Supplementary Figure S1. TIC807 coding sequence and flanking regions
1 GATCAGAAGCTTTGTAAGCATCTCAGAAGCGTTTTTGCCATCAAAGAAGGGTTAAGGGTA 60
61 TAGCCAGGTCTAAAAGTTTACTAGAAGGGCGAGAGCCGCAATTAAATGAAAGGGAGGAGT 120
121 TCATCAATCAAAAAGAATTGGATATTAGCTAGAGGGTGTTGCAAAACTCATAAATATCCA 180
181 AAAATGAATGCATAAAAAAATCCGTTCTTTTGGAAAAATGATAGTATCCCAAACTATCTC 240
241 CGGAAAGGACGGATTCACTAGTATGATAGCTAACTTTGATCAGAATCAATCCATGTTACA 300
301 GATTTTATTTGCATATGTAGACAGTTTAATCATTGGAGGCGTTCCGCCTTTTACAGGTCG 360
361 TCCACCTATATGTAGAAAGGCTTTATTAAAATGTTTTTTCTTGAAAACTGTATTCCAGAT 420
421 TGATTCTTTACGGAAATTAACTCGTTTTTTACATCAGTATCCTTCATTTCGTGTATCTTG 480
481 TGGCTTTTCTTCACTTCCTCATATCTCTACTTTTTCACGTGTAGGTACTTGGTTTCGTGA 540
541 AGAAGGTATTCCATTGATCCATAAGCAAATTCTTCACGAAATGAATTTCGGTTTAATTCC 600
601 GTGCGTTTTAATTGATCGTACAGCCTTACGGAGTAGTTTATATGATTCTCAAGCAAAATG 660
661 GGGGAAGTCGACTCGTTATGGTTGGTATAAAGGATATAAGGCCCATGTTTGTTCAACACC 720
721 AGAAGGTGTTGTGTTGTCTTACGCGTTTACAACAGCTAACATCCATGATAGCAAAATGGC 780
781 TCCTGTATTGCTTCAAAATATACAAGACCAAAATGTGTTGTTTTCTGTTGCAGATGCTGC 840
841 TTATGATAGTCAGCATATTTATGAAATTGCGCGAGCATGTAATATCTTTGCCATAAATCC 900
901 AATTAATCCAAGGAATGGTGAACAAAATAAGAGTACACATCGTCGTATATTATCTCAATT 960
961 TATACAAACTATCTTAGGTAAGCAGTTGATGAAGGAATGTGGAAAAATTGAACAACAGTT 1020
1021 TAGTAATCTCAAAGATAAAGGGCTGGAACAGCCACGTTGGTATGGAAAAAATCGTTATCT 1080
1081 ATTGCATGTTCAGCTCATTTTTCTGATTCATAACATTGCATATTTATTTTAGTTTTGCAA 1140
1141 CACCCTCAGCTATAAAGAATTAGAAGTGAAGTGAATAGCATGTCATTTCATTTCATTTGT 1200
1201 GTTCATAACTATAAACTATAATTTTATTTTAATGATAAAAGGAGAAATATATTATGCAAA 1260
1261 TTACGCTAGAACAAACTGTAAATGGAAAGAAATTTTTAAATATTAAGAATAAAAATTAAT 1320
1321 ATTTAAAATTGGTTCAAAAAACCAAAAACATACATATATTATTATTGTTGTTTTATTTTT 1380
1381 GAAAATTCAAAAATATATATTTTGTTCATATTTTGTAACGTTAATTATTTTATATTTTAA 1440
1441 ACAATTAGGAGATGATTTTTTTGGCAATTTTAGATTTAAAATCTTTAGTACTCAATGCAA 1500
M A I L D L K S L V L N A I
1501 TAAATTATTGGGGTCCTAAAAATAATAATGGCATACAGGGTGGTGATTTTGGTTACCCTA 1560
N Y W G P K N N N G I Q G G D F G Y P I
1561 TATCAGAAAAACAAATAGATACGTCTATTATAACTTTTACTCATCCTCGTTTAATTCCAT 1620
S E K Q I D T S I I T F T H P R L I P Y
1621 ATGATTTAACAATTCCTCAAAATTTAGAAACTATTTTTACTACAACTCAAGTATTAACAA 1680
D L T I P Q N L E T I F T T T Q V L T N
1681 ATAATACAGATTTACAACAAAGTCAAACTGTTTCTTTTGCTAAAAAAACAACGACAACAA 1740
N T D L Q Q S Q T V S F A K K T T T T T
1741 CTTCAACTTCAACTACAAATGGTTGGACAGAAGGTGGGAAAATTTCAGATACATTAGAAG 1800
S T S T T N G W T E G G K I S D T L E E
1801 AAAAAGTAAGTGTATCTATTCCTTTTATTGGAGAGGGAGGAGGAAAAAACAGTACAACTA 1860
