Patent Application: US-89364007-A

Abstract:
a method of activating the rice tungro bacilliform virus promoter in vivo is disclosed . the rtbv promoter is activated by exposure to at least one protein selected from the group consisting of rf2 a and rf2 b .

Description:
phloem specific expression of the rtbv promoter . rtbv is known to accumulate in phloem tissues in infected rice plants . similarly , the full length rtbv promoter ( fl ) and the e fragment of the promoter are expressed only in phloem tissues in transgenic rice plants ( 7 , 10 , 22 ). within the e fragment , there are several cis dna sequence elements that are conserved among vascular tissues ( 10 ). box ii is a cis element that is responsible for the basal behavior of the promoter . box ii shares homology with cis elements from other phloem and xylem specific promoters ( 10 ). data from 5 ′ deletion analyses showed that as long as box ii cis element is retained , the promoter is expressed in phloem tissues , although activity of the promoter is much lower in the absence of box ii than when it is present . it is known that some plant promoters retain specific expression patterns in different plant species ( 20 - 31 ) while others do not ( 32 , 33 ). rf2a can activate the promoter in cell types other than phloem cells . as we reported previously , the rtbv promoter contains several important cis elements , such as box ii , that contribute to expression of the promoter in vascular tissues . data from 5 ′ deletion analyses showed that as long as the box ii cis element is retained , the promoter is expressed in phloem tissues , although activity of the promoter is much lower in the absence of box ii than when it was present ( 10 ). he et al ( 15 ) reported that when the element , which contains box ii , was deleted or mutated in the context of the e fragment , the promoter activity dropped to basal levels . to better understand the interactions between rf2a and the rtbv promoter , the following plasmids were constructed : pga - fl :: gus , which contains the full length rtbv promoter and the uida reporter gene , uida ; pga - e :: gus , which contains the e fragment and the uida reporter gene ; and pet - rf2a , which contains the coding sequence for rf2a . to study the effects of co - expression of rf2a and the rtbv promoter , co - expression plasmids were constructed whereby the fusion gene p35s :: rf2a ( which contains the camv35s promoter , the coding sequence of rf2a and a nos terminator ) was cloned into pga - fl :: gus and pga - e :: gus . the resultant co - expression plasmids , each containing the box ii cis element , are pga - fl :: gus / p35s :: rf2a and pga - e :: gus / p35s :: rf2a . when plant cells were transformed with the co - expression plasmid , the pattern of gus activity in transgenic plants was constitutive rather than phloem specific . this is consistent with the known pattern of expression of the 35s promoter ( 17 ) and indicates that rf2a is involved in regulating expression of the rtbv promoter . many factors can influence the activity of transcription factors in controlling expression of the promoter , including affinity of binding of transcription factor with the dna element ( 35 ), the subcellular distribution of factors , post - transcriptional modifications of the factor ( 36 ), and synergistic interactions with other proteins ( 37 - 41 ). furthermore , it is common to construct promoters by combining multiple upstream activation sequences and by replicating activation domains on dna binding proteins . in the activation system we described here , a single copy of box ii together with rf2a was sufficient to increase expression of the rtbv promoter . in addition , the level of expression was positively correlated with the level of rf2a . other reports indicate that expression of p - 35s :: gus fusion genes in transgenic plants range from 321 u ( iu is equivalent to pmol 4 - mu min - − 1 mg - − 1 ) to 113 , 000 u ( 17 , 27 , 42 , 43 ). in our studies transgenic tobacco plants had gus activity between 4 , 000 u and 18 , 000 u when p - fl :: gus or p - e :: gus were activated by expression of the p35s :: rf2a enhancer gene , representing a 2 to 20 fold activation of the reporter gene . although we do not yet know the basis for the strong activation of the fl and e promoters by rf2a , there are several notable characteristics of this system . first , rf2a binds to a cis element ( box ii ) that is located very close to the tata box ( within approximately 7 nt ). it is not yet known if proximity of the cis element to the tata sequence is important for the activity of rf2a in expressing this promoter . second , the results of in vitro studies indicate that there is a direct physical interaction between tata binding protein ( tbp ), and rf2a ( q . zhu et al ., in preparation ). third , rf2a has three putative activation