Patent Application: US-80772001-A

Abstract:
this invention provides a novel method to confer disease resistance to plants . plant plastids are transformed using a plastid vector which contains heterologous dna sequences coding for a cytotoxic antimicrobial peptide . transgenic plants are capable of fighting off phytopathogenic bacterial infection .

Description:
this invention demonstrates the confering of phytopathogenic resistance in plants through plastid transformation . this invention includes the use of all plastids in plants , including chloroplasts , chloroplasts which are present in fruits , vegetables and flowers , amyloplasts which are present in tubers , proplastids in roots , lencoplasts in non - green parts of plants . in a preferred embodiment of the invention , the chloroplast genome is used . plastid transformation and expression vectors comprising heterologous dna encoding magainin and its analogues are provided . the anti - microbial peptide ( amp ) used in this invention is an amphipathic alpha - helix molecule that has an affinity for negatively charged phospholipids commonly found in the outer - membrane of bacteria . upon contact with these membranes , individual peptides aggregate to form pores in the membrane , resulting in bacterial lysis . because of the concentration dependent action of the amp , it was expressed via the plastid genome to accomplish high dose delivery at the point of infection . pcr products and southern blots confirmed plastid integration of the foreign genes and homoplasmy . growth and development of the transgenic plants was unaffected by expression of the amp within the plastids . in vitro assays with t 0 , t 1 and t 2 plants , confirmed the amp was expressed at levels high enough to provide 86 %( t 0 ), 88 %( t 1 ) and 96 %( t 2 ) inhibition of growth against pseudomonas syringae , a major plant pathogen . in situ assays resulted in intense areas of necrosis around the point of infection in control leaves , while transformed leaves showed no signs of necrosis . even when germinated in the absence of spectinomycin selection , t 2 generation plants showed 96 % inhibition of growth against p . syringae . msi - 99 is an analogue of a naturally occurring peptide ( magainin 2 ) found in the skin of the african frog . changes have been made to the amino acid sequence to enhance its lytic abilities . contrary to the prior knowledge in the art which proposed that anti - microbial peptides having high antibacterial activity also have a high potential for toxic activity against the plastid ( everett and nicholas , 1994 ), the transgenic plants of this invention grew , flowered and set seeds like the untransformed control . key features of cationic peptides such as msi - 99 are a net positive charge , an affinity for negatively charged prokaryotic membrane phospholipids over neutral - charged eukaryotic membranes , and the ability to form aggregates that disrupt the bacterial membrane ( houston et al ., 1997 ; matsuzaki et al ., 1999 ; biggin and sansom , 1999 ). given the fact that the outer membrane is an essential and highly conserved part of all bacterial cells , it is highly unlikely that bacteria would be able to adapt ( as they have against antibiotics ) and to resist the lytic activity of these peptides . in contrast to prokaryotic membranes , the thylakoid membrane consists of primarily glycolipids and galactolipids instead of phospholipids . monogalactosyldiacylglycerol ( mgdg ) makes up 50 % of membrane lipid and digalactosyldiacylglycerol ( dgdg ) 30 % ( siegenthaler et al ., 1998 ). both of these lipids are neutral . an object of this invention is to compartmentalize the expression of the msi - 99 within the plastid . compartmentalization of lytic enzymes is a natural occurrence in plants . compartmentalization serves two purposes : to increase the yield of the peptide and to deliver the peptide at the site of the infection . due to the high copy number associated with plastid expression , a larger amount of the peptide is produced . the higher yield is important due to the concentration - dependent action of the anti - microbial peptide . further , the peptide would be released at the site of infection during the hr response . when the hr response occurs , cells are lysed . this disrupts the osmotic balance and causes plastids to lyse . this would release the peptide at high concentration resulting in aggregation and formation of pores in the outer membrane of bacteria . this aids in the prevention of the spread of infection by bacteria . a high level of amp expression can be expected due to the following reasons . the nature of plastids to move from a somatically unstable heteroplasmic state to a state of homoplasmy itself lends to high expression ( brock and hagemann , 2000 ). the a + t % of msi - 99 is 51 . 