Biotechnology ammeriolation for viral diseases

1
TITLE:
“Molecular mechanism of viral diseases or
biotechnological interventions for ammeriolation of major
plant viral diseases.”
Name of the speaker : Amir Bashir Wani
Regd. No. : Fresh
Course No. : MBB-591
Seminar Incharge : Dr. Amjad M. Husaini
Assistant Professor,
Division of Bio-technology
Date of seminar : 18.11.2014

Virus
Sub microscopic entity consisting of
a single nucleic acid surrounded by a
protein coat and capable of
replication only within the living
cells of bacteria, animals or plants.

• VIRUSES
• Non-cellular form of life
• Obligate intracellular parasites
• Exist as inert particles (virions) outside the cell
• Virions harbor viral genome protected by protein
shell

Viral form of life
• CELLS VIRUSES
Reproduction Binary fission Assembly from the
pools of
components
Membrane All phases of the Only enveloped
life cycle viruses when
outside the cell
Translational All types of cells None of the viruses
machinery
Genome dsDNA ds or ss DNA or RNA

Virion Structure
Nucleic Acid
Spike
Projections
Protein
Capsid
Lipid Envelope
Virion
Associated
Polymerase

Basic Plant Virus Structures
Helix (rod)
e.g., TMV
Icosahedron
(sphere)
e.g., BMV

Virus Replication
1 Virus attachment
and entry
1 2 Uncoating of virion
2
3 Migration of
genome nucleic
acid to nucleus
3
4 Transcription
5 Genome replication
4
5
6 Translation of virus
mRNAs
6
7 Virion assembly
7
8 Release of new
virus particles
8

Acute Virus InfectionAmountofvirus
Time
Symptoms
Virus

Virus-induced transformation
Normal cells Transformed cells

• Important viral diseases of crops in India
Crop Disease Yield loss
(%)
Virus Virus group
Cassava Mosaic 18–25 Indian cassava
mosaic virus
Begomovirus
Cotton Leaf curl 68–71 Cotton leaf curl virus Begomovirus
Mungbean,
Blackgram &
Soybean Yellow
mosaic
21–70 Mungbean yellow
mosaic virus
Begomovirus
Potato Mosaic 85 Potato virus Y Potyvirus

Prevalent plant virus diseases
Crop Virus disease
Rice Rice Tungro Virus, Rice dwarf Virus
Maize Maize Leaf Fleck Mosaic Virus
Wheat Barley Yellow Dwarf Virus
Soybean Soybean Mosaic Virus, Tobacco Ring Spot Virus,
Mungbean Yellow Mosaic Virus
Cowpea Cowpea Aphid-borne Mosaic Virus, Cowpea Mosaic
Virus
Pea and
Broad bean
Pea Seed- borne Mosaic Virus
Cereals and Legumes:

Current status of major virus diseases in vegetable
crops:
Tomato
TLCV
– First reported in 1994 (Timila and
Joshi, 1994) after diagnosis using
cDNA hybridization in
collaboration with Dr. Maxwell
(Univ. of Wisconsin) and AVRDC.
• The virus is distributed in terai,
inner terai, valleys and foothills.
• Incidence ranged from 40-70%
(Joshi et al.1997) .
• Yield loss estimation in tomato
was 50% (Joshi and Shrestha,
1999), 40% in Western hills
(Ghimire et al., 2000).
• In recent years also, the disease
is severe in western terai.

 Tomato mosaic virus (ToMV):
Prevalent in tomato growing
areas causing poor plant growth
and also appeared in
combination with other viruses.

• Dmitriy Ivanovskiy (Russia)
And Martinus Beijerink (The Netherlands) describe
The first virus:
• Tobacco mosaic virus

CMV in Tomato CMV with other virus comlex
 Cucumber mosaic virus (CMV):
Widely distributed in tomato growing areas of
the mid-hills. Observed to be major. Severely
affected in some of the fields.

 Cucumber mosaic Virus (CMV):
Widely distributed in terai,
inner terai and midhills. The
incidence ranged from 50-80%
(SAVERNET II, midterm report.
1999). Presently also the
disease is problematic.

Cucurbit:
 CMV in cucumber , distributed
throughout cucumber cultivated
areas of mid-hills in severe form as
revealed by sample received at PPD,
Khumaltar and as observed in the
farmers’ fields, causing considerable
losses (PPD, 2003-2013).
 Complex infection of CMV, ZYMV,
SqMV, WMV 1 and 2, and CGMMV in
zucchini squash. Infection in some of
the fields, up to 100% incidence at
Kath. valley causing total crop failure
(PPD, 2006).