domains : acidic , glutamine rich , and proline rich domains ( 11 ), any or all of which may be important for the activity of rf2a . we propose that proximity of the cis element and the unique features of rf2a contribute to the strong activation of the promoter by rf2a . in transgenic plants in which the reporter genes are activated by the gene p35s :: rf2a , it is most likely that promoter activation is due to formation of homodimers of rf2a . however , the activation by heterodimerization of rf2a with homologous factors in tobacco cannot be completely excluded . the observation that e fragment and fl promoters are expressed in phloem tissues in tobacco plants suggests that there is / are rf2a - like transcription factor ( s ) in tobacco . a dominant negative mutant of rf2a affects plant development . in rice plants , rf2a acts as a transcription activator and its biological function is linked to the development of the vascular system : this conclusion was based upon the results of experiments with transgenic rice plants in which the levels of rf2a were reduced by an anti - sense approach ( 11 ). an alternative approach to determine gene function is to use directed mutagenesis of rf2a and determine the effects of mutations on the function of genes that require the activity of rf2a and proteins that form heterodimers with rf2a . mutants of bzip transcription factors have been used in such studies and found to act as dominant negative mutants ( 44 - 46 ). here , the mutant 3δ , which contains only the dna binding domain and the leucine zipper region , was created to test the biological function of rf2a - like transcription factors in tobacco , and the effect of 3δ on expression of the rtbv promoter . this study , showed that the 3δ mutant is capable of forming homodimers . the 3δ mutant also formed heterodimers with rf2a . both dimers bind to box ii in vitro . expression pattern of the rtbv promoter in transgenic tobacco plants . the rtbv full length ( fl ) promoter containing nucleotides − 731 to + 45 of the rtbv genome , is expressed exclusively in phloem tissues in transgenic rice plants ( 7 , 10 , 22 ). a fragment of the rtbv promoter , termed the e fragment and comprising nucleotides − 164 to + 45 , is also expressed exclusively in phloem tissues ( 7 ). within the e fragment four cis sequence elements that contribute to phloem specific gene expression have been described , including the gata motif , asl ( as - 1 like ) element , box ii and box i sequence elements ( as shown in fig1 a ) ( 10 ). since the host range of rtbv is limited to rice , we wished to determine whether the rtbv promoter is functional and maintains tissue specificity in transgenic tobacco plants . the rtbv promoter fl and the e fragment were ligated with the uida coding sequence to produce the chimeric genes p - fl :: gus and p - e :: gus . the genes were ligated to a t 1 , plasmid vector ( as shown in fig1 a ) and introduced into tobacco through agrobacterium - mediated transformation . 17 and 23 independent transgenic tobacco plants were developed for the p - fl :: gus and p - e :: gus constructs , respectively . a detailed histochemical analysis of gus expression patterns in transgenic tobacco plants showed that expression of p - e :: gus and p - fl :: gus were essentially the same ( as shown in fig2 ). in leaves of transgenic plants with p - e :: gus , strong gus activity was observed in vascular tissues ( as shown in fig2 a , c ) although in very young leaves there was a low amount of gus activity in mesophyll cells ( as shown in fig2 a ). cross sections through the midrib of more mature leaves showed gus activity only in phloem cells ( as shown in fig2 g ). these results are similar to those reported from studies of transgenic rice plants ( 10 ). modulation of phloem specific expression of the rtbv promoter by rf2a . rf2a , was isolated by virtue of its interaction with box ii dna ( 10 ); a cdna encoding rf2a was subsequently isolated and characterized . rf2a is a bzip protein with three potential functional domains : a proline - rich domain , an acidic domain and a glutamine - rich domain ( as shown in fig1 a ) ( 11 ). a mutant of rf2a that lacks these three putative functional domains was constructed and is referred to as “ 3δ ” ( as shown in fig1 a , b ). the 3δ protein contains the dna binding and leucine - zipper domains ( bzip ) of rf2a , including the putative nuclear localization signal . to determine the regulatory functions of rf2a and 3δ mutant on expression of the fl and e fragment promoters in transgenic tobacco plants , the following t 1 , binary plasmids were constructed : pga - e :: gus / p - 35s :: rf2a , pga - e :: gus / p - 35s :: 3δ , pga - fl :: gus / p - 35s :: rf2a , pga - fl :: gus / p - 35s :: 3δ ( as shown in fig1 b ). at least 15 independent transgenic tobacco lines with each construct were