Hypersensitive response elicitor fragments and uses thereof

专利摘要:
The present invention is directed to isolated fragments of an Erwiniahypersensitive response elicitor protein or polypeptide which fragments elicit a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode the Erwiniahypersensitive response eliciting fragment. Isolated fragments of hypersensitive response elicitor proteins or polypeptides, which elicit a hypersensitive response, and the isolated DNA molecules that encode them can be used to impart disease resistance to plants, to enhance plant growth, and/or to control insects on plants. This can be achieved by applying the hypersensitive response eliciting fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds. Alternatively, transgenic plants or plant seeds transformed with a DNA molecule encoding a hypersensitive response eliciting fragment can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
公开号:KR20010013226A
申请号:KR19997011216
申请日:1998-05-28
公开日:2001-02-26
发明作者:라비로날드제이.；위종-민；비어스티븐브이.
申请人:해우슬러 에이치 월터；코넬 리서치 파운데이션 인코포레이티드；리처드 에스. 카훈；브래들리 에스. 파웰；에덴 바이오사이언스 코포레이션；
IPC主号:

专利说明:

Hypersensitivity Response Factor Fragments and Their Uses {HYPERSENSITIVE RESPONSE ELICITOR FRAGMENTS AND USES THEREOF}
<110> CORNELL RESEARCH FOUNDATION, INC AND EDEN BIOSCIENCE CORPORATION
<120> HYPERSENSITIVE RESPONSE ELICITOR FRAGMENTS ELICITING A HYPERSENSI
TIVE RESPONSE AND USES THEREOF
<130> IPP996142US
<150> US 60 / 048,109
<151> 1997-05-30
<160> 30
<170> KOPATIN 1.5
<210> 1
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 1
gggaattcat atgagtctga atacaagtgg g 31
<210> 2
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 2
gggaattcat atgggcggtg gcttaggcgg t 31
<210> 3
<211> 29
<212> DNA
<213> Erwinia amylovora
<400> 3
ggcatatgtc gaacgcgctg aacgatatg 29
<210> 4
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 4
gggaattcat atgttaggcg gttcgctgaa c 31
<210> 5
<211> 29
<212> DNA
<213> Erwinia amylovora
<400> 5
ggcatatgct gaacacgctg ggctcgaaa 29
<210> 6
<211> 29
<212> DNA
<213> Erwinia amylovora
<400> 6
ggcatatgtc aacgtcccaa aacgacgat 29
<210> 7
<211> 27
<212> DNA
<213> Erwinia amylovora
<400> 7
ggcatatgtc cacctcagac tccagcg 27
<210> 8
<211> 34
<212> DNA
<213> Erwinia amylovora
<400> 8
gggaattcat atgcaaagcc tgtttggtga tggg 34
<210> 9
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 9
gggaattcat atgggtaatg gtctgagcaa g 31
<210> 10
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 10
gggaattcat atgaaagcgg gcattcaggc g 31
<210> 11
<211> 34
<212> DNA
<213> Erwinia amylovora
<400> 11
gggaattcat atgacaccag ccagtatgga gcag 34
<210> 12
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 12
gcaagcttaa cagcccacca ccgcccatca t 31
<210> 13
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 13
gcaagcttaa atcgttcagc gcgttcgaca g 31
<210> 14
<211> 34
<212> DNA
<213> Erwinia amylovora
<400> 14
gcaagcttaa tatctcgctg aacatcttca gcag 34
<210> 15
<211> 30
<212> DNA
<213> Erwinia amylovora
<400> 15
gcaagcttaa ggtgccatct tgcccatcac 30
<210> 16
<211> 34
<212> DNA
<213> Erwinia amylovora
<400> 16
gcaagcttaa atcagtgact ccttttttat aggc 34
<210> 17
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 17
gcaagcttaa caggcccgac agcgcatcag t 31
<210> 18
<211> 31
<212> DNA
<213> Erwinia amylovora
<400> 18
gcaagcttaa accgataccg gtacccacgg c 31
<210> 19
<211> 34
<212> DNA
<213> Erwinia amylovora
<400> 19
gcaagcttaa tccgtcgtca tctggcttgc tcag 34
<210> 20
<211> 25
<212> DNA
<213> Erwinia amylovora
<400> 20
gcaagcttaa gccgcgccca gcttg 25
<210> 21
<211> 338
<212> PRT
<213> Erwinia amylovora
<400> 21
Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser
1 5 10 15
Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser
20 25 30
Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr
35 40 45
Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln Gly Leu
50 55 60
Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser
65 70 75 80
Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys
85 90 95
Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp
100 105 110
Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln
115 120 125
Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly Asn Met
130 135 140
Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile Leu Gly
145 150 155 160
Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser Leu Gly
165 170 175
Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln Leu
180 185 190
Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala
195 200 205
Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val
210 215 220
Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp
225 230 235 240
Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly Trp
245 250 255
Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys
260 265 270
Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gln
275 280 285
Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr
290 295 300
Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile Asp Ala
305 310 315 320
Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala
325 330 335
Asn ala
<210> 22
<211> 2141
<212> DNA
<213> Erwinia amylovora
<400> 22
cgattttacc cgggtgaacg tgctatgacc gacagcatca cggtattcga caccgttacg 60
gcgtttatgg ccgcgatgaa ccggcatcag gcggcgcgct ggtcgccgca atccggcgtc 120
gatctggtat ttcagtttgg ggacaccggg cgtgaactca tgatgcagat tcagccgggg 180
cagcaatatc ccggcatgtt gcgcacgctg ctcgctcgtc gttatcagca ggcggcagag 240
tgcgatggct gccatctgtg cctgaacggc agcgatgtat tgatcctctg gtggccgctg 300
ccgtcggatc ccggcagtta tccgcaggtg atcgaacgtt tgtttgaact ggcgggaatg 360
acgttgccgt cgctatccat agcaccgacg gcgcgtccgc agacagggaa cggacgcgcc 420
cgatcattaa gataaaggcg gcttttttta ttgcaaaacg gtaacggtga ggaaccgttt 480
caccgtcggc gtcactcagt aacaagtatc catcatgatg cctacatcgg gatcggcgtg 540
ggcatccgtt gcagatactt ttgcgaacac ctgacatgaa tgaggaaacg aaattatgca 600
aattacgatc aaagcgcaca tcggcggtga tttgggcgtc tccggtctgg ggctgggtgc 660
tcagggactg aaaggactga attccgcggc ttcatcgctg ggttccagcg tggataaact 720
gagcagcacc atcgataagt tgacctccgc gctgacttcg atgatgtttg gcggcgcgct 780
ggcgcagggg ctgggcgcca gctcgaaggg gctggggatg agcaatcaac tgggccagtc 840
tttcggcaat ggcgcgcagg gtgcgagcaa cctgctatcc gtaccgaaat ccggcggcga 900
tgcgttgtca aaaatgtttg ataaagcgct ggacgatctg ctgggtcatg acaccgtgac 960
caagctgact aaccagagca accaactggc taattcaatg ctgaacgcca gccagatgac 1020
ccagggtaat atgaatgcgt tcggcagcgg tgtgaacaac gcactgtcgt ccattctcgg 1080
caacggtctc ggccagtcga tgagtggctt ctctcagcct tctctggggg caggcggctt 1140
gcagggcctg agcggcgcgg gtgcattcaa ccagttgggt aatgccatcg gcatgggcgt 1200
ggggcagaat gctgcgctga gtgcgttgag taacgtcagc acccacgtag acggtaacaa 1260
ccgccacttt gtagataaag aagatcgcgg catggcgaaa gagatcggcc agtttatgga 1320
tcagtatccg gaaatattcg gtaaaccgga ataccagaaa gatggctgga gttcgccgaa 1380
gacggacgac aaatcctggg ctaaagcgct gagtaaaccg gatgatgacg gtatgaccgg 1440
cgccagcatg gacaaattcc gtcaggcgat gggtatgatc aaaagcgcgg tggcgggtga 1500
taccggcaat accaacctga acctgcgtgg cgcgggcggt gcatcgctgg gtatcgatgc 1560
ggctgtcgtc ggcgataaaa tagccaacat gtcgctgggt aagctggcca acgcctgata 1620
atctgtgctg gcctgataaa gcggaaacga aaaaagagac ggggaagcct gtctcttttc 1680
ttattatgcg gtttatgcgg ttacctggac cggttaatca tcgtcatcga tctggtacaa 1740
acgcacattt tcccgttcat tcgcgtcgtt acgcgccaca atcgcgatgg catcttcctc 1800
gtcgctcaga ttgcgcggct gatggggaac gccgggtgca atatagagaa actcgccggc 1860
cagatggaga cacgtctgcg ataaatctgt gccgtaacgt gtttctatcc gcccctttag 1920
cagatagatt gcggtttcgt aatcaacatg gtaatgcggt tccgcctgtg cgccggccgg 1980
gatcaccaca atattcatag aaagctgtct tgcacctacc gtatcgcggg agataccgac 2040
aaaatagggc agtttttgcg tggtatccgt ggggtgttcc ggcctgacaa tcttgagttg 2100
gttcgtcatc atctttcrcc atcrgggcga cctgatcggt t 2141
<210> 23
<211> 403
<212> PRT
<213> Erwinia amylovora
<400> 23
Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser
1 5 10 15
Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg Gln
20 25 30
Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly Gly Gly Asn
35 40 45
Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu Leu Thr Gly Met Met
50 55 60
Met Met Met Ser Met Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu
65 70 75 80
Gly Gly Gly Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu
85 90 95
Gly Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr
100 105 110
Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Ser Thr Thr Asn Ser Pro
115 120 125
Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser
130 135 140
Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gln Gln
145 150 155 160
Leu Leu Lys Met Phe Ser Glu Ile Met Gln Ser Leu Phe Gly Asp Gly
165 170 175
Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gly Lys Gln Pro Thr Glu
180 185 190
Gly Glu Gln Asn Ala Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly
195 200 205
Leu Met Gly Asn Gly Leu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly
210 215 220
Gly Gly Gln Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu
225 230 235 240
Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln
245 250 255
Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln
260 265 270
Ala Leu Asn Asp Ile Gly Thr His Arg His Ser Ser Thr Arg Ser Phe
275 280 285
Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu Ile Gly Gln Phe Met
290 295 300
Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gln Tyr Gln Lys Gly Pro
305 310 315 320
Gly Gln Glu Val Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser
325 330 335
Lys Pro Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn
340 345 350
Lys Ala Lys Gly Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn
355 360 365
Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp
370 375 380
Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu
385 390 395 400
Gly ala ala
<210> 24
<211> 1288
<212> DNA
<213> Erwinia amylovora
<400> 24
aagcttcggc atggcacgtt tgaccgttgg gtcggcaggg tacgtttgaa ttattcataa 60
gaggaatacg ttatgagtct gaatacaagt gggctgggag cgtcaacgat gcaaatttct 120
atcggcggtg cgggcggaaa taacgggttg ctgggtacca gtcgccagaa tgctgggttg 180
ggtggcaatt ctgcactggg gctgggcggc ggtaatcaaa atgataccgt caatcagctg 240
gctggcttac tcaccggcat gatgatgatg atgagcatga tgggcggtgg tgggctgatg 300
ggcggtggct taggcggtgg cttaggtaat ggcttgggtg gctcaggtgg cctgggcgaa 360
ggactgtcga acgcgctgaa cgatatgtta ggcggttcgc tgaacacgct gggctcgaaa 420
ggcggcaaca ataccacttc aacaacaaat tccccgctgg accaggcgct gggtattaac 480
tcaacgtccc aaaacgacga ttccacctcc ggcacagatt ccacctcaga ctccagcgac 540
ccgatgcagc agctgctgaa gatgttcagc gagataatgc aaagcctgtt tggtgatggg 600
caagatggca cccagggcag ttcctctggg ggcaagcagc cgaccgaagg cgagcagaac 660
gcctataaaa aaggagtcac tgatgcgctg tcgggcctga tgggtaatgg tctgagccag 720
ctccttggca acgggggact gggaggtggt cagggcggta atgctggcac gggtcttgac 780
ggttcgtcgc tgggcggcaa agggctgcaa aacctgagcg ggccggtgga ctaccagcag 840
ttaggtaacg ccgtgggtac cggtatcggt atgaaagcgg gcattcaggc gctgaatgat 900
atcggtacgc acaggcacag ttcaacccgt tctttcgtca ataaaggcga tcgggcgatg 960
gcgaaggaaa tcggtcagtt catggaccag tatcctgagg tgtttggcaa gccgcagtac 1020
cagaaaggcc cgggtcagga ggtgaaaacc gatgacaaat catgggcaaa agcactgagc 1080
aagccagatg acgacggaat gacaccagcc agtatggagc agttcaacaa agccaagggc 1140
atgatcaaaa ggcccatggc gggtgatacc ggcaacggca acctgcaggc acgcggtgcc 1200
ggtggttctt cgctgggtat tgatgccatg atggccggtg atgccattaa caatatggca 1260
cttggcaagc tgggcgcggc ttaagctt 1288
<210> 25
<211> 341
<212> PRT
<213> Erwinia amylovora
<400> 25
Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met
1 5 10 15
Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser
20 25 30
Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met
35 40 45
Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala
50 55 60
Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val
65 70 75 80
Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe
85 90 95
Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met
100 105 110
Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu
115 120 125
Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met
130 135 140
Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro Ala Gln Phe Pro
145 150 155 160
Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe
165 170 175
Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile Ile
180 185 190
Gly Gln Gln Leu Gly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly
195 200 205
Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser
210 215 220
Val Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser
225 230 235 240
Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile Asp
245 250 255
Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val
260 265 270
Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gly Gly Gln Ser Ala Gln
275 280 285
Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala
290 295 300
Thr Leu Lys Asp Ala Gly Gln Thr Gly Thr Asp Val Gln Ser Ser Ala
305 310 315 320
Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg
325 330 335
Asn Gln Ala Ala Ala
340
<210> 26
<211> 1016
<212> DNA
<213> Erwinia amylovora
<400> 26
tcagtcttaa cagcagctcg ctgcaaaccc cggcaatggc ccttgtcctg gtacgtcctg 60
aagccgagac gactggcagt acgtcgagca aggcgcttca ggaagttgtc gtgaagctgg 120
ccgaggaact gatgcgcaat ggtcaactcg acgacagctc gccattggga aaactgttgg 180
ccaagtcgat ggccgcagat ggcaaggcgg gcggcggtat tgaggatgtc atcgctgcgc 240
tggacaagct gatccatgaa aagctcggtg acaacttcgg cgcgtctgcg gacagcgcct 300
cgggtaccgg acagcaggac ctgatgactc aggtgctcaa tggcctggcc aagtcgatgc 360
tcgatgatct tctgaccaag caggatggcg ggacaagctt ctccgaagac gatatgccga 420
tgctgaacaa gatcgcgcag ttcatggatg acaatcccgc acagtttccc aagccggact 480
cgggctcctg ggtgaacgaa ctcaaggaag acaacttcct tgatggcgac gaaacggctg 540
cgttccgttc ggcactcgac atcattggcc agcaactggg taatcagcag agtgacgctg 600
gcagtctggc agggacgggt ggaggtctgg gcactccgag cagtttttcc aacaactcgt 660
ccgtgatggg tgatccgctg atcgacgcca ataccggtcc cggtgacagc ggcaataccc 720
gtggtgaagc ggggcaactg atcggcgagc ttatcgaccg tggcctgcaa tcggtattgg 780
ccggtggtgg actgggcaca cccgtaaaca ccccgcagac cggtacgtcg gcgaatggcg 840
gacagtccgc tcaggatctt gatcagttgc tgggcggctt gctgctcaag ggcctggagg 900
caacgctcaa ggatgccggg caaacaggca ccgacgtgca gtcgagcgct gcgcaaatcg 960
ccaccttgct ggtcagtacg ctgctgcaag gcacccgcaa tcaggctgca gcctga 1016
<210> 27
<211> 344
<212> PRT
<213> Erwinia amylovora
<400> 27
Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln
1 5 10 15
Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser
20 25 30
Val Gln Asp Leu Ile Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile
35 40 45
Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly
50 55 60
Asn Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala
65 70 75 80
Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser
85 90 95
Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met
100 105 110
Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala
115 120 125
Ala Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys Gly Asn Gly Val
130 135 140
Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gln Gly Gly Leu Ala
145 150 155 160
Glu Ala Leu Gln Glu Ile Glu Gln Ile Leu Ala Gln Leu Gly Gly Gly
165 170 175
Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly
180 185 190
Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala
195 200 205
Asp Gly Gly Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn
210 215 220
Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp
225 230 235 240
Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu Asn
245 250 255
Ala Leu Val Gln Met Met Gln Gln Gly Gly Leu Gly Gly Gly Asn Gln
260 265 270
Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly
275 280 285
Ala Asn Pro Gly Ala Asn Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser
290 295 300
Gly Gln Asn Asn Leu Gln Ser Gln Ile Met Asp Val Val Lys Glu Val
305 310 315 320
Val Gln Ile Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln
325 330 335
Gln Ser Thr Ser Thr Gln Pro Met
340
<210> 28
<211> 1035
<212> DNA
<213> Erwinia amylovora
<400> 28
atgtcagtcg gaaacatcca gagcccgtcg aacctcccgg gtctgcagaa cctgaacctc 60
aacaccaaca ccaacagcca gcaatcgggc cagtccgtgc aagacctgat caagcaggtc 120
gagaaggaca tcctcaacat catcgcagcc ctcgtgcaga aggccgcaca gtcggcgggc 180
ggcaacaccg gtaacaccgg caacgcgccg gcgaaggacg gcaatgccaa cgcgggcgcc 240
aacgacccga gcaagaacga cccgagcaag agccaggctc cgcagtcggc caacaagacc 300
ggcaacgtcg acgacgccaa caaccaggat ccgatgcaag cgctgatgca gctgctggaa 360
gacctggtga agctgctgaa ggcggccctg cacatgcagc agcccggcgg caatgacaag 420
ggcaacggcg tgggcggtgc caacggcgcc aagggtgccg gcggccaggg cggcctggcc 480
gaagcgctgc aggagatcga gcagatcctc gcccagctcg gcggcggcgg tgctggcgcc 540
ggcggcgcgg gtggcggtgt cggcggtgct ggtggcgcgg atggcggctc cggtgcgggt 600
ggcgcaggcg gtgcgaacgg cgccgacggc ggcaatggcg tgaacggcaa ccaggcgaac 660
ggcccgcaga acgcaggcga tgtcaacggt gccaacggcg cggatgacgg cagcgaagac 720
cagggcggcc tcaccggcgt gctgcaaaag ctgatgaaga tcctgaacgc gctggtgcag 780
atgatgcagc aaggcggcct cggcggcggc aaccaggcgc agggcggctc gaagggtgcc 840
ggcaacgcct cgccggcttc cggcgcgaac ccgggcgcga accagcccgg ttcggcggat 900
gatcaatcgt ccggccagaa caatctgcaa tcccagatca tggatgtggt gaaggaggtc 960
gtccagatcc tgcagcagat gctggcggcg cagaacggcg gcagccagca gtccacctcg 1020
acgcagccga tgtaa 1035
<210> 29
<211> 26
<212> PRT
<213> Erwinia amylovora
<400> 29
Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala Ile Leu Ala
1 5 10 15
Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr
20 25
<210> 30
<211> 20
<212> PRT
<213> Erwinia amylovora
<400> 30
Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln
1 5 10 15
Leu Leu Ala Met
20
Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatibility with the development of intercellular bacteria, development of symptoms, and development of disease in host plants. : Pathogen-host interaction; And (2) non-compatible interactions that manifest as specific types of hypersensitivity reactions that occur without progressive disease symptoms. During conformance interactions with host plants, bacterial populations increase significantly and progressive symptoms occur. During incompatible interactions there is no increase in the population of bacteria and no progressive symptoms.
The hypersensitivity reaction is rapid and local necrosis associated with the active defense of plants against many pathogens (Kiraly, Z., "Defenses Triggered by the Invader: Hypersensitivity," page 201-224 in: Plant Disease: An Advanced Treatise , Vol 5, JG Horsfall and EB Cowling, ed.Academic Press New York (1980); Klement, Z., "Hypersensitivity," pages 149-177 in: Phytopathogenic Prokaryotes, Vol. 2, MS Mount and GH Lacy, ed. Academic Press, New York (1982). When high concentrations of pathogens with a limited host, such as Pseudomonas syringae or Erwinia amylovora, (10 ⁷ cells / ml or more) infiltrate the leaves of non-host plants, the hypersensitivity reactions induced by bacteria are easily observed as tissue collapse. (Necrosis occurs even at low levels of inoculum only in isolated plant cells) (Klement Z., "Rapid Detection of Pathogenicity of Phytopathogenic pseudomonas," Nature 199: 299-300; Klement, et al., "Hypersensitive Reaction Induced by Phytopathogenic Bacteria in the Tabacco Leaf, "Phytopathology 54: 474-477 (1963); Turner, et al.," The Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction, "Phytopathology 64: 885-890 (1974); Klement, Z., "hypersensitivity," 149-177 in Phytopathogenic Prokaryotes, Vol 2., MS Mount and GH Lacy, ed. Academic Press, New York (1982)). The ability to induce hypersensitivity reactions in nonhosts and the ability to be pathogenic in hosts seem to be correlated. Klement, Z., "Hypersensitivity," pages 149-177 in Phytopathogenic Prokaryotes, Vol. 2., MS Mount and GH Lacy, ed. As described by Academic Press, New York, these pathogens appear delayed in their interaction with a suitable host but cause physically similar necrosis. Moreover, the ability to produce hypersensitivity reactions or pathogenesis depends on a common set of genes named hrp (Lindgren, PB, et al., "Gene Cluster of Pseudomonas syringae pv. 'Phaseolicola' Conrtrols Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants, "J. Bacteriol. 168: 512-22 (1986); Willis, DK, et al.," Hrp Genes of Phytopathogenic Bacteria, "Mol. Plant-Microbe Interact. 4: 132-138 (1991). In conclusion, hypersensitivity reactions may include clues to plant defense properties and bacterial pathogenesis.
The hrp gene is widely distributed in Gram-negative plant pathogens, colonies, is conserved and in some cases interchangeable with the pathogens (Willis, DK, et al., "Hrp Genes of Phytopathogenic Bacteria," Mol.Plant-Microbe Interact. 4: 132-138 (1991); Bonas, U., "hrp Genes of Phytopathogenic Bacteria," pages 79-98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals-Molecular and Cellular Mechanism, JL Dangl, ed.Springer-Verlag, Berlin (1994). Several hrp genes code for components of the protein secretion pathway similar to Yersinia, Shigella and Salmonella spp., Which secrete essential proteins in animal diseases (Van Gijsegem, et al., "Evolutionary Conservation of Pathogenicity Determinants Among Plant and Animal Pathogenic Bacteria, '' Trends Microbiol. 1: 175-180 (1993). In E. amylovora, P. syringae, and P. solanacearum, the hrp gene is known to regulate the production and secretion of glycine-rich proteins that induce hypersensitivity reactions (He, SY, et al. "Pseudomonas Syringae pv. Syringae HarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants, "Cell 73: 1255-1266 (1993), Wei, Z.-H., et al.," Hrp I of Erwinia amylovora Functions in Secretion of Harpin and a Member of a New Protein family, "J. Bacteriol. 175: 7958-7967 (1993); Arlat, M. et al." PopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum, "EMBO J. 13: 543-553 (1994)).
The first of these proteins was found in the bacterium E. amylovora Ea321, which causes the fire blight of the Rosaceae, and was named harpin (Wei, Z.-M., et al, "Harpin, Elicitor of Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora, "Science 257: 85-88 (1992)). Mutations in the gene encoding hrpN have been shown to require harpin for E. amylovora to induce hypersensitivity in non-host tobacco leaves and to cause disease symptoms in highly sensitive fruit embryos. The P. solanacearum GMI1000 PopA1 protein has similar physical properties and induces hypersensitivity reactions in tobacco leaves that are not hosts of the strain (Arlat, et al. "PopA1, a Protein which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum "EMBO J. 13: 543-53 (1994)). However, P. solanacearum popA mutations still induce hypersensitivity in tobacco and disease in tomatoes. As such, the role of these glycine-rich hypersensitivity triggers is widely varied in Gram-negative pathogens.
Other plant pathogenic hypersensitivity triggers were isolated, cloned and sequenced. These are Erwinia chrysanthemi (Bauer, et al., "Erwinia chrysanthemi Harpin _Ech : Soft-Rot Pathogenesis," MPMI 8 (4): 484-91 (1995)); Erwinia carotovora (Cui, et al., "The RsmA Mutants of Erwinia carotovora subsp. Carotovora Strain Ecc71 Overexpress hrpN _Ecc and Elicit a Hypersensitive Reaction-like Response in Tabacco Leaves," MPMI 9 (7): 565-73 (1996)) ; Erwinia stewartii (Ahmad, et al., "Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize," 8th Int'l. Cong. Molec. Plant-Microb. Inter. July 14-19, 1996 and Ahmad, et al , "Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize," Ann. Mtg. Am. Phytopath. Soc. July 27-31, 1996); And Pseudomonas syringae pv. syringae (WO 94/26782 from Cornell Research Foundation, Inc.).
The present invention relates to the identification of hypersensitivity response inducer proteins or polypeptides that induce hypersensitivity reactions and to the use of these fragments.
The present invention relates to fragments that induce hypersensitive responses and uses thereof.
1 is a diagram showing the cleavage and proteolytic analysis of the Erwinia amylovora hypersensitivity response inducer (ie, harpin). A is the name of the harpin fragment. B represents the fragment length in amino acid residues. C indicates whether a detectable protein is produced. D indicates whether there is a hypersensitivity reaction (ie HR) that induces activity. The solid line indicates the presence of additional amino acids other than the encoded harpin, and the dashed line indicates the portion of the missing harpin. The number above the fragment in the box indicates the amino acid residue present at the end of the fragment; Residue # 1 is N-terminal and residue # 403 is C-terminal.
FIG. 2 shows Western blot results showing the specific secretion of Harpin _Ea but not Harpin _Ea C31. Lane A, Ea273 (pGP1-2) CFEP; Lane B, Ea273 (pGP1-2) (pCPP1104) CFEP; Lane C, E. coli DH5α (pCPP1107) CFEP Harpin standard size; Lane D, BioRad low molecular weight marker, lane E, Ea273 (pGP1-2) supernatant; Lane F, Ea273 (pGP1 2) (pCPP1104) supernatant. The blot was probed with anti-Harpin _Ea polyclonal antibody.
Figure 3 is a diagram showing the results of HR analysis for tobacco leaves infiltrated as follows. (1) A, harpin _Ea + raspberry IF; (2) B, harpin _Ea + apple IF; (3) C, harpin _Ea + tabacco IF; (4) D, harpin _Ea + endoproteinase Glu-C; (5) harpin _Ea + trypsin; (6) F, harpin _Ea ; (7) G, tabacco IF; (8) H, endoproteinase Glu-C; (9) I, trypsin; And (10) J, harpin _Ea . IF means intracelluar fluids.
4 is a diagram showing the results of cleavage of harpins using endoproteinase Glu-C. Lane A is harpin; Lane b is harpin + endoproteinase Glu-C; Lane C is a BioRad low molecular weight marker.
5A shows the results of protein degradation of harpins. Coomassie blue stained polyacrylamide gels showed the following results. A, BioRad low molecular weight marker; B, IF-apple; C, IF-raspberry; D, IF-tabacco; E, harpin _Ea ; F, harpin _Ea + IF-apple; G, harpin _Ea + IF-raspberry; H, harpin _Ea + IF-tabacco.
5B shows the Coomassie blue stained polyacrylamide gel analysis results as follows. A, IF-tabacco; B, IF-tabacco + harpin _Ea ; C, harpin _Ea ; D, BioRad low molecular weight markers; E, IF-tabacco + harpin _Ea + PMSF. The HR-induced activity of the sample after proteolysis is shown below the gel.
5C shows if proteolytic activity is present in IF in all tested plants. Intracellular fluids recovered from several plants were analyzed by PAGE on gels containing 0.1% copolymerized gelatin. Washed to remove SDS and allowed gelatin to be proteolyzed and then stained to see the presence or absence of gelatin degrading activity of the gel. A, IF-apple; B, IF-tabacco; C, IF-cotoneaster; D, BioRad mw; E, endoproteinase Glu-C; And F, pulverized leaf extract-tobacco.
FIG. 6 shows the results of refractionation of the trigger-activated peptide after proteolysis of harpin _Ea by tobacco IF. Absorbance was measured at 210 nm. Peak 1 comprises peptides P91 and P95; Peak 2 includes P65 and P69.
FIG. 7 shows the effect of these cleavage sites on the expected cleavage sites of the several tested proteins in harpins and the activity of active haffin fragments. Residues important for HR-induced activity based on degradation following degradation are indicated by arrows located from bottom to top.
8 is a diagram showing similarity near the N-terminus of the lower fin of Erwinia spp. Underlined residues are present in at least 4 of the 5 proteins tested (identical or similar). Nine of the first 26 residues are conserved in this way.
9 A-B shows the Kyte-Doolittle hydropathy plot of bacterial HR-derived proteins. Ea, E. amylovora EA321; Est, E. stewartii DC283; Ech, E. chrysanthemi AC4150; Ecc, E. cartovora subsp. cartovora; Rs, R, solanacearum; Pss, P. syringae pv. syringae.
10 shows truncated proteins of the hypersensitivity response inducer protein or polypeptide.
FIG. 11 shows a list of synthesized oligonucleotide primers for the structure of a truncated harpin protein. N represents the N-terminus (5 'region) and C represents the C-terminus (3' region). The primers correspond to sequence recognition numbers for this patent application: N1 (SEQ. ID. No. 1), N76 ((SEQ. ID. No. 2), N99 (SEQ. ID. No. 3), N105 ((SEQ.ID.No. 4), N110 (SEQ.ID.No. 5), N137 ((SEQ.ID.No. 6), N150 (SEQ.ID.No. 7), N169 ((SEQ. ID.No. 8), N210 (SEQ.ID.No. 9), N267 ((SEQ.ID.No. 10), N343 (SEQ.ID.No. 11), C75 (SEQ.ID.No. 12 ), C104 (SEQ. ID. No. 13), C168 (SEQ. ID. No. 14), C180 (SEQ. ID. No. 15), C204 (SEQ. ID. No. 16), C209 (SEQ. ID.No. 17), C266 (SEQ. ID. No. 18), C342 (SEQ. ID. No. 19), and C403 (SEQ. ID. No. 20).
The present invention relates to an isolated fragment of an Erwinia hypersensitivity trigger protein or polypeptide that induces hypersensitivity reactions in plants. It also includes an isolated DNA molecule that encodes such fragments.
Fragments of hypersensitivity response triggers can be used to confer disease resistance to plants, improve plant growth or control insects. This involves applying the fragments to a plant or plant seed in a non-infectious form, which gives the disease resistance to the plant or plant grown on the plant seed, enhances plant growth or insects. It is applied under conditions that are effective to suppress it.
Other methods of applying these fragments to plants or plant seeds to impart disease resistance to plants, to improve plant growth, and to inhibit insects include the use of transgenic plants or plant seeds. The use of a transgene plant provides a transgene plant transformed with a DNA molecule encoding a fragment of a hypersensitivity response inducer protein or polypeptide that induces a hypersensitivity reaction in the plant and confers disease resistance to the plant grown from the plant or plant seed. And growing the plant under conditions effective to enhance plant growth or inhibit insects. As an alternative to this, transgene plant seeds transformed with the DNA molecules encoding such fragments are provided and can be planted in the soil. The plants are then grown under conditions effective to increase disease resistance, improve plant growth or inhibit insects in plants or plants grown from plant seeds.
The present invention relates to an isolated fragment of a hypersensitivity response inducer protein or polypeptide, said fragment inducing a hypersensitivity reaction in a plant. The present invention also provides expression systems, host cells, and plants comprising the molecules, as well as DNA molecules encoding such fragments. The use of such fragments and DNA molecules encoding them is also provided.
Fragments of hypersensitivity trigger polypeptides or proteins according to the invention are derived from hypersensitivity trigger polypeptides or proteins of various fungal and bacterial pathogens. Local necrosis can be induced in plant tissues that have been contacted with such polypeptide or protein triggers. Examples of suitable bacterial sources of polypeptide or protein triggers include Erwinia, Pseudomonas, and Xanthamonas species (eg, Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris) Mixtures thereof).
An example of a fungus that is a source of a hypersensitivity response factor protein or polypeptide is Phytophthora. Examples of suitable Phytophthora are Phytophthora parasitica, Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora capsici, Phytophthora megasperma, and Phytophthora citrophthora.
The hypersensitivity response inducer polypeptide or protein of Erwinia chrysanthemi is shown in SEQ. ID. No. Has an amino acid sequence corresponding to 21:


