Scope of gene silencing in horticultural crops

1 of 50 13/10/2017 Kuldeep Singh Bhullar (L-2015-A-30-D)1 of 30
Scope of Gene Silencing in
Horticultural Crops
Kuldeep Singh Bhullar
L-2015-A-30-D

2 of 50 13/10/2017 Kuldeep Singh Bhullar (L-2015-A-30-D)
Outline
– Introduction 3
– Types of GS and their mechanisms
– Ribosomal Nucleic Acid Interference( RNAi)
– Virus Induced Gene Silencing (VIGS)
– Implications in horticulture
• Fasten breeding programme
• Nutraceautical properties
• Pest management
• Post-harvest
– Commercial application
– Opinion about the technology
– Conclusions

Introduction
Gene silencing (GS) is a phenomenon which
describes “switching off” of a gene by a
process other than genetic modification.

Types of GS
• Transcriptional
– Genomic Imprinting
– Paramutation
– Transposon silencing
– Transgene silencing
– Position effect
• Post-transcriptional
– RNA interference (RNAi)
– Virus Induced Gene Silencing (VIGS)

Types
• Transcriptional
– Genomic Imprinting
– Paramutation
– Transposon silencing
– Transgene silencing
– Position effect
– RNA-directed DNA
methylation
• Post-transcriptional
– RNA interference (RNAi)
– Virus Induced Gene
Silencing (VIGS)

Transcriptional Gene
Silencing

Genomic imprinting
The allele-specific expression of
a gene dependent on its
parent-of-origin
Independently evolved in
flowering plants and
mammals.
Kohler et al 2010

Paramutation
Kohler et al 2010
− Induced by allele interactions.
− Heritable
− A paramutable allele becomes a
paramutant (paramutated) in
response to a paramutagenic
allele if the two are in
heterozygous condition
− Seems to violate the Mendelian
principle of independent
assortment.

Transposon silencing
- Form of transcriptional
gene silencing targeting
transposons
- Transposon elements
are the jumping genes
Benjamin 2012

Transgene silencing
• Transgenes cause the silencing of endogenous plant
genes if they are sufficiently homologous. a
phenomenon known as transgene silencing or co-
suppression.

Position Effect
• Gene is expression can
depend as much on its
location within the genome
as its primary DNA
sequence.
• Proximity to enhancers and
suppressors outside the
core promoter can affect
gene expression,
• The most dramatic position
effects often involve
epigenetic silencing of
genes placed in proximity
to inactive or
heterochromatic regions of
the genome

Post-transcriptional
Gene Silencing

RNA interference
o A mechanism in which double-stranded RNA
triggers suppression of gene activity in a
homology-dependent manner
o Degradation of mRNA or translation inhibition.

Mechanism

Requirements of RNAi
Introduction of dsRNA:
 Target gene
 Homologous dsRNA
 Intron containing hairpin RNA (ihpRNA)
 Small interfering RNA (siRNA)
Conserved RNAi machinery:
• Argonaute (AGO)
• Dicer-like (DCL)
• RISC (RNA-induced silencing complex)
Vagner et al 2007

Asian Citrus Psyilid RNAi Pathway
Clauvis et al 2016

Virus Induced Gene silencing (VIGS)
o VIGS is the reverse genetics tool for analysis of gene
function
o It is based on the property of plant viruses to trigger a
defense mechanism related to PTGS
o Infection by a recombinant virus carrying a fragment
on a plant target gene will induce homology-dependent
silencing of the endogenous target gene.

18 of 50 13/10/2017 Kuldeep Singh Bhullar (L-2015-A-30-D) Stéphanie et al 2013

Implications in Horticulture
• Fasten breeding programme
• Enhancement of Nutraceautical
properties
• Pest management
• Post-harvest management

Fasten Breeding
Programme

Reduced generation time of apple seedlings
to within a year by plant virus vector
• Juvenile phase of apple (Malus x domestica)
generally lasts for 5–12 years
• Which is a serious constraint for genetic analysis
and for creating new apple cultivars through cross-
breeding.
• If modification of the genes involved in the
transition from the juvenile phase to the adult
phase can enable apple to complete its life cycle
within 1 year, as seen in herbaceous plants
• A significant enhancement in apple breeding will
be realized.
Noriko et al 2014

• OE of Arabidopsis FLOWERING LOCUS T
gene (AtFT)
• Silencing of apple TERMINALFLOWER 1
gene (MdTFL1-1)
• By using Apple latent spherical virus (ALSV)
vector (ALSV-AtFT/MdTFL1)
• To accelerate flowering time and life cycle in
apple seedlings.

