Patent Application: US-93032901-A

Abstract:
a novel method of over expressing genes in plants is provided . this method is based on the rna amplification properties of plus strand rna viruses of plants . a chimeric multicistronic gene is constructed containing a plant promoter , viral replication origins , a viral movement protein gene , and one or more foreign genes under control of viral subgenomic promoters . plants containing one or more of these recombinant rna transcripts are inoculated with helper virus . in the presence of helper virus recombinant transcripts are replicated producing high levels of foreign gene rna . sequences are provided for the high level expression of the enzyme chloramphenicol acetyltransferase in tobacco plants by replicon rna amplification with helper viruses and movement protein genes derived from the tobamovirus group .

Description:
the instant invention provides high level expression of foreign genes in plants by viral replicons wherein the replicons possess improved genetic stability . the replicons of the instant invention are produced in host plant cells by transcription of integrated transgenes . the replicons of the instant invention are derived , in part , from single stranded plus sense plant rna viruses . the replicons of the instant invention code for at least one foreign gene and possess sequences required in cis for replication (“ replication origins ”). fig2 ( c ). the replicons are produced by host cell transcription of a chromosomally integrated transgene to form an rna transcript . the transgene is a dna sequence that codes for the replicon and also contains a promoter and a transcription termination region . fig2 ( a ). the replicon is generated from an rna transcript of the transgene by rna processing and replication in the presence of a helper virus . fig2 ( b ). the replicons of the instant invention lack functional replication protein sequences . because the replicons of the instant invention lack replication protein sequences , they must rely on genetic complementation with helper viruses for replication . the replicon &# 39 ; s dependency on the helper virus for replication enables regulatable amplification of these replicons through the introduction of the helper virus . genetic complementation of the replicon with a helper virus provides many advantages over autonomous viral vectors for amplifying gene expression . each infected cell of a transgenic plant contains a correct master copy of the gene to be amplified . this reduces the effects of genetic drift in replicating rna populations that can result in sequence instabilities and point mutations after prolonged replication of an rna vector ( kearney , c . m ., et al ., virology ( in press )). in a further embodiment of the instant invention , the replicon codes for at least one sequence upon which the helper virus is dependent . thus , in this further embodiment , the replicon and the helper virus are mutually dependent . [ see fig3 ]. helper virus dependence on the replicon insures amplified expression of the replicon sequences by the helper virus in whole plants . in a further embodiment , the replicon codes for a functional movement protein such as the 30kda tmv movement protein . the helper virus used in this embodiment does not possess a functional movement protein . thus , the helper virus is dependent on the replicon for movement functionality . movement proteins are necessary for cell to cell movement in plants . by placing a functional movement protein sequence on the replicon and either deactivating or deleting the same sequence on the helper virus or by using a host species with helper virus encoded movement protein incompatibility , the helper virus &# 39 ; s dependency on the replicon enables systemic infection of the whole plant with the viral replicon plus helper virus . this embodiment of the instant invention has the further advantage that the only virus released into the environment will be a debilitated helper virus . thus , the helper virus will not be able to spread in plants that do not already contain a functional copy of the viral movement protein . this embodiment provides an option for more stringent levels of biological containment which may be desirable in some cases for large scale commercial production . in a preferred embodiment , the replicon is formulated such that the sequences encoding the replication origins and the movement functions are linked to the foreign gene sequences . the chromosomally integrated transgene that codes for the replicon is transcribed by host rna polymerase ii producing recombinant mrnas . in the presence of a helper virus , these transcripts are replicated as additional replicon components in a mixed population . during viral replication , subgenomic messenger rna may be produced from replicon rna resulting in amplified expression of foreign genes . the most preferred replicon gene arrangement places the foreign gene at the extreme 3 ′ end of the genome where the viral structural protein is normally encoded . see fig4 . this position for the foreign gene at the extreme 3 ′ end of the genome , as depicted in fig4 is critical for high level expression ( culver , j . n ., et al ., virology ( in press )). however , the protein coding sequences or other gene sequences located between the replication origins may be functional in any order . additional preferred embodiments of the replicon sequence