Patent Application: US-67936008-A

Abstract:
the invention relates to the use of a mutation for the generation of virus - resistant mutant plants and , specifically , to the use of mutation 8 . 1 of the atdbp1 gene of arabidopsis thaliana in order to modify the phenotype of the plant as a regulator of plant potyvirus resistance , as well as to the resulting genetically modified plants having greater potyvirus infection resistance than unmodified plants .

Description:
the potyvirus family represents a large number of plant viral pathogens that collectively may infect most cultivated species . infection by potyvirus may induce a variety of symptoms , including foliar mottling , the distortion of seeds and fruits , and may thus severely compromise the yield and / or quality of the crop . the potyvirus family , or potyviridae , includes the following definitive species : alstroemeria mosaic potyvirus , amaranthus leaf mottle potyvirus , araujia mosaic potyvirus , arracacha y potyvirus , artichoke latent potyvirus , asparagus 1 potyvirus , banana bract mosaic potyvirus , bean common mosaic necrosis potyvirus , bean common mosaic potyvirus , bean yellow mosaic potyvirus , beet mosaic potyvirus , bidens mosaic potyvirus , bidens mottle potyvirus , cardamom mosaic potyvirus , carnation vein mottle potyvirus , carrot thin leaf potyvirus , cassava brown streak potyvirus , cassia yellow spot potyvirus , celery mosaic potyvirus , chickpea bushy dwarf potyvirus , chickpea distortion mosaic potyvirus , clover yellow vein potyvirus , commelina diffusa potyvirus , commelina mosaic potyvirus , cowpea green vein - banding potyvirus , cowpea moroccan aphid - borne mosaic potyvirus , cowpea rugose mosaic potyvirus , crinum mosaic potyvirus , daphne y potyvirus , dasheen mosaic potyvirus , datura colombian potyvirus , datura distortion mosaic potyvirus , datura necrosis potyvirus , datura shoestring potyvirus , dendrobium mosaic potyvirus , desmodium mosaic potyvirus , dioscorea alata potyvirus , dioscorea green banding mosaic potyvirus , eggplant green mosaic potyvirus , euphorbia ringspot potyvirus , freesia mosaic potyvirus , groundnut eyespot potyvirus , guar symptomless potyvirus , guinea grass mosaic potyvirus , helenium y potyvirus , henbane mosaic potyvirus , hippeastrum mosaic potyvirus , hyacinth mosaic potyvirus , iris fulva mosaic potyvirus , iris mild mosaic potyvirus , iris severe mosaic potyvirus , johnsongrass mosaic potyvirus , kennedya y potyvirus , leek yellow stripe potyvirus , lettuce mosaic potyvirus , lily mottle potyvirus , maize dwarf mosaic potyvirus , malva vein clearing potyvirus , marigold mottle potyvirus , narcissus yellow stripe potyvirus , nerine potyvirus , onion yellow dwarf potyvirus , ornithogalum mosaic potyvirus , papaya ringspot potyvirus , parsnip mosaic potyvirus , passiflora ringspot potyvirus , passiflora south african potyvirus , passionfruit woodiness potyvirus , patchouli mosaic potyvirus , pea mosaic potyvirus , pea seed - borne mosaic potyvirus , peanut green mosaic potyvirus , peanut mottle potyvirus , pepper indian mottle potyvirus , pepper mottle potyvirus , pepper severe mosaic potyvirus , pepper veinal mottle potyvirus , plum pox potyvirus , pokeweed mosaic potyvirus , potato a potyvirus , potato v potyvirus , potato y potyvirus , primula mosaic potyvirus , ranunculus mottle potyvirus , sorghum mosaic potyvirus , soybean mosaic potyvirus , statice y potyvirus , sugarcane mosaic potyvirus , sweet potato feathery mottle potyvirus , sweet potato g potyvirus , swordbean distortion mosaic potyvirus , tamarillo mosaic potyvirus , telfairia mosaic potyvirus , tobacco etch potyvirus , tobacco vein - banding mosaic potyvirus , tobacco vein mottling potyvirus , tobacco wilt potyvirus , tomato peru potyvirus , tradescantia - zebrina potyvirus , tropaeolum 1 potyvirus , tropaeolum 2 potyvirus , tuberose potyvirus , tulip band - breaking potyvirus , tulip breaking potyvirus , tulip chlorotic blotch potyvirus , turnip mosaic potyvirus , ullucus mosaic potyvirus , vallota mosaic potyvirus , vanilla mosaic potyvirus , vanilla necrosis potyvirus , voandzeia distortion mosaic potyvirus , watermelon mosaic 1 potyvirus , watermelon mosaic 2 potyvirus , wild potato mosaic potyvirus , wisteria vein mosaic potyvirus , yam mosaic potyvirus , zucchini yellow fleck potyvirus , zucchini yellow mosaic potyvirus , and the following provisional species : asystasia gangetica mottle (?) potyvirus , celery latent (?) potyvirus , datura mosaic (?) potyvirus , endive necrotic mosaic (?) potyvirus , kalanchoe mosaic (?) potyvirus , konjak mosaic (?) potyvirus , nasturtium mosaic (?) potyvirus , patchouli mottle (?) potyvirus , shallot yellow stripe (?) potyvirus , sweet potato vein mosaic (?) potyvirus , welsh onion yellow stripe (?) potyvirus . the viral genome of potyviruses is composed of a simple rna molecule of positive polarity that encodes a large polyprotein , which is post - translationally processed in at least 10 mature proteins by three viral proteases . this viral rna is polyadenylated at the 3 ′- end ; and , unlike mrnas , it does not present a cap structure at the 5 ′- end , but , instead , it is substituted by a protein encoded by the virus called vpg (“ virus protein linked to the genome ”), which covalently binds to the 5 ′- end ( murphy et al ., virology . vol . 