Promotion of flowering with ALSV vectors
expressing a FT orthologue from various plants
Plant species FT orthologue Accession No. ALSV vector Flowering rate (%)
Arabidopsis thaliana AtFT AF152096 ALSV-AtFT 29.3 (17/58)*
A.thaliana AtTSF NM118156 ALSV-AtTSF 30.8 (8/26)
Citrusunshiu CiFT AB027456 ALSV-CiFT 0 (0/28)
Cucumissativus CsFT AB383152 ALSV-CsFT 0 (0/9)
C.moschata ComFT2 EF462212 ALSV-ComFT2 0 (0/9)
Glycinemax GmFT2a AB550122 ALSV-GmFT2a 10 (1/10)
G.max GmFT5a AB550126 ALSV-GmFT5a 0 (0/8)
Gentiana triflora GtFT1 AB605176 ALSV-GtFT1 7.7 (1/13)
G. triflora GtFT2 AB605177 ALSV-GtFT2 0 (0/7)
Ipomoea nil InFT1 EU178859 ALSV-InFT1 0 (0/11)
I. nil InFT2 EU178860 ALSV-InFT2 0 (0/5)
S.lycopersicum LeFT AY186735 ALSV-LeFT 0 (0/11)
Malus x domestica MdFT1 AB161112 ALSV-MdFT1 0 (0/31)
Malus x domestica MdFT2 AB458504 ALSV-MdFT2 0 (0/11)
Prunus mume PmFT AM943979 ALSV-PmFT 0 (0/8)
Pisum sativum PsFTa1 HQ538822 ALSV-PsFTa1 0 (0/9)
P. sativum PsFTc HQ538826 ALSV-PsFTc 0 (0/7)
Populus tremula PtFT1 DQ387859 ALSV-PtFT1 0 (0/10)
Vitis vinifera VvFT EF157728 ALSV-VvFT 0 (0/9)
–† None _ wtALSV 0 (0/50)

ALSV
vector
Expression
gene
Suppressio
n gene
Flowering
rate (%)
Leaf stage
of first
flowering
No. of
flowers
Growth after first
flowering
ALSV-AtFT* AtFT – 30 7–9 1 Vegetative
ALSV-
MdTFL1†
– MdTFL1-1 10 8–14 Multiple Continuous
flowering
ALSV-
AtFT/MdTF
L1
AtFT MdTFL1-1 >90 7–22 Multiple Continuous
flowering

(a):infected with ALSV-
AtTSF
(b ,c): ALSV-GtFT1
(d, e , f): ALSV-
AtFT/MdTFL1
(b) and (c) show the same
apple seedling on different
days postinoculation
Yellow arrowheads in (b)
and (c) indicate the same
position of flowering (only
one flower followed by
vegetative growth shoots)
Circles in (f) show
continuous flowering in an
apple seedling infected
with ALSV-AtFT/MdTFL1.
Promotion of flowering in apple seedlings

(a) 1.5 months postinoculation (mpi), (b) 3 mpi and (c) 7 mpi.
Arrows in (c) show continuous flowering, even after 7 mpi.
Continuous flowering with ALSV-AtFT/MdTFL1

Fruit production in apple seedlings infected with ALSV-AtFT/MdTFL1.
(a) Fruit on the apple seedling (9 mpi) 5 months after pollination
(b) Seedling (11 mpi) with four fruits 5 months after pollination
(c and d) Ripe fruits on apple seedlings (a) and (b) with viable seeds,
respectively.
(e) Seedlings germinated from seeds in

Precocious flowering of juvenile citrus
induced by a viral vector (CLBV)
• Target- Arabidopsis thaliana or Citrus FLOWERING
LOCUS T (FT) genes
• Transition from the vegetative to the reproductive
phase in juvenile citrus plants
• Citrus leaf blotch virus-based vector (clbvINpr-AtFT
clbvINpr-CiFT)
• Flowering startswithin 4–6 months
Karelia et al 2016

Graft inoculation

Nutraceautical
Properties

FaMYB1 negatively regulates the
anthocyanin synthesis in strwaberry
Kadomura et al 2015