This hypersensitivity response triggering polypeptide or protein has a molecular weight of 34 kDa, is heat stable, has a glycine content of at least 16% and does not contain cysteine. Erwinia chrysanthemi hypersensitivity inducer polypeptides or proteins are set forth in SEQ. ID. No. Encoded by a DNA molecule having a nucleotide sequence corresponding to 22:


Hypersensitivity response inducer polypeptides or proteins derived from Erwinia amylovora can be prepared by the following SEQ. ID. No. Has an amino acid sequence corresponding to 23:


This hypersensitivity response inducer polypeptide or protein has a molecular weight of about 39 kDa, a pi of about 4.3 and is heat stable at 100 ° C. for at least 10 minutes. This hypersensitivity response triggering polypeptide or protein does not contain cysteine. Hypersensitivity response inducer polypeptides or proteins derived from Erwinia amylovora are described in Wei, Z.-M. R.J.Laby, C.H. Zumoff, DWBauer, S.-Y.He, A. Collmer, and SVBeer, "Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora," Science 257: 85-88 (1992). , Which is hereby incorporated by reference. DNA encoding this polypeptide or protein is shown in SEQ. ID. No. Has a nucleotide sequence corresponding to 24:

The hypersensitivity response inducer polypeptide or protein derived from Pseudomonas syringae is shown in SEQ. ID. No. Has an amino acid sequence corresponding to 25:


This hypersensitivity trigger polypeptide or protein has a molecular weight of 34-35 kDa, is rich in glycine (about 13.5%) and lacks cysteine and tyrosine. For information on hypersensitivity response inducers derived from Pseudomonas syringae, see He, SY, HC Huang, and A. Colmer, "Pseudomonas syringae pv. Syringae Harpin _Pss : a Protein that is secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants , "Cell 73: 1255-1266 (1993), which are incorporated herein by reference. DNA molecules encoding the hypersensitivity response inducer obtained from Pseudomonas syringae are shown in SEQ. ID. No. Has a nucleotide sequence corresponding to 26:

The hypersensitivity response inducer polypeptide or protein derived from Pseudomonas solanacearum is represented by SEQ. ID. No. Has an amino acid sequence corresponding to 27:


This is shown in SEQ. ID. No. Encoded by a DNA molecule having a nucleotide sequence corresponding to 28:

For more information on hypersensitivity response inducing polypeptides or proteins derived from Pseudomonas solanacearum, see Arlat, M., F. Van Gijsegem, J.C. Huet, J.C. Pemollet, and C.A. Boucher, "PopA1, a Protein which Induces a Hypersensitive-like Response in Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J. 13: 543-533 (1994), incorporated herein by reference. Is cited.
Xanthomonas campestris pv. Hypersensitivity response inducer polypeptides or proteins obtained from glydines are described in SEQ. ID. No. Has an amino acid sequence corresponding to 29:

This sequence is shown in Xanthomonas campestris pv. hypersensitivity response factor of glycines is an amino acid sequence having only 26 residues from a polypeptide or protein. This is consistent with the fimbrial subunit protein determined in other Xanthomonas campestris pathovars.
Xanthomonas campestris pv. Hypersensitivity response inducer polypeptides or proteins from pelargonii are heat stable, protease sensitive and have a molecular weight of 20 kDa. This is shown in SEQ. ID. No. Contains the amino acid sequence corresponding to 30:

Methods for isolation of Erwinia carotovora hypersensitivity response factor proteins or polypeptides are described in Cui et al., "The RsmA Mutants of Erwinia carotovora subsp. Carotovora Strain Ecc71 Overexpress hrp N _Ecc and Elicit a Hypersensitive Reaction-like Response in Tabacco Leaves." MPMI, 9 (7): 565-73 (1996), incorporated herein by reference. Erwinia stewartii hypersensitivity trigger proteins or polypeptides are described in Ahmad et al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize," 8th Int'l. Cong. Molec. Plant-Microbe Interact., July 14-19, 1996 and Ahmad, et al., "Harpin is Not Necessary for the Pathogenicity of Erwinia stewartii on Maize," Ann. Mtg. Am. Phytopath. Soc., July 27-31, 1996, incorporated herein by reference.
Phytophthora: Most Specificity and Induction to Phytophthora: Phytophthora: Most Specificity and Phytophthora: Phytophthora cryptogea Fungal Phytopathogens, "Molec. Plant-Microbe Interact., 6 (1): 15-25 (1993), Ricci et al., "Structure and Activity of Proteins from Pathogenic fungi Phytophthora Eliciting Necrosis and Acquired Resistance in Tabacco," Eur. J. Biochem., 183: 555-63 (1989), Ricci et al., "Differential Production of Parasiticein, and Elicitor of Necrosis and Resistance in Tabacco, by Isolates of Phytophthora parasitica," Plant Path. 41: 298-307 (1992), Baillreul et al., "A New Elicitor of Hypersensitive Response in Tabacco: A Fungal Glycoprotein Elicits Cell Death, Expression of Defense Genes, Production of Salicylic Acid, and Induction of Systemic Aquired Resistance," Plant J., 8 (4): 551-60 (1995). And Bonnet et al., "Acquired Resistance Triggered by Elicitors in Tabacco and Other Plants," Eur. J. Plant Path., 102: 181-92 (1996), which is incorporated herein by reference.
The above triggers are one example. Other triggers can be identified by the growth of fungi or bacteria that induce hypersensitivity reactions when the gene encoding the trigger is expressed. Cell-free preparations obtained from culture supernatants can be tested for inducer activity (ie local necrosis) by infiltration into appropriate plant tissues.
As well as fragments of the hypersensitivity response triggering polypeptide or protein, fragments of the trigger having a full length obtained from other pathogens are included in the present invention.
Suitable fragments can be prepared by several different methods. The first known subclones of the trigger protein are generated by conventional molecular genetic engineering by subcloning the gene fragments. The subclones are then expressed in vitro or in vivo in bacteia cells to produce smaller proteins or peptides for testing for trigger activity according to the procedures described below.
Alternatively, proteolytic enzymes such as chymotrypsin or Staphylococcus proteinase A, or trypsin can be cleaved to full-length trigger proteins to produce fragments of trigger proteins. Different proteolytic enzymes degrade the trigger protein at different sites based on the amino acid sequence of the trigger protein. Some of the fragments resulting from proteolysis may be active triggers that are resistant.
In another method based on the primary structure of the analyzed protein, fragments of the causal protein gene can be synthesized using PCR with a specific series of primers selected to represent a particular portion of the protein. They can then be cloned into a suitable vector to express some cleaved peptide or protein.
Chemical synthesis can be used to make suitable fragments. Such synthetic methods are carried out using known amino acid sequences for the resulting triggers. Alternatively, fragments can be prepared by exposing the full length trigger to high temperatures and pressures. These fragments can be separated by conventional methods (eg chromatography, SDS-PAGE).
An example of a suitable fragment of Erwinia hypersensitivity triggers that induces hypersensitivity reactions is a fragment of Erwinia amylovora. Suitable fragments are shown in SEQ. ID. No. C-terminal of amino acid sequence of 23, SEQ. ID. No. The N-terminus of the amino acid sequence of 23, or SEQ. ID. No. Internal fragment of the amino acid sequence of 23; SEQ. ID. No. The C-terminal fragment having an amino acid sequence of 23 is SEQ. ID. No. 23 amino acids 105 and 403. SEQ. ID. No. The N-terminal fragment of the amino acid sequence of 23 is SEQ. ID. No. It may be in the range of amino acids of 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372. . ID. No. The internal fragment having an amino acid sequence of 23 is SEQ. ID. No. It may be in the following amino acid range of 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180. Other suitable Fragments can also be identified in accordance with the present invention.
Various variants can be prepared in various ways by deletion or addition of amino acids with minimal impact on the properties, secondary structure and hydrophobicity of the polypeptide. For example, a signal (or leader) sequence located at the N-terminus of the protein to be delivered simultaneously with or after translation of the protein can be bound to the polypeptide. Linkers or other sequences can be linked to the polypeptide to facilitate synthesis, purification or identification of the polypeptide.
The fragments of the invention are preferably produced in purified form (preferably at least about 60%, more preferably 80% pure form) by conventional methods. Generally, fragments of the present invention are produced but not secreted into the growth medium of recombinant host cells. In another method, the protein or polypeptide of the invention is secreted into the growth medium. For non-secreted proteins, host cells (eg E. coli) with recombinant plasmids are grown to isolate protein fragments, lysed by sonication, heat treatment or chemical treatment, and centrifuged to separate bacterial fragments. Remove it. The supernatant dmf is then heat treated and the fragments are separated by centrifugation. The upper fraction containing the fragments is separated by gel filtration on a moderately sized dextran or polyacrylamide column. If necessary, the protein fraction can be further purified by ion exchange or HPLC.
DNA molecules encoding fragments of the hypersensitivity response triggering polypeptide or protein can be introduced into cells using conventional recombinant DNA techniques. This method involves inserting the DNA molecule into an expression system in which the DNA molecule is heterologous (normally not present). The heterologous DNA molecule is inserted into an expression system or vector of the appropriate sense orientation and the correct reading frame. The vector contains necessary elements for the transcription or translation of the inserted protein-coding sequence.
U.S. Patent No. 4,237,224 to Cohen and Boyer describes the preparation of expression systems in the form of recombinant plasmids using conjugation by restriction enzymes and DNA ligase, which is incorporated herein by reference. These recombinant plasmids are then introduced by methods such as transformation and replicated in single cell cultures including eukaryotic cells and prokaryotic organisms growing in tissue culture.
Recombinant genes may also be introduced into viruses such as vaccina virus. Recombinant viruses can be produced by introducing plasmids into cells infected with the virus.
Suitable vectors include the following lambda vector systems: viral vectors such as gt11, gtWES.tB, Charon 4 and pBR322, pBR325, pVAYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, pBluescript II SK + Plasmid vectors such as /-or KS +/-, SV 40 (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif, incorporated herein by reference), pQE, PIH821, pGEX , pET series (FW Studier et al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990), which is incorporated herein by reference), and their Derivatives may be used, but are not limited to these. It can be introduced by transformation, in particular by transduction, conjugation, mobilization or electroporation. DNA sequences are cloned into vectors according to standard cloning methods in the art described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1989). Various host-vector systems can be used to express the protein-coding sequence (s). Firstly the vector system must be compatible with the host cell used. Host-vector systems include, but are not limited to, the following systems: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; Microorganisms, such as yeast, including yeast vectors; Mammalian cell systems infected with viruses (eg vaccinia virus, adenovirus, etc.); Insect cell systems infected with viruses (eg, baculovirus); And plant cells infected with bacteria. The expression factors of these vectors vary in their strength and specificity. Any of a number of suitable transcription and translation factors can be used depending on the host-vector system used.
Different genetic signals and processing phenomena regulate many stages of gene expression (eg DNA transcription and mRNA translation).
Transcription of DNA depends on the presence of a promoter, a DNA sequence that binds RNA polymerase to enhance mRNA synthesis. The DNA sequence of the eukaryotic promoter is different from that of the prokaryotic promoter. Moreover, eukaryotic promoters and accompanying genetic signals may not be recognized or function in prokaryotic systems. In addition, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
Translation of mRNA in prokaryotes depends on the presence of eukaryotic signals and other suitable prokaryotic signals. Translation of effective mRNA in prokaryotes requires ribosomal binding sites called Shine-Dalgarno (“SD”) sequences on the mRNA. This sequence is the short nucleotide sequence of the mRNA located before the start codon, AUG, which encodes the amino terminal methionine of the protein. The SD sequence is complementary to the 3 'end of the 16S rRNA (ribosome RNA), and forms a double helix with the rRNA to enhance the binding of mRNA to the ribosomes so that the ribosomes are correctly positioned. Methods for maximizing gene expression are described in Robert and Lauer, Method in Enzymology, 68: 473 (1979), incorporated herein by reference.
Promoters vary in their "strength" (ie, the ability to enhance transcription). It is desirable to obtain gene expression by high levels of transcription using strong promoters to express the cloned genes. Any of a number of suitable promoters may be used depending on the host cell system used. For example, when cloned from E. coli, its bacteriophage, or plasmid, the T7 phage promoter, the lac promoter, the trp promoter, the rec A promoter, the ribosomal RNA promoter, the collagen lambda P _R and P _L promoters and other lacUV5 Promoters such as, ompF, bla, lpp and the like can be used to induce high levels of transcription of adjacent DNA segments. In addition, hybrid trp-lacUV5 (tac) promoters or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be provided for transcription of inserted genes.
Bacterial host cell strains and expression vectors can be chosen to inhibit the action of the promoter unless specifically induced. In certain operations, the addition of specific inducers is required for effective transcription of the inserted DNA. For example, lac operon is induced by the binding of lactose or IPTG (isopropylthio-beta-D-galactoside). Various operons, such as trp and pro, are under different control.
Specific initiation signals are required for effective gene transcription and translation in prokaryotes. These transcription and translation initiation signals differ in intensity from each other as measured by the amount of synthesized gene specific mRNA and protein, respectively. DNA expression vectors comprising a promoter may comprise any combination of various “strong” transcriptional and / or translational initiation signals. For example, effective translation in E. coli requires an SD sequence located about 7-9 bases forward 5 'in the initiation codon ("ATG") to provide a ribosomal binding site. Thus any SD-ATG combination can be used if it can be used by the host cell ribosomes. Such combinations are not limited to SD-ATG combinations, and combinations can also be made from the cro or N genes of Coliphagia lambda, or E. coli tryptophan E, D, C, B or A genes. SD-ATG combinations produced by other techniques, including the introduction of recombinant DNA or synthetic nucleotides, can also be used.
The isolated DNA encoding the hypersensitivity response triggering polypeptide or fragment of the protein is cloned into the expression system and introduced into the host cell. Such introduction can be effected by various forms such as the transformations mentioned above depending on the vector / host cell system. Suitable host cells include, but are not limited to, bacteria, viruses, yeasts, mammalian cells, insects, plants, and the like.
The present invention also relates to a method capable of imparting disease resistance to plants, improving plant growth, and effectively inhibiting insects against plants. These methods apply fragments of the hypersensitivity response inducer polypeptide or protein that induces hypersensitivity reactions in uninfected form to some or all of the plant or plant seeds under conditions effective to confer disease resistance, improve growth and inhibit insects. It includes. Alternatively, fragments of the hypersensitivity response inducer protein or polypeptide can be applied to plants so that the seeds recovered from such plants themselves confer disease resistance to plants, improve plant growth, and effectively inhibit insects.
Transgenic plants or other methods of applying hypersensitivity triggers polypeptides or fragments of proteins to plants or plant seeds to impart disease resistance to plants, enhance plant growth and inhibit insects against plants grown from plants or seeds. Plant seeds can be used. When using the transgene plant, it provides a transgene plant transformed with a DNA molecule encoding a hypersensitivity response inducer polypeptide or fragment of a protein that induces a hypersensitivity reaction, the DNA molecule confers disease resistance to the plant, Growing the plant under effective conditions that enhance growth and inhibit insects. Alternatively, plant seeds transformed with DNA molecules can be planted in soil with a fragment of a hypersensitivity response inducing polypeptide or protein that induces hypersensitivity. The plant then grows from a seed planted under conditions that are effective for DNA molecules to impart disease resistance to the plant, enhance plant growth, and inhibit insects.
Embodiments of the invention in which a hypersensitivity response inducing polypeptide or protein is applied to a plant or plant seed include a number of applications including 1) application of isolated fragments or 2) application of bacteria transformed with the gene encoding the fragment without causing disease. It can be carried out by the method of. In embodiments carried out by the latter method, the fragment can be applied to a plant or plant seed by applying a bacterium comprising a DNA molecule encoding a hypersensitivity response inducer polypeptide or protein that induces a hypersensitivity reaction. Such bacteria must secrete or excrete fragments so that the fragments come into contact with the plant or plant seed cells. In these embodiments the fragments are produced by bacteria in the plant or in the seed or prior to introducing the bacteria into the plant or plant seed.
The method of the present invention can be used to treat various plants or plant seeds to confer disease resistance, improve growth, and inhibit insects. Suitable plants include dicotyledonous plants and monocotyledonous plants. Alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, kidney bean, pea, chicory, lettuce, endive, cabbage, cabbage, beet, parsnip, turnip, cauliflower, broccoli , Radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, tangerine, strawberry, grapes, raspberry, pineapple, beans, tobacco. Crops such as tomatoes, sugar cane, sugarcane, and sugarcane are particularly useful. Examples of suitable ornamental trees include Arabidopsis thaliana, Saintpaulia, Petunia, Pelargonium, Poinsettia, Chrysanthemum, Carnation, and Genia.
In conferring disease resistance, the use of the hypersensitivity response inducer protein or polypeptide fragments of the present invention does not result in absolute immunity to infection, but alleviates the severity of the disease and delays the development of symptoms. The damage number, the size of the damage and the degree of sporulation of the fungal pathogen are all reduced. This method of conferring disease resistance is an effective way to treat diseases that were previously incurable and to systematically treat diseases that could not be isolated because of cost, thus avoiding the use of infectious agents or environmentally harmful substances. .
Methods of imparting pathogen resistance to plants according to the invention are useful for imparting resistance to a variety of pathogens, including viruses, bacteria, and fungi. Among the viruses, in particular, resistance to tobacco mosaic virus and tomato mosaic virus can be obtained by the present invention. Among the bacteria, Pseudomonas solancearum, Pseudomonas syringae pv. tabaci, and Xanthamonas campestris pv. Resistance to pelargonii can be obtained by the present invention. Among the fungi, plants resistant to the fungus such as Fusarium oxysporum and Phytophthora infestans can be produced.
Various forms of plant growth enhancement or enhancement have been obtained in connection with the use of fragments of the hypersensitivity response inducer proteins or polypeptides of the invention to enhance plant growth. This may occur as soon as plant growth begins in the seed, or later in the plant's survival. For example, the plant growth according to the present invention may yield higher yields, increase the amount of seeds produced, increase the proportion of germinated seeds, increase plant size, larger biomass, larger and larger fruits, earlier fruits. Pigmentation, and maturation of earlier fruits and grains. As a result, the present invention provides significant economic benefits. Early germination and maturation, for example, allow crops to grow in areas where short growing seasons prevent them from growing in that place. Increasing seed germination improves crop stands and results in more efficient seed use. Increased yields, increased size, and improved biomass production make it possible to earn greater income from certain lands.
Another feature of the invention relates to a method of effectively inhibiting insects on plants. For example, insect suppression according to the present invention prevents insects from coming into contact with plants to which the hypersensitivity response factor is applied, injures them so that insects fall from such plants, kills insects in close proximity to such plants, Preventing insect larvae rearing, preventing insects from transplanting host plants, preventing insects from transplanting from releasing phytotoxins, and the like. The invention also aims to prevent disease damage to plants caused by insect infections.
The present invention is effective against various insects. European corn borers are the main pests of corn (dents and sweet corn), 200, including greens, waxes, lima beans and edible soybeans, peppers, potatoes, and tomatoes and many weeds. It grows on the plant of the species. Pests supplying additional insect larvae that damage various plant crops include beet armyworm, cabbage worms, larvae of large tobacco moths, fall armyworms, diamond-patterned moths, and cabbage root maggots. , Onion maggots, seed cone maggots, pickle bugs (melon beetles), pepper maggots, tomato larvae, and maggots. These groups of insect pests collectively represent the group of the most economically important pests for global plant production. The method of the present invention, including the application of a hypersensitivity response inducer polypeptide or a fragment of a protein that induces a hypersensitivity reaction, can be carried out through various procedures in the treatment of all or part of a plant, including leaves, stems, roots and the like. This may include but is not necessary to invade the plant with a hypersensitivity response inducing polypeptide or protein. Appropriate application methods include high pressure or low pressure spraying, injection and mouth wear at the time of trigger application. When treating (eg, cuttings) plant seeds or propagules according to the present invention, fragments of the hypersensitivity response triggering polypeptide or protein may be applied by low or high pressure spraying, coating, dipping or injecting methods. Can be. Other suitable application methods that can effectively contact the plant or plant seeds with the fragments can be practiced by one of ordinary skill in the art. Once treated with fragments of the hypersensitivity reaction trigger of the present invention, seeds can be planted in natural or artificial soil and cultivated by conventional methods to produce plants. After a plant has grown from seed according to the invention, the plant applies one or more fragments or whole triggers of a hypersensitivity trigger protein or polypeptide to confer disease resistance to plants, improve plant growth, and insects against plants. To suppress it.