include the use of regulatable promoters to control expression of the foreign gene and / or movement protein . one promoter for expression of a fusion protein containing the foreign protein or a series of subgenomic promoters may be employed . self - cleaving ribozymes or a polyadenylation region may also be employed as the transcription termination regions . the replicons are generated in vivo in plants through transcription of transgenes that are integrated into the host plant cell chromosome and through replication in the presence of a helper virus . the transgenes can be introduced into the host plant cell chromosome by known transformation methods using a variety of promoters . after the replicon has been introduced into the host , the resulting transgenic plants are grown to an optimized stage at which point a helper virus strain is added . the replicons are then amplified by the introduced helper virus and the foreign gene is expressed . the foreign gene product coded for and expressed by the replicon can be a very wide variety of rna or proteins products and include , for example , antisense and ribozyme rna , regulatory enzymes , and structural , regulatory and therapeutic proteins that may be expressed in their native form or as gene fusions . typical therapeutic proteins include members of the interleukin family of proteins and colony stimulating factors such as csf - g , csf - gm and csf - m . it is understood , however , that any therapeutic protein can be coded for and expressed in the instant invention . if expression of the foreign gene results in the accumulation of a protein or other material in the plant tissues , that resulting product may be harvested once the desired concentration of that product is achieved . significant quantities of recombinant proteins , nucleic acids or other metabolites can be inexpensively produced using this procedure . the low level of expression and wide variation that is observed in transgenic organisms chromosomally transformed with the same construct ( a phenomenon attributed to “ position effects ”), is avoided by this method . rna - based amplification is not critically dependent on initial transcript amounts . there is also no theoretical limit to the number of genes that can be amplified at the rna level . the target gene remains “ off ” before amplification because subgenomic mrna is only produced during viral replication . therefore this approach might be particularly appropriate for controlling complex biochemical pathways or producing products that are toxic to the plant . it would be feasible for example , to overexpress critical enzymes in a pathway and simultaneously down - regulate other genes by amplifying antisense rna only after inoculation with a helper virus . these types of manipulations are not possible using existing or proposed technologies for chromosomal transformation of plants or plant cell cultures or by using prior art viral vectors . construction of a transgene derived from tmv is set forth herein . the wild type tmv genome is set forth in fig1 . the construction of dna plasmids containing the 5 ′ replication origin fused to the camv 35s promoter are described in ( ow , d . w ., et al ., science 234 : 856 - 859 ( 1986 )) and the 3 ′ replication origin fused to a ribozyme termination region are described by turpen , t . h ., ph . d . disertation , university of california , riverside , pp . 88 - 105 ( 1992 ). the substitution of the coat protein gene for the coding sequence of cat is described in dawson , et al ., phytopathol . 78 : 783 - 789 ( 1988 ). these previously disclosed plasmids , pbgc43 , pbgc44 , pbgc75 ( turpen , t . h ., ph . d . disertation , university of california , riverside , pp . 88 - 105 ( 1992 )) and ptmvs3cat28 ( dawson , et al ., phytopathol . 78 : 783 - 789 ( 1988 )) are used as precursors for the construction of the desired transgene for synthesis of replicon rna ( fig5 ). in this construction , it is desired to place the 30 - kda movement protein gene at precisely the same position as the replicase gene ( relative to 5 ′ replication origin in the wild type tmv genome , see fig5 ). to accomplish this , a ndei site is introduced at the start codon of each gene by pcr - based mutagenesis using synthetic primers and unique adjacent cloning sites . a 270 bp mutagenesis product containing the internal ndei site from the pcr primer is subcloned using the ecorv site in the cauliflower mosaic virus 35s promoter and the hindiii site in the 30 - kda protein gene . the ligation product is then sequence verified . the 3 ′ segment of the replicon , containing the cat gene will be placed adjacent to the 3 ′- ribozyme as a hindiii - nsii fragment from the transient tmv vector ptmvs3cat28 ( fig5 ). in the final cloning step , the 5 ′ portion of the transgene and the 3 ′ portion will be subcloned into the unique bamhi site of the plant transformation vector pap2034 ( velton and schell , nar 13 : 6981 - 6998 ( 1985 ) as a bg1ii - bamhi fragment described previously ( turpen , t . h ., ph . d . disertation , university of california , riverside , pp . 88 - 132 ( 1992 )). the sequence of the replicon rna , produced by host transcription , rna processing , and replication in the presence of a helper virus , is given in fig6 . thus , the foreign gene ( cat ) is placed on a rna viral replicon , under control of the coat protein subgenomic promoter for messenger rna synthesis ( located at the 3 ′ end of the movement protein gene ). in one embodiment of this invention , agrobacterium tumefaciens is used for insertion of this sequence into the plant chromosome as described previously ( turpen , t . h ., ph . d . dissertation , university of california , riverside , pp . 