accumulation of heterologous proteins in plants.
first, we separated the cmv genomic rna cdnas onto two plasmids, one containing the rna  <dig> and  <dig> replication-associated genome components and the other containing the cmv rna  <dig> genome segment.
the cmv rna <dig> component is rather small in size,  <dig>  kb, and is easy to manipulate and to engineer to contain restriction enzyme sites to allow for easy removal of the cmv cp gene and replace it with any gene of interest.
the desired restriction enzyme sites were introduced into the intergenic region of rna  <dig>  as expected, these altered the 5′ untranslated leader sequence of the resulting mrna .
the sgrna promoter  is within the minus strand of rna  <dig> and is recognized by the rna-dependent rna polymerase and mrna transcription is initiated.
for cmv-q rna <dig>  the transcription initiation starts at nt position  <dig> in the intergenic region, which is upstream of the modified leader sequences.
our analyses demonstrated that the rna  <dig> modifications affected rna  <dig> and rna  <dig> accumulation, but showed even more unpredictable effects on resulting protein accumulation.
it does not appear that these can be attributed only to start codon context  <cit>  as the same construct  showed different protein yields whether the rna was delivered using cmvin vs.cmvar.
in contrast to cmviva, both cmvinii and cmvari and ii variants require mixing a. tumefaciens cells containing different plasmids which are then co-infiltrated into plants and t-dna from the different a. tumefaciens cells containing the cmv plasmids must be transferred to the same plant cell for the complete cmv amplicon.
for cmvinii variants this is then followed by induction using estradiol, which resulted in high level accumulation of the proteins tested here .
however, like for cmviva, the cmvinii rna  <dig> deletion does not allow for its replication, only translation of the newly transcribed mrna.
therefore we also developed the non-inducible autonomously replicating cmv-based system, cmvari and ii.
wildtype cmvar  replicated to very high levels and even caused systemic infections in plants.
however, when genes for gfp or e <dig> were substituted for the cp gene, both proteins were produced in plants within the infiltrated areas, and quantitative analyses showed that high levels of proteins accumulated for both cmvin vs.cmvar, particularly at 6 days post-infiltration.
although cmvinii e  <dig> and  <dig> variants gave slightly more active e <dig> in most experiments, cmvari and ii variants offers advantages in ease of use  and thus, two additional approaches to improve accumulation of the desired protein product were investigated.
like most viruses having genomes composed of multiple segments, cmv genomic rnas can be mixed  to achieve genetic diversity  <cit> , and this offers opportunities for using cmv to produce desirable proteins in different plant species, as has been demonstrated also by others  <cit> .
therefore, we generated cmv reassortant genotypes by substituting cmv subgroup i genomic rnas  <dig> and  <dig> derived from a more virulent cmv, with the original cmv q subgroup ii rna  <dig>  giving cmvari.
comparison of cmvari and cmvarii g, e variants showed higher gfp and e <dig> for cmvari g, e variants.
however, the cmvari ewt showed higher e <dig> accumulation than did the corresponding cmvinii ewt, but cmvinii e variants  <dig> and  <dig> gave the higher e <dig> accumulation than cmvar i  <dig> and  <dig> variants thus showing that reassortment alone was not sufficient.
as another alternative, we generated a mp c-terminal  <dig> amino acid deletion mutant.
cell-to-cell movement in cmv-infected plants requires interactions between the cp and mp  <cit> .
our cmv-based systems including cmviva, cmvari and ii and cmvinii are cell-to-cell movement deficient since they lack the cp and thus, desired recombinant proteins are produced only within infiltrated cells.
however, previous workers demonstrated that the cmv mp c-terminal  <dig> amino acids are essential to recognize and interact with the cp  <cit> .
when this region is deleted, the cmv infections can spread cell-to-cell even in the absence of cp  <cit> .
in support of this the cmvari and cmvarii 33 g variants showed high cmv-based gfp production .
when we created mp  <dig> amino acid deletion constructs and tested them, they showed increased production of not only gfp in cmvari and ii 33 g variants, but also of e <dig> in cmvari and ii 33e variants , and the highest yields of active e <dig> were obtained using the cmvari 33e variants.
other workers have produced versions of e <dig> in various transgenic plants with gene expression driven by different promoters.
for example, full-length e <dig> containing the catalytic domain, linker and carbohydrate binding domain has been previously produced in transgenic tobacco plants.
based on the resulting e <dig> activity, yields of up to  <dig> % on average of total leaf soluble proteins were shown with mac promoter, a chimeric promoter of the camv 35s and mannopine synthase gene  <cit> .
