Patent Application: US-91608606-A

Abstract:
the invention relates to nucleic acid , which codes for an autoactivated resistance protein for creating a resistance to pathogens in plants , characterized in that the nucleic acid has a limited portion of an nbs - lrr resistance gene , which extends from the 5 ′- end of the coded region of the nbs - lrr resistance downstream to the beginning of the nbs domain of the nbs - lrr resistance gene , the nbs - lrr resistance gene not being a tir - nbs - lrr resistance gene .

Description:
the invention as briefly described above is illustrated in greater detail in the following examples . verification of initiation of rapid resistance reaction in sugar beet leaves by overexpression of the gene bvkws3 — 133 the transient overexpression of the full - length cdna clone of the gene bvkws3 — 133 in sugar beet leaves by agrobacterium tumefaciens triggers a rapid cell death without visible necrosis formation . the cdna - clone bvkws3 — 133 was combined with the d35s promoter and inserted in the binary vector per - 34sluci ( fig1 ). the resulting vector was given the designation per133 - 34sluci . the vector per - 34sluci and per133 - 34sluci were transformed in the agrobacterium strain c58c1 ( an 1987 ). positive agrobacteria were cultured for the transient expression in 50 ml lb - medium with 100 mg / ml spectinomycin and 20 μm acetosyringon for 4 - 5 hours . subsequently , the bacteria were centrifuged and the precipitate was taken up in a solution of 10 mm mgcl 2 , 10 mm mes , 100 μm acetosyringon and adjusted to a bacteria density of od 600 = 0 . 1 . the bacteria suspension was allowed to rest for 2 - 3 hours and then injected into the leaves of old sugar beets with the aid of a 2 . 5 ml hypodermic needle via the underside of the leaf of 10 week old sugar beets . after incubation at 25 ° c . in an incubator , the photinus pyralis luciferase reporter gene activity was measured in the transformed leaves in the 1 , 2 and 3 days following inoculation . in addition , the luciferase activity was determined with the luciferase assay system ( promega , mannheim , germany ) in a sirius luminometer ( berthold detection system gmbh , pforzheim , germany ) according to the manufacturer &# 39 ; s specifications . for obtaining an enzyme suitable for the measurements , two leaf disks were stamped for each measurement interval . for each construct 8 measurement points were collected per measurement day . the leaf samples were homogenized in a mortise with addition of sea sand with the 10 - fold volume ( v / w ) of passive lysis buffer ( pbl ). the liquid supernatant was extracted and respectively 10 μl raw extract was employed for the photinus - luciferase activity measurement . sugar beet leaves which were transformed with the control construct per - 35sluci , showed on day 1 a small and on 2 and 3 a luciferase activity of 124 , 000 or as the case may be 116 , 000 rlu / mg of leave tissue . beet leaves , which were transformed with the construct per - 34sluci , showed an activity at all three measurement points which was greater than the mgcl 2 inoculated leaves ( fig2 ). thus , the transient expression of the cdna clone bvkws3 — 133 initiates a very rapid cell death in the inoculated beet leaves . the constitutive expression of the r - gene bvkw3 — 123 , bvkws3 — 133 and bvkws3 — 165 initiates a cell death in sugar beet leaves . the r - gene bvkws3 — 133 as well as the r - gene bvkws3 — 165 with the nucleotide sequence according to seq id no . 