106 - 132 ( 1992 )). the transformation vector pap2034 is a cointegrating type agrobacterium vector . pap2034 containing the transcription unit for the production of replicon rna is mobilized into a . tumefaciens by conjugation using the helper strain gj23 ( van haute , e ., joos , et al ., embo j . 2 : 411 - 417 ( 1983 )). transconjugants are selected and the structure of the cointegrate between donor plasmid and the disarmed ti plasmid pgv3850 ( zambryski , p ., et al ., embo j . 2 : 2143 - 2150 ( 1983 )) is confirmed by southern blot hybridization . a correct homologous recombination event places the transgene construct between the t - dna borders . axenic leaf segments of n . tabacum cv . xanthi are treated ( horsch , r . b ., et al ., leaf disc transformation , plant molecular biology manual . ( s . b . gelvin , r . a . schilperoort , and d . p . s . verma , eds . ), kluwer academic publishers , dordrecht , the netherlands , pp . a5 : 1 - 9 ( 1988 )) in the following sequence : day 1 ; leaf segments are dipped in a . tumefaciens liquid culture and placed on regeneration media ( rm ), day 3 ; explants are transferred to rm supplemented with cefotaxime ( 500 μg / ml ), day 5 ; explants are transferred to rm / cefotaxime ( 500 μg / ml )+ kanamycin ( 100 μg / ml ), day 30 - 40 ; shoots excised and placed onto rooting media containing cefotaxime ( 500 μg / ml ) and kanamycin ( 100 μg / ml ). cultures are maintained under continuous fluorescent light ( sylvania gte , gro - lux ws ) at 20 ° c . hardened plants are grown in commercial potting soil ( cascade forest products inc ., arcata , calif .) at a temperature of 21 - 29 ° c ., with a controlled release fertilizer ( osmocote , 14 - 14 - 14 ) using natural light ( vacaville , calif .) supplemented with fluorescent light on a 16 hr day length in an indoor greenhouse . the antibiotic resistance trait carried in transgenic lines is scored by germinating seedlings in sterile agar in the presence of 100 ug / ml kanamycin ( dunsmuir , p ., et al ., stability of introduced genes and stability of expression , plant molecular biology manual . ( s . b . gelvin , r . a . schilperoort , and d . p . s . verma , eds . ), kluwer academic publishers , dordrecht , the netherlands , pp . c1 : 1 - 17 ( 1988 )). the sequence of the replicon rna , produced by host transcription , rna processing , and replication in the presence of a helper virus , is given in fig6 . tobamoviruses with mutations or naturally occurring variation in the 30 - kda protein gene are deficient in cell - to - cell movement on specific host species . transgenic plants or alternate hosts can complement this defect . it will be appreciated to those skilled in the art that there are numerous methods of producing helper tobamoviruses by genetic engineering or by mutagenesis in addition to those helper variants or host species combinations occurring naturally . likewise , methods for producing transgenic plants which express 30 kda protein and which complement defective 30 kda containing viruses have been published . for example , movement deficient helper viruses can be synthesized by transcription of tmv with known mutations for the production of rna inoculum . transgenic plants expressing the 30 - kda protein complement this defect ( deom , c . m ., et al ., science 237 : 389 - 394 ( 1987 )). therefore , large quantities of a helper virus can be propagated . in one embodiment of this invention , a 30 - kda protein frameshift mutant , having a single base pair deletion at position 4931 thereby creating a ecorv site in the cdna , is used as helper virus . transgenic tobacco (˜ 100 plants ) are regenerated containing this replicon transgene construction and assayed for cat activity in the presence and absence of helper viruses using procedures described ( shaw , w . v ., chloramphenicol acetyltransferase from chloramphenicol - resistant bacteria , methods in enzymology , vol . 53 , ( s . fleischer and l . packer , eds . ), pp . 737 - 755 ( 1975 )). 200 mg of leaf tissue is macerated in assay buffer followed by the addition of 0 . 5 mm acetyl coa and 0 . 1 uci [ 14 c ] chloramphenicol , incubation for 45 min at 37 ° c ., extraction , resolution by thin - layer chromatography , and autoradiography . while the invention of this patent application is disclosed by reference to the details of preferred embodiments of the invention , it is to be understood that this disclosure is intended in an illustrative rather than limiting sense , as it is contemplated that modifications will readily occur to those skilled in the art , within the spirit of the invention and the scope of the appended claims . it is further understood that the instant invention applies to all viruses infecting plants and plants generally and is not limited to those plasmids , viruses or plants described herein . caauuacau aug gcu cua guu guu aaa gga aaa gug aau auc aau gag uuu 111 auc gac cug aca aaa aug gag aag auc uua ccg ucg aug uuu acc ccu 159 gua aag agu guu aug ugu ucc aaa guu gau aaa aua aug guu cau gag 207 aau gag uca uug uca gag gug aac cuu uuu aaa gga 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