178 , pp . 285 - 288 , 1990 ). the fact that the lack of atdbp1 translates into a reduction in the accumulation of said factor at the post - transcriptional level seems to indicate that atdbp1 may be exerting a control on the translation initiation factor . to this end , we decided to verify whether both proteins were capable of physically interacting . in this invention , said interaction is demonstrated ; therefore , it is very likely that this factor appears as a potential candidate for the atdbp1 - mediated dephosphorylation , thanks to the type -“ c protein phosphatase activity associated therewith . in mammals , three mechanisms for the regulation of the activity of eif4e are known . significant amongst these is the regulation of the transcription level of this gene ; in this case , the myc proto - oncogene binds to the promoter of this factor , thereby inducing the expression levels thereof . there is also a post - translational modification via phosphorylation through two kinases ( mnk1 and 2 ). this phosphorylation of eif4e seems to increase the translation , but the phosphorylated form has a lower affinity for the cap structure ; consequently , we propose that the phosphorylation could take place following the formation of the 43s pre - initiation complex , thereby promoting the release of eif4e and the subsequent “ scanning ” of the ribosome . finally , the main mechanism for the regulation of the activity of eif4e is through the interaction thereof with a family of repressive proteins called 4e - bps ( 4e - binding proteins ). the binding of these proteins to eif4e does not alter the binding to the cap structure , but prevents the interaction of eif4e with eif4g , thereby suppressing the formation of the eif4f complex . the interaction of 4e - bps with eif4e is controlled by the specific phosphorylation of serine and threonine residues in 4e - bp . the net effect of this phosphorylation of 4e - bp is the release of eif4e , which makes it possible for eif4e to actively bind to eif4g in order to form the eif4f complex and for the translation to proceed in a normal manner . this invention establishes the functional involvement of atdbp1 , a transcriptional regulator with protein phosphatase activity from the dbp family , in plant - potyvirus interactions as a factor required by the virus for the efficient replication and / or propagation thereof in the plant , and its use as a modulator of the defensive response . on the basis thereof , we show that the inhibition or absence of the expression of the atdbp1 gene ( seq . id . no . 9 ) and , therefore , the absence of the atdbp1 factor or protein makes it possible to obtain plants with a higher resistance to potyvirus infections . in order to determine the function of atdbp1 , plants were generated with a loss of function of the atdbp1 gene ( seq . id . no . 9 ). to this end , homozygotic plants were selected from a line with t - dna inserted in the atdbp1 structural gene ( salk — 005240 ; seq . id . no . 10 ), which carries an insertion in the atdbp1 gene that interrupts its encoding sequence and , consequently , must eliminate the expression thereof . this homozygotic line was called 8 . 1 line . the t - dna insertion is located on the second exon of the sequence that encodes atdbp1 ( fig1 a ), interrupting the n - terminal region of the protein at the dnc motif , which is involved in the specific binding to dna . consequently , in the event that there is expression of the atdbp1 gene in the 8 . 1 plants , the resulting protein would not be functional , since it would lack the phosphatase domain and the dna - binding capacity . in order to verify to what extent the t - dna insertion affected the expression of the atdbp1 gene , the accumulation of the corresponding mrna was analysed by means of rt - pcr in both col - 0 control plants and plants from the 8 . 1 line . the result of said analyses shows the absence of mrna corresponding to the atdbp1 gene in the 8 . 1 line ( fig1 b ). therefore , these results confirm that the expression of atdbp1 in 8 . 1 plants is inhibited as compared to the expression levels observed for the atdbp1 gene in wild col - 0 plants . the inhibition of the expression of atdbp1 triggers a lower accumulation of the eif ( iso ) 4e protein in the plant . as a part of the functional , molecular and genetic characterisation of the functional homologue of dbp1 in arabidopsis thaliana , and in order to identify the target genes of atdbp1 , a comparative analysis was performed between the proteome of the seedlings of the 8 . 