5 and the r - gene bvkws3 — 123 were combined with the doubled 35s promoter of the vector pcamv - 2 ( fig3 ). the resulting vectors carry the designation p70s - bvkws3 — 133 , p70s - bvkws3 — 165 and p70s - bvkws3 - 123 . in order to verify the functionality of the r - genes , the constructs p70s - bvkws3 — 133 , p70s - bvkws3 — 165 and p70s - bvkws3 - 123 were transiently expressed with the reporter gene vector p70s - luc in sugar beet leaves by biolistic transformation according to schmidt et al . ( 2004 ). as a positive control , the empty vector pcamv - 2 was used in combination with the reporter gene vector p70s - luc . in contrast to schmidt , et al . ( 2004 ), the use of a normalizing vector was dispensed with . the luciferase activity was determined with the luciferase assay system ( promega , mannheim , germany ) 20 hours after the transformation . the transformation experiments were repeated three times , wherein each experiment included nine test repetitions per construct . the development of the average value from the three experiments showed that in comparison to the luciferase activity of the positive control ( empty vector ) set at 100 %, the reporter gene activity for p70s - bvkws3 - 133 only 37 . 7 %, for p70s - bvkws3 — 165 only 66 % and for p70s - bvkws3 - 123 only 68 . 7 % ( fig4 ). the strong expression of the r - gene bvkws3 — 133 , bvkws3 — 165 and bvkws3 — 123 by the d35s promoter thus initiated cell death or as the case may be a hypersensitive reaction in one part of the transformed cells which prevented the co - expression of the simultaneously transformed reporter gene vector . therewith it was shown that the strong expression of the three r - genes led to a cell death or , as the case a hr , in the absence of a corresponding avirulence gene product . the 5 ′ area of the gene bvkws3 — 165 triggers a more rapid cell death than the full - length cdna clone bvkws3 — 165 beginning with full - length cdna clone bvkws3 . sub . — 165 with the nucleotide sequence according to seq id no . 5 in the construct p70s - bvkws3 . sub . — 165 , the 5 ′ area of the gene was amplified with the aid of the pfu - polymerase ( stratagene ) with use of the primer s316 ( ctcgagaattcgagctccaccgcgg ) with nucleotide sequence seq id no : 17 and s318 ( ctggatcctcacctccgttcttcatgttgctctacc ) with nucleotide sequence seq id no : 18 and simultaneously a stop codon was introduced in the coded area . the amplified area corresponded to the nucleotide sequence according to seq id no . 1 and encoded for the amino acid sequence 1 - 175 of bvkws3 . sub . — 165 ( fig1 ). the amino acid sequence included only the n - terminal area of bvkws3 . sub . — 165 and contained no nbs and no lrr domains ( fig1 ). the pcr product was cleaved with the restriction enzymes sacii and bamhi and cloned in the vector pcamv - 2 . the resulting vector was given designation p70s - bvkws3 . sub . — # 175 . the ability of the construct p70s - bvkws3 . sub . — 165 and p70 - 165 . sub . — # 175 to trigger a cell death in sugar beet leaves was tested quantitatively by transient biolistic transformation . for this , each vector was co - transformed with the reporter gene vector p70s - luc . as positive control the empty vector pcamv - 2 was used in combination with the reporter gene vector p - 70s - luc . in comparison to transformation of the empty vector ( pcamv - 2 ) the transformation of p70s - bvkws3 . sub . — 165 resulted in 65 % measurable reporter gene activity and the transformation of p70 - 165 -# 175 resulted in only 38 % measurable reporter gene activity ( fig5 ). this result showed that the exclusive expression of the 175 amino acid sized n - terminus of 165 . sub . — # 175 led to an intensive triggered of cell death in the transformed sugar beet leaves than the use of the 1066 amino acid sized full - length protein bvkws3 . sub . — 165 . by expression of 165 . sub . — # 175 more of the transformed leaf cells die off than in the case of the expression of bvkws3 . sub . — 165 . the cause for this difference is a new , more intensive form of the autoactivation of the r - protein by the shortening ( contraction ) at the n - terminus . the 5 ′- area of the gene bvkws3 — 135 triggers a more rapid cell death than the full - length cdna clone bvkws3 — 135 beginning with full - length cdna clone bvkws3 . sub . — 135 in the construct p70s - bvkws3 . sub . — 135 the 5 ′- area of the gene was amplified with the aid of the pfu - polymerase ( stratgene ) with use of the primer s316 ( ctcgagaattcgagctccaccgcgg ) ( seq id no : 17 ) and s330 ( ctggatcctcagggagaactccatctgggtggtcc ) with nucleotide sequence seq id no : 19 and simultaneously a stop codon was introduced in the coded area . the amplified area corresponds to the nucleotide sequence according to seq id no . 