K V S V S I P F I G E G G G K N S T T I
1861 TAGAAGCTAATTTTGCACATAACTCTAGTACTACTACTTTTCAACAGGCTTCAACTGATA 1920
E A N F A H N S S T T T F Q Q A S T D I
1921 TAGAGTGGAATATTTCACAACCAGTATTGGTTCCCCCAAGTAAACAAGTTGTAGCAACAT 1980
E W N I S Q P V L V P P S K Q V V A T L
1981 TAGTTATTATGGGAGGTAATTTTACTATTCCTATGGATTTGATGACTACTATAGATTCTA 2040
V I M G G N F T I P M D L M T T I D S T
2041 CAGAACATTATAGCCATTATAGTGGTTATCCAATATTAACATGGATATCGAGCCCCGATA 2100
E H Y S H Y S G Y P I L T W I S S P D N
2101 ATAGTTATAGTGGTCCATTTATGAGTTGGTATTTTGCAAATTGGCCCAATTTACCATCGG 2160
S Y S G P F M S W Y F A N W P N L P S G
2161 GGTTTGGTCCTTTAAATTCAGATAATACGGTCACTTATACAGGTTCTGTTGTAAGTCAAG 2220
F G P L N S D N T V T Y T G S V V S Q V
2221 TATCAGCTGGTGTATATGCCACTGTACGATTTGATCAATATGATATACACAATTTAAGGA 2280
S A G V Y A T V R F D Q Y D I H N L R T
2281 CAATTGAAAAAACTTGGTATGCACGACATGCAACTCTTCATAATGGAAAGAAAATATCTA 2340
I E K T W Y A R H A T L H N G K K I S I
2341 TAAATAATGTTACTGAAATGGCACCAACAAGTCCAATAAAAACAAATTAAGACTAAAATG 2400
N N V T E M A P T S P I K T N *
2401 AAAAAAGAATAAAAAAATAAAGCTCTTACTTTGTGTATATGATCACAACGTAGGAGCTTT 2460
2461 TTTCATTAGATTTGTTCATACAGCTTTCCTTGTATCCGGGGTAAACCCAGCATCTTCGAG 2520
2521 CAGAATGGACATTTTTAAATTTTCAGACAATTGATAGATTTTTTTATGGTTTTTACGGCA 2580
2581 CGAAATCATAGTTCATGCGTTCTGCGTCGCTTTTTTAAACTGTTAAAACATGAAATGAAC 2640
2641 CTGATGTTTAATTAATTTTGATGAAAAGTGTCACAGAATCATATTTCTATGTCACTTTTT 2700
2701 GTTTAAACTTATTAAATTTACACTGTTAAAATTCTGATTATTATGAAATGTCCGTTTTAC 2760
2761 TTGGAGATGCTGGTTCTACCCCTATCCGCCTGTTTTTATCTTTTAATCGCTTATTGTCAC 2820
2821 TTTCCAATTCTTTGATTATACAGAATAATTTTTCAAATGATTTTCACCTGATATAAGATC 2880
2881 GTACTCCATATGTGATTTATATATATTGTCTATTAGTAAAAATATTTGTGTCCTGTTTTT 2940
2941 ATTTGTTACACGTGATTAAGCATAAAAAGAGCAGAAAAGAGGAAAAAACAATACAACATC 3000
3001 ATAGAGCGAATGATTTATCAATTTTTTCTTGTGTGGGTCCGGTATGCGAAGATGAACTAC 3060
3061 ATCGTTTCCCTTTCTTTATAGCTGCTTCCAATTCAGATTTATCTTCATCTAAAAAAATAG 3120
3121 AGAAGCACTCTTTCCCTATTAATGCAGTACTTTCTTTTCCTAATAACATACTTCCAGCCT 3180
3181 GGTTTATATTTAATACGATACCGTTTGAATCAATCGTTAAAATAGGATC 3229
Supplementary Figure S2. Pair-wise alignment of TIC807 and Cry15Aa1
Program: needle
Aligned_sequences: 2
1: Cry15Aa1
2: TIC807
Matrix: EBLOSUM62
Gap_penalty: 10.0
Extend_penalty: 0.5
Length: 376
Identity:
Similarity:
Gaps:
Score: 258.5
Cry15Aa1
TIC807
Cry15Aa1
TIC807
Cry15Aa1
TIC807
Cry15Aa1
TIC807
Cry15Aa1
TIC807
Cry15Aa1
TIC807
Cry15Aa1
96/376 (25.5%)
148/376 (39.4%)
103/376 (27.4%)
1 MAIMN--DIAQDAARAWDIIAGPFIRPGTTPTNRQLFNYQIGNIEVEPGN
|||:: .:..:|...|
||....|....:
|.|.|...:::...