1 mutant line and the seedlings of the wild col - 0 line by means of two - dimensional electrophoresis . according to this analysis , amongst the differential spots observed between both genotypes , a polypeptide was identified which , following a mass - spectroscopy analysis of the fragments thereof by digestion with trypsin , was found to correspond to one of the isoforms of translation initiation factor 4e ( eif ( iso ) 4e ), which exhibited a low accumulation of the 8 . 1 mutant line in the seedlings . factors 4e and iso4e are a part of the 4f and iso4f complexes , respectively , the latter being exclusive of plants ( browning , plant mol . biol . vol . 32 , pp . 107 - 144 , 1996 ). these complexes , formed by factors 4a , 4g , 4e / iso4e , and factors pabp , are required , jointly with other initiation factors , for the initiation of the translation of proteins , allowing for the binding of mrna to the pre - initiation complex . specifically , translation initiation factor 4e is responsible for recognising and binding to the cap structures present at the 5 ′- end of mrnas . the low representation of factor eif ( iso ) 4e in the 8 . 1 mutant seems to indicate that atdbp1 positively contributes to the accumulation of eif ( iso ) 4e . in order to verify whether the control exerted by atdbp1 on said factor is at the transcriptional or the post - transcriptional level , the expression of eif ( iso ) 4e was analysed at the mrna level by means of rt - pcr and , at the protein level , by means of western - blot , using a specific polyclonal antibody . the results obtained indicate a lower accumulation of the eif ( iso ) 4e protein in the 8 . 1 mutant line as compared to col - 0 plants ( fig2 a ), whereas the level of mrna , analysed by rt - pcr , is similar in both genotypes ( fig2 b ). therefore , atdbp1 must affect the expression of eif ( iso ) 4e at the post - transcriptional level , which suggests a possible direct interaction between both proteins . in order to verify whether atdbp1 is capable of interacting with eif ( iso ) 4e , a two - hybrid assay was performed using translational fusions of both proteins to the dna - binding and transcription activation domains , respectively , of the yeast activator gal4 . the expression of atdbp1 , jointly with eif ( iso ) 4e in yeast , was capable of inducing the expression of the his3 marker gene , thereby confirming the specific interaction between both proteins ( fig3 a ). a point mutation in the c - terminal domain of atdbp1 that reduces the protein phosphatase activity thereof significantly weakened the interaction , which suggests that the isoform of translation initiation factor 4e could be a substrate susceptible to atdbp1 - mediated dephosphorylation . the interaction observed between atdbp1 and eif ( iso ) 4e was confirmed by means of co - immunoprecipitation . using a specific antibody against eif ( iso ) 4e , the presence of said factor was detected by means of western blot in the immunoprecipitate obtained with anti - ha monoclonal antibodies from transgenic plant extracts that expressed atdbp1 fused to the ha epitope under the control of the cauliflower mosaic virus ( camv ) 35s promoter , which is constitutively expressed ( fig3 b ). this shows that both proteins are capable of interacting in the plant in vivo . the inhibition of the expression of atdbp1 leads to a loss of susceptibility or increased resistance to potyvirus infection we attempted to determine whether the 8 . 1 mutants show specific recessive resistance to potyviruses . western - blot analyses revealed that these mutants present lower levels of eif ( iso ) 4e ; from this , we may conclude that the loss of function of this factor confers potyvirus resistance . although a total absence of the eif ( iso ) 4e protein was not observed in the 8 . 1 t - dna insertion line , the lower accumulation of eif ( iso ) 4e in these plants , as compared to col - 0 plants , suggests the possibility that the absence of atdbp1 ( or loss of function ) produces a phenotype with potyvirus resistance . for this reason , col - 0 plants and plants from the 8 . 1 line were inoculated with an infectious clone of the plum pox virus or sharka virus ( ppv ), a member of the potyvirus family . moreover , the viral clone used was a carrier of the gfp reporter gene , which allowed for an in vivo follow - up of the degree of the colonisation of the viral infection . initially , the accumulation of viral rna in non - inoculated systemic tissue was analysed by means of quantitative rt - pcr , using specific primers for the gfp marker gene . as shown in fig4 a , fifteen days after inoculation ( dpi ), a marked decrease in the accumulation of gfp mrna is observed in 8 . 1 plants as compared to col - 0 plants . this result correlates with the level of gfp protein detected by means of western - blot in systemic tissue from both genotypes , where a lower accumulation of the protein is also observed in 8 . 