2 and codes for the amino acid sequence 1 - 147 of bvkws3 . sub . — 135 ( fig1 ). the amino acid sequence includes only the n - terminal area of bvkws3 . sub . — 135 and contains no nbs and no lrr domains or , as the case may be , motifs from these domains . the pcr product was cleaved with the restriction enzymes sacii and bamhi and cloned in the vector pcamv - 2 . the resulting vector was given the designation p70s - 135 . sub . — # 147 . the ability of the construct p70s - bvkws3 . sub . — 135 and p70s - 135 . sub . — # 147 to trigger a cell death in sugar beet leaves was tested quantitatively by transient biolistic transformations . for this , each vector was co - transformed with the reporter gene vector p70s - luc . as positive control the empty vector pcamv - 2 was used in combination with the reporter gene vector p70s - luc . in comparison to transformation of the empty vector ( pcamv - 2 ), the transformation of p70s - bvkws3 . sub . — 135 led to 74 . 5 % measurable reporter gene activity and the transformation of p70s - 135 . sub . — # 147 led to only 58 % measurable reporter gene activity ( fig6 ). the result showed that the expression of the full - length clone bvkws3 . sub . — 135 led to triggering of cell death in the transformed tissue . however , the exclusive expression of the 147 amino acid sized n - terminus of 135 . sub . — # 147 brought about a more intensive cell death in the transformed sugar beet leaves than the use of the 844 amino acid sized protein bvkws3 . sub . — 135 . by expression of 135 . sub . — # 147 more transformed leaf cells died than in the case of the expression of bvkws3 . sub . — 135 . the cause of this difference is a new , more intensive form of the autoactivation of the r - protein by the contraction or shortening at the n - terminus the 5 ′- area of the gene bv13033 triggers a more rapid cell death than the full - length cdna clone bv13033 beginning with full - length cdna clone bv13033 in the construct p70s - bv13033 the 5 ′- area of the gene was amplified with the aid of the pfu - polymerase ( stratagene ) with use of the primer s316 ( ctcgagaattcgagctccaccgcgg ) ( seq id no : 17 ) and s333 ( ctggatcctcaagaacaagtctcaggccttctgtt ) with nucleotide sequence seq id no : 20 and simultaneously a stop codon was introduced in the coded area . the amplified area corresponds to the nucleotide sequence according to seq id no : 3 and codes for the amino acid sequence 1 - 159 of bv13033 ( fig1 ). the amino acid sequence includes only the n - terminus area of bv13033 and contains no nbs and no lrr domains or motifs from these domains . the pcr product was cleaved with the restriction enzymes sacii and bamhi and cloned in the vector pcamv - 2 . the resulting vector was given the designation p70s - 13033 . sub . — # 159 . the ability of the construct p70s - 13033 and p70s - 13033 . sub . — # 159 to trigger a cell death in sugar beet leaves was tested quantitatively by transient biolistic transformations . for this , each vector was co - transformed with the reporter gene vector p705 - luc . as positive control the empty vector pcamv - 2 was used . in comparison to transformation of the empty vector ( pcamv - 2 ), the transformation of p70s - 13033 led to 95 % measurable reporter gene activity and the transformation of p70s - 165 . sub . — # 175 led to only 68 % measurable reporter gene activity ( fig7 ). the results showed that the expression of the full - length clone bv13033 led to the triggering of only a weak cell death in the transformed tissue . the exclusive expression of the 159 amino acid sized n - terminus of 13033 . sub . — # 159 brought about on the other hand an intensive cell death in the transformed sugar beet leaves . the cause of this difference is a new , more intensive form of the auto - activation of the r - protein by the shortening at the n - terminus . triggering of cell death in sugar beet leaves by the 5 ′- area of the gene bv12069 the r - gene bv12069 with the nucleotide sequence according to seq id no . 