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Supplementary Figure S5: Structural annotation of inhibitor of apoptosis proteins (IAPs) encoded by transcripts differentially expressed following Diabrotica virgifera virgifera exposure to Cry3Bb1 or Gpp34/Tpp35Ab1. Annotations based on structure defined by Hay et al. (2000).

A) Annotation of D. v. virgifera IAP1 (DvvIAP1) isoforms encoded by transcripts DIAVI011972 (XP_028141650.1 isoform X1; DvvIAP1_X1) and DIAVI007715 (XP_028141655.1 isoform X5; DvvIAP1_X5). Two baculovirus inhibition of apoptosis protein repeat domains (BIR1 and BIR2) and RING finder domains characteristic of IAP family 1 proteins are underlined, and cysteine and histidine residues involved in tetrahedral coordination of Zn+ required to form the zinc-finger-like protein structure are highlighted grey. Table shows results sequence query of the conserved domain database (Marchler-Bauer A et al. 2017).

DIAVI011972: DvvIAP1_X1 significantly up-regulated in D. v. virgifera larvae exposed to the Bacillus thuringiensis toxin Cry34/35Ab1.

1-MTHIVKWLWTKLFHGTKMAVVQSNYIQNIPSFGCVDQPDNGSKTTRESLVEVSSSRPRQEDYSVYENRLA 71-SFTNWPNTQVSRESLARAGFIYTGQDDIVI C PI C KIEGYRWVSGDNPMDD H RVWNPN C PFLN RRDNIEHD 141-HSVGSRDTCGLFGIELLPNSVPEDNTSNLQKLGIQPGTGPQNQDKITLESRLATFQGWPKSIKQRPSELA 211-EAGFYYTGAGDQTV C FY C GGGLKDWDEGDDPWEQ H ALWFSK C VFLNL KKGKEFIDQVKRKADPQFSIPGP 351-SGTQAKEEPTATESSSDKQSETVKTKSDRESFATDTTLCKICFKNELGVVFLPCGHIVACVDCAAALKTC 421-AVCRKPLEATVRAFLS*

Results of querying the conserved domain database with the full translated DIAVI011972 protein sequence.

Feature Accessio n

Description Interval E-value

BIR1 cd00022 Baculoviral inhibition of apoptosis protein repeat domain 1 66-132 5.75e-34 BIR2 cd00022 Baculoviral inhibition of apoptosis protein repeat domain 2 189-257 3.08e-25 RING cl17238 The superfamily of Really interesting new gene (RING) finger domain

and U-box domain

315-366 5.29e-18

DIAVI007715: DvvIAP1_X5 significantly up-regulated in D. v. virgifera larvae exposed to the Bacillus thuringiensis toxin Cry3Bb1.

1-MAVVQSNYIQNIPSFGCVDQPDNGSKTTRESLVEVSSSRPRQEDYSVYENRLASFTNWPNTQVSRESLAR 71-AGFIYTGQDDIVI C PI C KIEGYRWVSGDNPMDD H RVWNPN C PFLN RRDNIEHDHSVGSRDTCGLFGIELL 141-PNSVPEDNTSNLQKLGIQPGTGPQNQDKITLESRLATFQGWPKSIKQRPSELAEAGFYYTGAGDQTV C FY 211-C GGGLKDWDEGDDPWEQ H ALWFSK C VFLNL KKGKEFIDQVKRKADPQFSIPGPSGTQAKEEPTATESSSD 351-KQSETVKTKSDRESFATDTTLCKICFKNELGVVFLPCGHIVACVDCAAALKTCAVCRKPLEATVRAFLS*

Results of querying the conserved domain database with the full translated DIAVI007715 protein sequence.

Feature Accession Description Interval E-value

BIR1 cd00022 Baculoviral inhibition of apoptosis protein repeat domain 1 49-115 1.99e-25 BIR2 cd00022 Baculoviral inhibition of apoptosis protein repeat domain 2 172-240 3.54e-34 RING cl17238 The superfamily of Really interesting new gene (RING) finger domain

and U-box domain

298-349 7.45e-18

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B) Annotation of D. v. virgifera IAP2 (DvvIAP2) encoded by transcript DIAVI011430. Three baculovirus inhibition of apoptosis protein repeat domains (BIR1, BIR2 and BIR3) and RING finder domains characteristic of IAP family 2 proteins are underlined, and cysteine and histidine residues involved in tetrahedral coordination of Zn+ required to form the zinc-finger- like protein structure are highlighted grey.

DIAVI011430: DvvIAP2 not significantly up-regulated in D. v. virgifera larvae exposed to the Bacillus thuringiensis toxin Cry3Bb1 or Cry34/35Ab1.