similar yields were shown also with camv 35s promoter  <cit> .
in transgenic z. mays seeds, the full-length e <dig> was produced using glob- <dig>  and yields up to 6% tsp were obtained  <cit> .
in transgenic rice  plants, 35s driven e <dig> lacking the carbohydrate binding domain but only containing the catalytic domain gave yields up to  <dig> % tsp  <cit> .
thus, our yields of up to  <dig> % tsp of intact e <dig> in nontransgenic n. benthamiana plants are similar to those achieved for intact e <dig> in transgenic tobacco, but less than those in more specialized systems.
furthermore, cmvin and cmvar-based production of the desired protein can be temporally regulated to give almost synchronous protein accumulation over a very short time period, even a few days.
CONCLUSIONS
our data demonstrate that the cmv-based systems, cmvin and cmvar, are good candidates for production of desired heterologous proteins in nontransgenic plants.
our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of their use, and reassortant genotypes and cmv movement protein deletions also allow for greater protein accumulation.
also, n. benthamiana, which is particularly suitable for agro infiltration, is a very good plant for protein production, but due to the wide host range of cmv, other plants may also prove to be useful for production of different proteins.
abbreviations
cmv: cucumber mosaic virus; tmv: tobacco mosaic virus; e1:acidothermus cellulolyticus endo- <dig>  4-β-glucanase; gfp: green fluorescent protein; cp: coat protein; orf: open reading frame; mp: movement protein; fw: fresh weight; tsp: total soluble protein; rfp: red fluorescent protein.
competing interests
the authors declare that they have no competing interests.
author’ contributions
msh was responsible for experiment design, execution, analysis and wrote the manuscript.
bel was responsible for experimental execution including enzymatic activity assays and interpretation.
kam helped to conceive the study, discussed, and helped edit the manuscript.
bwf helped to conceive the study, helped with organizing the experimental work, data interpretation, and helped to write and edit the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
figure s <dig>  construction of the pcmvar rna  <dig> plasmid.
click here for file
additional file 2
figure s <dig>  northern blotting analyses of cmvin ii variants.
click here for file
additional file 3
figure s <dig>  construction of the pcmvar g and pcmvar e variants for gfp and e <dig>
click here for file
additional file 4
figure s <dig>  construction of the pcmvar i and ii for subgroup i, ii rna  <dig> &  <dig>
click here for file
additional file 5
figure s <dig>  construction of the pcmvar 33g and pcmvar 33e for mp  <dig> amino acid deletion constructs.
click here for file
additional file 6
figure s <dig>  systemic symptoms in plants for cmvar ii variants.
click here for file
additional file 7
figure s <dig>  immunoblot analysis of e <dig> produced in infiltrated n. benthamiana leaves for cmvar i e and cmvar ii e variants.
click here for file
acknowledgements
we are extremely grateful to dr. john lindbo and to dr. mysore sudarshana for many helpful discussions.
we are especially grateful to bill adney, senior scientist at the national renewable energy lab, for providing the e <dig> antibody.
we also thank dr. shouwei ding, university of california, riverside, for the gift of pcassq <dig>  this work was supported in part by chevron technology ventures, a division of chevron, u.s.a., inc; and nsf cbet- <dig>

 using plant viruses as vehicles for foreign protein production in plants offers many advantages over more traditional prokaryotic-based, and even over transgenic plant-based protein production systems. for example, plants are relatively easy and inexpensive to grow, plants are able to perform post-translational protein modifications not possible with prokaryotes, and plant cells can secrete appropriately engineered proteins allowing for simplified product purification. transgenic plants engineered to produce desirable proteins offer some of these advantages, but engineered plants require substantial time, effort and cost to develop and do not offer flexibility for rapid change if modifications to the protein product are desired. by contrast, using plant viruses to produce desired proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. cucumber mosaic virus is the one of the viruses that has been used for protein production in plants. cmv has an extremely wide plant host range which opens the door for using plants other than only nicotiana spp. for producing proteins, and thus an optimized cmv-based protein production system would be very desirable. but cmv also has some potential drawbacks for foreign protein production. cmv has a tripartite single-stranded rna genome and each genomic rna is packaged separately within icosahedral capsids. cmv genomic rnas and encode the 1a and 2a proteins, respectively, which are involved in viral rna replication. rna also encodes a small protein called 2b, which affects virulence and is known to suppress the initiation of the plant defense, rna silencing, and to play a role in promoting cell-to-cell movement. rna also is bicistronic, encoding the cell-to-cell movement protein and the virion capsid protein. all three cmv genomic rnas are essential for the systemic plant infection and all five cmv-encoded proteins directly or