The fragment of the hypersensitivity factor polypeptide or protein according to the present invention may be applied alone or in admixture with other substances. Alternatively, the fragments may be applied to plants at different times apart from other substances.
Suitable compositions for treating plants or plant seeds in accordance with the methods of application of embodiments of the invention include fragments of hypersensitivity response inducing polypeptides or proteins that induce hypersensitivity reactions in a carrier. Suitable carriers include water, aqueous solutions, slurries or dried powders. In this embodiment the composition comprises at least 500 nM fragments.
Although not required, the composition may include additional additives including fertilizers, insecticides, fungicides, nematacides and mixtures thereof. Suitable fertilizers include (NH ₄ ) ₂ NO ₃ . Suitable pesticides include malathion. As a fungicide, Captan is useful.
Other suitable additives include buffers, wetting agents, coatings, and abrasives. These materials are used to facilitate the method of the present invention. Hypersensitivity reaction inducing fragments may also be applied to plant seeds in combination with conventional seed formulations and treatments including clays and polysaccharides.
In another embodiment of the invention using transgene plants and transgene seeds, fragments that induce hypersensitivity reactions do not generally need to be introduced into the plant or seed. Instead, transgene plants transformed with DNA molecules encoding such fragments are produced by methods known in the art.
The vectors described above can be microinjected directly into plant cells using micropipettes that mechanically deliver recombinant DNA (see Crossway, Mol. Gen. Genetic, 202: 179-85 (1985), which is herein incorporated by reference). Is cited for reference). The genetic material can be delivered to plant cells using polyethylene glycol (see Krens, et al., Nature, 296: 72-74 (1982), which is incorporated herein by reference).
Another method of transforming plant cells with genes that confer resistance to pathogens is particle bombardment (also called biolistic transformation) of host cells. This can be done in one of several ways. The first involves propelling inert or biologically active particles in the cell. This method is described in US Pat. Nos. 4,945,050, 5,036,006 and 5,100,792 to Sanford et al., Which are incorporated herein by reference. In general, these methods involve propelling inert or biologically active particles in a cell under conditions effective to penetrate and enter the outer surface of the cell. When inert particles are used, the vector can be introduced into the cell by coating the particle with a vector comprising heterologous DNA. Alternatively, the target cell may be surrounded by a vector to allow the vector to be introduced into the cell by wake of the particle. Biologically active particles (eg, dried bacterial cells comprising vector or heterologous DNA) can also be propagated into plant cells.
Another method of introduction is to fuse protoplasts with either minicells, cells, lysosomes or other fusionable lipid-surface materials (Fraley, et al., Proc. Natl. Acad. Sci. USA, 79). : 1859-63 (1982), which is incorporated herein by reference).
DNA molecules can be introduced into plant cells by electroporation (see Fromm et al., Proc. Natl. Acad. Sci. USA, 82: 5284 (1985), which is incorporated herein by reference). . In this method the plant protoplasts are electroporated in the presence of a plasmid comprising an expression cassette. High field strength electric shocks make the biomembrane reversibly permeable, allowing plasmids to be introduced. Electroporated plant protoplasts reshape, divide and regenerate cell walls.
Another way to introduce DNA molecules into plant cells is to infect plant cells comprising Agrobacterium tumefaciens or A. rhizogenes, which have been previously transformed with the gene. Under suitable conditions in this field, the transformed plant cells form shoots and roots and grow further into plants. Generally this process inoculates plant tissues with a suspension of bacteria and incubates the tissues for 48 to 72 hours in regeneration medium at 25-28 ° C. without antibiotics.
Agrobacteria is a representative species of Gram-negative Rhizobiaceae. These species are responsible for A. tumefaciens and hairy root disease (A. rhizogenes). Myocarcinoma tumors and plant cells in the hair follicles are induced to produce amino acid derivatives known as opines, which are metabolized only by bacteria. Bacterial genes for expressing opines provide a convenient control element for chimeric expression cassettes. Assays for the presence of opines can also be used to identify transformed tissues.
The heterologous gene sequence can be introduced into suitable plant cells by Ti plasmid of A. tumefaciens or Ri plasmid of A. rhizogenes. The Ti or Ri plasmids are delivered to plant cells infected by Agrobacteria and stably integrated into the plant genome (J. Schell, Science, 237: 1176-83 (1987), which is incorporated herein by reference). .
After transformation, the transformed plant cells must be reproduced.
Plant reproduction from cultured protoplasts is described by Evans et al., Handbook of Plant Cell, Vol. 1: (MacMillan Publishing Co., New York, 1983); And Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which is incorporated herein by reference.
Virtually all plants can be reproduced in, but not limited to, cultured cells or tissues, including sugarcane, sugar beets, cotton, fruit trees, and all major species of legumes.
Reproduction means vary from plant to plant, but generally are primarily provided with a petri dish containing a suspension of transformed protoplasts or a transformed explant. Union tissues are formed, branches are derived from the union tissues, and then roots are formed. Alternatively, embryo formation may be induced in the callus tissue. These pears germinate as naturally formed pears to form plants. The culture medium generally contains various amino acids and hormones such as auxin and cytokinin. Also for species such as corn and alfalfa, it is particularly advantageous to add glutamic acid and proline to the medium. Effective reproduction will depend on the medium, genotype, medium. If these three parameters are adjustable, reproduction is reproducible and repeatable.
The expression cassettes are stably introduced into transgenic plants and then transferred to other plants by sexual mating. Many standard breeding methods can be selected and used depending on the species being crossed.
When these types of transgenic plants are produced, the plants themselves are cultivated by conventional methods such that there is a gene encoding a hypersensitivity response inducing fragment which confers disease resistance, increased plant growth, and / or insect suppression on the plant. Can be. In an alternative method, transgene seeds or propaglu (eg cuttings) are recovered from the transgene plant. The seeds are then planted in soil and grown according to conventional methods to produce transgenic plants. This transgenic plant propagates from transgenic seeds planted under conditions effective to confer disease resistance to plants, improve plant growth and inhibit insects. While not wishing to be bound by any theory, such things as disease resistance, increased growth, and / or insect inhibition may be mediated by RNA and may also be due to expression of polypeptides or protein fragments.
When a transgene plant or plant seed is used according to the invention, the transgene plant or plant seed is additionally treated with the same material used to treat the plant and seed to which the hypersensitivity reaction inducing fragment is applied. These other materials, including hypersensitivity reaction inducing fragments, can be applied to transgene plants and plant seeds by the methods described above, including high or low pressure spraying, injection, coating, and dipping. Similarly, after a plant has grown in a transgenic plant seed, the plant can be treated to apply one or more hypersensitivity reaction fragments to confer plant resistance, enhance growth and inhibit insects. Such plants may also be treated with conventional plant treatments (pesticides, fertilizers, etc.).
Example 1- Strains and Plasmids Used
The strains and plasmids used are as described in Table 1 below.
TABLE 1
Example 2 Molecular Biological Techniques
Several methods have been used to obtain truncated or other variants of E. amylovora harpins. Such methods include the following: (iii) subcloning into restriction vectors of restriction fragments comprising a portion of the gene encoding the hypersensitivity response inducer protein or polypeptide of Erwinia amylovora (ie hrpN) (Sambrook, et al. ., Molecular Cloning: a Laboratory Manual, 2 ^nd ed. Cold Spring Harbor, Laboratory, "Cold Spring Harbor, NY (1989), incorporated herein by reference); (ii) hrpN of Ω-fragment. Insertion into Fellay, et al., "Interposon Mutagenesis of Soil and Water Bacteria a Family of DNA Fragments Designed for in vitro Insertional Mutagenesis of Gram-Negative Bacteria," Gene 52: 147-154 (1987), supra (I) Site-specific mutagenesis (Innis, et al., PCR Protocols.A Guide to Methods and Applications, Academic Press San Diego, CA (1990); Kunkel, et al., "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection," Proc. Nat. Acad. Sci. USA 82: 488-492 (1985), which is incorporated herein by reference; (iii) generation of nested deletion (Erase-a-Base ^™ kit; Promega, Madison, WI) C-terminal loss analysis of the hypersensitivity response inducer protein or polypeptide of Erwinia amylovora in pCPP1084 (harpin _Ea ) could not be performed due to the location of restriction enzyme cleavage sites in pCPP1084. For N-terminal loss, pCPP1084 DNA was prepared using a Qiagen midiprep column (Qiagen, Chatsworth, Calif.), Cut to sst I and cut to EcoR I. The cleaved DNA was then cut with exonuclease III, conjugated and transformed into E. coli BL21 (DE3). Lost size was measured by agarose gel electrophoresis. The Harpin fragment was named for the portion of the missing Harpin (ie harpin _Ea C82 lacks the C-terminal 82 amino acid residues in full length harpin _Ea ).
Example 3 Protein Expression
To express from the T7 promoter, a T7 RNA polymerase-dependent system was used. These systems are described in strain E. coli BL21 (DE3) (Studier, et al., "Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-Level Expression of Cloned Gene," J. Mol. Biol. 189: 113-130 (1986). ), Or the plasmid pGP1-2 (Tabor, et al., "A Bacteriophage T7 DNA Polymerase / Promoter System for Controlled Exclusive Expression of Specific Genes," Proc.) In E. coli DH5α. Natl.Acad.Sci., USA 82: 1074-1078 (1985), which is incorporated herein by reference. Expression of hrpN from the T7 promoter was induced by the addition of IPTG with a final concentration of 0.4 mM. To express in E. amylovora Ea321 (ie harpin _Ea ) or Ea273, pGP1-2 was introduced by transformation or electroporation (Bioras Gene Pulser ^™ ) by 42 ° C heat shock for 10 minutes. Hypersensitivity reactions (HR) -induced activity are shown in tabacco cv. In planta elution from Xanthi leaves (He, et al., "Pseudomonas syringae pv. Harpin _Pss : a Protein That is Secreted Via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73: 1255-1266 (1993) ), Or the preparation of boiled or non-boiled "CFEPs" (Wei, et al., "Harpin, Elicitor of Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora," Science 257: 85-88 (1992), which is incorporated herein by reference.
Example 4 Proteolysis of Harpins in Vitro
Proteolysis of harpin _Ea in vitro is preferably performed using Staphlococcus V8 proteinase (endoproteinase Glu-C), trypsin, pepsin and papain at 20-37 ° C. for 2-16 hours ( Scopes, et al., Protein Purification: Principles and Practice, 2 ^nd ed.Springer-Verlag.New York (1987), which is incorporated herein by reference). Cleavage by the endoproteinase Glu-C is 50 mM ammonium bicarbonate, pH 7.8 where cleavage occurs only after glutamic acid, or 50 mM potassium phosphate xm, where cleavage is glutamic acid Takes place behind aspartic acid).
Example 5 Plant-induced Proteinases
Intracellular fluids (IF) were obtained from tobacco, tomatoes, apples, raspberries, and cotoneasters by vacuum filtration of intracellular spaces using high purity water (Hammond-Kosack, et al., "Preparation and Analysis of Intercellular Fluid, "p. 15-21 In SJ Gurr, MJ McPherson, and DJ Bowels (ed.), Molecular Plant Pathology A Practical Approach, 2 ^nd ed.The Practical Approach Series, IRL Publishers, Oxford (1992) Which is incorporated herein by reference). IF and harpin _Ea were mixed in the same volume to perform proteolysis of PAGE-purified harpin _Ea at pH, 20-37 ° C. for 2-16 hours. Whole leaf extracts were obtained by grinding tobacco leaf panels using mortar and pestle in 5 mM potassium phosphate. The extract was centrifuged, filtered, and the purified milled leaf extract was used in the same manner as IF. Proteinase inhibitors include pepstatin A (final concentration 1 μM), E-64 (1 μM), aprotinin (2 μg / ml), o-phenanthroline (1 mM), and p-mercurybenzoate (PCMB) (Sigma, St. Louis, MO) was used.
Example 6-Peptide Purification
Peptide fragments of Harpin obtained after digestion with tobacco fluid (tabacco IF) were sorted by reverse phase HPLC on a Vydac C18 column using a 2-60% acetonitrile gradient in 0.1% trifluoroacetic acid. The fractions were lyophilized, resuspended in 5 mM potassium phosphate and infiltrated into tobacco leaf panels. The fractions with the highest HR-inducing activity were reclassified by the above method using an acetonitrile gradient of 35-70% and the purity of each fraction was analyzed by gas chromatography-mass spectroscopy in a Cornell biotechnology program core laboratory. (GC-MS) and N-terminal protein sequencing.
Example 7 Proteinase Activity-Stained Gel
Proteinase activity of IF was characterized by copolymerization with 0.1% gelatin (Heussen, et al., "Electrophoretic Analysis of Plasminogen Activator in Polyacrylamide Gels containing Sodium Dodecyl Sulfate and Copolymerized Substrate," Anal. Biochem. 102: 196-202 (1980 ), Which is incorporated herein by reference). Laemmli, "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4," Nature 227: 680-685 (1970). , Which is incorporated herein by reference). After electrophoresis, each gel was washed strongly to remove SDS to allow the proteinase in the gel to refold. After further allowing proteolysis to occur, the gel was stained with 0.1% Amido Black in 30% methanol / 10% acetic acid. Except where the proteinase degraded gelatin, each gel was dyed black (due to the presence of copolymerized gelatin), and the band without color was shown to have proteinase activity.
Example 8 Lost Hatin Retains HR-Induced Activity
The stability and HR-inducing activity of proteins encoded by various DNA constructs is shown in FIG. 1. Many DNA constructs that encode portions of harpin _Ea or harpin _Ear induce expression in the T7 promoter-polymerase system due to instability of the encoded protein (Tabor, et al., “Bacterioharge T7 DNA Polymerase / Promoter System for Controlled Exclusive Expression of Specific Genes, "Proc. Natl. Acad. Sci. USA 82: 1074-1078 (1985), which is incorporated herein by reference), and produce detectable protein products after analysis of cell extracts by PAGE. I could not let you. None of the DNA constructs (eg Erase-a-Base ^™ protocol) produced detectable protein products that showed N-terminal loss of the entire protein. Stable but inactive proteins have not been identified. Several constructs encoding proteins containing C-terminal loss and additional vector-encoded amino acids produced detectable products (eg harpin _Ea C82). In contrast, the construct that codes for the 321 N-terminal amino acid residues of the same harpin _Ea , but produces a protein lost due to the presence of Ω-fragment (harpin _Ea C82 Ω), was unstable (ie, no product was detected). . The construct encoding the harpin _Ea fragment (harpin _Ea I175) with large internal loss was also used to successfully express the protein. HR-induced activity was tested against these various cleaved proteins. The N-terminal harpin _Ea fragment (harpin _Ea C305) with 98 residues was the peptide produced in the smallest bacteria exhibiting HR-inducing activity.
Example 9 Secretion of harpin _Ea with Modified C-terminus
The effect of the disappearance of the C-terminus of harpin on its secretion was examined. Harpin C31 contains 372 amino acids of the N-terminal end of Harpin and lacks the 31 residues of the C-terminal, which are replaced by 47 residues encoded by the vector, resulting in a slightly larger protein than the wild type harpin _Ea . The C31 protein retains HR-inducing activity and is stable and detected by Western analysis or PAGE, but no longer secreted into the supernatant in culture as wild type protein (FIG. 2). The presence of harpin _Ea C31 does not inhibit the secretion of wild type harpins, and wild type harpins are found in CFEP and supernatants in culture. However, harpin _Ea C31 is only found in CFEP.
Example 10 Effect of Protein Degradation on HR Induced Activity of Harpin _Ea
To produce additional harpin _Ea fragments, purified full-length proteins were digested in vitro by several proteinases, such as endoproteinases Glu-C, trypsin, pepsin, and papain (FIG. 3 and FIG. 4). All of the hafpin solutions digested with trypsin or papain lost activity. In contrast, HR-induced activity was maintained after digestion with endoproteinase Glu-C. Large peptides larger than 6 kD were evident on PAGE after digestion with trypsin. Endoproteinase Glu-C degradation produced a fragment of about 20 kD larger than expected when all cleavage sites were cut, indicating that the degradation was incomplete (FIG. 4).
Example 11 Apoplastic Fluids (IF) Have Harpin-Degradable Proteolytic Activity
Apoplast fluid (intracellular fluid; IF) from tobacco or other plants was used to break down the harpin. Each tested IF resolved the purified harpin _Ea by IF overnight and then detected detectable harpin as well as the presence of multiple active-stained bands in the polyacrylamide gel comprising copolymerized gelatin (FIGS. 5A-5C). _Ea was not shown to have proteolytic activity as can be seen (Schagger, et al., "Tricine-Sodium Dodecyl Sulfate Gel Electrophoresis for the Separation of Proteins in the Range From 1 to 100 kDa," Anal. Biochem. 166: 368-379 (1987), which is incorporated herein by reference). Proteinase activity was greater at 37 ° C. than at 20 ° C., and was higher at pH 8.5 than pH 7. Several inhibitors were used to determine the proteolytic activity of IF. None of the single proteinase inhibitors used inhibited the degradation of harpin _Ea . However, a mixture of inhibitors of pepstatin A (1 μM), E-64 (1 μM), aprotinin (2 μg / ml), and o-phenanthroline (1 mM) may be used for acid proteinases, cysteine proteins, and serine pros. Tenases and metalloproteinases, respectively, were inhibited and partially inhibited proteolysis.
Harpin _Ea degraded by proteolysis present in the apoplasts of plants maintained HR-inducing activity (FIG. 3). On the other hand, harpin _Ea proteolyzed by the purified extract produced by crushing tobacco leaf tissue using mortar and pestle lost HR-induced activity. To determine if affine degradation of hafnium is necessary for HR-induced activity, which is required for leaf collapse when either intact hafpin or hafpin pre-digested with tobacco IF is infiltrated into tobacco leaf panels. The time was compared. Both induced HR at similar times (12-18 hours depending on test conditions).