1-MHQEINRLQTFSEWPSDAQVSPQRIAKAGFFATKQGLEVE C FA C HAKISEWNYGDQVMTR H IALNRD C PF 71-VLNPSTSGNVPITSSRVPSTSINMYRSSETRLASFENWPAADIVTPDSLVQAGFYYLKEGDNTQ C AF C KG 141-VVRAWEVGDDPDTE H QRHFPN C PFVMAV INPRLQARRGSNDRNNPENNQIVKDSFPNINVVGTEQNLGEL 211-GVQAHRGPKKSNFATVEARLRSYVGWSSDLIQTPEVLAEAGFYYEGMGDQVR C FH C DGGLRTWDPHDDPW 281-TE H ARWFPN C SFVKL VKGQDFVTACTIGQTTDSSVRPSAQRIQTTRIRREVTEREIQSYLTSPQALAALS 351-IGLNVERVKRAIREKLEQTGRAYSQPDALVEAALNLQHEEEDPNSHEHYTPIDRSLRNVVCAAMEECIDR 421-QPEQVQQPEPAQQPEMDEAFEVSPTTAPDGTPQLHYQLVKTVSLEEENRILKEARL C KI C MDSEVGIVFL 491-PCGHLAT C VN C APNLED C PV C RSTIKATVRTFFS *

Results of querying the conserved domain database with the full translated DIAVI011430 protein sequence.

Feature Accession Description Interval E-value

BIR1 cd00022 Baculoviral inhibition of apoptosis protein repeat domain; Found in inhibitors of apoptosis ...

5-73 9.93e-24 BIR2 cd00022 Baculoviral inhibition of apoptosis protein repeat domain; Found in inhibitors

of apoptosis ...

99-167 4.92e-30

BIR3

cd00022 Baculoviral inhibition of apoptosis protein repeat domain; Found in inhibitors of apoptosis ...

227-295 7.23e-32 UBA_IAPs cd14321 ubiquitin-association(UBA) domain found in inhibitor of apoptosis proteins

(IAPs); IAPs are frequently overexpressed ...

343-386 4.56e-11 RING-

HC_BIRC2_3_

7

cd16713 RING finger, HC subclass, found in apoptosis protein c-IAP1, c-IAP2, livin, and similar ...

471-524 1.10e-27

(3)

C) Multiple protein alignment of baculoviral inhibition of apoptosis protein repeat (BIR) domains from Drosophila melanogaster inhibitor of apoptosis protein 1 and 2 [DIAP1 (AAF49548.1) and DIAP2 (AAF58095.1)] with putative Diabrotica virgifera virgifera IAP1 orthologs encoded by isoforms X1 (DvvIAP1_X1) and X5 (DvvIAP1_X5), and IAP2 (DvvIAP2). Conserved cysteine and histidine residues involved in tetrahedral coordination of Zn+ required to form the zinc-finger-like structure are highlighted yellow, and remaining conserved residues with 100% conservation are in dark grey and ≥50% in light grey.

Two BIR repeats from DIAP1 orthologs

DIAP1A_BIR1 -ETRLKTFTDWPLDWLDK-RQLAQTGMYFTHAGDKVKCFFCGVEIGCWEQEDQPVPEHQRWSPNCPLLRR 68 DvvAIP1X1_BIR1 -ENRLASFTNWPNTQVSR-ESLARAGFIYTGQDDIVICPICKIEGYRWVSGDNPMDDHRVWNPNCPFLN- 68 DvvAIP1X5_BIR1 -ENRLASFTNWPNTQVSR-ESLARAGFIYTGQDDIVICPICKIEGYRWVSGDNPMDDHRVWNPNCPFLN- 68 DIAP1A_BIR2 ETARLRTFEAWPRNLKQKPHQLAEAGFFYTGVGDRVRCFSCGGGLMDWNDNDEPWEQHALWLSQCRFVKL 70 DvvAIP1X1_BIR2 -ESRLATFQGWPKSIKQRPSELAEAGFYYTGAGDQTVCFYCGGGLKDWDEGDDPWEQHALWFSKCVFLNL 69 DvvAIP1X5_BIR2 -ESRLATFQGWPKSIKQRPSELAEAGFYYTGAGDQTVCFYCGGGLKDWDEGDDPWEQHALWFSKCVFLNL 69 Three BIR repeats from DIAP2 orthologs