indirectly affect the movement of cmv within the plant host. still, cmv genome segments and have been modified in some cases for insertion of specific sequences which can give foreign protein production in plants. in our previous work we engineered a binary plasmid to contain modified complementary dnas representing the complete cmv tripartite genome, in which the cmv coat protein gene was replaced by the gene encoding α-1-antitrypsin. we deleted a region of the cmv rna leader sequence to ensure that the viral replicase was not able to replicate the truncated rna and since coat protein was lacking, infectious cmv was not generated thereby eliminating possible unwanted spread of the recombinant cmv. furthermore, because one of the key cmv-encoded protein components of the viral replicase is under the control of a relatively tightly regulated chemically inducible promoter, recombinant viral amplicons were produced intracellularly only after addition of the inducer. the high efficiency and specificity are among the major advantages of the xve system, and thus it provides a potent tool for research in plant biotechnology. despite the advantage of having all cmv components on a single plasmid, the cmviva plasmid proved to not be easy for subsequent manipulation. its size alone made subsequent cloning manipulations difficult. therefore, here we explored development of new cmv-based smaller-sized variants by separating genome components onto different plasmids to give a bipartite inducible and tripartite, autonomously replicating forms of cmv. we also assessed the effects of mrna leader sequence variants and compared two cmv genotypes for their abilities to give in planta production of two proteins, the green fluorescent protein and the acidothermus cellulolyticus endo- 4-β-glucanase, a cellulose degrading enzyme. this heat-stable,  mw well-studied endoglucanase has been produced previously in different species of transgenic plants, and is believed to have potential application for cellulose biomass conversion to sugars and use in biofuel production. here we show that active e can be rapidly produced in non-transgenic plants by using agroinfiltration with the cmv-based systems. this reduces the time and energy required to generate transgenic plants and still gives the comparable yields of active e to those obtained previously by others. methods plants and photography three-week-old nicotiana benthamiana plants and nine day old zucchini squash plants were used for virus inoculations or agroinfiltration. plants were photographed with a cannon g digital camera equipped with a tiffen deep yellow filter. for photographing gfp expression, plants were illuminated with a hand-held long-wave uv lamp. cloning and plasmid construction in order to develop cmvin and cmvar, the gene-of-interest was inserted into the coat protein coding region of cmv-q rna to give pcmvar rna we modified the cmv rna intergenic region, which also gives rise to the mrna leader sequence, by pcr primer tagging to introduce additional restriction enzyme sites for easier cloning. this was done by pcr amplifying the cp coding region using tagged forward primers and the reverse primer, and goflexi taq dna polymerase. amplified fragments were transferred to pgem-t easy and sequences were verified. plasmids were then digested by pst i and tth i, and the desired fragment was transferred to pqa. then sequences containing the camv 35s promoter, rna and 35s terminator were pcr amplified using primers 35spfwd and 35strev and pfu dna polymerase, and the resulting fragments were ligated into the sma i site of the mini binary vector, pcb. these were then used as the rna source for cmvar and cmvin variants. the higher producing constructs were selected for further experiments. the e sequence used here was first codon-optimized for dicots and constructed to contain the rice alpha amylase signal peptide at its n’-terminus, and a 6-his tag at its c’ terminus. the green fluorescent protein and e coding sequences were pcr amplified and cloned into the cp coding region of pcmvar rna first, primer sets downstreamfwd and the rna4wtrev, rna42rev, rna46rev, rna48rev, were used for reverse pcr to remove the cp coding region. the e gene was amplified by pcr using specific primers set and ligated into coat protein gene-deleted pcmvar rna by blunt end ligation, yielding pcmvar e. the gfp coding sequence was pcr amplified from pcmviva gfp using the specific primer sets, and cloned into the coat protein region of pcmvar rna using the same methods as for e resulting in pcmvar g. to generate the cmvar replicating constructs, cmv rna and rna segments were pcr amplified using the specific forward and reverse primer sets. the rna region of pcmvar rna was removed and replaced by the rna or genome segments and gave i and ii pcmvar rna and. the subgroup i rna and rna were originally from a california cmv, and the subgroup ii rna and rna were from cmv-q. for the cmvin system, rna and segments came from pduxlr1r. pcr products were eluted from an agarose gel and self ligated to make pcmvar 33 g wt and pcmvar 33e wt variants, respectively. table  shows the names, activities and genotypes for the expression system variants used in this paper. 