developed through agrobacterium - mediated transformation . the integration of the full - length t - dna in transgenic plants was confirmed by southern blot hybridization analysis ( data not shown ). histochemical analysis of gus activity in the ti progeny showed that expression of the reporter genes was substantially altered by co - expression of rf2a . in the plants that contained either p - e :: gus / p - 35s :: rf2a or p - fl :: gus / p - 35s :: rf2a , gus activity was detected throughout the cotyledonary leaves ( as shown in fig2 b ) and true leaves ( as shown in fig2 c ). cross sections through the leaf midrib of these plants showed very strong gus activity in phloem cells , the epidermis and trichomes ( compare g and h in fig2 ). the palisade and spongy mesophyll cells of the leaf lamina also exhibited intense staining ( compare fig2 l and k ) as did parenchyma cells of the midrib ( compare i and j in fig2 ). guard cells showed strong gus activity when the fl :: gus gene was co - transformed with p - 35s :: r - f2a ( not shown ). in the root tissues of transgenic plants that contained p - 3 :: gus / p - 35s :: rf2a or p - fl :: gus / p35s :: rf2a , there was a high level gus activity throughout the cortex ( as shown in fig2 f ), and very strong levels of gus activity in root tips . in contrast , root tissues of transgenic plants that were transformed only with reporter genes revealed gus activity only in the vascular cylinder ( as shown in fig2 e ). gus activity in the leaves of t 0 transgenic tobacco plants was quantified using mug in a fluorescence assay . as anticipated , there were variations in the amount of gus activity among transgenic lines for each construct . the gus activity of transgenic plants with p - fl :: gus was higher than the activity in plants with p - e :: gus genes ( as shown in fig3 ) when rf2a was co - expressed with either p - e :: gus or pfl :: gus , the gus activity was increased by 2 to 20 fold compared with plants that lacked rf2a ( as shown in fig3 ). the increase of gus activity is in agreement with the observation that the pattern of gus activity was constitutive when rf2a was co - expressed with the reporter gene ( as shown in fig2 ). to establish a correlation between the gus activity and the amount of rf2a , elisa reactions were carried out to quantitatively measure the amount of rf2a in individual transgenic plants . soluble protein samples of t 0 plants were triturated and aliquots of soluble proteins were tested in quantitative elisa reactions using anti - rf2a antibodies . aliquots of the same set of extracts were also used in quantitative analyses of gus activity . as shown in fig4 , the results of these assays showed a positive correlation between the amount of rf2a in the samples and gus activity . dna binding and heterodimerization of rf2a and 3δ . the gene encoding the mutant protein 3δ , which lacks the three putative regulatory domains of rf2a , was constructed to determine if it can restrict expression of the rtbv promoter . it has been reported that sequences outside of the leucine zipper region of such proteins can contribute to dimer stabilization ( 28 ). in these cases deletions of other ( putative ) domains can affect the ability of the protein to form homo - or heterodimers , and to bind dna . to test the dna binding ability of mutant protein 3δ , emsas were carried out ( as shown in fig5 ). rf2a and 3δ can each bind 32p - labeled boxiiml , presumably by forming homodimers ( lanes 2 and 5 ). when both proteins are added , a band with mobility intermediate with the mobilities of the probe to which rf2a and 3δ homodimers are bound was observed . this band is presumed to correspond to the binding of the heterodimer rf2a / 3δ with the probe ( lane 4 ). all of these complexes can be competed by 80 × molar excess of unlabeled cold probe ( lane 3 ). morphological changes of transgenic plants with 3δ . a gene encoding 3δ protein was introduced along with the reporter gene into tobacco ( fig1 b ), and 15 independent lines carrying either the p - e :: gus / p - 355 :: 3 δ or the p - fl :: gus / p - 35s :: 3δ genes were developed . of these , 7 lines carrying the p - e :: gus / p - 35s :: 3δ genes , and 6 lines carrying the p - fl :: gus / p - 35s :: 3δ genes exhibited abnormal phenotypes . in contrast , no abnormal phenotypes were observed in transgenic lines with reporter gene constructs alone , or reporter genes plus the rf2a gene . the abnormal t o transgenic lines carrying p - e :: gus / p - 35s :: 3δ or p - fl :: gus / p - 35s :: 3δ genes were characterized by downward curving of the mid - vein ( fig6 d , e , f ), and the most severely affected plants were stunted ( as shown in fig6 a ). two of the affected plants exhibited abnormal floral development : these lines did not produce seeds . to confirm that the abnormal phenotypes were due to the transgene , we analyzed the t 1 , generation of the abnormal plant lines that gave seeds . among all of the 11 lines tested , the abnormal phenotype was inherited through the second generation . line 1 of plants carrying p - fl :: gus / p - 35s :: 3δ showed an abnormal segregation pattern , while the segregation of phenotype in other lines , for the most part , followed classical , single locus segregation patterns . abnormal plants grew much more slowly than the normal plants in t 1 progeny , and were characterized by stunting of shoots and roots ( as shown in fig6 a , q ). the abnormal phenotype of t 1 plants that contained the p - e :: gus / p - 35s :: 3δ and p - fl :: gus / p - 35s :: 3δ genes can be characterized as either mild ( as shown in fig6 b ) or severe ( as shown in fig6 f ). plants with the mild phenotype grew much more slowly than non - transgenic control plants but they ultimately achieved normal height . these plants had wrinkled or distorted leaves ( as shown in fig6 d , e ) with yellow and green mosaic leaf color and exhibited reduced apical dominance and the appearance of lateral shoots ( as shown in fig6 b , see arrows ). plants that exhibited a severe phenotype were stunted with very short internodes , thick leaf lamina , and an increased number of side shoots ( as shown in fig6 f ). the developmental problems in plants were severe and the plants did not produce fertile flowers . expression of the 3δ gene in the t 1 progeny was confirmed by northern blot analysis ( data not shown ). there was no correlation between severity of the abnormal phenotype and the accumulation of mrna derived from the transgene . gus activity of transgenic lines with the p - e :: gus / p - 35s :: 3δ or p - fl :: gus / p - 35s :: 3δ genes were analyzed . the transgenic lines that did not show an abnormal phenotype had the same levels of gus activity as the lines that contained only the reporter gene ( i . e . p - e :: gus or p - fl :: gus ) and there was no clear indication of gene activation or repression . some of the lines that exhibited abnormal morphology ( i . e ., lines j , n , o of p - e :: gus / p35s :: 3δ ; lines e , h , k of pfl :: gus / p - 35s :: 3δ ) had a similar level of gus activity as transgenic lines that contained only the reporter genes . no correlation was established between severity of plant phenotype and gus activity . in some lines with severely abnormal phenotypes , gus activity increased with the age of the plants . these plants were physiologically different from normal plants and we suggest that factors other than those related to the 3δ mutant protein may affect expression of the rtbv promoter in these plants . plasmid constructions . the complete rtbv promoter ( nucleotides − 731 to 45 ) ligated with the uida coding sequence was released from plasmid pmb9089 ( 7 ) by digestion with xbai and kpni . the chimeric gene comprising the e fragment of the rtbv promoter ( nucleotides − 164 to + 45 ) and the uida coding sequence was released from plasmid pmb9089 ( 7 ) by digestion with hind iii and kpn i . inserts were purified and ligated into the binary vector pga482 through xba i and kpn i or hind iii and kpn i sites . the resultant plasmids are named pga - fl :: gus and pgae :: gus , respectively ( as shown in fig1 b ). to construct plasmids for in vitro expression of rf2a and the bzip dna binding domain of rf2a ( referred to as 3δ ) ( as shown in fig1 a ) the plasmid pet - 12a , which contains the cdna encoding rf2a , ( 11 ) was used . the rf2a coding sequence was amplified by pcr from the starting plasmid by using primers 5 ′ rf2a ( gccgcc catatg gagaagatgaacagggagaaatcc ) ( seq id no : 3 ) and 3 ′ rf2a ( cg cgga tcc tcagttgccgctgcttcctga ) ( seq id no : 4 ). ndel and bamhi sites were introduced into the 5 ′ and 3 ′ primers , respectively ( underlined ). the coding sequence for 3δ , comprising amino acids 108 to 283 of rf2a ( 11 ), was amplified by pcr from the rf2a cdna by using primers 5 ′ δpδqδa - rf2a ( gccgcc catatg gagaagatgtccgccgccgccca ) ( seq id no : 5 ) and 3 ′ δ - rf2a ( cg cggatcc tcagtgtggcatgccaccgaa ) ( seq id no : 6 ). as described above , nde l and bam hi sites were introduced into the primers . pfu dna polymerase ( stratagene ) was used in all pcr reactions . the pcr products were digested with nde i and bam hi and inserted into pet28a ( novagen ) restricted by the same enzymes . the new constructs were named pet - rf2a and pet - 3δ the sequence of inserts in each plasmid was confirmed by dna sequencing . plant transformation vectors for co - expression of the reporter gene and the effector proteins were constructed . the coding sequences of rf2a and 3δ insert were released from pet - rf2a and pet - 3a with nde i and bam hi . the dna fragments were then cloned into the plant expression vector pmon999 ( a gift from monsanto co . ), which contains the camv 35s promoter ( p35s ) and nos terminator , after the plasmid was restricted by xba i ( blunted with klenow plus dntps ) and bam hi . the fusion genes named p35s :: rf2a or p35s :: 3δ were released by noti and blunted with klenow polymerase plus dntps . the two fusion genes were then cloned into binary vectors pga - e :: gus or pgafl :: gus through the blunted clal site . in the co - expression vectors , the reporter gene and effector genes are in head to tail orientation . the final plasmids were named pga - e :: gus / p35s :: rf2a , pga - fl :: gus / p - 35s :: rf2a , pga - e ::/ gus / p - 35s :: 3δ and pga - fl :: gus / 35s .. 