• FaMYB1 negatively controls anthocyanin
content
• Down-regulation of FaMYB1 exhints a
significant increase in anthocyanin
content by decrease in the amount of
transcripts of LAR
• Overexpression of FaMYB1 resulted in a
decrease in anthocyanin content
• Overexpression of FaMYB1 also
significantly reduced expression of genes
encoding ANS and FGTs

Knockdown of ccd4 gene via VIGS Confers
Yellow Coloration in Peach Fruit
Songling et al 2016

35 of 50 13/10/2017 Kuldeep Singh Bhullar (L-2015-A-30-D) Javiera et al 2015
• ccd4 is proposed to be the key factor responsible for
carotenoid degradation in white flesh peaches
• Knocked down the ccd4 gene in the white flesh
peaches (“Akatsuki” and “Manami”)
• There was yellow pigmentation and increased
contents of carotenoids including lutein, ß-carotene,
ß-cryptoxanthin, zeaxanthin, and violaxanthin
• which evident that ccd4 is a determinant for
yellow flesh trait of peach.

Pest Management

Silencing awd of ACP Disrupts Adult Wing
Dev. and Increases Nymph Mortality
o Target- Abnormal wing disc (awd)
o Transcription of awd is development-dependent
o It is max. level in the last instar (5th)
o Micro-application of dsRNA to 5th instar of nymphs
caused significant nymphal mortality and adult wing-
malformation.
Ibrahim et al 2013

Micro-application of awd-dsRNA and its effects on ACP wing formation.
El-Shesheny I, Hajeri S, El-Hawary I, Gowda S, Killiny N (2013) Silencing Abnormal Wing Disc Gene of the Asian Citrus
Psyllid, Diaphorina citri Disrupts Adult Wing Development and Increases Nymph Mortality. PLOS ONE 8(5): e65392.
https://doi.org/10.1371/journal.pone.0065392
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065392

RNAi- Reverse insecticide resistance
o Insecticides are main tool for ACP management
o CYP4 confers insecticide resistande
o RNAi shut down translation of mRNA into enzymes
o It Reverse the insecticide resistance.
o Rendered ACP more susceptible to insecticides.
Elizabeth et al 2017

Oral delivery of dsRNAs induces mortality in
nymphs and adults of the ACP
• Target: Chitin synthase, Cathepsin D, inhibitor
of apoptosis genes
• Insects were fed with corresponding dsRNA
• Both nymphs and adult ACP exhibited significantly
increased mortality over time compared with that
of the controls.
• Confirmation with qRT-PCR was also done for
dsRNA-mediated RNAi effects on target mRNAs.
• So RNAi can be a powerful tool for HLB control.
Diogo et al 2017

Post harvest

PrupeSEP1 regulation during ripening and
softening in peach
Jinjin et al 2017

Phenotypes a) TRV2 control b) TRV2-SEP1

Commercial application

Non Browning apple-Arctic apple
• Browning is mediated by polyphenol oxidase (PPO)
• PPO can take a variety of chemical precursors
(such as catechol or 4-metholcatechol in apples)
and convert them to other compounds
• The consumers don’t prefer if it is not perfect
Andrew 2015

NON-BROWNING POTATOES – INNATE
• Developed by J. R. Simplot Company.
– Phytophthora infestans
– The presence of high amounts of reducing sugars which cause
brown spots on accumulation
– Enzymatic browning (browning also occurs non-enzymatically
because of the partial degradation of starch into sucrose and
fructose)
– Presence of high level of non-essential free amino acid
asparagine which gets oxidized to form carcinogenic
compound acrylamide upon baking or frying
• Used RNAi to silence 4 different genes
Rebecca 2014

Opinion about the technology
Positive
1. Specific, efficient, flexible, Stable and long-term silencing
2. Easily target the gene of interest
3. Plant virus vector system can be used to add new traits to
plants without altering the host genome
4. Thus, despite utilization of a genetically modified virus,
technique does not pass genetic modification to the next
generation and will serve as a new plant-breeding
technique
Negative
1. Off-target effects (Yuriy et al 2006)
2. Introduction of long double-stranded external RNA is a
very difficult process

Conclusions
o RNA interference is a sequence-specific mechanism
o VIGS-Fast, Efficient and Powerful functional
genomics tool.
o Increasing number of gene silenced in various
plants using newly designed VIGS vectors is a
testimony to its usefulness.
o It can be a magical tool for horticultural crops
o Conventional breeding techniques can be employed
in horticulture by using this technique

THANKS

Scope of gene silencing in horticultural crops

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Scope of gene silencing in horticultural crops

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