1all rna constructs are from subgroup ii cmv-q. 2rna and sources are subgroup i or subgroup ii as indicated. cells containing required plasmids were mixed and infiltrated into non-transgenic n. benthamiana plants as described. agroinfiltration binary plasmids purified from e. coli cultures were transformed into agrobacterium tumefaciens gv or eha cells using electroporation. transformed a. tumefaciens cells were plated on luria-bertani plates containing rifampicin and gentamycin for gv and kanamycin for specific constructs, and rifampicin and tetracycline for eha and gentamycin for specific constructs, respectively. for agroinfiltration, a single colony was inoculated into 5 ml l-mesa media, μl  m acetosyringone) and grown to an od of. cells were harvested by centrifuging for 10 min at  g and resuspended in induction media, μl  m acetosyringone), and allowed to sit at room temperature for 3 hrs before infiltration. when mixtures of a. tumefaciens cells were infiltrated into plants, cultures were prepared separately in induction medium and combined immediately before infiltration. for inoculating small sugar pumpkin plants, a. tumefaciens cells containing the constructs were infiltrated into n. benthamiana plants. leaves were harvested 6 days after infiltration, and used for standard rub inoculation. rna extraction and realtime rt-pcr samples for rna and protein extraction were harvested from infiltrated and non-infiltrated leaves at 6 days after infiltration. total rna was extracted using the rneasy kit following the manufacturer’s instructions. complementary dna synthesized from dnase-digested total rna was used for reverse transcription using the rna 3end primer as listed in table  and superscript ii reverse transcriptase, as described by the manufacturer. realtime pcr was performed using gene specific primers for each cmv rna segment. real-time pcr was performed using sybr green pcr master mix in an abi prism sequence detection system under standard amplification conditions. the threshold cycle is defined as the fractional cycle number at which the fluorescence exceeded the fixed threshold. statistical analyses were performed using the bonferroni t test using the sas program. protein extraction and immunoblotting proteins were extracted from leaves using protein extraction buffer, 10 mm edta, 5 mm dtt, 150 mm nacl, % triton x- 1x protease inhibitor and tissue maceration using a bead-beater. samples were centrifuged at  g for 20 min to remove cell debris, and protein concentrations were determined by bradford assay using coomassie plus with bovine serum albumin as the standard. proteins were analyzed by sds-page in 12% polyacrylamide gels and transferred to hybond-c extra membranes. membranes were incubated with rabbit cmv anti-cp polyclonal antibody at 1: dilution, followed by goat anti-rabbit igg-alkaline phosphatase conjugate at 1: dilution. for e detection, membranes were incubated with specific mouse monoclonal igg anti-e antibody at 1: dilution, followed by goat anti-mouse igg alkaline phosphatase conjugate at 1: dilution. after washing with tris-buffered saline with % tween- for three times, the membrane was developed to a purple color using colorimetric ap conjugate substrate reagent kit including premixed bcip and nbt substrate solutions. endoglucanase activity assays endoglucanase activity assays were done as described. a 60 μl aliquot of diluted supernatant containing e was added to 540 μl of acetate buffer and 200 μl of substrate ). then, 200 μl of the reaction was sampled at time zero and after 30 min, and added to 800 μl of stop buffer. the change in fluorescence as released methylumbelliferine over time was measured with a versafluor fluorometer. fluorescence was converted to activity, and specific activity was determined as described.  modified rna leader sequences affect mrna and coat protein levels to facilitate cloning desired genes into the cmv cp region, we first created a separate cmv rna 3-based plasmid and modified the intergenic nucleotide sequence upstream of the cp orf start codon to contain desired restriction endonuclease sites. this also resulted in changes to the mrna nucleotide leader sequence immediately preceding the aug start codon and therefore could affect mrna translation efficiency. therefore, we first compared the respective cmvin and cmvar variants for their ability to replicate and to express cmv cp within infiltrated leaves. we used real-time pcr to quantify levels of progeny rnas and plus. when we compared the cmvinii variants with cmviva, all showed accumulation of genomic and subgenomic rnas, and of cmv cp. because we are most interested in protein production from rna comparison of the rna & data show that the modified variants all showed slightly more rnas and than did the wildtype cmvinii, but none were higher than cmviva. although the levels of rnas and were not statistically different, comparison of cmv cp accumulation showed more cmv cp was detected for cmvinii variants and cmviva, than was for the wildtype cmvinii. cmviva and cmvinii variants both have nucleotide deletions in the inducible cmv rna such that although it is transcribed and the resulting rna serves for translation to yield the 1a protein, the rna genome segment is not replicated as are genome segments rnas and. because the 1a component of the replicase complex is under the control of a relatively tightly regulated chemically inducible promoter, the recombinant viral amplicons are only produced under induction conditions. when we compared the cmvarii variants, all showed accumulation of all cmv rnas, and so long as