Various virus (Complex) disease symptoms in
Zucchini squash
Bottle gourd

Broad leaf mustard:
Turnip mosaic virus
(TuMV): Observed
mainly in broad leaf
mustard but radish and
turnip also affected.
Widely distributed in
the mid hills. Incidence
up to 100%, causing
total crop failure in
some of the fields.

Beans:
Bean common mosaic virus
(BCMV) has been observed in
Bean fields in low incidence. Its
distribution mainly in the mid-
hills (as observed in the field
visits)
Bean common mosaic necrosis
virus (BCMNV) recently reported
from Kaski and Chitwan in Sweet
bean with incidence ranging
from 60-70% (Pudasaini et al.,
2013).

Okra:
Mainly distributed in
terai, inner terai and
valleys of the mid hills.
up to 70% incidence
observed (Dahal,
1990). At present this
disease is under high
priority also.

Potato: reported six viruses
Potato leaf roll virus and potato
virus y: distribution high in terai
and moderate in midhills. They
could cause yield loss 12-50%
and 80% respectively.
PVX, PVA, PVS and PVM, their
distribution ranged from 24-
27% higher in the Terai (24, 25,
27, 25%) but lesser than PLRV
and PVY), Comparatively, PVX
and
PVS higher in high hills also.
(Source, Dhital et al., 2010)

Cardamom:
Prevalence of Large cardamom
chhirke virus (LCCV) and Cardamom
bushy dwarf virus (CBDV) have been
reported in the eastern hills with
incidence ranging from 15-20% and
10% repectively (Srivastav, 2012).
Low incidence of CBDV observed in
our survey.
(These viruses used to be the issues for
cardamom crop, & losses not
estimated yet)

PLANT VIRUS SYMPTOMS
• Viruses rarely kill plants
• Most severe disease usually in least well-adapted
host/pathogen systems
• Levels of tissue specificity differ among plant viruses
– Some infect all or most tissues
– Most cause symptoms only in aerial portions
– Some accumulate only in roots
– Symptoms in fruit or flowers may be most harmful
• Systemic symptoms only in developing tissue
• Local necrotic lesions undetectable in natural infections

PLANT VIRUS SYMPTOMS
• Stunting - common
• Mosaics & mottles - common
• Ringspots - common
• Abnormal growth/tumors/enations - rare
• Blights - rare
• Wilts – rare
• Systemic necrotic lesions – relatively rare

lStem pitting – usually results in Loss of woody perennials

Deformities
Phyllody – tissue destined to
develop flower parts instead
develops leaves

Mosaics – very common
Tomato mosaic
Alfalfa mosaic

Mosaics on monocots are streaks or stripes
Maize streak Maize mosaic

Necrotic lesions
SystemicLocal
“Local lesion” or “hypersensitive” response is an apoptotic response. Cells within a short
distance of the initially inoculated cell begin to undergo programmed cell death in advance
of virus invasion, preventing further virus spread.

Hypersensitive response – an apoptotic
reaction to infection
May be viral coat
protein or another
viral gene product

VIRUS TAXONOMY AND NOMENCLATURE
• Modified binomial is used
• Taxonomy depends on particle properties, nucleic acid
properties and especially sequence
• Family is the highest taxonomic level that is commonly
used; ends in viridae, e.g., Bromoviridae
• Genus ends in suffix virus, e.g., Bromomovirus
• Species is usually the commonly used virus name; it is
italicized in formal usage, e.g., Brome mosaic virus
• Small genome sizes, gene shuffling make broad
taxonomic schemes difficult (above Family level)

Virus properties: Plant viruses are often simpler
than animal viruses
• Genome sizes 0.3 - 300 kb; plant viruses 0.3-30 kb
• May have single-stranded or double-stranded RNA or
DNA genome; most plant viruses ssRNA
• If RNA, may be + or – sense; most plant viruses + sense
ssRNA
• May have one or many proteins in particles; most plant
viruses have 1-2
• May or may not have lipid envelope; most plant viruses
do not

Types of plant virus genomes
• double-stranded (ds) DNA (rare)
• single-stranded (ss) DNA (rare)
• ssRNA, negative sense (rare)
• ssRNA, positive sense (common)
• dsRNA (rare)