Example 12 Characterization of HR-Induced Active Peptide Fragments
Peptides resulting from degradation by apoplast plant proteinase (s) were sorted by reverse phase HPLC (Vydac C18 column) and tested for activity. After treatment of circular harpin _Ea with tobacco IF, three fractions contained HR-induced activity against tobacco. Two of the three showed weak activity and fewer proteins were present. These are more specific. As well as most proteins, sort 19 with strong activity was reclassified using a lower elution gradient (FIG. 6). Reclassification, N-terminal protein sequencing, and molecular weight analysis by mass spectroscopy reveal that there are four broadly overlapping peptides. Peak 19-1 comprises peptides P91 and P95 corresponding to harpin _Ea residues 110-200 and 110-204; Peak 19-2 includes peptides P64 and P68 corresponding to harpin _Ea residues 137-200 and 137-204. 19-1 and 19-2 each had HR-inducing activity. Thus the smallest peptide identified as having activity consisted of residues 137-204. Two peptides at each peak did not separate under the conditions used. These active fragments are distinguished from the smallest active N-terminal fragment (harpin _Ea C305), and at least one portion of harpin _Ea appeared to exhibit in planta activity. Further degradation with trypsin abolished the HR-induced activity of 19-2. This proteinase cleaves P64 and P68 as shown in FIG. 7. Further degradation with endoproteinase Glu-C in ammonium bicarbonate buffer extinguished the HR-inducing activity of 19-1. Endoproteinase Glu-C is expected to degrade P91 and P95 as shown in FIG. 7. Further degradation of these peptides with endoproteinase Glu-C or trypsin lost induction-activity.
Example 13-E. Similarity with other proteins of amylovora harpin
The predicted protein sequences of other proteins known or believed to be secreted by proteinase HR and type III secretion pathways obtained from several different bacterial plant pathogens were compared with the sequences of harpin _Ea . Erwinia chrysanthemi EC16 (harpin _Ech ) (Bauer, et al., “Erwinia chrysanthemi harpin _Ech : An Elicitor of Hypersensitive Response That Contributes to Soft) when harpin _Ea is compared with the trigger from E. amylovora Ea246 (harpin _Ear ). -Rot Pathogenesis, "Mol. Plant-Microbe Interact 8: 484-491 (1995), which is incorporated herein by reference), Erwinia carotovora subsp. carotovora (harpin _Ecc ) (Mukherjee, et al., Presented at the 8 ^th International Congress Molecular Plant-Microbe Interactions, Knoxville, TN (1996), incorporated herein by reference), Erwinia stewartii (harpin _Es ) Frederick, et al., "The wts Water-Soaking Genes of Erwinia stewartii are Related to hrp genes," Presented at the Seventh International Symposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland (1994), supra Ralatonia (Pseudomonas) solanacearum (PopA) (Arlat, et al., "PopA1, a Protein Which Induces a Hypersensitivity-Like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J. 13: 543-553 (1994), which is incorporated herein by reference), Pseudomonas syringe 61 (harpin _Pss ) (He, et al., "Pseudomonas syringe pv. Syringe harpin _Pss : a Protein That is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants, "Cel 73: 1255-1266 (1993), which is incorporated herein by reference), Pseudomonas syringe pv. tomato (harpin _Pst ) (Preston, et al., "The HrpZ Proteins of Pseudomonas syringe pvs. syringe, glycinea, and tomato Are Encoded By An Operon Containing Yersinia ysc Homologs and Elicit the Hypersensitive Response in Tomato But Not Soybean," Mol. Plant-Microbe Interact 8: 717-732 (1995), which is incorporated herein by reference), Erwinia-derived harpins included significant regions of similarity at the C-terminus. All triggers were also glycine rich, secreted, and heat stable. Limited similarities between harpin _Pss and harpin _Ea have been previously reported (He, et al., "Pseudomonas syringe pv. syringe harpin _Pss : a Protein That is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants," Cell 73: 1255-1266 (1993), which is incorporated herein by reference), (Laby, et al., Presented at the Seventh International Symposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland (1994), supra Is incorporated herein by reference). harpin _Ea with other harpins frm Erwinia spp. Limited regions with similarities between were evident at the end of the N-terminus of each protein, with 9 of the first 26 residues conserved (FIG. 8). Kyte-Doolittle hydrophobic plots for each haffin generated from different Erwinia spp. Are shown in FIG. 9. The Erwinia harpins used in the experiments generally showed similar hydrophobic plots for the full length protein. This is distinct from the profile by PopA and harpin _Pss and does not have the symmetry that is evident in the profiles of these two proteins. The hydrophobic profile of each Erwinia harpin is generally similar to the others, but differs from that reported by harpin _Pss (Alfano, et al., "Analysis of the Role of the Pseudomonas Syringe HrpZ harpin in Elicitation of the Hypersensitive Response to Tabacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated hrpZ Fragments, and hrmA Mutations, Mol Microbiol 19:... 715-728 (1996), supra is herein incorporated herein by reference) _Ecc harpin is significant near residues 54-143 It has a hydrophobic domain such portion of the protein (Mukherjee, et al., Presented at the 8 th International Congress Molecular Plant-Microbe Interactions, Knoxville, TN (1996), supra, see herein incorporated by herein). FIG harpin _It is the most hydrophobic region of _Ea and harpin _Es , with the remainder of each protein having good hydrophilicity.
Harpin's missing proteins and fragments obtained by digesting full length proteins show some unexpected features of harpin _Ea HR-induced activity. These harpin fragments have HR-induced activity in certain regions of the protein, with relatively small fragments at or below 68 residues of the protein being sufficient for this activity. Fragments of other plant pathogen-derived trigger proteins also have biological activity, including Avr9 of Caldosporium fulvum (Van den Ackervecken, et al., "The AVR9 Race-Specific Elicitor of Caldosporium fulvum is Processed by Endogenous and Plant Proteases, "Pl. Physiol. 103: 91-96 (1993), incorporated herein by reference), Pep-13 of Phytophthora megasperma (Nurnburger, et al.," High Affinity Binding of a Fungal Oligopeptide Elicitor to the Parsely Plasma Membranes Triggers Multiple Defense Response, "Cell, 78: 449-460 (1994), which is incorporated herein by reference), and P. syringe pv. Harpin _Pss (Alfano, et al., "Analysis of the Role of the Pseudomonas Syringe HrpZ harpin in Elicitation of the Hypersensitive Response to Tabacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated HrpZ Fragments, and hrmA Mutations, Mol. Microbiol. 19 : 715-728 (1996), which is incorporated herein by reference.
Expression of the missing harpin fragments and proteolysis of the full-length harpins showed that the two separated fragments had HR-inducing activity. The primary sequence of each active peptide did not show markedly identified similarities to each other or to other factor-active peptides. However, cleavage sites with trypsin and endoproteinase Glu-C suggested that portions of each fragment were required for activity. Certain modifications of amino acids near these cleavage sites have shown interesting results to see if the HR-induced activity is altered or lost. In addition, harpin _Ea P64 and P68 showed clear hydrophobicity in reverse phase HPLC (FIG. 6), which corresponded to the hydrophobic peak in the kyte-Doolittle plot (FIG. 9). The putative role of this hydrophobic domain can be tested by mutagenesis, or by the synthesis of modified peptides. However, the fact that multiple fragments independently have HR-inducing activity complicates full-length proteolysis.
The fact that fragments of these proteins have HR-inducing activity also means that at least two apoplast proteinase activities that are distinct from intracellular plant proteinase activity are allowed. Two apoplast plant proteins (obtained from soybeans) are being studied further. SMEP, metalloproteinases (Huangpu, et al., "Purification and Developmental Analysis of Extracellular Proteinase From Young Leaves of Soybean," Plant Physiol. 108: 969-974 (1995); McGeehan, et al. ., "Sequencing and Characterization of the Soybean Leaf Metalloproteinase," Plant Physiol. 99: 1179-1183 (1992), which is incorporated herein by reference, is believed to degrade in G / L and G / I. . The original harpin _Ea has 19 possible SMEP cleavage sites, but only one of them is located inside fragments P91 and P95 and not inside fragments P64 and P68 (FIG. 7). P91 and P95 may thus represent a partial degradation product of SMEP-like proteinases in apoplasts of tobacco. Sobin apoplast proteins, SLAP and sulfhydryl proteins that are sensitive to p-chloromercurybenzoic acid (pCMB) have also been studied (Huangpu, et al., "Purification and Developmental Analysis of an Extracellular Proteinase"). From Young Leaves of Soybean, "Plant Physiol. 108: 969-974 (1995), incorporated herein by reference. There is some evidence that multiple proteolytic activity in IF degrades harpin _Ea . PMSF, a serine protease inhibitor, reduces but does not completely block harpin _Ea degradation (FIG. 5C); Any single proteinase inhibitor tested also blocked the degradation of haffin, with dissimilarity of the cleavage site after the 109, 136, 200 and 204 residues. Endoproteinases Glu-C do not destroy the activity of full-length harpins, but do extinguish the activities of P91 and P95 (presumably P64 and P68); Trypsin abolishes the activity of P64 and P68 (presumably P91 and P95). These final degradations appear to present a specific portion of each distinct HR-inducing peptide that is presumed necessary for activity as described above.
Apoplast activity degrades harpins without disrupting HR-inducing capacity as compared to the intracellular proteolytic activity present in the pulverized leaf-extracts that deactivate the activity. This raises the question of whether plants use these harpin fragments as trigger-signals. The leaves were infiltrated with tobacco leaves in order to determine the timing of HR that had been previously degraded by the full-length harpin and tobacco intracellular fluid. HR induced by each preparation occurred 12-18 hours after infiltration. Although limited proteolytic activity can be ruled out, co-infiltration of proteinase inhibitors with harpins with tobacco leaves, especially since at least two or more apoplast proteins are present in tobacco. Did not affect HR induced activity. Since pre-decomposed heartins induce HR less quickly than undegraded proteins, proteolytic activity does not appear to be a rate-limiting step required for HR to occur. The role of these apoplast proteins, which can partially hydrolyze harpins but do not extinguish the HR-inducing activity of harpins in tobacco, is still unclear. Salzer et al., "Rapid Reactions of Spruce Cells to Elicitors Released From the Ectomycorrhizal Fungus Hebeloma crustuliniforme and Inactivation of These Elicitors by Extracellular Spruce Cell Enzymes," Planta 198: 118-126 (1996), which is incorporated herein by reference. C) regulates the level of fungal cell membrane triggers released by Hebeloma crustuliniforme, an ectomycorrhizal fungus, by spruce (Picea abies (L.) Karst.) Inactivating these molecules in apoplasts. It is described as. The document suggests that Picea regulates the level of triggers as part of a symbiotic relationship with fungi. Similarly, PGIP of Phaseolus vulgaris has been reported to modulate levels of trigger-activated oligogalacturonides present during plant-parasitic interactions in soybeans (Desiderio, et al., "Polygalasturonase, PGIP"). , and Oligogalacturonides in Cell-Cell Communication, "Biochem. Sci. Trans. 22: 394-397 (1994), which is incorporated herein by reference). Retaining HR-induced activity by harpin fragments is believed to confer plants with the ability to recognize the presence of pathogens. In this regard, whether transgenic host and non-host plants designed for apoplast expression of harpin activity-degrading proteinases show reduced or increased sensitivity of E. amylovora as compared to undesigned plants. It is very interesting to find out.
Despite many attempts, only a few missing derivatives of harpin _Ea and harpin _Ear were successfully expressed in some hrpNs. Problems related to protein stability have been clarified in terms of several lost haffin instability and difficulty in purification. In addition, the expression of missing harpins can be harmful to bacteria. However, the lost harpin _Ea C31 is stable and easily purified but not secreted, suggesting that the C-terminal sequence is involved in the secretion of harpin. Unfortunately the presence of the vector-encoded amino acids of this protein further complicates this conclusion. All attempts to clone the β-galactosidase-hapin fusion protein were not as successful as attempts to clone and express the hrpN downstream of the lacZ promoter in commonly used vectors such as pBluescript. Expression of such constructs was detrimental to bacterial strains.
Wei, et al., "Harpin, Elicitor of the Hypersensitive Response Produced By the Plant Pathogen Erwinia amylovora," Science, 257: 85-88 (1992), found that harpin _Ea , such as keratin and glycine-rich cell wall proteins, has been reported by BLAST. It has been previously reported that it has some similarity with several other glycine rich proteins, which is incorporated herein by reference. However, this is thought to be due to the high content of glycine and harpin _Ea does not present itself as an act of harpin _Ea. Examination of the N-terminal sequence from several HR-derived proteins produced by phytopathogenic bacteria showed significant similarity. But that area was very short. The putative primary sequence similarity region is limited to the first 26 residues at the N-terminus, and its role remains unclear. Surprisingly, E. carotovora harpin _Ecc appeared to be more similar to E. amylovora and E. stewartii harpins than E. chrysanthemi harpins, and closely related to the pathogenic mechanism (ie soft-rots) as well as the taxonomic location. It was related. Furthermore, although primary structural similarity was strong only at the third C-terminus of each protein, Erwinia harpins had a similar hydrophobic profile over the entire length (FIG. 9). Based on the hydrophobic profile, Alfano, et al., "Analysis of the Role of the Pseudomonas Syringe HrpZ harpin in Elicitation of the Hypersensitive Response to Tabacco Using Functionally Nonpolar hrpZ Deletion Mutations, Truncated HrpZ Fragments, and hrmA Mutations, Mol. Microbiol. 19 : 715-728 (1996) describes that harpin _Pss may have amphotericity, which is incorporated herein by reference, but the Erwinia harpin's profile did not match that of harpin _Pss .
Many other secreted glycine-rich pathogenicity has recently been described in relation to proteins, inducers of HR, or other plant-protective hypersensitivity reactions from other plant pathogenic bacteria and fungi (Boller, "Chemoperception of Microbial Signals in Plant Cells, "Ann. Rev. Plant Physiol. Plant Molec. Biol. 46: 189-214 (1996), incorporated herein by reference), examples of which include Phytophthora megasperma (Ballieul, et al., "A New Elicitor of the Hpersensitive Response in Tabacco: a Fungal Glycoprotein Elicits Cell Death, Expression of Defense Genes, Production of Salicylic Acid, and Induction of Systemic Acquired Resistance," Plant Journal 8: 551-560 (1995); Nurnburger, et. al., "High Affinity Binding of a Fungal Oligopeptide Elicitor to the Parsley Plasma Membranes Triggers Multiple Defense Response," Cell 78: 449-460 (1994), incorporated herein by reference) and Magnaporthe grisea (Sweigar). d, et al., "Identification, Cloning, and Characterization of PWL2, a Gene For Host Species Specificity in the Rice Blast Fungus," Plant Cell 7: 1221-1223 (1995), which is incorporated herein by reference. There is). Proteinase HR-induced factors are also obtained in Phynshosporium secalis (Rohe, et al., "The Race-Specific Elicitor, NIP1, From the Barley Pathogen, Phynshosporium secalis, Determines Avirulence on Host Plants of Rrs1 Resistance Genotype," EMBO Journal 14: 4168-4177 (1995), P. infeatans (Pieterse, et al., "Structure and Genomic Organization of ipiB and ipiO Gene Clusters Phytophthora infeatans," Gene, 138: 67-77 (1994), supra Cited herein by reference generate a glycine-rich pathogenic-associated protein with unknown function, since the primary amino acid sequence of each trigger protein or peptide fragment does not show obvious similarities to others. It is unclear whether it interacts with the same target in or on a plant cell, plasma membrane, or cell wall, and in this regard, testing whether one of these molecules inhibits the activity of another molecule Maze will wool plant-defense hypersensitivity reaction-inducing activity (HR or other activity) for having Pep13, Avr9, P68, and the increased availability of the peptide, such as harpin _Ea C305 are not those of the active mechanism is similar, are different or overlapping that determine Not only can they allow accurate probing of their targets in or on plant cells.
Example 14 Bacteria Strains and Plasmids
As E. coli strains used in the examples below, DH5α and BL21 (DE3) purchased from Gibco BRL and Stratagene were used. pET28 (b) vector was purchased from Novagen. Eco DH5α / 2139 contained the complete hrpN gene. The 2139 structure was produced by D. Bauer of Cornell University. The hrpN gene was digested with Hind III restriction enzymes, digested from 2139 plasmids, and then purified from agarose gels to provide the missing hrpN clone as a DNA template for PCR synthesis. These clones were inserted into the (His) ₆ vector pET28 (b) containing the Kan ^r gene for selection of the transformants.
Example 15-DNA Operation
Restriction enzymes were purchased from Boehringer Mannheim or Gibco BRL. T4 DNA ligase, bovine intestinal alkaline phosphatase (CIAP), and PCR Supermix ^™ were purchased from Gibco BRL. QIAprep Spin Miniprep Kit, Qiagen Plasmid Mini Kit, and QIAquick PCR Purification Kit were purchased from Qiagen. PCR primers were synthesized by Lofstand Labs Limited (Gaithersburg, MD). Oligopeptides were synthesized by Bio-Synthesis (Lewisville, TX). All manipulations of DNA such as plasmid isolation, restriction enzyme digestion, DNA conjugation, and PCR were performed according to standard methods (molecular cloning) or procedures provided by the manufacturer.
Example 16 Fragmentation of the hrpN Gene
A series of hrpN genes and internal fragments with missing N- and C-terminus were prepared by PCR (FIG. 10). The full length hrpN gene was used as the DNA template and the 3 'and 5' primers were designed for each missing clone (Figure 11). The 3 'primer contained an Nde I enzyme cleavage site comprising the initiation codon ATG (methionine) and the 5' primer contained a Hind III enzyme cleavage site for conjugation to the stop codon TAA and pET28 (b) vectors. PCR was performed in 0.5 ml tubes in GeneAmp ^™ 9600 or 9700. 45 μl of Supermix ^™ was mixed with 20 pmole of DNA primer, 10 ng of full length Harpin DNA, and diH ₂ O to bring the total volume to 50 μl. After heating the mixture at 95 ° C. for 2 minutes, PCR was performed in 30 cycles for 1 minute at 94 ° C., 1 minute at 58 ° C. and 1.5 minutes at 72 ° C. PCR products were analyzed on 6% TBE gels (Novex). Amplified DNA was purified using QIAquick PCR Purification Kit, digested with NdeI and Hind III for 5 hours at 37 ° C, extracted once with phenol: chloroform: isoamyl alcohol (25: 25: 1), and precipitated with ethanol. . 5 μg of pET28 (b) vector DNA was digested with 15 units of NdeI and 20 units of Hind III for 3 hours at 37 ° C., followed by CIAP treatment to reduce product by incomplete single enzyme digestion. The digested vector DNA was purified by QIAquick PCR Purification Kit and these were used directly for conjugation. ca. Conjugation was carried out at 14-16 ° C. for 5-12 hours in 15 μl of a mixture containing 200 ng of digested pET28 (b), 30 ng of the target PCR fragment, and 1 unit of T4 DNA ligase. 5-7.5 μL of the conjugate solution was added to 100 μL of DH5α cells in 15 mL Falcon tubes and incubated on ice for 30 minutes. After 45 seconds of heat shock at 42 ° C., 0.9 ml of SOC solution or 0.45 ml of LB medium was added to each tube and incubated at 37 ° C. for 1 hour. 20,100 and 200 μl of transformed cells were placed on LB agar containing 30 μg / ml kanamycin and incubated overnight at 37 ° C. Single colonies were transferred to 3 ml of LB-medium and incubated overnight at 37 ° C. Plasmid DNA was prepared from 2 ml of culture containing the QIAprep Miniprep Kit. DNA from the transformed cells was analyzed by restriction enzyme digestion or partial sequencing and these results showed that the transformation was successful. The plasmids described above having the desired DNA sequence were transferred to the BL21 strain using standard chemical transformation methods. Clones containing full length harpins in the pet28 (b) vector were prepared as positive controls and clones with only pET28 (b) were prepared as negative controls.
