Biotechnology Strategies for irna for Phytosanitary and Pest Control

Transcription

1 Biotechnology Strategies for irna for Phytosanitary and Pest Control Non-transgenic Methods of Delivery Wayne Hunter, & Eduardo Chumbinho de Andrade Embrapa Labex USA-BRAZIL USDA, ARS, U.S. Horticultural Res. Lab, 2001 South Rock Rd, Ft. Pierce, FL, USA

2 By 2050 we ll need to feed two Billion more people. The Future of Food How can we do that without overwhelming the planet?

3 Fiber EARTH Livestock BioPharma Crops Gene-based Biotechnologies Forests Aquaculture Biomaterials Human Innovation Agrarian societies Domestication of Animals Discovery of DNA, Develop Computers Genomics Bioinformatics Chemical technologies

4 PROBLEM Huanglongbing Pathosystem Tanaka e Kitajima Photos: Juliana Freitas-Astúa Photo: Jeff Lotz and David Ziesk Photo from California Citrus Research Board

5 HLB impact in Sao Paulo Huanglongbing (HLB), Citrus greening Disease Pathogen Candidatus Impact USA, 2005 detected in Florida 2.28 Liberibacter asiaticus 37.4 millions of plants eliminated 2.2 * Source: CDA-SP Losses over $5.6 billion and Item 8,000 jobs since US$/ha HLB inspection (4 to 6/year) 24,31 to 52,30 Psyllid control ( 8 to 16 sprays/year) 74,18 to 274,14 Plant removal 4,57 to 12,35 Replanting 0,00 to 74,18 Losses: US$ /ha to US$ /ha Source: Fundecitrus

6 WHY try RNA interference? Cell Diagram: Central Dogma Molecular Biology from Nuclear DNA to Protein, Transcription Translation

7 INNOVATION First evidence Introduction of a transgenic copy of the Chalcone synthase in petúnia plants resulted in a suppression of the endogenous gene. Co-suppression Napoli et al., Plant Cell 2:279, 1990

8 RNAi - mechanism Natural mechanism in eukaryotic cells; Gene regulation and antiviral defense; Degradation of mrnas in a sequence specific manner to modulate proteins. dsrn A Enables silencing genes in a specific way (surgically) using dsrnas. Stop or Reduce Protein production

9 RNAi products are coming! Provide Safer, Highly Specific, Greater Adaptability

10 The Agroindustry Companies are investing heavily ($ Millions) in development of RNAi products, and Plants.

11 RNAi Highly Specific Pest Control Advantages using RNAi for pest control: Bt corn represented a breakthrough on pest control, but RNAi would take the concept to a new level by targeting harmful insects with surgical precision. RNAi mechanism is a natural Process we can use to modulate Natural protein expression 1) Specificity to the target insect; 2) Customize to control different insect pests, or pathogens; 3) Adaptability for non-transgenic products for use in annual or perennial crops to control insects and pathogens. Monsanto spent $29.2 millions for IP. in RNAi (gene Snf7 of WCR) from Alnylam Pharmaceuticals: Development of STAX-Maize GMO corn expressing First insect targeting Snf7 dsrna; Development of Topical dsrna insecticide spray; Biodirect

12 Some of the Problems in Chemical Insect pest management: *Insecticides are broad spectrum, kill many insect species, including beneficials. *Development of insecticide resistance, within 2-4 years of heavy use. *Emergence of secondary insect pests, due to loss of parasitoids and predators. What would be a better pest management strategy? 1) Something that is more specific to the target 2) Not generate resistance development 3) Not affect beneficials, protects Pollinators- like honey bees, Predators, and Parasitoids.

13 Q: What is needed? Hunter, Shatters, Hall Psyllid Genome Ver. 2.0 Completed 2013 (ARS, CRB, LANL) Identified 18,600 genes For use in RNAi research. Three datasets Eggs, Nymphs, and Adults Researchers using for RNAi against : - Development Answer: Genetic Sequences - Digestion - Pathogen interactions - Feeding Reese, et al., Characterization of the Asian citrus psyllid transcriptome. Jour. Genomics 2013(2): doi: /jgen Intl Psyllid Genome Consortium

14 RNAi strategies to reduce Huanglongbing Identification of psyllid target genes Transcriptome of all life stages Egg, Nymphs, Adults Selection based on current Knowledge of cellular functions, examine pathways for redundancy (gene families difficult) Test to make sure no effects on non-target insects Broad application (closely related insects); No Side-effects on beneficial insects (Bees). Test to make sure no Traits change in the plant Distribution Systemic or localized Persistance dose needed?

15 Bioassay in plant system Hunter and Andrade 2013 in plant system: Bioassay for plant-feeding Hemipteran; Plant flushes were washed and placed in water (control) or dsrna solution; Mortality NT 24 h.p.t stem leaf stem leaf 40 d.p.t - leaves Days post feeding Psyllid detection NT + Plant tissue ~0.25 grams/ treated with 10, 50, or 100 nm dsrna

16 RNAi In plant system validation Mortality Mortality over time 90,00 80,00 70,00 60,00 50,00 40,00 70,00 60,00 50,00 40,00 30,00 * 30,00 20,00 10,00 0,00 Arg. Kin. Arg. Kin - sirna GFP - sirna GFP - dsrna Water 20,00 10,00 0,00 Arg. Kin Arg. Kin - sirna GFP - dsrna GFP - sirna (5ug) sirna = 21 nucleotides long dsrna= 240 nucleotides long {Consistently better} Equal molecules, at a concentration of 100 ng final conc. Absorbed into 0.25 grams of citrus new growth tissues. water Andrade and Hunter 2015

17 Insect Target genes - Mortality Mortality Mortality over time ,00 90,00 80,00 70,00 60,00 50,00 40,00 30,00 20,00 10,00 0,00 Systems Targeted-- Detoxifying, Metabolism, Developmental, Receptors, Nerves, Reproduction

18 Target evalulation Natural system LESS MORE Different Targets and treatments at various concentrations Can produce highly variable mortality!

