Crop improvement using small RNAs: applications and predictive ecological risk assessments

1. Crop improvement using small RNAs: applications and predictive ecological risk assessments Chairman Presented By Dr. N. Kumaravadivel Mamta Kumari Associate Professor 08-807-001

2. Overview • Small RNA Family • Mechanism of action • Application of small RNAs in crop improvement • GE crops in USA using RNAi • Risk assessment • Case study I and II • Questions and concerns about RNAi and HD-RNAi crops • conclusions • Future prospects

3. RNA Family RNA Non-coding RNAs Coding RNA mRNA Transcriptional RNAs Non transcriptional RNAs rRNA tRNA miRNA (stRNA) snoRNA siRNA snRNA

4. Types of small silencing RNAs

5. Mechanism of action Makoto Kusaba,2004

6. Cloning strategies for three RNAi methods. For hpRNAi For VIGS Ossowski et al., 2008

7. Cont… For amiRNAs Alternative approach for amiRNAs Ossowski et al., 2008

8. Application of small RNAs in crop improvement • Crop quality traits : Sunilkumaret al., 2006. reduced the toxic terpenoid gossypol in cotton seeds and cotton oil by engineering small RNAs for the cadinenesynthase gene in the gossypol biosynthesis pathway. • Virus resistance : the toxic terpenoid gossypol in cotton seeds and cotton oil by engineering small RNAs for the cadinenesynthase gene in the gossypol biosynthesis pathway. • Protection from insect pests : • Baum et al. 2007. showed that silencing of a vacuolar ATPase gene (V-type ATPase A gene) in midgut cells of western corn rootworm (WCR) led to larval mortality and stunted growth. • Researchers identified a cytochrome P450 monooxygenase (CYP6AE14) gene important for larval growth expressed in midgut cells with a causal relationship to gossypol tolerance. Transgenic tobacco and Arabidopsis producing CYP6AE14 dsRNA were fed to larvae, successfully decreasing endogenous CYP6AE14 mRNA in the insect, stunting larval growth and increasing sensitivity to gossypol.

9. Cont… • Nematode resistance : • Yadavet al., 2006. showed transgenic tobacco having dsRNAtargetting two Meloidogyne (root knot) nematode genes had more than 95% resistance to Meloidogyne incognita. • Huang et al., 2006. showed that Arabidopsis plants expressing dsRNA for a gene involved in plant–parasite interaction (16D10) had suppressed formation of root galls by Meloidogyne nematodes and reduced egg production. • Bacterial and fungal risistance : • Little progress. • Escobar et al. 2001. showed that silencing of two bacterial genes (iaaM and ipt) could decrease the production of crown gall tumors (Agrobacteriumtumefaciens) to nearly zero in Arabidopsis, suggesting that resistance to crown gall disease could be engineered in trees and woody ornamental plants.

10. Case study ISilencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol Mao et al.,2007, Nature Biotechnology.

11. Vector construct • The dsRNA construct pBI121-dsCYP6AE14 for wild-type and pCAMBIA1300-dsCYP6AE14 for wild-type or the dcl2 dcl3 dcl4 triple mutant plants. • Contained a 35S promoter, • A sense fragment of CYP6AE14 cDNA, • A 120-nucleotide intron of A. thaliana RTM1 gene, • The CYP6AE14 fragment in antisense orientation, and a NOS terminator.

12. Analysis of Larval Tolerance to Gossypol Diet supplement with different concentration of gossypol for 5d to third instar larvae Fifth-instar larvae were fed an artificial diet with or without piperonyl butoxide (PBO), gossypol, or both, for 2 d.

13. Correlation of CYP6AE14 Expression with larval growth qRT-PCR analysis of CYP6AE14 transcripts Immunohistochemical localization of CYP6AE14 proteins in the fifth-instar larval midgut RT PCR (30 cycles) (midgut 1), fatty body 2), malpighian tube (3), ovary (4) and brain 5) of the 3rd instar larvae growing on artificial diet. qRT-PCR analysis of CYP6AE14 transcript

14. Cont……. Western blot analysis RT PCR with insect larva fed with other chemicals

15. suppression of CYP6AE14 expression by ingestion of dsRNA-producing plant material 2 d after transfer RNA blot analysis