Virus types, by nucleic acid
DNA RNA
ss ds ss ds
env naked env naked env naked env naked
0 5 9 12 9 14 2 5
0 100 200 300 200 600 10 300
Families
Species
Host type
Vertebrate
Invertebrate
Plant
Fungus
Bacteria
- + ++ ++ ++ ++ - ++
- + ++ - ++ ++ - ++
- ++ - + + +++ - +
- - - + + + + +++
- + + +++ - + + -

Plant viruses are diverse,
but not as diverse as
animal viruses – probably
because of size constraints
imposed by requirement to
move cell-to-cell through
plasmodesmata of host
plants
Plant viruses often contain
divided genomes spread
among several particles

Helical symmetry
• Tobacco mosaic virus is typical, well-
studied example
• Each particle contains only a single
molecule of RNA (6395 nucleotide
residues) and 2130 copies of the coat
protein subunit (158 amino acid
residues; 17.3 kilodaltons)
– 3 nt/subunit
– 16.33 subunits/turn
– 49 subunits/3 turns
• TMV protein subunits + nucleic acid
will self-assemble in vitro in an
energy-independent fashion
• Self-assembly also occurs in the
absence of RNA
TMV rod is 18 nanometers (nm)
X 300 nm

Cubic (icosahedral) symmetry
• Tomato bushy stunt virus
is typical, well-studied
example
• Each particle contains
only a single molecule of
RNA (4800 nt) and 180
copies of the coat protein
subunit (387 aa; 41 kd)
• Viruses similar to TBSV
will self-assemble in vitro
from protein subunits +
nucleic acid in an energy-
independent fashion
TBSV icosahedron is 35.4 nm in
diameter
Protein Subunits Capsomeres
T= 3 Lattice
C
N

Plant virus genome organizations
• Very compact
• Most are +sense RNA viruses, so translation
regulation very important
• Use various strategies for genome expression
• Only a few genes absolutely required:
– Replicase
– Coat protein
– Cell-to-cell movement protein
• Other genes present in some viruses

Plant viruses have members in all 3 supergroups of + strand RNA viruses
From Principles of Virology,
Academic Press 1999

Genome expression of + strand RNA viruses
• Most use more than one strategy
– Polyprotein processing
– Subgenomic RNA
– Segmented genome
– Translational readthrough
– Frameshift
– Internal initiation of translation (without scanning)
– Scanning to alternative start site (truncated product)
– Alternative reading frame (gene-within-a-gene)

Polyprotein processing
• Post-translational cleavage of viral proteins may occur in
cis or in trans
• Some viruses use polyprotein processing exclusively to
regulate gene expression
• Many viruses use polyprotein processing as one of
several regulation mechanisms
• Examples:
– Potyviruses*
– Comoviruses*
– Closteroviruses
– Carlaviruses

Subgenomic RNA
• Similar to traditional mRNA, but synthesized from
an RNA template
• Many viruses use polyprotein processing as one of
several regulation mechanisms
• Examples:
– Tobamoviruses (TMV)*
– Bromoviruses (BMV)*
– Tombusviruses (TBSV)
– Potexviruses (PVX)

Segmented genome
• Positive sense RNA genomes are usually
encapsidated in separate particles
• Segmented genomes lend themselves to
recombination
• Examples:
– Bromoviruses (Brome mosaic virus, BMV)*
– Dianthoviruses (Red clover necrotic mosaic virus,
RCNMV)*
– Hordeiviruses (Barley stripe mosaic virus, BSMV)

Translational readthrough
• Usually UAG codon is read through using suppressor
tyrosine tRNA
• Common mechanism in plant viruses
• Examples:
– Tobamoviruses (Tobacco mosaic virus, TMV)*
RCNMV)*
– Hordeiviruses (Barley stripe mosaic virus, BSMV)

Translational frameshift
• Typically +1 or -1
• Common mechanism in plant viruses
• Examples:
– Luteoviruses (Barley yellow dwarf virus, BYBV)*
RCNMV)*
– Closteroviruses (Beet yellow vein virus, BYVV)

Internal initiation
• Cap-free translation
• Less complex in plant viruses than in animal
viruses
• Examples:
– Potyviruses (Tobacco etch virus, TEV)*
– Sobemoviruses (Southern bean mosaic virus,
(SBMV)*

Tobacco mosaic virus is a typical positive-sense RNA
plant virus with a 6.4 kilobase genome

Plant Virus Life Cycle
• Virus entry into host
– no attachment step with plant viruses
– by vector, mechanical, etc. – must be forced
– requires healable wound – delivery into cell
• Uncoating of viral nucleic acid
– may be co-translational for + sense RNA viruses
– poorly understood for many
• Replication
– replication is a complex, multistep process
– viruses encode their own replication enzymes

