Patent Application: US-81324904-A

Abstract:
the invention relates to a method for efficient rna silencing of target genes in eucaryotic cells , particularly plant cells . consequently , the method can be used to reduce the phenotypic expression of an endogenous gene in a plant cell . furthermore , the method can be applied in a high throughput screening for mutant phenotypes as a result of rna silencing of any endogene .

Description:
a post - transcriptionally silenced inverted repeat transgene locus can trigger silencing of a reporter gene producing non - homologous transcripts . we studied the interaction between three transgene loci x 1 , y 1 and z 1 ( fig2 . for a detailed description of all loci and constructs , see materials and methods ) to address the question whether or not a stepwise homology between loci can lead to silencing . it has been demonstrated previously that the post - transcriptionally silenced nptii genes in locus xi are capable to in trans silence transiently expressed genes with partial transcript homology to their nptii transcripts ( van houdt et al ., 2000 b ). we subsequently found that also a stably expressed β - glucuronidase ( gus ) gene ( in locus y 1 ), with partial transcript homology to the nptii transcripts of the silencing inducing locus x 1 , becomes efficiently silenced in trans ( fig2 : x 1 and y 1 and table 1 : x 1 y 1 compared to y 1 ). on the contrary , the nptii genes of locus x 1 are not able to trigger silencing of the gus genes in locus z 1 which is expected as the genes of both loci produce transcripts without significant homology ( fig2 ). the homology between the two transcripts of x 1 and y 1 is mainly situated in the 3 ′ untranslated region ( 250 nucleotides ), but also the 5 ′ untranslated sequences show a small region of homology ( 29 nucleotides ). these results demonstrate that the in trans silencing effects are not triggered by promoter homology . when y 1 and z 1 loci are combined in so called y 1 z 1 hybrids both types of gus genes , having transcript homology in the gus coding sequence of 1809 nucleotides , remain highly expressed as reflected in the normal gus activity showing that the rna silencing mechanism does not become activated ( table 1 : y 1 z 1 compared to y 1 and z 1 ). surprisingly , upon creation of a stepwise homology between x 1 and z 1 by introducing locus y 1 , the new observation described here is that also the gus expression in locus z 1 is reduced in x 1 y 1 z 1 plants ( table 1 : x 1 y 1 z 1 compared to y 1 z 1 ). thus , creating a stepwise homology between a silenced locus and a target gene by introducing a recombinant gene is sufficient to trigger silencing of the target . silencing inducing transgene loci can trigger silencing of a non - homologous endogene . we further assessed the universality and the usefulness in high throughput functional gene analyses of silencing elicited by a stepwise homology in trans , called domino silencing . therefore , we evaluated whether the expression of the tobacco endogenous catalase1 ( cat1 ) genes is reduced in plants carrying a silencing locus ( x locus ) showing no significant homology with the catalase endogene by introducing a recombinant gene ( y construct ). as silencing locus we used either x 1 or x 2 ( fig2 : locus x 1 , fig3 : locus x 2 ), in either case containing the 3 ′ chalcone synthase sequences of anthirrinum majus ( 3 ′ chs ). as transmitter for silencing we constructed a recombinant gene composed of the catalase1 coding sequence and the 3 ′ chs region under control of the 35s promoter ( p35s ) ( residing on t - dna pps35scat1s3chs , fig2 and 3 : t - dna in y 2 ). the recombinant cat1 3 ′ chs genes ( y2 ) were introduced in tobacco leaves bearing locus x 1 ( or x 2 ) via agrobacterium injection . as a negative control , we introduced a recombinant gene in which the cat1 coding sequence is replaced by the gus coding sequence ( pguschss , t - dna construct as in locus y 1 fig1 ). in this case , no stepwise homology is created between the silencing inducing locus and the target catalase endogenes . as a positive control , the recombinant construct y 2 was also introduced in transgenic tobacco with silenced catalase1 genes by the presence of a catalase1 antisense construct ( cat1as in champnongpol et al ., 1996 ). sixteen days after agrobacterium injection , the catalase activity was determined in protein extracts of injected leaf tissue and compared with the activity in non - injected wild type ( sr1 ) leaf tissue ( table 2 ). the results indicate that domino silencing is