Next generation rootworm management tools from North America and their. applicability to Brazil. Renato Carvalho. Entomology Development Lead

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1 Next generation rootworm management tools from North America and their /////////// applicability to Brazil Renato Carvalho Entomology Development Lead

2 Agenda RNAi Mode of Action RNAi against CRW Safety of RNAi Key conclusions 2

3 Regulating Gene Expression using RNAi DNA RNA Protein Transcription Translation RNA interference (RNAi) A natural process cells use to suppress the activity of certain genes 3

4 Early observations of RNAi in plants were in transgenic co-suppression hints of exquisite regulation Transcription Star-patterned petunia: natural RNAi in horticulture In early 1990s, Jorgensen and others observed unexpected white patterning when trying to overexpress chalcone synthase High expression levels and/or duplicated integration architectures sometimes lead to RNAdependent RNA polymerase (RdRP) activity that created templates for small RNA production (in this case sirna s) Where suppression occurred flowers became white sirna (small interfering RNA) 4

5 RNAi - Two Key Complexes Dicer - an endogenous enzyme that cleaves dsrna molecules into short sirna duplexes RNA-Induced Silencing Complex (RISC) -contains Argonaute and cleaves the mrna molecule 5

6 Nontransgenic Tomato Once the mechanism of RNAi in plants was known more efficient approaches could be engineered Tethering two complementary sequences in the same transcript obviates the need for RdRP and efficiently generates small RNAs Low-expressing and tissue specific promoters become more tractable for engineering First approved biotech product (Flavr Savr tomato) and some sister events created an inverted repeat structure of the polygalcturonase (PG) antisense construct upon integration Flavr Savr Tomato Efficient generation of Dicer substrate dsrna PG mrna PG sirnas sirna 6 Krieger et al. (2008) Hort Sci 43: 962

7 RNA-based products have been approved by regulatory agencies First products appeared in the mid-90 s and have gained international approvals Flavr Savr tomato Virus resistance Soybean composition Potato composition Calgene 1994/1995 (U.S., Canada, Mexico) Squash 1994 (U.S., Canada) Papaya 1996 (U.S., Canada) Potato 1998 (U.S., Canada, Mexico, Australia, New Zealand, Japan, S. Korea, Philipines) Plum 2007 (US) Common Bean 2011 (Brazil) DuPont 1997/2011 (U.S., Canada, Mexico, Australia, New Zealand, S. Korea, China, Taiwan) Monsanto 2011/2012 (U.S., Canada, Mexico, Australia, New Zealand, Japan, S. Korea, Columbia) BASF amylopectin 2010 (EU) Simplot Innate 2014 (U.S.) 7

8 November 2007, Volume 25 No 11 pp Control of coleopteran pests through RNA interference James A Baum, Thierry Bogaert, William Clinton, Gregory R Heck, Pascale Feldmann, Oliver Ilagan,Scott Johnson, Geert Plaetinck, Tichafa Munyikwa, Michael Pleau, Ty Vaughn & James Roberts Western Corn Rootworm Coleoptera:Chrysomelidae Diabrotica virgifera virgifera 8

9 Agenda RNAi Mode of Action RNAi against CRW Safety of RNAi Key conclusions 9

10 DvSnf7 dsrna The first totally new MOA against CRW since Cry34/35 Cry3Bb, Cry34/35, mcry3a, ecry3.1ab, DvSn7 dsrna 2017 Cry3Bb, Cry34/35, mcry3a, ecry3.1ab 2014 Cry3Bb, Cry34/35, mcry3a 2007 Cry3Bb, Cry34/ No cross-resistance between dsrna and Cry3Bb1 in WCR Moar et al. PLoS ONE, (12(1): e doi: /journalpone Cry3Bb

11 DvSnf7 Targets and Suppresses Snf7, an Essential Enzyme Component for Cellular Protein Transport Endosomal Sorting Complex Required for Transport (ESCRT) is required for sorting of membrane receptors for recycling or degradation in the lysosome. Large DvSnf7 dsrna enters the insect cell Snf7 suppression occurs via the endogenous RNAi pathway in the insect Observed cellular defects are consistent with the suppression of Snf7, producing the expected phenotype Yeast ESCRT pathway ESCRT III Nature Rev. Mol. Cell Biol. (2002) 3:

12 DvSnf7 RNA is Orally Delivered to Suppress Gene Expression in CRW Plant Cell 21-24mers Ingestion (oral uptake) Corn Rootworm Cell Dicer Plant expresses the DvSnf7 RNA hairpin Dicer stable transgene 21-24mers RISC Cleavage 5 cap AAAAA mrna recognition transcription target gene Relies on natural ability of CRW to respond to environmental RNA DvSnf7 RNA sequence carefully selected for specificity to CRW Activity tied closely to phylogenetic relatedness, strong evidence demonstrates low risk outside of closely related beetles 12

13 Key MOA Messages The insecticidal activity of the CRW3 dsrna occurs through the RNAi pathway within the insect Cellular uptake of large dsrna (not the sirna defines test substance) Snf7 suppression occurs specifically at both RNA and protein levels via the RNAi pathway Cellular defects are consistent with the expected phenotype for Snf7 suppression Systemic spread: Snf7 mrna levels decrease in multiple tissues. Larval death 13

14 Characterizing the Mechanism of Action against CRW dsrna uptake, target mrna and protein suppression, systemic spreading, growth inhibition and mortality 14

15 New Mode of Action to Preserve Existing Bt Technology RNAi mode of action different from Bt proteins; combined with existing Bt s for superior efficacy Corn rootworm is a significant insect pest that can cost > $1 billion annually to growers Next generation product in BRZ: Two modes of action against targeted Coleopteran pests and three modes of action against Lepidopteran pests Lepidopteran above ground protection includes Cry1A.105, Cry2Ab2 and Vip3A Bt proteins Coleopteran below ground protection includes Cry3Bb1 Bt protein and DvSnf7 RNA 15

16 Agenda RNAi Mode of Action RNAi against CRW Safety of RNAi Key conclusions 16

17 RNA has a favorable profile for mammalian consumers Many barriers contribute to protection X RNA in its many forms is a common part of diets from both plant and animal sources (Ivashuta, et al., 2009; Jensen, et al., 2013; Petrick et al., 2013) RNA experiences many challenges in the mammalian digestive tract and unlike in some invertebrates, does not encounter a competent uptake system Such barriers contribute to mammalian safety of dsrna, and have also thwarted oral delivery of RNA-based pharmaceuticals Multiple studies looking for the uptake and potential function of RNA from food have failed to find significant presence and regulation in mammals (Witwer et al, 2013; Snow et al., 2013; Dickinson et al., 2013; Tosar et al., 2014) 17

18 Multiple species are being evaluated for specificity 18

19 RNA breaks down rapidly in the environment dsrna can be accurately measured and evaluated from a variety of sources Baseline data about presence and fate of dsrna will be broadly useful for exposure analyses dsrna quickly degrades (less than 24 hours in soil, 72 hours in hives) Early studies show degradation comparable/better than Bt proteins Pollen Grain Soil Hive Armstrong et al., J Ag & Food Chemistry 61: 12557; Dubelman et al., PLoS ONE 9: e

20 RNA-based Technologies - Key Conclusions RNA-based suppression is based on natural mechanisms, and has been used in biotechnology as a longstanding approach to achieve gene regulation RNA has a history of safe consumption RNA offers highly-specific gene suppression that greatly reduces potential impact on non-target organisms RNA based pest suppression can be highly efficacious 20

21 Renato Carvalho Thank you! ///////////