39 %, which is compatible with the nicotiana tobacum plastid 61 % a + t content ( bogorad et al ., 1991 ; shimada et al ., 1991 ). also , published reports from our lab report expression of cry2a operon ( a + t content of 65 %) at levels as high as 46 % total soluble protein ( decosa et al ., 2000 ). msi - 99 was most effective against p . syringae , evidenced by total inhibition of 1000 p . syringae cells with only 1 μg / 1000 bacteria ( smith et al . unpublished data ). because the lytic activity of antimicrobial peptides is concentration dependent , the amount of antimicrobial peptide required to kill bacteria was used to estimate the level of expression in transgenic plants . based on the minimum inhibitory concentration , it was estimated that transgenic plants expressed msi - 99 at 21 % of the total soluble protein . without the availability of antibody for msi - 99 , other direct methods of protein estimation were not feasible . plastid vectors and plant transformation : the synthetic peptide used in this invention ( msi - 99 ), is an analogue of the naturally occurring 23 amino acid peptide , magainin ii . msi - 99 is a 22 amino acid sequence with an overall charge of + 6 as shown in fig1 . the gene cassette used for transformation consisted of the 16s rrna promoter , the aada gene , which confers resistance to spectinomycin , the msi - 99 gene and the psba ( photosynthetic binding protein ) terminator . the gene construct may contain , in addition to the msi - 99 gene , another heterologous dna sequence coding for a gene of interest . flanking sequences are from the petunia plastid genome as shown in fig1 a . transformation efficiency was much lower ( 7 %) than that observed using the pld vector ( 91 %), which contains tobacco homologous flanking sequences . other vectors that are capable of plastid transformation may be used to deliver the gene cassette into the plastid genome of the target plant cells . such vectors do include plastid expression vectors such as puc , pbluescript , pgem , and all others identified by daniell in u . s . pat . nos . 5 , 693 , 507 and 5 , 932 , 479 . these publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference . the vectors preferably include a ribosome binding site ( rbs ) and a 5 ′ untranslated region ( 5 ′ utr ). a promoter operably in green or non - green plastids is to be used in conjunction with the 5 ′ utr ) the number of transformants from the total number of shoots determined percent of transformants . out of 55 spectinomycin resistant shoots screened , only 4 were transformants with the msi - 99 gene and the rest were mutants . all transformants grew healthy with no apparent morphological effects to t 0 and t 1 , generations as shown in fig2 a . t 1 , seeds germinated in the presence of spectinomycin produced healthy green seedlings , while control seedlings were bleached as shown in fig2 b . foreign gene integration , homoplasmy and copy number : pcr was performed by landing one primer on the 5 ′ end of the aada coding sequence , not present in native plastid and the 3 ′ end of the 16s rdna ( fig3 a ). pcr products of t 0 , t 1 , and t 2 generations yielded the same size product as the plasmid ( msi - 99 ) as shown in fig3 b , c , d confirming integration of the foreign genes . the probe used for the southern analysis was a 2 . 3 kb fragment from the 5 ′ end of the tmi ( bamhi ) to the 3 ′ end of the 16srdna ( noti ) ( fig4 a ). the plant dna was digested with bamhi . dna from untransformed plants produced a 3 . 269 kb fragment and transformed plant dna produced a 4 . 65 kb fragment . southern analysis confirmed integration of foreign genes for t 0 and t 1 , as shown in fig4 b , c . untransformed dna showed a 3 . 2 kb fragment while the transformed contained a 4 . 65 kb fragment . presence of some wild type fragments in t 0 transgenic samples indicated some heteroplasmy as shown in fig4 b . however , dna from t 1 , generation produced only the 4 . 