Example 17 Expression of Lost Harpin Proteins
Escherichia coli BL21 (DE3) strains containing hrpN clones were incubated at 37 ° C. overnight at 37 ° C. with Luria juicy medium containing 30 μg / ml kanamycin (g / L Difco yeast extract, 10 g / L Difco tryptone, 5 g / L NaCl, and 1 mM NaOH). The bacteria were then inoculated in 100 volumes of the same medium to grow to OD ₆₂₀ of 0.6-0.8 at 37 ° C. The bacteria were then inoculated in 250 volumes of the same medium and ca. It was grown to an OD ₆₂₀ of 0.3 or 0.6-0.8. 1 mmol of IPTG was added and the culture was allowed to grow overnight at 19 ° C. (ca. 18 h). Not all clones were successfully expressed by this method. Some clones were incubated in Terrific gravy (12 g / L Bacto tryptone, 24 g / L Bacto yeast, 0.4% glycerol, 0.17 MK ₂ HPO ₄ ) and then selectively grown at 37 ° C. after IPTG induction, and also Optionally recovered earlier overnight.
TABLE 2
Expression of Missing Harpin Proteins
General expression method: Escherichia coli BL21 (DE3) strain containing hrpN subclone was cultured overnight at 37 ° C with Luria juicy medium containing 30 μg / ml kanamycin (g / L Difco yeast extract, 10 g / L Difco). Tryptone, 5 g / L NaCl, and 1 mM NaOH). The bacteria were then inoculated in 100 volumes of the same medium to grow to OD ₆₂₀ of 0.6-0.8 at 37 ° C. The bacteria were then inoculated in 250 volumes of the same medium to grow at 37 ° C. to specific induced OD ₆₂₀ . 1 mmol of IPTG was added and the culture was allowed to grow at a temperature appropriate for protein expression and then recovered at the time to recover the highest level of protein.
Example 18 Small Scale Purification of Lost Harpin Proteins (Verification of Expression)
50 ml cultures of hrpN clones were cultured as above to induce the expression of missing proteins. Upon recovery of the culture, 1.5 ml of the cell suspension was centrifuged at 14,000 rpm for 5 minutes, resuspended in urea lysis buffer (8M urea, 0.1M Na ₂ HPO ₄ , and 0.01M Tris-pH 8.0) and at room temperature. Incubated for 10 minutes, centrifuged again at 14,000 rpm for 10 minutes and the supernatant was stored. Equilibrium (His) and 50% slurry _6-binding nickel agarose was a water droplet 50㎕ (aliquot) of Los resin was added to the supernatant and mixed for 1 hour at 4 ℃. Nickel agarose was then washed three times with urea wash buffer (8M urea, 0.1M Na ₂ HPO ₄ , and 0.01M Tris-pH 6.3) and centrifuged at 5,000 rpm for 5 minutes between each wash. Protein was eluted from the resin with 50 μl of urea elution buffer (8M urea, 0.1M Na ₂ HPO ₄ , 0.01M Tris and 0.1M EDTA-pH 6.3). The eluate was hung on 4-20%, 16%, or 10-20% Tris-glycine pre-cast gels depending on the size of the protein lost to verify expression.
Example 19 HR Induction in Cigarettes
1.5 mL drops from the cultured 50 mL cultures were centrifuged at 14,000 rpm for 4 minutes and resuspended in the same volume of 5 mM potassium phosphate buffer (pH 6.8) to purify the lost protein in small scale. The cell suspension was ca. Sonication for 30 seconds followed by dilution of 1: 2 and 1:10 with phosphate buffer. A hole is made in one compartment in one leaf and the needle is infiltrated into the intracellular leaf space using a syringe without needles to remove the two dilutions and the neat cell lysate 10. -15 leaves Infiltrated 4th to 9th leaves of tobacco plants. HR response was recorded 24-48 hours after infiltration. Tobacco (Nicotiana tabacum v. Xanthi) seedlings in 12-h light / 12-h dark photoperiod and ca. Growing in an environmental chamber at 20-25 ° C. with 40% RH. Cell lysates were used in the initial HR assay because small urea tablets produced very little protein denatured by the purification process (to screen for lost protein for HR activity).
Example 20 Large-scale Native Purification of Lost Harpin Proteins for Comprehensive Biological Activity Assay
Six 500 ml cultures of hrpN clones were grown as described above to induce the expression of lost protein. Upon recovery of the culture, the cells were centrifuged at 7,000 rpm for 5 minutes and resuspended in imidazole lysis buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris) and 0.05% Triton X-100 and 0.1 mg / ml in lysozyme. And sonicated for 2 minutes, then centrifuged again for 20 minutes and the supernatant stored. 50 μl drop of equilibrium (His) ₆ -bonded nickel agarose resin of 50% slurry was added to the supernatant and ca. Mix for 4 hours. Nickel agarose is then washed three times with imidazole wash buffer (20 mM imidazole, 0.5 M NaCl, and 20 mM Tris), centrifuged at 5,000 rpm for 5 minutes between each wash, and then placed on a chromatography column. I was. Centrifuge the column at 1100 rpm for 1 minute to remove residual wash buffer, then incubate the column with elution buffer for 10 minutes at room temperature and centrifuge the column at 1100 rpm for 1 minute to 4 ml of imidazole elution buffer. (1M imidazole, 0.5M NaCl, and 20 mM Tris) eluted the protein from the resin. The eluate was hung on a 4-20%, 16%, or 10-20% tris-glycine pre-cast gel, depending on the size of the protein lost to verify expression. The concentration of the protein was determined by comparing the protein bands with Mark 12 molecular weight markers.
Example 21 Large-scale Urea Purification of Missed Harpin Proteins for Comprehensive Biological Activity Assay
Urea lysis buffer, wash buffer, and elution buffer were used and performed in the same manner as large-scale natural purification except that the cells were not sonicated as in natural purification. After purification, the protein was regenerated by dialysis over 8 hours to dialyzate low concentrations of urea and then dialysis overnight with 10 mM Tris / 20 mM NaCl. The regeneration process allowed the N-terminal protein to precipitate. 100 mM Tris-HCl was added at pH 10.4 and then ca. The protein was heated for 1 hour to dissolve the precipitated 1-168 protein. The concentration of the protein was determined by comparing the protein bands with Mark 12 molecular weight markers. Since the 1-75 and 1-104 protein fragments were not readily soluble, sonication in 100 mM Tris-HCl at pH 10.4 allowed the protein to dissolve as much as possible and exposed the active site of the protein for biological activity analysis.
Example 22 Expression of Lost Harpin Proteins
Small scale expression and purification of fragment proteins to screen expression and HR activity (Table 3).
TABLE 3
Expression and HR Activity of Lost Harpin Proteins (Small Screening)
All cloned fragment proteins were expressed to some extent with the exception of three small fragments (amino acids 169-209, 150-209, and 150-180). The fragments were expressed at various levels. Fragments 210-403 and 267-403 were well expressed and produced high concentrations of protein by small scale purification, showing significant protein bands in SDS gel electrophoresis. Other fragments (amino acids (a.a.) 1-168 and 1-104) produced much less protein, showing a faint protein band in electrophoresis. It was difficult to determine whether fragment 343-403, the smallest C-terminal protein, was expressed because of the presence of several background proteins on the gel in addition to the expected 343-403 protein. Expression and HR activities were tested for positive and negative control proteins consisting of full length proteins and only basic proteins, respectively.
Large-scale expression and purification of fragment proteins were performed to determine levels of expression and HR activity (Table 4).
TABLE 4
Expression and HR Titers of Missing Harpin Proteins (Large Purification)
Due to time constraints, not all proteins were expressed on a large scale. Lost proteins were selected which are considered to be the most important for characterizing harpins. Positive controls (full length harpins) were expressed at relatively high levels of 3.7 mg / ml. All C-terminal proteins were expressed at relatively high levels of 2-5 mg / ml except for the above mentioned proteins 343-403. The N-terminal fragment was also well expressed, but during the purification the protein precipitated and a very small amount of redissolved. The concentrations in Table 3 reflect only the dissolved protein. Internal fragments were expressed in the range of 2-3.6 mg / ml. Since the protein bands on the SDS gel were not well stained in size, it was quite difficult to determine the concentration of fragments 105-168 (the concentration was supposed to be much higher than indicated). Negative controls included several basic proteins as expected, and apparently did not induce major proteins
Example 23-Derivation of HR in Tobacco
The full length control protein induced HR only as low as 5-7 μg / ml. Negative control (pET 28) imidazole purified “protein” —containing only basic protein—induced a low HR response with a 1: 2 dilution, which was analyzed because 1: 1 and 1: 2 dilutions were not used. The sensitivity of the is lowered. The HR of this error was presumed to be due to the affinity of the imidazole used during the purification process, which binds to one or several basic proteins and does not completely dialysis. ca. The imidazole at 60 mM concentration did not induce a false HR response.
One clear domain containing a small internal region of a protein with only 44 amino acids of amino acids 137-180 (SEQ. ID. No. 23) was identified as the smallest HR domain. Another possible HR domain was thought to be located at the N-terminus of amino acids 1-104 (possibly amino acids 1-75) (SEQ. ID. No. 23) protein. Difficulties in purification of these fragment proteins made it difficult to identify and narrow the N-terminal HR domain. N-terminal fragment proteins were purified using urea because none of the proteins were recovered when negative purification was used. As a result, these proteins precipitated during the regeneration process, making it difficult or nearly impossible to return to solution, making it difficult to run the protein through HR analysis because only soluble proteins can induce HR. . In addition to the difficulty in narrowing the N-terminal HR domain, there is a problem that the negative control induces false HR at low dilution levels, thereby reducing the sensitivity of the assay.
The internal domain protein induced a HR response of 5-10 μg / ml like the positive control, and the N-terminal domain protein induced a HR response of 1-3 μg / ml lower than the positive control.
Surprisingly, when the internal HR domain was degraded between amino acids 168 and 169 (fragments 76-168 and 105-168) (SEQ. ID. No. 23), the fragments lost HR activity. This indicates that the HR activity of fragments 1-168 (SEQ. ID. No. 23) will not affect the internal HR domain but will have a second HR domain found in the N-terminal region of the protein by affecting other domains. The guess is reached. However, it was difficult to confirm this conjecture as described above.
Harpin C-terminus (amino acids 210-403 (SEQ. ID. No. 23)) did not contain an HR domain. This did not induce HR to detectable levels in HR analysis. Even the large C-terminus of amino acids 169-403 (SEQ. ID. No. 23) did not induce HR even if it contained part of the internal HR domain. As mentioned above, proteins of amino acids 168-169 (SEQ. ID. No. 23) resulted in loss of HR activity.
Since some of the 61 a.a or smaller cloned proteins were not expressed, oligopeptides with 30 amino acids were synthesized to narrow the functional region of the internal HR domain. The oligopeptides were synthesized in the range of amino acids 121-179 (SEQ. ID. No. 23). However, these oligos did not elicit an HR response. Since these fragments do not contain the essential amino acids 168 and 169 (SEQ. ID. No. 23), it is believed that there will be HR responses from oligos 137-166, 121-150 and 137-156 (SEQ. ID. No. 23). It was not expected. Oligo 150-179 (SEQ. ID. No. 23) was speculated to induce an HR response. 30 amino acids are so small that the protein lacks folding and thus lacks binding to induce activity, or that important amino acids are missing during the synthesis of the peptide (by synthesis or by the selection of 30 amino acids to be synthesized) , Fragments could not induce HR. There is also a small possibility that these small proteins may undergo some form of post-translational modifications that were not included in the synthesis, making them unable to induce an HR response.
Example 24-Biological Activity of HR Induced Fragments
SEQ. ID. Two N-terminal harpin fragments ranging from No 24 nucleotides 1-104 and nucleotides 1-168 exhibited the effect of inducing tobacco resistance to TMV in the same way as the full length harpin protein. SEQ. ID. Internal fragments ranging from No 24 nucleotides 76-209 and nucleotides 105-209 also showed the effect of inducing TMV resistance. In addition, these same four fragments gave plant growth enhancement (“PGE”) in tomatoes, such as increasing plant height 4-19% greater than buffered control plants. Full length harpin protein increased growth by 6% or more than the buffer control. Negative controls did not induce TMV resistance or growth.
TABLE 5
TMV Resistance and PGE Activity of Harpin-Derived HR Induced Fragments
snippet#Amino Acid (SEQ.ID.No.23)HR activeTMV resistantPGEht ＞ Buffer Control 1 (+ control)1-403++6% 2 (-control)----2% 91-104++4-8% 101-168++5-13% 1376-109++4-18% 15105-209++6-19%
The invention has been described in detail for purposes of illustration, and modifications of the invention can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence table
(1) General Information
(i) Applicant: Cornell Research Foundation, Inc. and
Eden Bioscience Corporation
(ii) Name of the invention: hypersensitivity reaction inducing factors that induce hypersensitivity reactions and
Their use
(iii) number of sequences: 30
(iv) communication address:
(A) To: Nixon, Hargrave, Devans & Doyle LLP
(B) Street: Clinton Square, P.O. Box 1051
(C) City: Rochester
(D) State: New York
(E) Country: U.S.A.
(F) Zip code: 14603
(v) computer readable form:
(A) Medium form: floppy disk
(B) Computer: IBM PC Compatible
(C) working system: PC-DOS / MS-DOS
(D) Software: PatentIn Release # 1.0, Version # 1.30
(vi) Current application data:
(A) Application number:
(B) filing date:
(C) Classification:
(vii) Priority Resources:
(A) Application number: 60 / 048,109
(B) Application date: May 30, 1997
(viii) Agent Information:
(A) Name: Goldman, Michael L.
(B) Registration Number: 30,727
(C) Reference / Document Number: 19603/1302
(ix) Communications Information:
(A) Telephone: (716) 263-1304
(B) Fax: (716) 263-1600
(C) Telex:
(2) Information about SEQ ID NO: l
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) Sequence description: SEQ ID NO: l:
GGGAATTCAT ATGAGTCTGA ATACAAGTGG G
(2) information on SEQ ID NO: 2
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 2:
GGGAATTCAT ATGGGCGGTG GCTTAGGCGG T
(2) information on SEQ ID NO: 3
(i) Sequence Features:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 3:
GGCATATGTC GAACGCGCTG AACGATATG
(2) information on SEQ ID NO: 4
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 4:
GGGAATTCAT ATGTTAGGCG GTTCGCTGAA C
(2) information on SEQ ID NO: 5
(i) Sequence Features:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 5:
GGCATATGCT GAACACGCTG GGCTCGAAA
(2) information on SEQ ID NO: 6
(i) Sequence Features:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 6:
GGCATATGTC AACGTCCCAA AACGACGAT
(2) information on SEQ ID NO: 7
(i) Sequence Features:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 7:
GGCATATGTC CACCTCAGAC TCCAGCG
(2) information on SEQ ID NO: 8
(i) Sequence Features:
(A) Length: 34 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 8:
GGGAATTCAT ATGCAAAGCC TGTTTGGTGA TGGG
(2) information on SEQ ID NO: 9
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 9:
GGGAATTCAT ATGGGTAATG GTCTGAGCAA G
(2) Information about SEQ ID NO: 10
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 10:
GGGAATTCAT ATGAAAGCGG GCATTCAGGC G
(2) information on SEQ ID NO: 11
(i) Sequence Features:
(A) Length: 34 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 11
GGGAATTCAT ATGACACCAG CCAGTATGGA GCAG
(2) information on SEQ ID NO: 12
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 12:
GCAAGCTTAA CAGCCCACCA CCGCCCATCA T
(2) information on SEQ ID NO: 13
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 13:
GCAAGCTTAA ATCGTTCAGC GCGTTCGACA G
(2) information on SEQ ID NO: 14
(i) Sequence Features:
(A) Length: 34 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 14:
GCAAGCTTAA TATCTCGCTG AACATCTTCA GCAG
(2) information on SEQ ID NO: 15
(i) Sequence Features:
(A) Length: 30 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 15:
GCAAGCTTAA GGTGCCATCT TGCCCATCAC
(2) information on SEQ ID NO: 16
(i) Sequence Features:
(A) Length: 34 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 16:
GCAAGCTTAA ATCAGTGACT CCTTTTTTAT AGGC
(2) information on SEQ ID NO: 17
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 17:
GCAAGCTTAA CAGGCCCGAC AGCGCATCAG T
(2) Information about SEQ ID NO: 18
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 18:
GCAAGCTTAA ACCGATACCG GTACCCACGG C
(2) information on SEQ ID NO: 19
(i) Sequence Features:
(A) Length: 34 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 19:
GCAAGCTTAA TCCGTCGTCA TCTGGCTTGC TCAG
(2) information on SEQ ID NO: 20
(i) Sequence Features:
(A) Length: 25 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular Form: cDNA
(xi) SEQ ID NO: SEQ ID NO: 20:
GCAAGCTTAA GCCGCGCCCA GCTTG
(2) information on SEQ ID NO: 21
(i) Sequence Features:
(A) Length: 338 amino acids
(B) type: amino acid
(C) Chain form:
(D) topology: on board
(ii) Molecular Form: Protein
(xi) SEQ ID NO: SEQ ID NO: 21:
(2) Information about SEQ ID NO: 22
(i) Sequence Features:
(A) Length: 2141 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular form: DNA (genome)
(xi) SEQ ID NO: SEQ ID NO: 22:
(2) information on SEQ ID NO: 23
(i) sequence characteristics
(A) Length: 403 amino acids
(B) type: amino acid
(C) Chain form:
(D) topology: on board
(ii) Molecular Form: Protein
(xi) SEQ ID NO: SEQ ID NO: 23:
(2) Information about SEQ ID NO: 24
(i) Sequence Features:
(A) Length: 1288 amino acids
(B) type: amino acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular form: DNA (genome)
(xi) SEQ ID NO: SEQ ID NO: 24:
(2) information on SEQ ID NO: 25
(i) Sequence Features:
(A) Length: 341 amino acids
(B) type: amino acid
(C) Chain form:
(D) topology: on board
(ii) Molecular Form: Protein
(xi) SEQ ID NO: SEQ ID NO: 25:
(2) information on SEQ ID NO: 26
(i) Sequence Features:
(A) Length: 1026 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular form: DNA (genome)
(xi) SEQ ID NO: SEQ ID NO: 26:
(2) information on SEQ ID NO: 27
(i) Sequence Features:
(A) Length: 344 amino acids
(B) type: amino acid
(C) Chain form:
(D) topology: on board
(ii) Molecular Form: Protein
(xi) SEQ ID NO: SEQ ID NO: 27:
(2) information on SEQ ID NO: 28
(i) Sequence Features:
(A) Length: 1035 base pairs
(B) Type: nucleic acid
(C) Chain form: single
(D) topology: on board
(ii) Molecular form: DNA (genome)
(xi) SEQ ID NO: SEQ ID NO: 28:
(2) information on SEQ ID NO: 29
(i) Sequence Features:
(A) Length: 26 imino acids
(B) type: amino acid
(C) Chain form:
(D) topology: on board
(ii) Molecular Form: Protein
(xi) SEQ ID NO: SEQ ID NO: 29:
Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala Ile Leu Ala Ala Ile
1 5 10 15
Ala Leu Pro Ala Tyr Gln Asp Tyr
20 25
(2) Information about SEQ ID NO: 30
(i) Sequence Features:
(A) Length: 20 amino acids
(B) type: amino acid
(C) Chain form:
(D) topology: on board
(ii) Molecular Form: Protein
(xi) SEQ ID NO: SEQ ID NO: 30:
Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln Leu Leu
1 5 10 15
Ala met
20