inocula contained all three cmv genomic rnas, accumulation of rnas and was not significantly different for the different variants. however, realtime pcr analysis showed lower accumulation of rnas 3 +  for cmvarii variant when compared to the monopartite pq. all cmvarii variants showed much less cp accumulation than was seen for pq and cmvarii and showed less cp than did cmvarii wt. however, all cmvarii variants were able to initiate systemic infections in n. benthamiana and zucchini squash plants. rt-pcr and nucleotide sequence analysis of the cmv progeny showed that the modified leader sequences were retained in rnas extracted from systemically-infected leaves. cmvinii and cmvarii variants yield high gfp fluorescence to assess foreign protein production we first cloned the gene for gfp into the cp coding region for the cmvinii and cmvarii variants. non-transgenic n. benthamiana plants were infiltrated and leaves were examined for fluorescence at and 10 days post-infiltration. at 6 days post-infiltration bright gfp fluorescence was seen in the infiltrated regions. in general, the regions of the leaves infiltrated with cmvinii and cmvarii variants showed very bright fluorescence. by 10 days post-infiltration, bright gfp fluorescence was observed for the cmvinii and cmvarii variants, regardless of leader sequence. despite the fact that cmvarii variants producing the cmv cp spread within plants giving systemic infections, cmvarii variants producing gfp did not, and fluorescence was localized to the infiltrated areas. this is most likely because these variants do not produce cmv cp, which is known to be a determinant of cmv systemic spread in plants, and thus these infections were localized to the infiltrated regions of the treated leaves. we also tested another reporter protein, the red fluorescent protein, for expression using cmvinii and cmvarii and obtained essentially identical results to those shown for the variants expressing gfp. e was produced in plants using both cmvinii and cmvarii variants our intent is to develop easy-to-use cmv variants that give efficient production of desirable proteins in non-transgenic plants, including proteins with potential biofuel applications. therefore, we next assessed e accumulation in leaves of infiltrated n. benthamiana plants. we first used immunoblot analysis to detect total e accumulation in leaves infiltrated with the cmvinii and cmvarii variants. e was detected in the infiltrated leaves for both cmvinii and cmvarii wild type variants. the leader sequence cmvinii variants and showed higher e compared to the wildtype cmvinii. for cmvarii variants, the wild type showed higher e accumulation, but the and variants also gave good e accumulation. interestingly, the intact e migrated as a ca. kda protein as shown in figure  and additional file 7: figure s even though the calculated mw of e is kda, including the histidine tag and rice amylase signal peptide. similar reports for anomalous e migration in sds-page have been previously reported. the immunoblot experiments showed e protein accumulation but our interest is in production of enzymatically active e therefore we used activity assays on plant extracts to estimate yields of active e in repeated experiments, the cmvinii e and e consistently yielded to almost fold higher relative e accumulation than wildtype cmvinii e, while the yield for cmvinii e was negligible. by contrast, the wildtype cmvarii e consistently yielded more e than the other cmvarii variants. cmvarii e gave negligible e accumulation while cmvarii e and e showed low yields, but much less than wildtype cmvarii e. we had anticipated that cmvarii would give higher protein accumulation in plants because of rna replication, which is lacking in cmvinii, but this proved not to be the case, the highest overall yields were obtained with cmvinii e and the cmvarii is easier to use since there is no requirement for adding the rna inducer, estradiol, and if we could achieve higher protein accumulation with cmvarii this would be our choice. therefore we next attempted to increase cmvar-driven protein accumulation by two additional approaches: to increase cmv rna replication and to increase cmv spread within plants. 1numbers indicate e production as μg/g fresh weight. average ± standard deviations are shown. samples were collected 6 days post-infiltration, using plants, leaves from each plant for each biological trial. results for three different biological trials are shown for each test. the identities of variants tested are given in table  group superscript letters next to the numbers represent different statistical groups, means with the same letter are not significantly different by the bonferroni t test using the sas program. reassortant cmvari variants yield more protein compared to cmvarii variants cmv is one of the world’s most widespread plant viruses, and has many genetic variants which are primarily divided into the taxonomic subgroups i and ii. in general, subgroup i cmvs show more severe symptoms in plants than do subgroup ii cmv isolates, which can show mild or even symptomless infections. this is suggested to be associated with the 2b protein as a silencing suppressor, and effects can vary in different plant hosts. therefore, we generated and compared cmv subgroup i and ii genomic reassortants for their abilities to give greater replication and protein production. all reassortants contained