DIAP2A_BIR1 -SVRLATFGEWPLNAPVSAEDLVANGFFATGNWLEAECHFCHVRIDRWEYGDQVAERHRRSSPICSMVLA 69 DvvAIP2_BIR1 -INRLQTFSEWPSDAQVSPQRIAKAGFFATKQGLEVECFACHAKISEWNYGDQVMTRHIALNRDCPFVLN 68 DIAP2A_BIR2 -EANRLVTFKDWPNPNITPQALAKAGFYYLNRLDHVKCVWCNGVIAKWEKNDNAFEEHKRFFPQCPRVQM 69 DvvAIP2_BIR2 -ETRLASFENWPAADIVTPDSLVQAGFYYLKEGDNTQCAFCKGVVRAWEVGDDPDTEHQRHFPNCPFVMA 69 DIAP2A_BIR3 -EARLRTFTDWPISNIQPASALAQAGLYYQKIGDQVRCFHCNIGLRSWQKEDEPWFEHAKWSPKCQFVLL 69 DvvAIP2_BIR3 -EARLRSYVGWSSDLIQTPEVLAEAGFYYEGMGDQVRCFHCDGGLRTWDPHDDPWTEHARWFPNCSFVKL 69

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>DvvAIP1X1_BIR1

ENRLASFTNWPNTQVSRESLARAGFIYTGQDDIVICPICKIEGYRWVSGDNPMDDHRVWNPNCPFLN

>DvvAIP1X1_BIR2

ESRLATFQGWPKSIKQRPSELAEAGFYYTGAGDQTVCFYCGGGLKDWDEGDDPWEQHALWFSKCVFLNL

>DvvAIP1X5_BIR1

ENRLASFTNWPNTQVSRESLARAGFIYTGQDDIVICPICKIEGYRWVSGDNPMDDHRVWNPNCPFLN

>DvvAIP1X5_BIR2

ESRLATFQGWPKSIKQRPSELAEAGFYYTGAGDQTVCFYCGGGLKDWDEGDDPWEQHALWFSKCVFLNL

>DIAP1A_BIR1

ETRLKTFTDWPLDWLDKRQLAQTGMYFTHAGDKVKCFFCGVEIGCWEQEDQPVPEHQRWSPNCPLLRR

>DIAP1A_BIR2

ETARLRTFEAWPRNLKQKPHQLAEAGFFYTGVGDRVRCFSCGGGLMDWNDNDEPWEQHALWLSQCRFVKL

>DIAVI011430_DvvAIP2_BIR1

NRLQTFSEWPSDAQVSPQRIAKAGFFATKQGLEVECFACHAKISEWNYGDQVMTRHIALNRDCPFVLN

>DIAVI011430_DvvAIP2_BIR2

ETRLASFENWPAADIVTPDSLVQAGFYYLKEGDNTQCAFCKGVVRAWEVGDDPDTEHQRHFPNCPFVMAV

>DIAVI011430_DvvAIP2_BIR3

ARLRSYVGWSSDLIQTPEVLAEAGFYYEGMGDQVRCFHCDGGLRTWDPHDDPWTEHARWFPNCSFVKL

>DIAP2A_BIR1

RLATFGEWPLNAPVSAEDLVANGFFATGNWLEAECHFCHVRIDRWEYGDQVAERHRRSSPICSMVLA

>DIAP2A_BIR2

EANRLVTFKDWPNPNITPQALAKAGFYYLNRLDHVKCVWCNGVIAKWEKNDNAFEEHKRFFPQCPRVQM

>DIAP2A_BIR3

ARLRTFTDWPISNIQPASALAQAGLYYQKIGDQVRCFHCNIGLRSWQKEDEPWFEHAKWSPKCQFVLL

>AAF49548.1_DIAP1A

MASVVADLPSYGPIAFDQVDNNTNATQLFKNNINKTRMNDLNREETRLKTFTDWPLDWLDKRQLAQTGMY FTHAGDKVKCFFCGVEIGCWEQEDQPVPEHQRWSPNCPLLRRRTTNNVPINAEALDRILPPISYDICGAN DSTLEMREHAYAEGVIPMSQLIQSIGMNAVNAAGSVTGTAAPQPRVTVATHASTATQATGDVQPETCRPS AASGNYFPQYPEYAIETARLRTFEAWPRNLKQKPHQLAEAGFFYTGVGDRVRCFSCGGGLMDWNDNDEPW EQHALWLSQCRFVKLMKGQLYIDTVAAKPVLAEEKEESSSIGGVAVASTQASEEEQQTSLSSEEAVSGDV APSVAPTAATRIFNKIVEATAVATPSTNSSGSTSIPEEKLCKICYGAEYNTAFLPCGHVVACAKCASSVT KCPLCRKPFTDVMRVYFS

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