65 kb fragment confirming homoplasmy . as shown in fig4 c . a cell is said to be homoplasmic when all of the plastid are uniformly transformed . if only a fraction of the genomes was transformed , the copy number should be less than 10 , 000 ( bendich , 1987 ). by confirming that the msi - 99 integrated genome is the only one present in transgenic plants ( homoplasmy ), one could estimate that the msi - 99 gene copy number could be as many as 10 , 000 per cell . bioassays : t 0 in situ assays in potted plants ( 6 to 7 months old ) resulted in areas of necrosis surrounding the point of infection in untransformed control , while transgenic leaves showed no areas of necrosis ( fig5 ). even inoculation of 8 × 10 5 cells resulted in no necrosis in transgenic leaves ( fig5 a ), suggesting the local concentration of the antimicrobial peptide to be very high . however , untransformed plants inoculated with 8 × 10 3 cells displayed intense necrosis as shown in fig5 b . cell free extracts of t 0 , t 1 , and t 2 transgenic plants displayed a strong ability to inhibit growth of p . syringae in vitro by 84 %, 86 % and 96 % compared to untransformed plants as shown in fig6 . the increase in growth inhibition from t 0 to t 2 can be attributed to heteroplasmy in the t 0 generation that was eliminated in subsequent generations . this indicates the peptides retained their lytic activity and successfully passed on the trait to the subsequent generations . the control had less growth than the buffer only . this is most probably due to natural defense peptides such as defensins and thionins produced by plants ( mourgues et al ., 1998 ). when performing in vitro bioassays against p . aeruginosa , results were similar with t 1 , generation showing 96 % inhibition of growth ( fig7 ). absorbance readings as shown in fig8 a from transgenic plants germinated in the absence of spectinomycin , displayed 96 % inhibition of growth that is comparable to transgenic plants germinated in the presence of spectinomycin . plated cells of bioassay samples from t2 plants germinated in the absence of spectinomycin as shown in fig8 b showed 83 % inhibition of growth compared to the control . the marginal degree of difference between the plating results and the bioassay results ( 13 %) can be explained by the difference in environment . while the plated bacteria were no longer exposed to active peptides , bacteria in the liquid media were constantly surrounded by active peptides . protein estimation : the plate with 10 − 5 dilution had 43 cfus . the equated to 43 × 10 6 cfu / ml . the count was adjusted to reflect the 5 μl of culture used . this resulted in a count of 21 , 500 bacterial cells in the initial 5 μl of culture incubated with the peptide . using 1 μg to kill 1000 p . syringae cells as the reference ( smith et al . unpublished data ), the estimated expression of msi - 99 was 21 . 5 μg in 100 μg soluble protein ( 21 . 5 %). the initial low rate of transformation was most likely due to less than 100 % homology between the petunia flanking sequences and the tobacco plastid genome . this is not surprising because very low transformation efficiency was also observed when tobacco plastid flanking sequences were used to transform potato plastid genome ( sidorov et al ., 1999 ). also , other projects in our lab that use the pld vector ( has tobacco flanking sequences ) obtained transformation efficiency of 91 % transformants to mutants . t 0 and t 1 transgenic plants were healthy and showed no morphological or developmental abnormalities . retention of lytic activity was evident in the sharp decrease in bacterial growth in the in vitro bioassays ( 84 to 96 %). when comparing southern blots to lytic activity , lytic activity increased as homoplasmy was reached . equal lytic activity was also observed in transgenic plants germinated in the absence of spectinomycin ( 96 % inhibition of growth ). transgenic plants transferred to potting soil for 5 to 6 months after being removed from spectinomycin selection , displayed similar antimicrobial properties against inoculations of p . syringae . these observations eliminate the possibility that spectinomycin absorbed into the plant tissue during germination of seeds , may be responsible for the growth inhibition in the in vitro and in situ bioassays . also , the observation that msi - 99 was equally active in transgenic plants germinated in the presence or absence of spectinomycin shows the stability of the introduced trait in the absence of any selection pressure . plastid expression in crops such as tobacco should allow for mass production of the peptide at a lower cost compared to chemical synthesis or production in e . coli . this invention thus demonstrates another option in the on going battle against pathogenic bacteria . plant transformation : for plant transformation , nicotiana tabacum var . petit havana seeds were germinated on mso media at 27 ° c . with photoperiods of 16 hour light and 8 hour dark . sterile leaves were bombarded using the bio - rad helium driven pds - 1000 / he system . after bombardment , leaves were wrapped and kept in the dark for 48 hours . leaves were then cut into 1 cm 2 squares and placed on a petri dish containing rmop media with 500 μg / ml spectinomycin ( first round of selection ). four to six weeks later , shoots were transferred to fresh media and antibiotic ( second round of