Chairman Presented By Dr. N. Kumaravadivel Mamta Kumari Associate Professor 08-807-001

Crop improvement using small RNAs: applications and predictive ecological risk

assessments

Overview

Small RNA FamilyMechanism of actionApplication of small RNAs in crop improvementGE crops in USA using RNAiRisk assessmentCase study I and IIQuestions and concerns about RNAi and HD-RNAi cropsconclusionsFuture prospects

RNA Family

snRNA

Transcriptional RNAs

tRNA

Non transcriptional RNAs

rRNA

Non-coding RNAs

RNA

mRNA

Coding RNA

miRNA(stRNA)

siRNA snoRNA

Types of small silencing RNAs

Name Organism Length(nt)

Proteins Source of trigger Function

miRNA Plants,algae,animals,viruses, protists

20-25 Drosha (animals only) + Dicer

Pol II transcription (pri-miRNAs Regulation of mRNA stability,Translation

casiRNA Plants 24 DCL3 Transposons, repeats Chromatin modification

tasiRNA Plants 21 DCL4 miRNA-cleaved TAS RNAs Post transcriptional regulation

natsiRNA Plants primarysecondary

2421

DCL2DCL1

Bidirectional transcripts induced by stress

Regulate stress response genes

Exo -siRNA Animals, fungi, protists, plants

21-24 Dicer Transgenic, viral or otherexogenous dsRNA

Post transcriptional regulation,antiviral defense

Endosi RNA Plants,algae,animals,fungi, protists

21 Dicer Structured loci, convergent and bidirectional transcription, mRNAs paired to antisense pseudogene transcripts

Post transcriptional regulation oftranscripts and transposonsTranscriptional gene silencing

piRNA germ line Drosophila melanogaster,mammals, zebrafish

24-30 Dicer- independent Long, primary transcripts Transposon regulation, unknownfunctions

piRNA like Drosophila melanogaster

24-30 Dicer- independent In ago2 mutants in Drosophila

Unknown

21U-RNA piRNAs Caenorhabditis elegans

21 Dicer- independent Individual transcription of eachpiRNA

Transposon regulation ,unknownfunctions

26G RNA Caenorhabditis elegans

26 RdRP Enriched in sperm Unknown

Mechanism of action

Makoto Kusaba,2004

Cloning strategies for three RNAi methods.

For hpRNAi

For VIGS

Ossowski et al., 2008

Cont…

For amiRNAs

Alternative approach for amiRNAs

Ossowski et al., 2008

Application of small RNAs in crop improvement

Crop quality traits : Sunilkumar et al., 2006. reduced the toxic terpenoid gossypol in cotton seeds and cotton oil by engineering small RNAs for the cadinene synthase gene in the gossypol biosynthesis pathway.

Virus resistance : the toxic terpenoid gossypol in cotton seeds and cotton oil by engineering small RNAs for the cadinene synthase 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.

Cont…

Nematode resistance : Yadav et al., 2006. showed transgenic tobacco having dsRNA targetting 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 (Agrobacterium tumefaciens) to nearly zero in Arabidopsis, suggesting that resistance to crown gall disease could be engineered in trees and woody ornamental plants.

Case study ISilencing a cotton bollworm P450 monooxygenase gene

by plant-mediated RNAi impairs larval tolerance of gossypol

Mao et al.,2007, Nature Biotechnology.

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.

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 artificialdiet with or without piperonyl butoxide (PBO), gossypol, or both, for 2 d.

Correlation of CYP6AE14 Expression with larval growth

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 transcripts

Immunohistochemicallocalization of CYP6AE14 proteins in the fifth-instar larval midgut

qRT-PCR analysis of CYP6AE14 transcript

Cont…….

Western blot analysis

RT PCR with insect larva fed with other chemicals

suppression of CYP6AE14 expression by ingestion of dsRNA-producing plant material

2 d after transferRNA blot analysis

CYP6AE14 suppression reduced the larval tolerance to gossypol

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

Suppression of CYP6AE14 by dcl2 dcl3 dcl4 triple mutant plantsexpressing dsCYP6AE14

Case study IIEngineering broad root-knot resistance in transgenicplants by RNAi silencing of a conserved and essential

root-knot nematode parasitism gene Guozhong 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)

vectorpeptide 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 .

In Vitro RNAi of 16D10

Fluorescence microscopy showing ingestion of Full length and truncated parasitism gene in the treated J2

Overexpression of 16D10 dsRNA in Arabidopsis

RNAi silencing of 16D10 in Arabidopsis

GE crops in USA using RNAi

Predictive Ecological Risk Assessments

Risk assessment

EcologicalRiskcharacterization

Potential exposure pathwaysPollen- mediated gene flowPlants escaped from cultivationRoots exudates /plant debries in soilPlant debriws in waterPlant debries/pollen in air

Potential ecological hazardsGene flow to related plant speciesOff target effectsNon- target effects on herbivoresTri- trophic effectsIncreased plant fitness/ weediness

Monitering/identity preservation/segregation:PCR with sequence specific primersELISA no longer useful (e.g. quick check stripes)

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?

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

Ecological risk assessment: comparison

Bt endotoxin GE crop ERA information

HD-RNAi GE crop

ERA information Challenges and questions

Molecular characterizationof active molecule

Gene coding for Bt endotoxin protein Gene coding for small RNA molecules (20–24 nucleotides)

Limited genomic databases make comparative analysis for sequence homology in non-target species Difficult

Mode of action Bt endotoxin protein binds to insect gut membrane receptor proteins resulting in cell lysis; action localized to insect mid-gut

Multistep process involving small RNAs from crop plant and insect protein complexes leading to insect mRNAcleavage and gene silencing

Multiple modes of action are known in Arabidopsis, but these are poorly understood in most crop species Lack of benchmarks or normalization for small RNA activity limits the ability to conduct comparative Assessments

Toxicity testing Testing on non-target organisms, often using a tiered approach Allergenicity potential evaluated

Toxicogenomics analysis of off-target and non-target effects Testing on non-target organisms, often using a tiered approach Allergenicity probably not an issue

Lack of normalized genomic libraries and DNA arrays for ecotoxicological model organisms Validation might be needed for tiered testing of crops with RNA-mediated traits

Exposure assessment Includes environmental fate estimates (crop gene flow, protein half-life in soil and water), methodology for tracking the Bt protein and its gene (lateral flow strips, ELISA, quantitative PCR), and measurement of Bt toxin distribution in plant Tissues

Includes environmental fate estimates for small RNA (crop gene flow, small RNA half-life in soil and water), potential for uptake by non-target organisms, and characterization of systemic gene silencing (if present)

Persistence and fate of small RNAs in ecosystems (e.g. soil, water) are largely unknown Extraction and identification of small RNAs for environmental monitoringcan be very difficult

Crop plant productsustainability

Analysis of the development of insect resistance (e.g. predictive, deterministic or stochastic models). Resistance management plans Transgene stability over several crop plant generations

Analysis of the development of insect resistance (e.g. predictive deterministic or stochastic models). Transgene stability over several crop plant generations

Resistance development models for RNA-mediated traits have not been developed but might not be necessary to characterize risk Mutation rates in genes for small RNAs can be high relative to protein-codinggenes

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

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.