19 Target evalulation natural system 100% Mortality Adults 643 psyllids dead

20 Specificity Test Bioassay RNAi by oral feeding trial (Evans et al, 2009, USDA: Hunter, Andrade, Evans, 2014). Newly emerge nurse bees. (Honey bee, Apis mellifera L.) Double-blind study, water, GFP, and Dc-dsRNA s 10 ng dsrna in sucrose solution ingested. Feed 25 bees each treatment. Bees marked and put back into hive for d. Open and record number of survivors. No Significant difference between dsrna treated and controls.

21 OFF Target effects - Beneficial Honey bee (Apis melifera) Newly emerged bees (30 / group) were handfed 5 ul sucrose (10ng of dsrna); Bees were marked by paint, mixed and placed back into colony; Collected 15 days after treatment; Compound Color code Recaptured Arg. Kin. (BZ 1) yellow/pink 25 Detox 1 (BZ 2) white/green 24 Met 1 (BZ 3) orange/yellow 22 GFP (BZ 4) pink/green 23 ddh2o white/yellow 21 Collaborator: Jay Evans - USDA-ARS Bee Research Lab (Beltsville MD)

22 Field Trial Delivery Systems for dsrna to Citrus Trees yr old Valencia trees, Collaboration: USDA- Shatters, Hall, Stover, Monsanto, and Andrade- EMBRAPA, Preliminary Data RNAi treated Citrus more fruits per tree. {~20} June 2014 Field Tree RNAi Trial. Delivery Systems. Less CLas-like Symptoms {Testing trees for CLas levels} {Testing trees for CTV levels} No difference in psyllid loads? {No choice trials this year} Need to repeat another year to Validate effects! {Examine tree gene expression} Delivery trial: Foliar sprays Root drench Trunk Injections

23 Delivery by sprays on Soil and Foliar SOIL analyses: Soil Drench: Small Citrus Seedlings in 1 gal pots: Treated 200 mg dsrna In 300 ml of water, Detectable dsrna in leaves still at 5 weeks post treatment Environmental Fate: Preliminary results dsrna degrades and Undetectable in 48 hrs Brian Pearson, Univ. FL

24 Stopping Citrus Root Weevil Diaprepes abbreviatus Andrade & Hunter USDA Agricultural Research Service

25 Bioassay Foliar Topical Application Spray dsrna on leaf surface Leaf bouquet transferred to a cage with 20 adult weevils Measurements: 1. Leaf damage 2. Weevil mortality 3. Oviposition

26 Reduction on leaf damage over time Water GFP DRW_01 dsrna 0 days Day 0 5 Day days 5 Day days Andrade and Hunter 2015

27 Weevil target genes - Mortality Andrade and Hunter 2015

28 Insect mortality reduced leaf damage Water GFP dsrna DRW_01 dsrna 10% 20% 5% 100% 85% 85% Andrade and Hunter 2015

29 RNAi Impact on Weevil Fecundity DRW_Control Water Control dsrna DRW_01 dsrna Many Eggs Few Eggs Number of eggs Andrade and Hunter 2015

30 RNAi APPLIED TO PEST CONTROL - challenges Efficiency X Cost Delivery strategies will play a central role Delivered by: Transgenic plants; Topical (sprayable); Viral vectors. other...

31 International Collaboration: Dr. Eduardo Andrade, EMBRAPA, Brazil LabEx-USA Program, Soil application of dsrna s - Citrus field trees. $30,000 worth of dsrna if Made with kits. 200 ml of 200 mg dsrna 2014 Commercial Companies Now selling less $500 US AgroRNA, LLC, S. Korea

32 Advances in Production = Shrinking Costs Towards Commercial Product Costs over $575,000 to make 40 g dsrna used kits to produce this amount. Cost ~$5,745/ tree. ( if treating 100 trees with 200 mg) Method Developed- Beeologics, LLC. ~$200,800 for 40 grams dsrna, for 100 trees would be about $2,500 per tree Another production breakthrough- Cost about $4 per tree. Research & Development Field trial test of RNAi, about Costs $1 per tree Reduce to 0.50 cents per tree.costs keep dropping! Improved Production & Commercial Companies

33 Future of RNAi Products: RNAi products ARE COMING! Pest management ALSO--Use to Protect Beneficials Feasible, Affordable, RNAi production (0.50/tree) ALL BIG Agro-Industries, portfolio s include RNAi Regulatory Pathway by Agencies, EPA, FDA RNAi delivery as Topical Non-transgenic or Transgenic modes of applications {Plants or Virus}

34 Biotechnology working to develop crops needing: LESS Water, LESS Fertilizer LESS Insecticides Food Security Better Environment More Nutritious

35 SOUR RNAi Modify Plant Traits SWEET Delicious Apples RNAi Bitter melon

36 Plant Trait Modification Seedy Seedless RNAi See a problem and Produce a Solution

37 Other uses for RNAi strategies INNOVATION Medical or Chemical Production

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