the same cmv subgroup ii wildtype rna or variant constructs for gfp or e gfp fluorescence was brighter for all cmvari variants compared with the respective cmvarii variants. we next compared production of active e among the cmvari and ii e variants by immunoblotting and found that the reassortant wildtype cmvari e gave more active e than did cmvarii e in side-by-side experiments. by contrast, cmvari e variants and gave very low e accumulation. however, wildtype cmvari e gave relatively high e accumulation, similar to that for cmvinii e variants and the mp c-terminal amino acid deletion constructs showed increased yields compared to the intact mp constructs cmv requires both the mp and cp for cell-to-cell movement in plants, both of which are encoded by rna. thus for both cmvar and cmvin variants, when foreign sequences are cloned into the cp coding region, there is no cell-to-cell movement due to lack of the cmv-encoded cp, and the desired recombinant proteins accumulate only in the initially-infected cells. however, it was shown previously that when the cmv mp was mutated so as to lack the c-terminal amino acids, cmv infections were able to move cell-to-cell in plants even in the absence of cp. therefore, we deleted the mp c-terminal amino acids and compared e and gfp accumulation in plants using the cmvari and cmvarii variants. gfp fluorescence was high for all variants with the amino acid truncated mp. however, comparing e accumulation for all variants, the highest levels of active e were obtained for the amino acid truncated cmvari ewt variant. our assays were for intact, enzymatically active e and we obtained yields up to 21 μg/g of active e corresponding to ~ % of tsp. furthermore, unlike for the wildtype mp variants, the cmvinii 33e variants and gave relatively low accumulation of e discussion several different viruses have been used for protein production in plants, and each has advantages as well as disadvantages. many of the “first generation” plant virus vectors utilized whole plant systemic virus infections to give desired proteins. while many of these have proven to be very useful there are some significant drawbacks. systemic infections can take several days to fully develop. protein production is then asynchronous and yields can vary in different tissues. recombinant viruses also show size constraints for the inserted sequence, often coding sequences of only 1 kb or less can be inserted. then as the infection develops the viruses partially or completely excise the inserted recombinant sequence, leading to loss of the desired intact protein product. furthermore, some viruses may have even more severe size constraints if rna encapsidation is a requirement for development of the systemic infection. then if the coding sequence for the desired protein is large, insertion into the viral rna may preclude encapsidation, thereby preventing efficient spread. recent progress in developing agrobacterium tumefaciens delivered plant virus-based protein production systems has been made by several research groups using different plant viruses. these plant virus-based amplicon systems offer many advantages including the fact that non-transgenic plants can be used, the desired protein production is rapid, the product can accumulate to high levels, and virus-based expression can be temporally regulated to be almost synchronous in all infiltrated areas. because a majority of the infiltrated cells become simultaneously infected, virus movement to new cells is not necessary, encapsidation of recombinant rnas is not an issue. these “second generation” virus-based systems also can retain larger foreign coding sequences and thus produce larger proteins in plants, here we produced enzymatically active  mw e in our previous work, we used the estradiol-inducible, cmv-based cmviva to produce α anti-trypsin in non-transgenic n. benthamiana plants. cmviva has all three cmv genome components in one large kbp plasmid, which, due to its large size is difficult to manipulate. thus, here we took approaches to develop cmv-based inducible as well as autonomously replicating systems, both of which are more easily manipulated and might be able to give high accumulation of heterologous proteins in plants. first, we separated the cmv genomic rna cdnas onto two plasmids, one containing the rna and replication-associated genome components and the other containing the cmv rna genome segment. the cmv rna component is rather small in size,  kb, and is easy to manipulate and to engineer to contain restriction enzyme sites to allow for easy removal of the cmv cp gene and replace it with any gene of interest. the desired restriction enzyme sites were introduced into the intergenic region of rna as expected, these altered the 5′ untranslated leader sequence of the resulting mrna. the sgrna promoter is within the minus strand of rna and is recognized by the rna-dependent rna polymerase and mrna transcription is initiated. for cmv-q rna the transcription initiation starts at nt position in the intergenic region, which is upstream of the modified leader sequences. our analyses demonstrated that the rna modifications affected rna and rna accumulation, but showed even more unpredictable effects on resulting protein accumulation. it does not appear that these can be attributed only to start codon context as the same construct showed different protein yields whether the rna was delivered