4 codes for the 166 amino acid sized n - terminus of r - protein . the protein bv12069 contains no nbs and no lrr domains , however , evidences a distinct homology to the 175 , 147 and 159 amino acid sized n - termini of the autoactivated r - proteins 165 — # 175 , 135 — # 147 , 13033 — # 159 ( fig1 ). the cdna clone was combined with doubled 35s promoter of vector pcamv - 2 ( fig3 ) to form the vector p70s - 12069 . in order to check the functionality of the gene bv12069 , the construct p70s - 12069 was expressed in combination with the reporter gene vector p70s - luc in sugar beet leaves transiently by biolistic transformation . the reporter gene activity in the leaves transformed with p70s - 12069 and p70s - luc amounted in three independent tests to 51 % of the activity which could be measured in the positive control ( empty vector pcamv - 2 and p70s - luc ) ( fig8 ). the expression of the 166 amino acid sized protein bv12069 therewith triggered a cell death in sugar beet cells . the shortening of the gene bvkws3 — 135 results in an autoactivated r - protein , however not the mutagenesis of the mhd domains the inventive mechanism of the autoactivation by condensation of a r - protein of the nbs - lrr type to the nbs - and lrr free n - terminus was compared with the method of autoactivation by mutagenesis of the mhd motif . the mutagenesis of the mhd motif of the rx - gene of the potato and the l5 gene of flax led to an autoactivation of the indicated gene ( bendahamane et al ., 2002 ; howes et al ., 2005 ). the cdna clone bvkws3 — 135 does for the mhd motif equivalent to the vhd motif , a motif that besides the mhd motif is likewise frequently found is r - gene ( howles et al ., 2005 ). the corresponding mutation was , as described in bendahmane et al . ( 2002 ), introduced in the full - length clone bvkws3 — 135 . for this , the amino acid aspartate in the vhd motif of the gene bvkws33 — 135 was exchanged with the amino acid valine . the resulting gene was given designation bvkw3 — 135_d480v . the effectiveness of the gene 135 — # 147 , bvkws3 — 135 - d408v and the non - modified gene bvkws3 — 135 were tested by agrobacterium tumefaciens initiated transient overexpression in sugar beet leaves . for this the cdna clone bvkws3 — 135 was combined with the d35s promoter and inserted in the binary vector per - 34sluci . the resulting vector was given the designation per135 - 34sluci . similarly there were processed the shortened cdna clone 135 — # 147 with a nucleotide sequence according to seq id no . 2 as well as with the mutantgenized cdna clone bvkws3 — 135_d408v . the resulting vectors were given designation per135 — # 147 - 35sluci and per135_d480v - 35sluci . the vectors were transformed in agrobacterium of strain or line c58c1 as described and injected in sugar beet leaves simultaneously with the control per - 35sluci . the photinus pyralis luciferase - reporter gene activity was measured 1 , 2 and 3 days post inoculation in the transformed leaves . sugar beet leaves , which were transformed with the control construct per - 35sluci showed on day 1 a small and the 2 nd and 3 rd day a luciferase activity of 299 , 000 and 433 , 000 rlu / mg leaf tissue . beet leaves which were transformed with the construct per135 - 35sluci showed on day 2 and day 3 a luciferase activity of 190 , 000 and 245 , 000 rlu / mg leaf tissue and therewith , in comparison to the positive control per - 35sluci , a measurable cell death . the reported gene activity of the construct per — 135_d480v - 35sluci amounted on day 2 and day 3 to 188 , 000 and 206 , 000 rlu / mg ( fig9 ). accordingly the introduction of the mhd mutation in the gene bvkws3 — 135 resulted in no , or a barely measurable , autoactivation . the r - gene 135 — # 147 shortened in accordance with this process showed on day 2 and 3 a reporter gene activity of 90 , 000 and 63 , 000 rlu / mg ( fig9 ) and therewith a significantly stronger cell death initiation