Plant Virus Life Cycle 2
• Cell-to-cell movement
– cell-to-cell movement through plasmodesmata
– move as whole particles or as protein/nucleic acid
complex (no coat protein required)
• Long distance movement in plant
– through phloem
– as particles or protein/nucleic acid complex (coat
protein required)
• Transmission from plant to plant
– requires whole particles

Typical RNA-containing plant virus replication cycle
From Shaw, 1996 Ch. 12 in Fundamental Virology (Academic Press)
2. RNA is released;
translates using
host machinery
3. Replication in
cytoplasm
4b. New virus particles
are assembled
1. Virus particle enters
first cell through
healable wound
4a. Infectious TMV
RNA is shuttled to
adjacent cell through
plasmodesmata, by
virus-coded
movement protein

• Virus Life Cycle
1 Invasion
• through leaves:
vectoring insects;
mechanical damage
Invasion through roots:
vectoring nematodes or fungi;
mechanical damage
• Exception: seed and pollen transmission

• Virus Life Cycle 2
Genome uncoating, expression and replication
uncoating
Translation
Replication

Cell-to-cell movement
VIRION
PLASMODESMA
cw

Systemic transport through phloem

Plant-to-plant transmission

Cell-to-Cell Movement of Plant Viruses
• Plant viruses move cell-to-cell slowly through
plasmodesmata
• Most plant viruses move cell-to-cell as complexes of
non-structural protein and genomic RNA
• The viral protein that facilitates movement is called
the “movement protein” (MP)
• Coat protein is often dispensable for cell-to-cell
movement

Cell-to-Cell Movement of Plant Viruses
• Several unrelated lineages of MP proteins have
been described
• MPs act as host range determinants
• MP alone causes expansion of normally constricted
plasmodesmata pores; MPs then traffic through
rapidly
• MPs are homologs of proteins that naturally traffic
mRNAs between cells
• MPs may act as suppressors of gene silencing

Plant cells are bound by rigid cell walls and are interconnected
by plasmodesmata, which are too small to allow passage of
whole virus particles.
Plasmodesma

Understanding virus
infection and
movement through
plants requires
understanding
architecture of
dicotyledonous
plants and the
connections
between different
cell types.
This has been
studied extensively
with GFP-labeled
virus

Some plant viruses radically modify plasmodesmata, allowing
for cell-to-cell movement as whole particles

Systemic spread of
plant viruses is
primarily through
vascular tissue,
especially phloem

Plant Virus Transmission
• Generally, viruses must enter plant through healable
wounds - they do not enter through natural
openings (no receptors)
• Insect vectors are most important means of natural
spread
• Type of transmission or vector relationship
determines epidemiology
• Seed transmission is relatively common, but specific
for virus and plant

Plant Virus Transmission
• Mechanical transmission
– Deliberate – rub-inoculation
– Field – farm tools, etc.
– Greenhouse – cutting tools, plant handling
– Some viruses transmitted only by mechanical
means, others cannot be transmitted
mechanically

• Transmission by vectors: general
– Arthropods most important
– Most by insects with sucking mouthparts
• Aphids most important, and most studied
• Leafhoppers next most important
– Some by insects with biting mouthparts
– Nematodes are important vectors
– “Fungi” (protists) may transmit soilborne viruses
– Life cycle of vector and virus/vector relationships
determine virus epidemiology
– A given virus species generally has only a single type of
vector
Plant Virus Transmission by Vectors

• Insect transmission (vectors)
– Aphids most important
– Leafhoppers
– Whiteflies
– Thrips
– Mealybugs
– Beetles
– Mites (Arachnidae)
– Ants, grasshoppers, etc. – mechanical
– Bees, other pollinators – pollen transmission

Types of vector relationships
Terms apply mainly, but not exclusively, to aphid
transmission
• Non-persistent transmission
– virus acquired quickly, retained short period
(hours), transmitted quickly
– “stylet-borne” transmission
– virus acquired and transmitted during exploratory
probes to epidermis

• Persistent transmission
– virus acquired slowly, retained long period
(weeks), transmitted slowly
– circulative or propagative transmission
– virus acquired and transmitted during feeding
probes to phloem

• Semi-persistent transmission
– virus acquired fairly quickly, retained moderate
period (days), transmitted fairly quickly
– virus acquired and transmitted during exploratory
probes