selection ). shoots that appeared during the second selection were transferred to bottles containing mso and spectinomycin ( 500 82 g / ml ). plants were screened via pcr for transformation . those that were pcr positive for the presence of the msi - 99 gene were transferred to pots and grown in chambers at 27 ° c . with photoperiods of 16 - hour light and 8 - hour dark . after flowering , seeds were harvested and sterilized with a solution of i - part bleach and 2 - part water with 1 drop of tween - 20 . seeds were vortexed for 5 minutes then washed 6 times with 500 μl of dh 2 0 and dried in speed vac . t 1 , and t 2 seeds were germinated on mso + 500 μg / ml spectinomycin . untransformed petit havana seeds were germinated on the same media as a control to ensure the spectinomycin was active . pcr conformation plant dna extraction on t 0 , t 1 , and t 2 was performed using the qiagen dneasy mini kit on putative transgenic samples and untransfon - ned plants . pcr primers were designed using primer premier software and made by gibco brl . primer ( 8p : 5 ′ atcaccgcttccctcataaatccctccc3 ′) anneals with the 5 ′ end of the aada and primer ( 8m : 5 ′ ccacctacagacgctttacgcccaatca3 ′) anneals with the 3 ′ end of 16srdna as shown in fig3 . pcr was carried out using the gene amp pcr system 2400 ( perkin - elmer ). samples were run for 29 cycles with the following sequence : 94 ° c . for 1 minute , 65 ° c for 1 minute and 72 ° c . for 3 minutes . the cycles were proceeded by a 94 ° c . denaturation period and followed by a 72 ° c . final extension period . a 4 ° c . hold followed the cycles . pcr products were separated on agarose gels . southern analysis : integration of foreign genes for t 0 and t 1 , was determined by southern blot analysis . dna from transformed and untransformed plants was digested with bamhi and run on a 0 . 7 % agarose gel . the dna was then transferred to a nylon membrane by capillary action . the probe was digested with bamhi and notl and was labeled with 32 p using the probe quant ™ g - 50 micro colurnis and protocol ( amersharn ). labeled probe was hybridized with the nylon membrane using the stratagene quick - hyb hybridization solution and protocol . membrane was exposed to film , and developed . in vitro bioassay : p . syringae and p . aeruginosa were cultured overnight prior to the assay . 50 mg of leaf tissue ( minus mid - rib ) was grounded in a micro - centrifuge containing 150 μl of phosphate buffer ph5 . 5 with 5 mm pmsf and 5 mm with a plastic pestle . samples were centrifuged for 5 minutes at 10 , 000 × g at 4 ° c . supernatant was transferred to a fresh tube and kept on ice . protein concentration was determined by bradford assay . one hundred μg of total plant protein was mixed with 5 μl of bacteria from overnight culture in a falcon tube . initial absorbency ranged from 0 . 1 to 0 . 3 ( a 600 ). tubes were incubated for 2 hours at 25 ° c . on a rotary shaker at 125 rpm . one ml of lb broth was added and tubes were allowed to incubate for 18 hours at 27 ° c . for p . syringae and 37 ° c . for p . aeruginosa on a rotary shaker at 125 rpm . absorbance ( a 600 ) was read for each tube . results were statistically analyzed using graphpad prism . to rule out spectinomycin as the cause of growth inhibition , the same experiment with p . syringae was repeated using t 2 plants that were geminated on mso with no spectinomycin . for confirmation of the absorption readings , a serial dilution was made of samples after the initial 2 - hour incubation . dilutions of 10 − 3 to 10 − 5 were plated onto lb plates and incubated overnight at 27 ° c . the next morning a count of viable cfus were made using the bio rad gell dock . to estimate the level of protein expression , a serial dilution was prepared from the starting bacterial culture ( absorbance 600 , 0 . 1 - 0 . 3 ) used for the in vitro bioassay . fifty μl of each dilution was plated on lb medium and incubated overnight at 27 ° c . the following morning , cfus were counted using the bio rad gel dock and the amount of cells used in the bioassay was calculated . the minimum inhibitory concentration of iμg / 1000 p . syringae cells was used to determine antimicrobial peptide concentration in 100 μg of cell free plant extracts . in situ bioassay : p . syringae was cultured overnight prior to the assay . five to seven mm areas of t 0 transformants and untransformed petit havana leaves were scraped with fine grain sandpaper . ten μl of 8 × 10 5 , 8 × 10 4 , 8 × 10 3 and 8 × 10 2 cells from an overnight cultur syringae were added to each prepared area . photos were taken 5 days after inoculation . baker b , zambryski p , staskawicz s p , dinesh - 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