using cmvin vs.cmvar. in contrast to cmviva, both cmvinii and cmvari and ii variants require mixing a. tumefaciens cells containing different plasmids which are then co-infiltrated into plants and t-dna from the different a. tumefaciens cells containing the cmv plasmids must be transferred to the same plant cell for the complete cmv amplicon. for cmvinii variants this is then followed by induction using estradiol, which resulted in high level accumulation of the proteins tested here. however, like for cmviva, the cmvinii rna deletion does not allow for its replication, only translation of the newly transcribed mrna. therefore we also developed the non-inducible autonomously replicating cmv-based system, cmvari and ii. wildtype cmvar replicated to very high levels and even caused systemic infections in plants. however, when genes for gfp or e were substituted for the cp gene, both proteins were produced in plants within the infiltrated areas, and quantitative analyses showed that high levels of proteins accumulated for both cmvin vs.cmvar, particularly at 6 days post-infiltration. although cmvinii e and variants gave slightly more active e in most experiments, cmvari and ii variants offers advantages in ease of use and thus, two additional approaches to improve accumulation of the desired protein product were investigated. like most viruses having genomes composed of multiple segments, cmv genomic rnas can be mixed to achieve genetic diversity, and this offers opportunities for using cmv to produce desirable proteins in different plant species, as has been demonstrated also by others. therefore, we generated cmv reassortant genotypes by substituting cmv subgroup i genomic rnas and derived from a more virulent cmv, with the original cmv q subgroup ii rna giving cmvari. comparison of cmvari and cmvarii g, e variants showed higher gfp and e for cmvari g, e variants. however, the cmvari ewt showed higher e accumulation than did the corresponding cmvinii ewt, but cmvinii e variants and gave the higher e accumulation than cmvar i and variants thus showing that reassortment alone was not sufficient. as another alternative, we generated a mp c-terminal amino acid deletion mutant. cell-to-cell movement in cmv-infected plants requires interactions between the cp and mp. our cmv-based systems including cmviva, cmvari and ii and cmvinii are cell-to-cell movement deficient since they lack the cp and thus, desired recombinant proteins are produced only within infiltrated cells. however, previous workers demonstrated that the cmv mp c-terminal amino acids are essential to recognize and interact with the cp. when this region is deleted, the cmv infections can spread cell-to-cell even in the absence of cp. in support of this the cmvari and cmvarii 33 g variants showed high cmv-based gfp production. when we created mp amino acid deletion constructs and tested them, they showed increased production of not only gfp in cmvari and ii 33 g variants, but also of e in cmvari and ii 33e variants, and the highest yields of active e were obtained using the cmvari 33e variants. other workers have produced versions of e in various transgenic plants with gene expression driven by different promoters. for example, full-length e containing the catalytic domain, linker and carbohydrate binding domain has been previously produced in transgenic tobacco plants. based on the resulting e activity, yields of up to % on average of total leaf soluble proteins were shown with mac promoter, a chimeric promoter of the camv 35s and mannopine synthase gene. similar yields were shown also with camv 35s promoter. in transgenic z. mays seeds, the full-length e was produced using glob- and yields up to 6% tsp were obtained. in transgenic rice plants, 35s driven e lacking the carbohydrate binding domain but only containing the catalytic domain gave yields up to % tsp. thus, our yields of up to % tsp of intact e in nontransgenic n. benthamiana plants are similar to those achieved for intact e in transgenic tobacco, but less than those in more specialized systems. furthermore, cmvin and cmvar-based production of the desired protein can be temporally regulated to give almost synchronous protein accumulation over a very short time period, even a few days.  our data demonstrate that the cmv-based systems, cmvin and cmvar, are good candidates for production of desired heterologous proteins in nontransgenic plants. our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of their use, and reassortant genotypes and cmv movement protein deletions also allow for greater protein accumulation. also, n. benthamiana, which is particularly suitable for agro infiltration, is a very good plant for protein production, but due to the wide host range of cmv, other plants may also prove to be useful for production of different proteins. abbreviations cmv: cucumber mosaic virus; tmv: tobacco mosaic virus; e1:acidothermus cellulolyticus endo- 4-β-glucanase; gfp: green fluorescent protein; cp: coat protein; orf: open reading frame; mp: movement protein; fw: fresh weight; tsp: total soluble protein; rfp: red fluorescent protein. competing interests the authors declare that they have no competing interests. author’ contributions msh was responsible for experiment design, execution, analysis and wrote the manuscript. bel was responsible for experimental execution including enzymatic activity assays and interpretation. kam