Viroids
• Very small, covalently closed, circular RNA molecules
capable of autonomous replication and induction of
disease
• Sizes range from 250-450 nucleotides
• No coding capacity - do not program their own polymerase
• Use host-encoded polymerase for replication
• Mechanically transmitted; often seed transmitted
• More than 40 viroid species and many variants have been
characterized
• “Classical” viroids have been found only in plants

Viroids are divided into two groups, based on site and
details of replication

Viroid Diseases
• Potato spindle tuber viroid
(PSTVd)
– May be limiting to potato
growers
– First viroid characterized
– Many variants described
– Control with detection in
mother stock, clean seed
PSTVd in tomato
PSTVd in potato

Viroid Diseases
• Citrus exocortis viroid (CEVd)
– Causes stunting of plants, shelling
of bark
– May result in little yield loss
– May be useful to promote dwarfing
for agronomic advantage
– Transmitted though stock, graft
– Control by removal of infected
plants, detection, clean stock
Citrus exocortis viroid

Apple crinkle fruit viroid Avocado sun blotch viroid
Citrus exocortis viroid
Healthy Infected

Potato spindle tuber viroid (PSTVd) is the most thoroughly
characterized viroid disease
(From R. Owens, USDA, Beltsville)

Viroid structures
-All are covalently closed circular RNAs fold to tightly base-paired structures
-Two main groups of viroids: self-cleaving and non-self-cleaving
-Non-self cleaving viroids replicate in nucleus and fold into “dog bone” or rod-like structure
-Five domains identifiable in non-self-cleaving
-Left hand (LH) and right hand (RH) domains are non-base-paired loops
-Single mutations to pathogenic domain often alter virulence
-Mutations to conserved central domain are often lethal
-Mutations to variable domain are often permitted

Minor variations in viroid sequence, and presumably attendant
RNA structure changes, are associated with virulence differences
(From R. Owens, USDA, Beltsville)

Viroid replication
• In nucleus or chloroplasts, depending on class of viroid
• Chloroplast-associated viroids process into monomers by
ribozyme-mediated cleavage; nucleus-associated viroids process
into monomers by using host-derived enzyme
• In both classes, host DNA-dependent RNA polymerase is the
performs RNA polymerization on + and – strand RNA templates
Ribozyme-mediated Cleavage by host-factor
RZ RZ
RZ RZ HF HF

• Virions under EM
• Geminiviruses)
(ssDNA within
a double sphere)

Tobacco mosaic virus
(ssRNA within a
helical rod)

Transgenic virus resistant papaya: Main
hope for controlling papaya ringspot
virus in Hawaii

Generating high-yielding varieties by genetic
manipulation of plant architecture…………...
The major factor that contributed to the success of the green revolution
was the introduction of high-yielding semi-dwarf varieties of wheat and
rice, in combination with the application of large amounts of nitrogen
fertilizer.
DR. M. S. Swaminathan
World Food Prize - 2003

 Field Efficacy of chitinase gene against Black sigatoka
 28 events, 1200 plants planted on 30th Nov. 2007
 Trial effectively challenged with sigatoka disease
through natural infestation
 Data collection ongoing
First CFT of GM banana in Africa

Virus resistance technologies
continue to grow
Pathogen Derived Resistance
Protein Mediated Resistance
Coat Protein Mediated Resistance
Replicase Mediated Resistance
Movement Protein Mediated Resistance
RNA-mediated resistance
sRNAs that target vRNAs for degradation
Non-Pathogen, Protein Mediated Resistance
Transcription regulators: TFs, and sZFPs
DNA binding proteins
Interferon-like strategies; ds-RNA degrading enzymes
Translation initiation factors/co-factors

Virus Resistant Crops Commercialized
to Date:
• Papaya: resistance to PRSV, developed in public sector
• Squash: resistance to potyviruses, developed in private
sector
• Plum: resistance to ‘Sharka’ disease: PPV, USDA
• Tomato, sweet peppers, cucumbers: resistant to
different viruses, developed and released in China in
mid-’90s.
• Nothing released yet in developing economies

sunflower
TSV on Other Crops
Marigold

Resistance To Tomato
Spotted Wilt Virus in
tobacco
AgriBioInstitute
University of Sofia,
Bulgaria
Alternative:
insecticides to control
insect vector

Resistance to Bean Golden Mosaic Geminivirus in Brazil; EMBRAPA, Aragio et al
Disease is white-fly borne, controlled in part by insecticides