权利要求:
Claims (43)
[1" claim-type="Currently amended] An isolated fragment of an Erwinia hypersensitive response inducer protein or polypeptide that induces hypersensitivity in plants.
[2" claim-type="Currently amended] The isolated fragment of claim 1, wherein said hypersensitivity trigger factor protein or polypeptide is from Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, or Erwinia stewartii.
[3" claim-type="Currently amended] The isolated fragment of claim 2, wherein the hypersensitivity response inducer protein or polypeptide is from Erwinia amylovora.
[4" claim-type="Currently amended] The method of claim 3, wherein the fragment is SEQ. ID. No. The C-terminal fragment of amino acid sequence of 23, SEQ. ID. No. The N-terminal fragment of the amino acid sequence of 23, and SEQ. ID. No. An isolated fragment selected from the group consisting of internal fragments of amino acid sequence of 23.
[5" claim-type="Currently amended] The method of claim 4, wherein said fragment is SEQ. ID. No. SEQ ID NO: 23 of amino acids 105 through 403. ID. No. An isolated fragment, which is a C-terminal fragment of 23 amino acid sequence.
[6" claim-type="Currently amended] The method of claim 4, wherein said fragment is SEQ. ID. NO. SEQ ID NO: 23 within the range of the following amino acids. ID. NO. Isolated fragments that are N-terminal fragments of the amino acid sequence of 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 and 372 .
[7" claim-type="Currently amended] The method of claim 4, wherein said fragment is SEQ. ID. NO. SEQ ID NO: 23 within the range of the following amino acids. ID. NO. Separate fragments, which are central fragments having the amino acid sequence of 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180 .
[8" claim-type="Currently amended] An isolated DNA molecule encoding a fragment according to claim 1.
[9" claim-type="Currently amended] The isolated DNA molecule of claim 8, wherein said hypersensitivity trigger protein or polypeptide is derived from Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, or Erwinia stewartii.
[10" claim-type="Currently amended] The isolated DNA molecule of claim 9, wherein said hypersensitivity trigger protein or polypeptide is derived from Erwinia amylovora.
[11" claim-type="Currently amended] The method of claim 10, wherein said fragment is SEQ. ID. No. The C-terminal fragment of amino acid sequence of 23, SEQ. ID. No. The N-terminal fragment of the amino acid sequence of 23, and SEQ. ID. No. An isolated DNA molecule selected from the group consisting of internal fragments of 23 amino acid sequences.
[12" claim-type="Currently amended] The method of claim 10, wherein said fragment is SEQ. ID. No. SEQ ID NO: 23 of amino acids 105 through 403. ID. No. An isolated DNA molecule, which is a C-terminal fragment of 23 amino acid sequence.
[13" claim-type="Currently amended] The method of claim 10, wherein said fragment is SEQ. ID. NO. SEQ ID NO: 23 within the range of the following amino acids. ID. NO. Isolated DNA molecules that are N-terminal fragments of the amino acid sequence of 23: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1 and 266, 1 and 342, 1 and 321, and 1 372.
[14" claim-type="Currently amended] The method of claim 10, wherein said fragment is SEQ. ID. NO. SEQ ID NO: 23 within the range of the following amino acids. ID. NO. Isolated DNA molecules that are internal fragments with amino acid sequences of 23: 76 and 209, 105 and 209, 99 and 209, 137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180, and 105 and 180 .
[15" claim-type="Currently amended] An expression system transformed with a DNA molecule according to claim 8.
[16" claim-type="Currently amended] The expression system of claim 15, wherein said DNA molecule is of the appropriate sense orientation and correct reading frame.
[17" claim-type="Currently amended] A host cell transformed with the DNA molecule according to claim 8.
[18" claim-type="Currently amended] 18. The host cell of claim 17, wherein said host cell is selected from the group consisting of plant cells and bacterial cells.
[19" claim-type="Currently amended] The host cell of claim 17, wherein the DNA molecule has been transformed with an expression system.
[20" claim-type="Currently amended] A transgenic plant transformed with the DNA molecule according to claim 8.
[21" claim-type="Currently amended] 21. The plant of claim 20, wherein the plant is alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, kidney bean, pea, chicory, lettuce, endive, cabbage, cabbage, beet, Parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, tangerine, strawberry, grape, raspberry, Pineapple, Beans, Tobacco. A transgenic plant selected from the group consisting of tomatoes, sugar cane (sorghum), and sugar cane.
[22" claim-type="Currently amended] 21. The transgenic plant of claim 20, wherein said plant is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, Petunia, Pelargonium, Poinsettia, Chrysanthemum, Carnation, and Genia.
[23" claim-type="Currently amended] A transgenic plant seed transformed with the DNA molecule of claim 8.
[24" claim-type="Currently amended] 24. The plant of claim 23 wherein the plant is alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, kidney bean, pea, chicory, lettuce, endive, cabbage, cabbage, beet, Parsnip, turnip, cauliflower, broccoli, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, tangerine, strawberry, grape, raspberry, Pineapple, Beans, Tobacco. Transgenic plant seeds selected from the group consisting of tomatoes, sugar cane (sorghum), and sugar cane.
[25" claim-type="Currently amended] The transgene plant of claim 23, wherein said plant is selected from the group consisting of Arabidopsis thaliana, Saintpaulia, Petunia, Pelargonium, Poinsettia, Chrysanthemum, Carnation, and Genia.
[26" claim-type="Currently amended] Subjecting the plant to disease resistance comprising applying a non-infectious fragment of a hypersensitivity inducer protein or polypeptide that induces a hypersensitivity response to the plant or plant seeds under conditions to confer disease resistance. Way.
[27" claim-type="Currently amended] 27. The method of claim 26, wherein said method is applied to a plant.
[28" claim-type="Currently amended] 27. The method of claim 26, further comprising: planting the seed treated with the fragment of the hypersensitivity reaction in natural or artificial soil; And
Propagating the plant from the seed planted in the soil
A disease resistance grant method applied to plant seeds further comprising.
[29" claim-type="Currently amended] A method of enhancing plant growth, comprising applying, in a non-infectious form, a hypersensitive response inducer protein or polypeptide fragment that induces a hypersensitive response to a plant or plant seed under conditions for imparting plant growth.
[30" claim-type="Currently amended] The method of claim 29, which is applied to a plant.
[31" claim-type="Currently amended] The method of claim 29, further comprising: planting the seed treated with the fragment of the hypersensitivity reaction in natural or artificial soil; And
Propagating the plant from the seed planted in the soil
Plant growth enhancement method applied to the plant seeds comprising more.
[32" claim-type="Currently amended] A method of inhibiting an insect against a plant, the method comprising applying a non-infectious form of a hypersensitivity inducer protein or polypeptide that induces a hypersensitivity response to a plant or plant seed under conditions for effectively controlling the insect.
[33" claim-type="Currently amended] 33. The method of claim 32, which is applied to a plant.
[34" claim-type="Currently amended] 33. The method of claim 32, further comprising: planting the seed treated with the fragment of the hypersensitivity reaction in natural or artificial soil; And
Propagating the plant from the seed planted in the soil
Insect suppression method applied to plant seeds further comprising.
[35" claim-type="Currently amended] Providing a transgenic plant or plant seed transformed with a DNA molecule encoding a fragment of the hypersensitivity response inducer protein or polypeptide that induces a hypersensitivity response; And
Growing the transgene plant or the transgene plant produced from the transgene plant seed under conditions effective to confer disease resistance.
How to impart disease resistance to the plant comprising a.
[36" claim-type="Currently amended] 36. The method of claim 35, wherein said method is provided to provide a transgenic plant.
[37" claim-type="Currently amended] 36. The method of claim 35, wherein said method is provided to provide a transgenic plant seed.
[38" claim-type="Currently amended] Providing a transgenic plant or plant seed transformed with a DNA molecule encoding a fragment of the hypersensitivity response inducer protein or polypeptide that induces a hypersensitivity response; And
Growing the transgene plant produced from the transgene plant or the transgene plant seed under conditions effective to enhance plant growth.
How to improve plant growth comprising a.
[39" claim-type="Currently amended] The method of claim 38, wherein the plant growth enhancement method provides a transgenic plant.
[40" claim-type="Currently amended] The method of claim 38, wherein the plant growth seed is provided.
[41" claim-type="Currently amended] Providing a transgenic plant or plant seed transformed with a DNA molecule encoding a fragment of the hypersensitivity response inducer protein or polypeptide that induces a hypersensitivity response; And
Growing the transgene plant or the transgene plant produced from the transgene plant seed under conditions for effectively inhibiting insects
Method for inhibiting insects on plants, including.
[42" claim-type="Currently amended] 41. The method of claim 40, wherein the insect inhibition plant provides a transgenic plant.
[43" claim-type="Currently amended] 41. The method of claim 40, wherein the insect suppressant provides transgenic plant seeds.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Choi et al.2013|Harpins, multifunctional proteins secreted by gram-negative plant-pathogenic bacteria

