Sip1A: TIC901a novel secreted protein from Bacillus thuringiensis. David Bowen Agronomic Traits - Discovery Monsanto Company

Transcription

1 Sip1A: TIC901a novel secreted protein from Bacillus thuringiensis David Bowen Agronomic Traits - Discovery Monsanto Company 1

2 Insect protection Insecticidal proteins from Bacillus thuringiensis 2

3 Multiple protein families with diverse activities Cyt2Aa1 Cry37Aa1 Cry23Aa1 Cry22Aa1 Li et al. (1996) J. Mol. Biol. 257:129. Cry1Aa1 1 2 Vip2Aa Vip2A 3 Han et al., (1999) Nat. Struct. Biol. 6(10):932. Grochulski et al. (1995) J. Mol. Biol. 254:447 3

4 Insect protected crops: the early years Barton, K. A., H. R. Whiteley, and N. Yang Bacillus thuringiensis - endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects: Plant. Physiol. 85: Vaeck, M., A. Reynaerts, H. Hofte, S. Jansens, M. DeBeuckeleer, C. Dean, M. Zabeau, M. Van Montagu, and J. Leemans Transgenic plants protected from insect attack. Nature 328:33-37 Corn Bt event 176 Cry1Ab Lepidopterans 1996 Bt11 Cry1Ab Lepidopterans 1997 (NKBT11) YieldGard Cry1Ab Lepidopterans 1997 (MON810) Starlink Cry9Ca Lepidopterans 1998 Cotton Bollgard Cry1Ac Lepidopterans

5 Second-generation insect-protected crops General features Contain multiple or novel insecticidal protein genes Encode engineered or natural proteins Provide expanded control of insect pest species Some offer improved insect resistance management by providing a different mode-of-action Insect protection traits are stacked with herbicide tolerance traits Glyphosate tolerance (cp4 gene from Agrobacterium) Glufosinate tolerance (phosphinothricin-n-acetyltransferase PAT gene from Streptomyces) 5

6 Insect Control Products of Today are Driven by Bacillus thuringiensis Derived Proteins Company Product Name Event Gene (s) Pest Commercial Year Monsanto Corn YieldGard CB MON810 cry1ab Lepidopterans (2) 1997 YieldGard RW MON863 cry3bb Coleopterans (3) 2004 YieldGard VT RW MON88017 cry3bb Coleopterans (3) 2007 YieldGard VT PRO MON89034 cry1a.105 and cry2ab2 Lepidopterans (6) 2009 Cotton Bollgard MON531 cry1ac Lepidopterans (4) 1996 Bollgard II MON15985 cryiac and cry2ab Lepidopterans (5) 2003 DOW/DuPont Corn Herculex I 1507 cry1fa2 Lepidopteran (1) 2003 Herculex RW cry34ab1 and cry35ab1 Coleopterans (3) 2006 DOW Corn N/A DAS cry1f Lepidopterans (5) (?) Cotton Widestrike cry1ac Lepidopterans (5) 2005 Widestrike cry1f Lepidopterans (4) 2005 Syngenta Corn NaturGard KnockOut 176 cry1ab Lepidopterans (1) 1996 AgriSure CB BT11 cry1ab Lepidopterans (1) 1997 TBA MIR162 VIP3A Lepidopterans 2010 AgriSure RW MIR604 mcry3a Coleopterans (3) 2007 Cotton TBA COT67B Cry1Ab Lepidopterans? VipCot COT102 VIP3A Lepidopterans (8)? Bayer (Aventis CropScience) Corn StarLink CBH-351 cry9c Lepidopterans (1) 1998 Cotton BTX Stacked TBA Cry proteins Lepidopterans

7 Corn Rootworm Species in the U.S. Diabrotica virgifera virgifera D. undecimpunctata D. barberi D. virgifera zea 7

8 An example of rootworm feeding damage NIS 1 NIS 2 NIS 3 Iowa State 0 3 node injury scale (Root Damage Rating) One entire node of roots (or equivalent) pruned % of a 2nd node of roots (or equivalent) pruned 8

9 YieldGard RW: the first corn variety offering protection from corn rootworm feeding damage (2004) Specific regions targeted for oligonucleotide-directed mutagenesis Proteins screened by insect bioassay Cry3Bb1 variants identified with ~10X improvement in the LC50. LC50 ug/well (95% CI) Cry3Bb variant Cry3Bb variant Wild-type Cry3Bb1 Fold increase over WT Cry3Bb ( ) 63.5 (46-91) 10.1 X Cry3Bb ( ) 24.2 (13-40) 5.4 X Cry3Bb ( ) 32.2 (14-67) 12.9 X Cry3Bb C Mlu I 149 Hind III nptii 35S nptii npt II Mlu I 4840 Hind III 4834 ori-puc tahsp17 3' PV-ZMIR bp NOS 3' 4-AS1 wt CAB leader ract1 intron EcoR V 1773 Hind III 1794 Root-enhanced promoter cry3bb 317 cry3bb English et al. U. S. Patent

10 YieldGard Plus/YieldGard RW root protection Increasing Root Damage YieldGard Plus/ YieldGard Rootworm Lorsban Aztec Force Check Source: 2001 and 2002 academic trials: 35 trials conducted with universities and private consultants with RDR > Each mean = a minimum of 650 individual roots. YieldGard Plus/YieldGard Rootworm and Check treated with 0.16 mg/seed of Gaucho. e.25 Economic Threshold 10