and autoactivation than the construct per135 - 35sluci and per — 135_d480v - 35sluci . identification of common amino acid motifs in the n - termini of the r - protein of bvkws3 — 165 , bvkws3 — 135 . bv13033 and bv12069 und str3a a homology comparison between the 175 , 147 , 159 und 166 amino acid sized n - terminus of the r - proteins bvkws3 — 165 , bvkws3 — 135 , bv13033 and bv12069 and the 155 amino acid sized n - terminus of the r3a gene of the potato ( huang et el ., 2005 ) was carried out in order to identify common sequence motifs . the comparison lead to the identification of multiple consensus sequences in the n - termini of the autoactivated r - protein . the common sequence motifs are highlighted as consensus sequences in fig1 a ). one consensus sequence corresponds to the amino acid sequence according to seq id no : 13 : avlxdaexkqxx xxxlxxwlxd lkdxvydxdd ilde . another consensus sequence corresponds to the amino acid sequence according to seq id no : 14 : ixeixxkldd l both consensus sequences in the described form are contained only in such n - termini of cc - nbs - lrr r - proteins in which the expression leads to an autoactivation . thus , the 160 amino acid sized cc - domain of the rx - gene is not capable of initiating cell death or , as the case may be , a hypersensitive reaction ( bendahmane et al ., 2002 ). the transient expression of the 177 amino acid sized n - terminus of the r - gene bvkws3 — 133_e08 of sugar beet and the 540 amino acid sized n - terminus of the r1 gene of the potato ( ballvora et al ., 2002 ) initiated in comparison to the full - length r - gene bvkws3 — 133_e08 no amplified or , in the case of the r1 gene , no cell death ( data not shown ). the amino acid comparison of the n - termini of the autoactivated proteins bvkws3 — 165 — # 176 , bvkws3 — 135 — # 147 , bv13033 — # 159 , bv12069 und str3a -# 1 - 155 with the amino acid sequences of the cc - domain of the rx -, str1 - und bvkws3 — 133 — # 177 - protein show the absence of the above described consensus sequences in the not autoactivated n - termini ( fig1 b ). in particular the sequence motif dae is an important aid of identification of r - proteins , of which the n - terminus is autoactive . with the aid of the sequence motif dae in the consensus sequence suitable r - genes for an autoactivation can be found in numerous plant species , as shown in fig1 c for examples of arabidopsis thaliana ( atab028617 ), bean ( pvulgarisj71 ), rice ( osativaap003073 ), soy bean ( gmaxkr4 ) und tomato ( tomato - i2 ). the amino acid sequence of 147 - 175 is important for the autoactivation of the r - protein 165 — # 175 in order to identify the amino acid section in the protein 165 — # 175 which is important for the autoactivation of the n - terminus of nbs - lrr proteins , the encoding region of the cdna clone 165 — # 175 was shortened . the cdna clones 165 — # 93 and 165 — # 146 coded for the amino acid 1 - 93 or as the case may be 1 - 146 of the protein 165 — # 175 . the transient biolistic test of the constructs p70s — 165 — # 93 , p70s — 165 — # 146 and p70s — 165 — # 175 showed that only the protein 165 — # 175 , however not 165 — # 93 and 165 — # 146 , triggered a strong cell death ( fig1 ). accordingly the sequence region of 146 - 175 is essential for the autoactivation of nbs - lrr proteins . in this region there lies a sequence motif conserved in all examined proteins ( fig1 a ). rapid activation of the synthetic pathogen inducible promoters 2xs - 2xd and 2xw2 - 2xd by fungal infestation for the pathogen induced overexpression of complete or partial resistance genes , particularly suited are synthetic promoters of type nxs - mxd , nxw2 - mxd and nxgst1 - mxd , wherein n = 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 and m = 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . for example , promoters of type 2xs - 2xd according to seq id no . 10 , 2xw2 - 2xd according to seq id no . 11 as well as 2xgst1 - 2xd according to seq id no . 