also applicable to endogenes since the catalase activity is clearly reduced in 6 out of 7 samples , while it remains high in the negative controls . in conclusion , not only an inverted repeat - bearing silencing - inducing transgene locus , but also a silencing - inducing locus in which the two residing chimeric genes give rise to transcripts with complementarity in the 3 ′ utr ( 3 ′ chs )( fig3 : x 2 ), is able to trigger domino silencing reducing endogenous catalase expression . [ 0028 ] table 2 results of a catalase - activity determination in protein extracts of leaf tissue harvested from agrobacterium injected tobacco leaves . genotype injected construct introduced via catalase activity 16 days plant agrobacterium injection after injection ( 60 μg tsp ) wt ( sr1 ) -( non - injected ) − 0 . 2116 2 100 % 3 x 1 pguschss − 0 . 2556 121 % x 1 y 2 − 0 . 0589 27 % x 1 4 y 2 − 0 . 0698 33 % x 2 pguschss − 0 . 1782 84 % x 2 y 2 − 0 . 0641 30 % x 2 y 2 − 0 . 0987 47 % x 2 4 y 2 − 0 . 0914 43 % x 2 4 y 2 − 0 . 1996 94 % x 2 4 y 2 − 0 . 0627 30 % cat1as y 2 − 0 . 0439 21 % pps35scat1s3chs : the t - dna of this plasmid is schematically shown in fig3 : y 2 and the nucleotide sequence is depicted in seq id no : 1 of the accompanying and incorporated herein sequence listing . locus x 1 harbors an inverted repeat about the right t - dna border of construct pgvchs287 , carrying a neomycin phosphotransferase ii ( nptii ) gene under the control of the cauliflower mosaic virus 35s promoter ( p35s ) and the 3 ′ signalling sequences of the anthirrinum majus chalcone synthase gene ( 3 ′ chs ). the nptii genes are post - transcriptionally silenced and can trigger in trans silencing and methylation of homologous target genes ( van houdt et al ., 2000 a and b and fig2 ). locus y 1 contains a single copy of the pguschss t - dna , containing a gus gene under the control of p35s and 3 ′ chs ( in transformant guschss29 ) and shows normal levels of gus expression ( fig2 ). locus z 1 contains more than one copy of the pxd610 t - dna , harboring the gus gene under control of p35s and the 3 ′ untranslated region ( utr ) of the nopaline synthase gene ( 3 ′ nos ), ( in plant lxd610 - 2 ) and shows normal gus expression ( de loose et al ., 1995 and fig2 ). locus x 2 contains a single copy of both the pguschss and pguschsas t - dna ( in transformant guschss + guschsas11 ) and triggers silencing in cis of the gus genes , but also in trans of ( partially ) homologous genes ( fig4 ). x 1 and z 1 hemizygous plants were obtained as hybrid progeny of the crossing of tobacco plants homozygous for locus x 1 (= holo1 ; van houdt et al ., 2000 a and b ) and homozygous for locus z 1 (= lxd610 - 2 / 9 de loose et al ., 1995 ) to wild type sr1 respectively . y 1 hemizygous plants were obtained by crossing the hemizygous primary tobacco transformant guschss29 to sr1 and selecting for the presence of locus y 1 in the hybrid progeny . x 1 y 1 and y 1 z 1 hemizygous plants are the hybrid progeny plants of the cross between holo1 and guschss29 and between guschss29 and lxd610 - 2 / 9 respectively that are selected for the presence of y 1 . x 1 z 1 hemizygous plants are the hybrid progeny of the cross between holo1 and lxd610 - 2 / 9 . x 1 y 1 z 1 hemizygous plants were obtained by crossing x 1 y 1 hemizygous plants to lxd610 - 2 / 9 ; as we only selected for the presence of y 1 in the hybrid progeny both y 1 z 1 and x 1 y 1 z 1 hemizygous plants were obtained . the agrobacteria c58c1rif r ( pgv2260 ) ( pguschss ) cb r , ppt r or c58c1rif r ( pmp90 )( pps35scat1s3chs ) gm r , ppt r were mainly grown as described by kapila et al ., 1997 except that the agrobacteria were resuspended in mma to a final od 600 of 1 . greenhouse grown plants of 10 to 15 cm in height were used . half of the third top leaf was injected via the lower surface using a 5 ml syringe while the leaf remained attached to the plant . the plants were kept in the greenhouse and 16 days after injection three to four discs of 11 mm in diameter were excised from the injected tissue for the preparation of a fresh protein extract to determine the catalase activity . preparation of the protein extracts and gus - activity measurements were done as previously described ( van houdt et al ., 2000 b ). preparation of the protein extracts for catalase - activity measurement and the spectrophotometric catalase - activity determination was done according to champnongpol et al ., 1996 . van houdt , h ., kovarik , a ., van montagu , m ., and depicker , a . 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