1 plants as compared to col - 0 plants at 15 dpi ( fig4 b ). in turn , the gfp marker gene makes it possible to perform a follow - up of the movement and distribution of the virus by means of fluorescence microscopy . this demonstrated a significant delay in viral movement in 8 . 1 plants as compared to col - 0 plants , where both the viral accumulation in the vascular tissue and the movement of the virus through the petiole toward distal tissues takes place earlier on ( fig4 c ). furthermore , for each of the times analysed following inoculation , it is observed that the non - inoculated systemic tissue analysed in col - 0 plants exhibits a greater viral invasion , which reaches almost the entire leaf , whereas in the equivalent tissue of 8 . 1 plants the dispersion of the virus predominantly affected the vascular tissue . therefore , the loss of expression and , consequently , the loss of function of atdbp1 exhibited by the 8 . 1 mutant slow down and attenuate the viral infection . due to the absence of symptoms in infections by ppv , the response of 8 . 1 plants to another member of the potyvirus family that causes symptoms in arabidopsis , the turnip mosaic virus ( tumv ), was also analysed . this would make it possible to determine whether the loss of susceptibility observed for ppv could be extended to other members of the family . as shown in fig5 , inoculation with tumv caused a much less severe symptomatology in 8 . 1 plants than in control col - 0 plants . the symptomatology shown in fig5 could be specified by means of visual analysis only in the leaves , since an induction of chlorosis and subsequent necrosis , which leads to the collapse of foliar tissue due to infection of the tissue by the virus , is generated in the wild plant , but not in the 8 . 1 plant , given its special resistance . ultimately , said chlorosis and necrosis , in the case of severe infections or high titres of viruses , extends to the entire foliar tissue and eventually leads to general plant collapse ; this collapse and death of the plant are characteristic of pathogenic infections as severe as those studied in this case . therefore , the manipulation of the function of atdbp1 seems to trigger an increase in resistance or a loss of susceptibility to several members of the potyvirus family . the vegetable materials used were arabidopsis thaliana ( l .) heynh plants of the col - 0 ecotype and the 8 . 1 mutant line , also in a col - 0 genetic background , which corresponds to the salk — 005240 t - dna insertion line from the salk institute ( la jolla , usa ). the seeds from these plants were stratified for 3 days at 4 ° c . following imbibition . subsequently , the plants were grown in jiffy - 7 compacted substrate ( clause - tezier ibérica , valencia , spain ) at 23 ° c ., with a photoperiod of 10 hours of light and 14 hours in the dark . the inoculum used consisted of a suspension of agrobacterium tumefaciens carrying an infectious clone of the ppv virus ( plum pox virus or sharka virus ) with the gfp marker gene . the bacterial culture was re - suspended in 10 mm mes buffer , ph 5 . 6 , 10 mm mg 2 cl , 150 μm acetosyringone , at an optical density of 0 . 5 at 600 nm , and kept at ambient temperature for several hours . five - week - old plants were inoculated with 20 μl of said inoculum , by infiltrating the distal half of one leaf per plant through the back part of the leaf . subsequently , the inoculated plants were kept under the same light and temperature conditions in order to follow up the viral infection at different times . for the rna extractions , trizol ( invitrogen ) was used , following the manufacturer &# 39 ; s recommendations . the quality and integrity of the extracted rna was analysed by means of spectrophotometry and agarose gel electrophoresis . in order to evaluate the degree of expression of certain genes , the semi - quantitative rt - pcr technique was used . the corresponding cdna was obtained from the total rna by reverse transcription with oligodt , using the “ revertaid h minus first strand cdna synthesis kit ” ( fermentas ). the cdna obtained was used as a template in pcr reactions with specific primers for the gene of interest in a ptc - 100 peltier thermal cycler . the final products were separated by means of agarose gel electrophoresis . the primers used ( seq . id . nos . 1 to 6 ) are shown in table 1 : the samples were analysed in triplicate and the real - time pcr reactions were performed using sybr green pcr master mix ( applied biosystems ) in an abi prism 7000 sequence detector . the direct and reverse primers were designed using the primer express computer package . the sequences of the primers used ( seq . id . nos . 7 and 8 ) are shown in table 2 : in order to determine whether factor eif ( iso ) 4e is capable of specifically interacting with atdbp1 , the yeast two - hybrid system was used . to this end , translational fusions were generated , of atdbp1 to the dna - 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