12 were combined with the luciferase gene from photinus pyralis , transformed in sugar beets and analyzed in reaction to fungal infestation . for the plant transformation the binary vectors 2xs - 2xd - luc - kan , 2xw2 - 2xd - luc - kan , and 2xgst1 - 2xd - luc - kan were found to be useful . the binary vectors were transformed in the agrobacterium tumefaciens type c58c1 with the resident plasmid pgv2260 by a direct dna - transformation process ( an , 1987 ). the selection of recombinant a . tumefaciens clones occurred using the antibiotic kanamycin ( 50 mg / l ). the transformation of the sugar beets occurred according to lindsey et al . ( 1991 ) using the antibiotic kanamycin . the transgenecity of the plants was tested by pcr . the use of the primer gtggagaggctattcggta and ccaccatgatattcggcaag lead to the amplification of the 553 base pair sized dna - fragment from the nptii - gene . the pcr was carried out using 10 ng genomic dna , a primer concentration of 0 . 2 μm at an annealing temperature of 55 ° c . in a mutli - cycler ptc - 200 ( mj reasearch , watertown , usa ). in order to analyze the pathogen inducibility of the promoter , the transgenic sugar beets were infected under in - vitro conditions with a leaf spot inducer of sugar beets , cercospora beticola . respectively 4 plants of a transgenic line dipped in a suspension of c . beticola mycelium fragments ( 400 , 000 fragment / ml ) and 4 plants were dipped for control purposes in dilute vegetable juice . infected plants and control plants were subsequently incubated at 25 ° c . and 16 h illumination in a culture cabinet . infected and non - infected leaf material was removed 1 , 2 , 3 , 4 and 6 - 7 days subsequent to the inoculation and the luciferase reporter gene activity was determined with the luciferase assay system ( promega , mannheim , germany ) as described . both the 2xs - 2xd as well as the 2xw2 - 2xd promoter showed a rapid and strong pathogen inducibility in the early phase of the infection , differed however in base activity and promoter strength ( fig1 - 13 ). the 2xs - 2xd - promotor was rapidly induced in the case of the transgenic lines pr39 / 11 , pr39 / 48 and pr39 / 49 , already 11 - 59 fold on the first day after inoculation and 21 - 384 fold on the second day in comparison to the non - infected plants ( fig1 ). while day 1 is still characterized by a growth of the fungal hyphae on the epidermis , on day 2 there is a penetration of the leaves through the stomata and therewith a penetration into the leaf tissue . in the late phase of the infection at day 7 , a 113 - 792 fold induction of the promoter was measured with a visible development of the necrosis . the base activity of the 2xs - 2xd promoter measured as reported gene activity of the non - infected plats is very small and amounted to only the 1 - 10 fold of the luciferase activity measurable in the non - transgenic plants . the activation of the 2xw2 - 2xd promoter progressed somewhat slower than that of the 2xs - 2xd promoter . on the first infection day the 2xw2 - 2xd promoter exhibited only a 2 - 11 fold , and on the second infection day a 5 - 56 fold , pathogen induction . with the occurrence of the necrosis on day 7 , a maximal 318 - 672 fold pathogen induction was achieved ( fig1 ). the base activity of the 2xw2 - 2 - d promoter , having a 10 - 50 fold of reporter gene activity measurable in comparison to the non - transgenic plants , is higher than in the case of the 2xs - 2xd promoter . significantly the 2xw2 - 2xd promoter exceeds the 2xs - 2xd promoter by its approximately 10 - fold higher promoter strength . the characteristic of a synthetic promoter of type nxs - mxd , nxw2 - mxd and nxgst1 - mxd with n = 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 and m = 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 can be modulated and optimized by variation of the number of the cis - elements according to the requirements of the gene expression . this is shown for illustrative purposes for the promoter type nxs - mxd . besides the binary vector 2xs - 2xd - luc - kan the binary vectors 4xs - 2xd - luc - kan , 2xs - 4xd - luc - kan