3δ ( as shown in fig1 b ). tobacco transformation . gene constructs containing pga482 derived plasmids ( as shown in fig1 b ) were introduced into agrobacterium tumefaciens strain lba4404 by electroporation and used for agrobacterium - mediated transformation . leaf discs from nicotiana tabacum cv . xanthi nn were used for transformation following the protocol described by horsch et al ( 25 ). at least 14 independent transgenic lines for each construct were produced and grown in a greenhouse . the transgenic plants were self - fertilized and t 1 seeds were collected . for the analysis of the t 1 generation , seeds were germinated on murashige and skoog ( ms ) medium ( 26 ) with or without kanamycin ( 100 mg / l ) selection and the seedlings were grown in a greenhouse . analysis of gus activity . histochemical analysis of β - glucuronidase ( gus ) activity was performed essentially as described by jefferson et al ( 27 ). hand - cut fresh tissue sections of leaves and stems of primary transformants t 0 or t 1 progeny were incubated at 37 ° c . for 4 to 12 hours in reaction buffer containing 1 mm 5 - bromo - 4 - chloro - 3 - indolyl glucuronide ( x - gluc ) ( research organic ), 100 mm sodium phosphate buffer ph 7 , 2 mm potassium ferrocyanide and potassium ferricyanide , 0 . 1 % triton x - 100 and 20 % methanol . for analysis of young t 1 seedlings , whole platelets were collected about one week after germination and immersed in the buffer containing x - gluc . after vacuum infiltration , incubation was carried out overnight at 37 ° c . samples were cleared by several washes with 70 % ethanol . stained sections were immersed in 75 % glycerol and visualized by optical microscopy . quantitative gus analyses using the substrate 4 , methylumebelliferone - β - s glucuronide ( mug ) were performed as described by jefferson et al . ( 27 ). elisa . to detect rf2a in extracts of transgenic tobacco leaves , sandwich elisas were performed . 96 - well microfilter plates ( nunc , maxisorp ) were coated overnight at 4 ° c . with protein a at 1 ug / ml in pbs , 100 ul / well ( pierce ). plates were incubated for 1 hour at room temperature in blocking buffer [ 1 × pbs containing 10 % ( v / v ) fetal bovine serum . ( fbs )]. plates were incubated with the purified anti - rf2a antibody ( 7 ) at 1 ug / ml in 1 × pbs plus 1 % ( v / v ) fbs , for 1 hour at room temperature . plant extracts in 50 mm tris - hcl , ph 7 . 5 , 0 . 1 % v / v ) triton x 100 were added and the plates were incubated overnight at 4 ° c . residual protein a binding sites were blocked with protein a at 1 ug / ml in 1 × pbs plus 1 % ( v / v ) fbs . plates were incubated again with the purified anti - rf2a antibody , 1 ug / ml in 1 × pbs plus 1 %. ( v / v ) fbs , for 1 hour at room temperature , and then with protein - a - hrp ( pierce ) at 1 ug / ml in 1 × pbs plus 1 % ( v / v ) fbs and developed with tmb ( kpl , maryland , usa ). plates were read at 650 nm with an absorbency plate reader . between each incubation step , five washes with 1 × pbs were performed . purified rf2a was used to create a standard curve to quantify the amount of rf2a in each sample . production of rf2a and 3δ in e . coli . pet - rf2a and pet - 3δ were transformed into e . coli bl21 ( de3 )/ plyse . cells were grown at 37 ° c . to an od 600 of 0 . 5 - 0 . 6 , induced by 0 . 5 mm isopropyl - 8 - d - thiogalactopyranoside ( iptg ) for 3 h at 250 c . the cell pellets were resuspended in 20 mm tris - hcl ph 8 , 500 mm nacl , 0 . 1 % np40 , 1 mm pmsf , 1 mg / ml lysozyme , 10 ug / ml dnaase and 10 ug / ml rnase . lysis was completed using a french press . under these conditions the target proteins were soluble , and were dialyzed against silvana and stored at − 80 ° c . electrophoretic mobility shift assays ( emsas ). emsas were carried out essentially as described by yin and beachy ( 7 ). 500 ng of e . coli protein extracts were incubated with 32 p labeled dna probes comprising box iiml that was constructed using annealed oligonucleotides ( 10 ). for competition emsa , unlabeled oligonucleotides were added to the binding reactions at 80 fold molar excess relative to the labeled probe ( see fig5 ). 1 . roeder , r . g . 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