1 MAILDLKSLVLNAINYW----GPKNNNGIQGGD---FGYPISEKQIDTSI
48
49 LNFS---VVP---------ELDFSVSQDLFNNTSVQQSQTASFNESRTET
:.|:
::|
|..|:.:|.|.|||.:|||||.||.:..|.|
44 ITFTHPRLIPYDLTIPQNLETIFTTTQVLTNNTDLQQSQTVSFAKKTTTT
86
87 TSTAVTHGVKSGVTVSASAKFNAKILVKSI-----EQTITTTVSTEYNFS
|||:.|:|...|..:|.:.:....:.:..|
:.:.|...:..:|.|
94 TSTSTTNGWTEGGKISDTLEEKVSVSIPFIGEGGGKNSTTIEANFAHNSS
132 STTTRTNTVTRGWSIAQPVLVPPHSRVTATLQIYKGDFTVPVLL-----S
:||.:..:....|:|:|||||||..:|.|||.|..|:||:|:.|
|
144 TTTFQQASTDIEWNISQPVLVPPSKQVVATLVIMGGNFTIPMDLMTTIDS
43
93
131
143
176
193
177 LRVYGQTG-----TLAGNPS------FPSLYAATYENTLLGRIREHIAPP
...|....
|...:|.
|.|.|.|.:.|.
|
194 TEHYSHYSGYPILTWISSPDNSYSGPFMSWYFANWPNL-----------P
215
216 ALFRASNAYISNGVQAIWRGTATTRVSQGLYSVVRIDERPLAGYSGETRT
:.|...|: .|.| .:.|:..::||.|:|:.||.|:..:.......:|
233 SGFGPLNS--DNTV--TYTGSVVSQVSAGVYATVRFDQYDIHNLRTIEKT
265
TIC807
266 YYL-PVTLSNSSQILTPGSLGSEIPIINPVPNASCKKENSPIIIHHDREK
:|. ..||.|
|.:|.|.|....|
..|||..:
279 WYARHATLHN----------GKKISINNVTEMA----PTSPIKTN
Cry15Aa1
315 HRERDYDKEHICHDQAEKYERDYDKE
340
TIC807
310
309
232
278
314
309
Supplementary Figure S3. Coding region for a transposase adjacent to the TIC807 gene
Query= pMON96219 insert
>gb|AAZ99889.1| transposase [Bacillus thuringiensis serovar tenebrionis]
Length: 289
E value = e-148
87% sequence identity
Query:
263 MIANFDQNQSMLQILFAYVDSLIIGGVPPFTGRPPICRKALLKCFFLKTVFQIDSLRKLT 442
MI NFDQNQS+LQILFAYVDSL IGGVPP TGRPPICRKALLKCFF+KTVFQI+SLRKLT
Sbjct:
Query:
1 MIINFDQNQSVLQILFAYVDSLTIGGVPPLTGRPPICRKALLKCFFVKTVFQINSLRKLT 60
443 RFLHQYPSFRVSCGFSSLPHISTFSRVGTWFREEGIPLIHKQILHEMNFGLIPCVLIDRT 622
RFLHQYPSFRVSCG S +PHISTFSRVG WFR EGIP+IHKQ L EMN GLIPCVLI+ T
Sbjct:
61 RFLHQYPSFRVSCGLSFVPHISTFSRVGNWFRNEGIPMIHKQTLEEMNLGLIPCVLINST 120
Query:
623 ALRSSLYDSQAKWGKSTRYGWYKGYKAHVCSTPEGVVLSYAFTTANIHDSKMAPVLLQNI 802
ALRSSLYDSQAKWGKSTRYGWYKGYKAHVCSTPEG+VLSY+FTT N+H SKMAPVLL++I
Sbjct:
