Download Use of PVX vector as expression system.ppt (Read

3/8/11
Post-Bt Scenario
Use of PVX vector as expression
system and mealybug as a model
target insect to resistance against
sucking pests
The real challenge is sucking pests
Mealybug
Whiteflies
Aphids
Jassids
Thrips
Muhammad Ashfaq
M. Shaheen, A. M. Khan, S. Zaid, A. Raza, M. Ashfaq,
I. Amin, Z. Mukhtar, S. Mansoor,
Genetically engineered resistance
Approaches for insect control
ü Biological control
ü Natural host resistance
ü Control through chemical pesticides
ü Genetically-engineered resistance
Proteins and RNAi
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Application approaches
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Virus vector
Feeding
Transgenic plants
In vitro synthesis of dsRNA
Bt
VIPs
Lectins
Animal origin (Hvt etc)
RNAi
RNAi can be achieved by dsRNA!
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Injection
Ingestion
Soaking
Transgene
Transgenic vectors
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Insect control ????
Reported issues with insects
RNAi and insects
Drosophila melanogaster
(Kennerdell and Carthew, 1998)
Anopheles gambiae
(Paskewitz et al., 2006)
Caterpillar Manduca sexta
(Vermehren et al., 2001)
Milkweed bug Oncopeltus fasciatus
(Hughes and Kaufman, 2000)
German Cockroach Blatella germanica
(Cruz et al., 2006)
Triatomine bug Rhodnius prolixus
(Araujo et al., 2006)
Apple brown moth Epiphyas postvittana
(Turner et al., 2006)
Ladybird beetle Harmonia axyridis
(Kuwayama et al., 2006)
Pea aphid Acyrthosiphon pisum
(Mutti et al., 2006)
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RNAi varies among insect orders and species
Insects lack RdRP
Drosophila lacks sid-1 gene
Sid-1 homologues identified in a number of insect
species
•  Another mechanism, receptor-mediated endocytosis
By injection
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RNAi-mediated gene knockdown reported in:
Diptera
Coleoptera
Hymenoptera
Orthoptera
Blattodea
Lepidoptera
Hemiptera
Phenotypes of parental RNAi (paRNAi) and embryonic RNAi (emRNAi) against Gryllus
bimaculatus caudal (Gb cad). (a) Eggs obtained by paRNAi against Gb cad in which
only the head region is formed (also see d and d ),
indicating that the expression of Gb cad in the caudal region of the embryo is required for
the formation of regions other than the head (Shinmyo et al. unpublished). (Mito et al.
2011).
Beye et al. 2002.
Specific developmental gene silencing in the honey bee
using a homeobox motif
Gene: E30 homeobox motif
Injections of dsRNA with an Oxford micromanipulator, a
microinjector, and an ordinary stereomicroscope.
The phenotype of nymphal RNAi (nyRNAi) against Gryllus bimaculatus laccase 2
lacks tanning (top). This result indicates that the expression of laccase 2 is crucial for
cuticle tanning in G. bimaculatus. After injection of the dsRNA for laccase 2 into
nymphs at the fifth instar, the nymphs were reared to be adults. The bottom shows a
control cricket. Bar represents 1 cm. (Mito et al. 2011)
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By feeding
By transgene
Target genes:
Cell-1
Hex-2
Zhou et al. 2008. IMB
Can we engineer insect resistance
by expression of insect-specific
double stranded RNA in plants???
Use of hairpin transgene in the host
Putting RNAi in plants
•  Use of dsRNA for RNAi ??
•  Use of hairpin transgenes in the host ü
Control of coleopteran insects through RNAi
Baum et al.
Nature Biotechnology November, 07
The approach
Developed Western corn borer cDNA library
Selected 290 target genes
125 showed significant mortality at 52ng/cm
67 showed mortality at 5.2 ng/cm
14 at less than 5.2 ng/cm
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Silencing bollworm P450 by plant mediated RNAi (Gossypol)
Plants expressing a V-ATPase A dsRNA are
protected from feeding damage
Objectives
Ø To establish the use of a plant virus vector for
expression of insecticidal genes by expression of
cry1Ac or HVt and Lectin from PVX.
Ø To target insecticidal gene constructs based on RNA
silencing from PVX vector and check insecticidal
activity.
Ø To determine the target specificity by checking some
of the non target insects for insecticidal gene construct.
Methods
•  PVX (pgR107) transformed in Agrobacterium
strain GV3101.
•  Tobacco plants inoculated through infiltration
and on symptoms arrival on plants to get VIGS
well established phenomena.
•  Bioassay on Mealybug conducted to reveal
RNAi trigger to respective gene and phenotypic
abnormality in development and mortality
Mao et al. 2007.
Idea behind using PVX vector
Potato Virus X is used as a screening tool for expression
of insecticidal protein in phloem of the model plants to
specifically target the sucking insects because PVX
replicates and moves systemically through the phloem.
PVX a single stranded RNA virus being used to trigger a
plant mediated Systemic RNAi in insects via feeding
siRNA homologous to insect genes generated by the
plants in response to viral induced gene silencing.
Target genes
Ø Elongation factor 1a (EF-1a): 325bp
Ø Ca-channel gene: 152bp
Ø CHS1: 510bp
Ø Bursicon: 550bp
Ø V-ATPase
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Why mealybug (Phenacoccus
solenopsis)
Virus vector
(PVX)
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Sucking insect
Convenience of rearing
High fecundity
Multiple host plants
Available round the year
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(B)
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Bioassay on Mealybug RNAi Calcium channel
Transgenic-PVX inoculated Nicotiana
benthamiana
Experimental plants in insect rearing
cages
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(A)
(B)
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Bioassay on Mealybug RNAi Elongation factor1alpha
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Bursicon RNAi in P. solenopsis:
CHS1 RNAi in P. solenopsis:
1.  Control
1.  Control
2, 3. Fed on transgenic PVXinoculated plants
2, 3. Fed on transgenic PVXinoculated plants
Figure: Suppression of CHS1 gene as a result of RNAi
L- Ladder, 1,2 CHS1-Treated (RNAi); 3,4 CHS1- Control
5-8 B-Actin
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Take home message
Insects genes can be suppressed by expressing
insect-specific dsRNA in transgenic plants
Thank you
RNAi protect against insect damage
Why important?
Alternative to toxins
More acceptable than toxin proteins in food crops
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