helped to conceive the study, discussed, and helped edit the manuscript. bwf helped to conceive the study, helped with organizing the experimental work, data interpretation, and helped to write and edit the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 figure s construction of the pcmvar rna plasmid. click here for file additional file 2 figure s northern blotting analyses of cmvin ii variants. click here for file additional file 3 figure s construction of the pcmvar g and pcmvar e variants for gfp and e click here for file additional file 4 figure s construction of the pcmvar i and ii for subgroup i, ii rna & click here for file additional file 5 figure s construction of the pcmvar 33g and pcmvar 33e for mp amino acid deletion constructs. click here for file additional file 6 figure s systemic symptoms in plants for cmvar ii variants. click here for file additional file 7 figure s immunoblot analysis of e produced in infiltrated n. benthamiana leaves for cmvar i e and cmvar ii e variants. click here for file acknowledgements we are extremely grateful to dr. john lindbo and to dr. mysore sudarshana for many helpful discussions. we are especially grateful to bill adney, senior scientist at the national renewable energy lab, for providing the e antibody. we also thank dr. shouwei ding, university of california, riverside, for the gift of pcassq this work was supported in part by chevron technology ventures, a division of chevron, u.s.a., inc; and nsf cbet- 
also, n. benthamiana, which is particularly suitable for agroinfiltration, is a very good plant for transient protein production. quantitative rt-pcr and immunoblot analysis showed good levels of cmv rna and coat protein accumulation for some variants of both cmvin and cmvar. when genes for e or gfp were inserted in place of the cmv coat protein, both were produced in plants as shown by fluorescence and immunoblot analysis. this reduces the time and cost compared to that required to generate transgenic plants and still gives the comparable yields of active e our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of use. the objective of this work was the development of advanced cmv-based protein production systems to produce acidothermus cellulolyticus endo- 4-β-glucanase in non-transgenic plants. enzymatic activity assays showed that active e was produced in plants with yields up to ~ 11 μg/g fresh weight for specific variant constructs. we used two new cucumber mosaic virus -based vector systems for producing the green fluorescent protein and more importantly, the acidothermus cellulolyticus endo- 4-β-glucanase in non-transgenic nicotiana benthamiana plants. using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. we modified a binary plasmid containing the complete cmv rna cdna to facilitate insertion of desired sequences, and to give modifications of the subgenomic mrna leader sequence yielding several variants. taken together specific variant constructs yielded up to ~21 μg/g fw of e in non-transgenic plants. intact, active e was rapidly produced in non-transgenic plants by using agroinfiltration with the cmv-based systems.
 using plant viruses to produce desirable proteins in plants allows for using non-transgenic plant hosts and if necessary, the ability to make rapid changes in the virus construct for increased or modified protein product yields. the objective of this work was the development of advanced cmv-based protein production systems to produce acidothermus cellulolyticus endo- 4-β-glucanase in non-transgenic plants.  we used two new cucumber mosaic virus -based vector systems for producing the green fluorescent protein and more importantly, the acidothermus cellulolyticus endo- 4-β-glucanase in non-transgenic nicotiana benthamiana plants. these are the inducible cmvin and the autonomously replicating cmvar systems. we modified a binary plasmid containing the complete cmv rna cdna to facilitate insertion of desired sequences, and to give modifications of the subgenomic mrna leader sequence yielding several variants. quantitative rt-pcr and immunoblot analysis showed good levels of cmv rna and coat protein accumulation for some variants of both cmvin and cmvar. when genes for e or gfp were inserted in place of the cmv coat protein, both were produced in plants as shown by fluorescence and immunoblot analysis. enzymatic activity assays showed that active e was produced in plants with yields up to ~ 11 μg/g fresh weight for specific variant constructs. we also compared in vitro cmv genomic rna reassortants, and cmv rna mutants which lacked the c’ terminal amino acids of the 3a movement protein in attempts to further increase e yield. taken together specific variant constructs yielded up to ~21 μg/g fw of e in non-transgenic plants.  intact, active e was rapidly produced in non-transgenic plants by using agroinfiltration with the cmv-based systems. this reduces the time and cost compared to that required to generate transgenic plants and still gives the comparable yields of active e our modifications described here, including manipulating cloning sites for foreign gene introduction, enhance the ease of use. also, n. benthamiana, which is particularly suitable for agroinfiltration, is a very good plant for transient protein production. cucumber mosaic virusprotein productionendoglucanaseagrobacterium tumefaciencesviral vectortransient protein expressionnicotiana benthamiana 