and 4xs - 4xd - luc - kan were constructed and transformed in sugar beets . the transgenic plants were infected with c . beticola as described and reporter gene activity was measured daily subsequent to fungal inoculation . the test results from 13 independent 2xs - 2xd - luc lines , 14 independant 4xs - 2xd - luc lines , 15 independant 2xs - 4xd - luc lines as well as 15 independent 4xs - 4xd - luc lines were determined and the measurement values were compared in their promoter strength , pathogen induction and base activity . the comparison of the 2xs - 2xd promoter characteristics with the variants 2xs - 4xd , 4xs - 2xd and 4xs - 4xd showed that the average promoter strength was increased by the use of tetramers in comparison to the promoters constructed of dimers ( fig1 ). in addition , the pathogen inducibility of dimer - dimer promoter ( 2xs - 2xd ) climbed above the tetramer - dimer and dimer - tetramer promoters ( 4xs - 2xd , 4xs - 2xd ) to the tetramer - tetramer promoter ( 4xs - 4xd ) at all measurement intervals ( table 1 ). parallel with the increase in the promoter strength and the pathogen inducibility there results an increase in the base activity of the promoters which contain the tetramers ( table 2 ). shown is the average value of the base activity of 13 - 15 independent transformants ( lines ) per promoter construct , which were measured in the 4 day infection experiment as non - infected controls . the base activity provides the behavior or relationship of the reporter gene activity of the transgenic plants in comparison to the non - specific background activity of non transgenic plants . this example shows that the promoter characteristics important to the concept , such as promoter strength , pathogen inducibility and base activity , can be regulated by the number of the cis - elements and that optimal promoter variants can be created for the respective technical conversion . the optimal number of cis - elements of pathogen inducible promoters is , in the experimental example , with regard to the pathogen inducibility , greater than the dimer solution described by rushton et al ., 2002 . producing fungus resistant by transformation of the pathogen inducible resistance gene . for increasing the fungal resistance of sugar beets the promoters 2xs - 2xd or , as the case may be , 2xw 2 - 2xd were respectively combined with the four r - genes bvkws3 — 123 , bvkws3 — 133 , bvkws3 — 135 and bvkws3 — 165 and transformed in sugar beets . then the 13 , 959 or , as the case may be , 13 , 969 kb sized binary vectors 2xs - 2xd - luc - kan and 2xw2 - 2xd - luc - kan were cleaved with sac1 and the cleaved locations were filled by t4 - dna polymerase treatment . subsequently the vectors were re - sectioned with xhol , electrophoretically separated and the 12 , 284 or , as the case may be , 12 , 294 kb size vectors were separated from the 1 , 675 kb size luciferase gene and isolated . the isolation of the zr resistance gene occurred from the vectors p70s - bvkws3 — 123 , p70s - bvkws3 — 133 , p70s - bvkws3 — 135 and p70s - bvkws3 — 165 . for this , the vectors were first linearized with not1 and the cleavage points were filled by klenow treatment . the vectors were then cut with xhol and the r - gene isolated . the resulting vectors were given the designations 2xs - 2xd - bvkws3 — 123 , 2xs - 2xd - bvkws3 — 133 , 2xs - 2xd - bvkws3 — 135 und 2xs - 2xd - bvkws3 — 165 or , as the case may be , 2xw 2 - 2xd - bvkws3 — 123 , 2xw 2 - 2xd - bvkws3 — 133 , 2xw 2 - 2xd - bvkws3 — 135 and 2xw 2 - 2xd - bvkws3 — 165 ( fig1 - 18 ). the binary vectors were used as described for the production of transgenic sugar beets . identification of fungal resistant sugar beets by resistance testing with plant pathogenic fungi cercospora beticola . the elevated fungal resistance of the plants was observed in a fungal resistance test which is described in the following for exemplary purposes for the resistance testing for the sugar beet with respect to cercospora beticola . for the infection of sugar beets with the leaf spot inducer