Transcript Crop improvement using small RNAs: applications and predictive

Crop improvement using small RNAs:
applications and predictive ecological risk
assessments
Chairman
Dr. N. Kumaravadivel
Associate Professor
Presented By
Mamta Kumari
08-807-001
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
RNA Family
RNA
Non-coding RNAs
Coding RNA
mRNA
Transcriptional RNAs
rRNA
Non transcriptional RNAs
tRNA
siRNA
miRNA
(stRNA)
snoRNA
snRNA
Types of small silencing RNAs
Name
Organism
Length
(nt)
Proteins
Source of trigger
Function
miRNA
Plants,algae,animals,v
iruses, 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 primary
secondary
24
21
DCL2
DCL1
Bidirectional transcripts induced by
stress
Regulate stress response genes
Exo -siRNA
Animals, fungi,
protists, plants
21-24
Dicer
Transgenic, viral or other
exogenous dsRNA
Post transcriptional regulation,
antiviral defense
Endosi RNA
Plants,algae,animals,f
ungi, protists
21
Dicer
Structured loci, convergent and
bidirectional transcription, mRNAs
paired to antisense pseudogene
transcripts
Post transcriptional regulation of
transcripts and transposons
Transcriptional gene silencing
piRNA germ line
Drosophila
melanogaster,
mammals, zebrafish
24-30
Dicer- independent
Long, primary transcripts
Transposon regulation, unknown
functions
piRNA like
Drosophila
melanogaster
24-30
Dicer- independent
21U-RNA piRNAs
Caenorhabditis
elegans
21
Dicer- independent
Individual transcription of each
piRNA
Transposon regulation ,unknown
functions
26G RNA
Caenorhabditis
elegans
26
RdRP
Enriched in sperm
Unknown
In ago2 mutants in
Drosophila
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 I
Silencing 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 artificial
diet with or without piperonyl butoxide
(PBO), gossypol, or both, for 2 d.
Correlation of CYP6AE14 Expression with larval growth
qRT-PCR analysis of CYP6AE14 transcripts
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
Immunohistochemical
localization of CYP6AE14 proteins
in the fifth-instar larval midgut
Cont…….
Western blot analysis
RT PCR with insect larva fed
with other chemicals
suppression of CYP6AE14 expression by ingestion of dsRNAproducing plant material
2 d after transfer
RNA blot analysis
CYP6AE14 suppression reduced the larval tolerance to gossypol
Plant-mediated insect RNAi as a functional tool in H. armigera
gene suppression
Suppression of CYP6AE14 by dcl2 dcl3 dcl4 triple mutant plants
expressing dsCYP6AE14
Case study II
Engineering broad root-knot resistance in transgenic
plants 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 50011
Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and
approved August 8, 2006 (received for review June 8, 2006)
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 .
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
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
Monitering/identity preservation/segregation:
PCR with sequence specific primers
ELISA no longer useful (e.g. quick check stripes)
Ecological
Risk
characterization
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
characterization
of 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
mRNA
cleavage 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 nontarget 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 monitoring
can be very difficult
Crop plant product
sustainability
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 RNAmediated 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.