11 94% of YieldGard Plus/YieldGard Rootworm Root Damage Scores were Below the Economic Threshold Compared to 69% for the Best Insecticide 100% % of Roots per Category 80% 60% 40% 20% 0% 94 YieldGard YieldGard Plus/ Plus YieldGard Rootworm Aztec Force Lorsban Check <0.25 Source: 2001 and 2002 academic trials: 35 trials conducted with universities and private consultants with RDR > Each mean = a minimum of 650 individual roots. YieldGard Plus/YieldGard Rootworm and Check treated with 0.16 mg/seed of Gaucho. 11

12 Larger Root System Provides Higher Yield Potential LSD.05 = 3.2 Bushels/Acre Untreated Insecticide Treated YieldGard Rootworm Source: Monsanto Company Field Trials. Seventy Three Monsanto comparisons of YieldGard Rootworm Hybrids to genetically similar hybrids at sixty-six locations. All locations >0.25 Iowa State Node-Injury Scale in the check. Insecticides: Aztec 23, Capture 6, Counter 5, Force 27, Fortress 1, Lorsban 9, Regent - 1. Data as of November

13 A new class of insecticidal proteins B. thuringiensis strains grown under conditions that delay sporulation Culture supernatants from stationary-phase cultures showed activity against a coleopteran insect pest (Colorado potato beetle) DEAE column chromatography Bt strain Bioassay-guided fractionation N-terminal sequence used to design a degenerate oligonucleotide probe for gene cloning TIC

14 Five distinct subclasses Pairwise alignments of the mature proteins A B C TIC 407 TIC 417 TIC 431 TIC 901 TIC 1201 TIC407 75% 75% 80% 82% TIC417 95% 79% 81% TIC431 79% 80% TIC901 91% RFLP variants identified among known coleopteranactive Bt strains Five distinct subclasses identified among ~7500 strains of B. thuringiensis TIC1201 Proteins recovered from culture supernatants are clipped at their N-termini 14

15 What are they related to? Psi Blast search with TIC901 against the non-redundant protein database Search results from two iterations of Psi Blast: (nr.aa.58) Hit ID Hit Description Score (bits) E Value Mtx3 mosquitocidal toxin [Bacillus sphaericus] E Cry14-4 [Bacillus thuringiensis] E Mtx2 mosquitocidal toxin [Bacillus sphaericus] E epsilon toxin [Clostridium perfringens] E alpha-toxin [Clostridium septicum] E parasporal crystal protein C53 [Bacillus thuringiensis] E natterin 4 precursor [Thalassophryne nattereri] Cry15Aa crystal protein [Bacillus thuringiensis]

16 Signal peptides for secretion SignalP 3.0 prediction for signal peptide cleavage sites in gram-positive bacteria Predicted Determined TIC417sig MKYKSSKVAMCTLSALMLS--TIATPSISVFAAETTSSHAV-TNQQTITQRAESYIDIVHN-RM- TIC431sig MKYKSSKVAMCTLSALMLS--TIATPSISVFAAETTASHKV-TNQQTIAQRAESYIDIVHN-RM- TIC407sig MKYKSSKVAICTLSALMLS--TIGTSSMSTFAAETTLPGQT-LKEQSITPRAESYIDIVQD-RM- TIC901sig MKNRFSKVALCTVPILMVS--TFASSSMSAFAAEAKSPDLN-VSQQVIGPYAESYIDIVQD-RM- TIC1201sig MKYKFSKVVKCTLPALMIT--TFVTPSMAVFAAETKSPNLN-ASQQAITPYAESYIDTVQD-RM- Mtx3 MMKNKAKVILMG-ATIGLS--LLSSP- IAMAANGDSNVKENQSIANFSPIKNSFPDAANGSRFL Consensus Mk...sKV..ct...lm.s..t...s...fAAe...q.i...aeSyiD.v...Rm. 16

17 Phylogram of Cry23Aa/Mtx2 family proteins 595 Cry23Aa1 ET75 B. thuringiensis 899 CRY15AA CRY33AA1 BTC35 Crystal proteins Cry23Aa NT32KD AAA Mtx2 Mtx3 B. sphaericus TIC417 TIC431 TIC407 Secreted proteins TIC901 TIC

18 Overproduction of TIC901-type proteins in B. thuringiensis EG2158 Bt ori44 OR-Bt.ori44-0:1:1 BglII 8284 BamHI 1 XhoI 7 CR-Bt.TNPase-0:1:8 TIC901 CR-Bt.TIC901-0:1:1 HindIII 1972 EcoRI 1978 Toxin-host TIC901 TIC1201 pic17048 JABAUM pic bp CR-Ec.lacZ-1:1:1 P-Ec.lac-1:1:1 CR-Ec.lacI-1:1:2 StuI 5784 NcoI 5587 cat CAT:1:3 CR-Ec.lacZ-1:1:2 OR-f1.f1-0:1:1 CR-Ec.bla-1:1:3 OR-Ec.ori-ColE1-1:1:2 18

19 Toxicity towards corn rootworm larvae Percent mortality TIC1201 TIC Percent mortality Cry3Bb TIC Log concentration (ppm) Log concentration (ppm) 19

20 Characterization of the TIC901 protein family < Large MW oligomers? Crystal for structure determination Monomer Formation of high molecular weight oligomers upon storage Large conductances in planar lipid bilayers >>>>> TIC901 Conductance up to 22.5 ns Time, min 36 20

21 Recent Publications: 21

22 Acknowledgements: Monsanto Biotechnology Agronomics Traits - Insect Control: -Molecular -Entomology Jim Baum Ty Vaughn Bill Donovan Tom Clark Karina Krasomil-Osterfeld 22

23 Thanks! 23