c . beticola , use was made of , besides the transgenic plants , sugar beets of the genotype 3dc4156 used for the transformation , in a greenhouse . two weeks prior to the plant inoculation vegetable juice plates ( 40 % albani - vegetable juice ) were spiked with the aggressive c . beticola isolate ahlburg and incubated at 25 ° c . directly prior to inoculation the agar with growing fungi is scratched off with the aid of an object carrier and some water . the concentration of mycellular fragments and fungal spores is determined using a counting cell chamber . the inoculum density is adjusted by dilution with water to a concentration of 20 , 000 fragments / ml . for infection the 10 - 12 week old plants were dipped inverted in a 5 l glass beaker filled with the inoculum . per line to be examined , 30 plants were inoculated and the plants were set up randomized in the greenhouse . the plants were incubated following inoculation for 4 days at 28 ° c . and 95 % humidity in a greenhouse . after the fourth day the humidity was reduced to 60 - 70 %. two , three and four weeks following inoculation the leaf drop is optically evaluated using the kleinwanzlebener saatzucht ( kws ) rating scheme ( 1970 ) ( 1 = healthy leaves , 9 = 100 % destroyed leaves ). transgenic lines , which were transformed with the constructs 2xs - 2xd - bvkws3 — 123 , 2xs - 2xd - bvkws3 — 133 , 2xs - 2xd - bvkws3 — 165 , 2xw 2 - 2xd - bvkws3 — 123 , 2xw 2 - 2xd - bvkws3 — 133 , 2xw 2 - 2xd - bvkws3 — 135 or 2xw 2 - 2xd - bvkws3 — 165 , showed , in comparison the control , an elevated fungal resistance ( table 3 ). 2 audpc ( area under disease progress curve ) value determined over 3 rating periods ( t1 - t3 ). the audpc encompasses the progression of the strength of infestation of multiple rating time points into a single value . the analysis of the time progression of the infestation development in the transformants pr68 - 6 and pr70 - 32 over the three rating periods shows that with advance of experiment duration the difference in the infestation development between control and transgenic lines increases ( fig1 and 20 ). these results show that the induced expression of different r - genes of the sugar beet leads , with the aid of the pathogen specific promoter , to an elevated fungal resistance . producing fungal resistant plants by transformation of the n - terminal area of the r - gene under the control of pathogen responsive promoters . in order to produce fungal resistant plants with use of the n - terminal section of the r - gene , the condensed or shortened r - genes 13033 — # 159 , 135 — # 147 , 165 — # 175 and bv12069 were combined with the promoters 2xs - 2xd and 2xw2 - 2xd and transformed in sugar beets . for this , the 13 , 959 or , as the case may be , 13 , 969 kb sized binary vectors 2xs - 2xd - luc - kan and 2xw2 - 2xd - luc - kan were cleaved with saci and the cleavage points were filled by treatment with t4 - dna polymerase . subsequently , the vectors were further cut with xhol , gel - electrophoretically separated , and the 12 , 284 or , as the case may be , 12 , 294 kb size vectors were separated from the 1 , 675 size luciferase gene and isolated . the isolation of the shortened r - gene occurred from the vectors p70s - 12069 , p70s - 13033 — # 159 , p70s - 135 — # 147 and p70s - 165 — # 175 . the vectors were first linearized with xbal , the dna ends were filled by klenow treatment , and the vectors were further cut with xhol . the isolated r - gene fragments were then cloned in the prepared binary vectors . the resulting vectors were given the designations 2xs - 2xd - 12069 , 2xs - 2xd - 13033 — # 159 , 2xs - 2xd - 135 — # 147 , 2xs - 2xd - 165 — # 175 or , as the case may be , 2xw2 - 2xd - 12069 , 2xw2 - 2xd - 13033 — # 159 , 2xw2 - 2xd - 135 — # 147 , 2xw2 - 2xd - 165 — # 175 ( fig2 - 22 ). the binary vectors were transformed as described in sugar beets and the fungal resistant plants were identified by a cercospora beticola resistance test . altschul , s . f . et al . 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