16. CYP6AE14 suppression reduced the larval tolerance to gossypol

17. Plant-mediated insect RNAi as a functional tool in H. armigeragene suppression

18. Suppression of CYP6AE14 by dcl2 dcl3 dcl4 triple mutant plantsexpressing dsCYP6AE14

19. Case study IIEngineering broad root-knot resistance in transgenicplants by RNAi silencing of a conserved and essentialroot-knot nematode parasitism geneGuozhong Huang*, Rex Allen*, Eric L. Davis†, Thomas J. Baum‡, and Richard S. Hussey**Department of Plant Pathology, University of Georgia, Athens, GA 3060, 7274;Department of Plant Pathology, North Carolina State University,Raleigh, NC 27695-7616; and ‡Department of Plant Pathology, Iowa State University, Ames, IA 50011Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved August 8, 2006 (received for review June 8, 2006)

20. vector peptide coding or full-length 16D10 dsRNA molecule driven by the cauliflower mosaic virus 35S promoter using the pHANNIBAL vector was used for gene silencing. Forty-two base pair (the peptide-coding region, 16D10i-1) and 271-bp sequences (the full-length sequence excluding AT-rich regions at the 5’ and 3’ ends, 16D10i-2) of parasitism gene 16D10 were amplified from the full-length cDNA clone by using the primers 16D10T7F1 and16D10T7R1 and 16D10T7F2 and 16D10T7R2 .

21. In Vitro RNAi of 16D10 Fluorescence microscopy showing ingestion of Full length and truncated parasitism gene in the treated J2

22. Overexpression of 16D10 dsRNA in Arabidopsis

23. RNAi silencing of 16D10 in Arabidopsis

24. GE crops in USA using RNAi

25. Predictive Ecological Risk Assessments

26. Risk assessment Potential exposure pathways Pollen- mediated gene flow Plants escaped from cultivation Roots exudates /plant debries in soil Plant debriws in water Plant debries/pollen in air Potential ecological hazards Gene flow to related plant species Off target effects Non- target effects on herbivores Tri- trophic effects Increased plant fitness/ weediness Ecological Risk characterization Monitering/identity preservation/segregation: PCR with sequence specific primers ELISA no longer useful (e.g. quick check stripes)

27. Questions and concerns about RNAi and HD-RNAi crops • What off-target effects could occur within the crop or in organisms? • What non-target effects could create a hazard in the environment? • How persistent are small RNA molecules in the environment? • What will be the effect of mutations and polymorphisms in the crop plant and organisms consuming the crop? • What tools will be useful for rapidly detecting and tracking these crops and their derived products? And • How should uncertainty in risk assessments be expressed?

28. Off-target effects: study in HD-RNAi nematode-resistant tobacco, Fairbairn et al. searched a genomic database for homologies between nematode and plant genes. No homologies were found. • Non-target effects: research has shown that insect pests consuming small RNA molecules could be killed (or stunted) by cleaving mRNA of the vacuolar ATPase housekeeping gene. • Environmental persistence of small RNA molecules • Effects of mutations and polymorphisms : mutations and polymorphisms could affect the efficacy and stability of small RNAs, • mutations in the GE crop • mutations and polymorphisms in plant pest populations (e.g. viruses, insects), and • mutations occurring in non-target organisms (e.g. beneficial insects), • Tracking RNAi and HD-RNAi crops: Crop identity preservation, monitoring and segregation are important • Uncertainties

29. Ecological risk assessment: comparison

30. conclusions • Recent advances have created high expectations for the future role of RNA-mediated traits in GE crops. • The most important applications will be in altering crop–pest interactions so that plants are protected from insects, nematodes or pathogens. • It has been suggested that plants could serve as biological factories for small RNAs that could become therapeutic treatments for viral pathogens in humans and animals

31. Future prospects • Most RNAi research has been carried out in Arabidopsis, there are substantial gaps in our knowledge about the RNAi mechanisms at work in all of the economically important crops and host–pest interactions, so substantial research is needed. • In the future, the predictive ERA process will need to be flexible and adaptable for analysis of the next generation of crops engineered using RNAi and HD-RNAi. • As a first step, regulatory agencies and risk analysts need to become familiar with the science of RNAi and its application to plant biotechnology.

32. THANK YOU