---

US9546378B2|2017-01-17|Hemipteran-and coleopteran active toxin proteins from Bacillus thuringiensis

---

US8759620B2|2014-06-24|Transgenic plants expressing modified CRY3A

---

AU2012214420B2|2017-03-02|Pesticidal nucleic acids and proteins and uses thereof

---

Joosten et al.1999|The Tomato–C ladosporium F ulvum Interaction: A Versatile Experimental System to Study Plant-Pathogen Interactions

---

Yeh et al.1997|Sweet potato | trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura

---

Wang et al.2008|A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis

---

He et al.1993|Pseudomonas syringae pv. syringae harpinPss: a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants

---

Thilmony et al.1995|Expression of the tomato Pto gene in tobacco enhances resistance to Pseudomonas syringae pv tabaci expressing avrPto.

---

Bauer et al.1994|Erwinia chrysanthemi hrp genes and their involvement in soft rot pathogenesis and elicitation of the hypersensitive response

---

Yang et al.1995|Xanthomonas avirulence/pathogenicity gene family encodes functional plant nuclear targeting signals

---

US5750874A|1998-05-12|Fungus-responsive chimaeric gene

---

ES2289743T3|2008-02-01|Transgenic plants expressing dna constructions understanding a plurality of genes to provide a viral resistance.

---

US5750386A|1998-05-12|Pathogen-resistant transgenic plants

---

Pontier et al.1998|Activation of hsr 203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death

---

Osusky et al.2004|Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot

---

US6174717B1|2001-01-16|Elicitor of the hypersensitive response in plants

---

Kessmann et al.1994|Activation of systemic acquired disease resistance in plants

---

Shao et al.2008|Expression of a harpin‐encoding gene in rice confers durable nonspecific resistance to Magnaporthe grisea

---

Williamson et al.1996|Nematode pathogenesis and resistance in plants.

---

Peng et al.2004|Expression of harpinXoo in transgenic tobacco induces pathogen defense in the absence of hypersensitive cell death

---

Bartetzko et al.2009|The Xanthomonas campestris pv. vesicatoria Type III effector protein XopJ inhibits protein secretion: evidence for interference with cell wall–associated defense responses

---

Chao et al.1999|Leucine aminopeptidase RNAs, proteins, and activities increase in response to water deficit, salinity, and the wound signals systemin, methyl jasmonate, and abscisic acid

---

Sun et al.2006|Within-species flagellin polymorphism in Xanthomonas campestris pv campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2–dependent defenses

---

US6916970B2|2005-07-12|Transgenic plants comprising antifungal polypeptides from alfalfa and methods for controlling plant pathogenic fungi

同族专利:

---

公开号 | 公开日

---

US6583107B2|2003-06-24|

---

WO1998054214A2|1998-12-03|

---

FI992545A|

---

NZ501138A|2002-11-26|

---

AU750732B2|2002-07-25|

---

CN1265145A|2000-08-30|

---

CA2289905A1|1998-12-03|

---

FI19992545A|2000-01-28|

---

WO1998054214A3|1999-03-04|

---

US7132525B2|2006-11-07|

---

PL337094A1|2000-07-31|

---

JP2002501388A|2002-01-15|

---

US20010011380A1|2001-08-02|

---

AU7700498A|1998-12-30|

---

US20030182683A1|2003-09-25|

---

EP0996729A2|2000-05-03|

---

BR9809699A|2000-07-11|

---

WO1998054214A9|1999-04-01|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
1997-05-30|Priority to US4810997P

---

1997-05-30|Priority to US60/048,109

---

1998-05-28|Application filed by 해우슬러 에이치 월터, 코넬 리서치 파운데이션 인코포레이티드, 리처드 에스. 카훈, 브래들리 에스. 파웰, 에덴 바이오사이언스 코포레이션

---

1998-05-28|Priority to PCT/US1998/010874

---

2001-02-26|Publication of KR20010013226A

优先权:

---

申请号 | 申请日 | 专利标题

---

US4810997P| true| 1997-05-30|1997-05-30|

---

US60/048,109|1997-05-30|

---

PCT/US1998/010874|WO1998054214A2|1997-05-30|1998-05-28|Hypersensitive response elicitor fragments and uses thereof|