121 ALRSSLYDSQAKWGKSTRYGWYKGYKAHVCSTPEGIVLSYSFTTVNVHGSKMAPVLLRDI 180
Query:
803 QDQNVLFSVADAAYDSQHIYEIARACNIFAINPINPRNGEQNKSTHRRILSQFIQTILGK 982
QD+NVLFSVADAAYDSQHIYEIAR
NIFA+NPINPRNGEQ KSTHRR+LS F+QT+ GK
Sbjct:
181 QDRNVLFSVADAAYDSQHIYEIARISNIFAVNPINPRNGEQIKSTHRRVLSHFVQTLFGK 240
Query:
983 QLMKECGKIEQQFSNLKDKGLEQPRWYGKNRYLLHVQLIFLIHNIAYLF 1129
QLMKE GKIEQQFSNLKDKGLEQPRWYG+NRYLLHVQL+FLIHNIAYLF
Sbjct:
241 QLMKERGKIEQQFSNLKDKGLEQPRWYGQNRYLLHVQLVFLIHNIAYLF 289
Supplementary Figure S4. Coding sequence for the plant-expressed TIC807 protein
ATGGCTATCCTAGACCTTAAGTCCCTCGTGCTGAACGCCATTAACTACTGGGGCCCTAAGAACA
ACAACGGCATCCAGGGCGGTGACTTCGGCTACCCCATCTCTGAGAAGCAGATCGACACTAGCAT
CATTACCTTCACCCACCCTCGCTTGATCCCCTACGATCTTACTATCCCGCAGAACCTTGAGACC
ATCTTCACCACAACGCAGGTGCTCACCAATAACACTGACCTCCAGCAATCCCAGACCGTGAGCT
TTGCGAAGAAGACCACTACCACGACCTCAACTAGCACGACCAACGGTTGGACAGAAGGAGGCAA
GATCAGCGACACGCTGGAGGAGAAAGTTTCGGTTAGCATTCCGTTCATCGGTGAGGGTGGCGGG
AAGAACTCGACTACCATAGAGGCCAACTTCGCACACAACTCTAGCACCACTACCTTCCAGCAAG
CAAGCACTGACATTGAGTGGAACATTAGCCAACCGGTGCTGGTTCCTCCCTCTAAACAAGTTGT
CGCGACCCTTGTGATCATGGGAGGCAACTTTACCATCCCTATGGACTTGATGACCACGATTGAT
AGTACAGAGCACTACTCCCACTACTCCGGTTACCCTATCCTCACCTGGATCTCGTCCCCAGATA
ACTCTTACTCCGGTCCCTTTATGTCATGGTACTTTGCAAACTGGCCTAACCTTCCGAGTGGATT
CGGCCCACTGAATAGTGATAACACGGTCACATACACTGGCTCTGTCGTGTCCCAAGTTTCGGCC
GGTGTCTACGCTACCGTCCGGTTCGATCAGTATGACATTCACAATCTCCGTACTATCGAGAAGA
CTTGGTATGCTCGCCATGCGACGCTGCATAATGGCAAGAAGATTTCTATCAACAATGTCACGGA
AATGGCTCCAACATCCCCTATCAAGACAAATTGA
TIC807 (5 ng)
negative plant
ovary
anther
pistil
bract
petal
leaf
boll
TIC807 (5 ng)
Supplementary Fig. S5. Western blot analysis of floral tissues from a cotton plant transformed
with pMON105864. Each lane was loaded with the equivalent of 1 mg fresh tissue.
Supplementary Table S1. LC50 determination for Lygus hesperus
N
Slope + SE
LC50 (95% CL)
Chi square
262
1.77 + 0.268
72.9 ppm (50.2- 116.4)
43.28 (P<0.001)