• Resistance employed by plants to combat infection by pathogens from a
broad range of species is frequently mediated by resistance genes (R
genes). While R genes are known to be involved in pathogen recognition,
how they convey this message to host defence machinery is not completely
understood. These studies employ genetics and cell biology to evaluate the
interaction of pathogen infection in resistant and susceptible host plants.
The system used here is the I locus of Phaseolus vulgaris L. and the
Potyvirus, Bean common mosaic virus (BCMV). Near isogenic lines for the I
locus were challenged with BCMV at 20°C, 26°C and 34°C, and assayed over
time using a number of different techniques. A protoplast system was
developed for use in transfection experiments for determination of viral
replication in the presence of the I allele. Confocal laser scanning
microscopy was used in combination with fluorescence immunostaining to
localize viral coat protein in resistant and susceptible responses. Genes that
are differentially expressed in these isolines at 26°C and 34°C following
inoculation with BCMV were detected using cDNA-AFLP. Protoplast
experiments revealed that BCMV is able to accumulate in genotypes
containing zero, one, or two copies of the I allele, although at different
rates. Results from microscopic observations support the protoplast data
and show that BCMV infects II, Ii and ii plants but that movement is
restricted in resistant genotypes (II and Ii). cDNA-AFLP analysis revealed 20
genes that are differentially expressed during the infection process.
Sequence analysis demonstrated that several of these genes are Phaseolus
homologs of those known to be involved in plant defence responses in
other well-characterized systems.

• Bean common mosaic virus
• Bean common mosaic virus is a typical Potyvirus at 750 nm x 15 nm and a
genome size of 9.6 Kb (Bos, 1971; Urcuqui-Inchima et al., 2001). Its virion is
a flexuous rod that is made up of approximately 2000 CP monomers
encapsidating the RNA genome (Urcuqui-Inchima et al., 2001). BCMV is an
important pathogen of Phaseolus vulgaris genotypes worldwide and can
infect a wide range of crop legume species (Bos, 1971). Seed transmission is
an important source of initial infections with up to 83% of the seed from an
infected plant carrying the virus (Bos, 1971). Several strains of BCMV exist
with different virulences and have been categorized into pathogenicity
groups I (NL 1, US 1, PR 1), II (NL 7), III (NL 8), IVa (US 5), IVb (US 4;, US 3, NL
6), Va (US 2), Vb (NL 2), VIa (NL 3), VIb (NL 5), and VII (US 6, NL 4) based on
their virulence on 11 differential cultivars established by Drijfhout
(Drijfhout, 1978). These strains fall into two different serogroups, type A
including NL 8, NL 3 and NL 5, while type B encompasses the remainder
(Vetten et al., 1992). BCMV serotype A has been renamed as Bean common
mosaic necrotic virus (BCMNV) based on serological and symptomatic
differences between the two groups (McKern et al., 1992a; Vetten et al.,
1992).
• In Phaseolus spp. BCMV produces several distinct symptoms. In susceptible
genotypes at typical growing temperatures (26-28°C), a severe mosaic,
curling of the 9
• leaves, vein banding and mottled and malformed pods can appear (Figure
1.2) (Bos, 1971). At elevated temperatures (above 30°C)

Figure 1.2. Symptoms
typical of I gene-
containing germplasm
when infected with
Bean common mosaic
virus NY15. Images are
of ‘Black Turtle Soup’
near isogenic lines.
Subscripts denote
genotype at the I locus.
34ºC image by M.M.
Jahn.

• Protein mediated resistance (PMR)
• The initial report on PMR used Tobacco mosaic
virus (TMV) CP gene expression to produce the
resistance in tobacco plants (Powell et al, 1986).
Since then, a number of studies have used PMR to
confer plant resistance to a variety of viruses (Miller
and Hemenway, 1998; Tepfer, 2002; Gharsallah
Chouchane et al, 2008). Viral coat protein-mediated
resistance can provide either broad or narrow
protection (Tepfer, 2002). Thus, the CP gene
ofPotato mosaic virus (PMV) strain N605 provides
resistance in transgenic potato plants against this
virus strain and also to the related strain 0803
(Malnoe et al, 1994).

Post-transcriptional gene
silencing
Sanjeev Sharma*, Aarti Bhardwaj$, Shalini Jain# and Hariom Yadav#
*Animal Genetics and Breeding Division, #Animal Biochemistry Division,
National Dairy Research Institute, Karnal-132001, Haryana, India
$College of Applied Education and Health Sciences, Meerut, U.P.

Posttranscriptional gene silencing
Promoters active
Gene hypermethylated
in coding region
Purpose - Viral
immunity?
S. Grant (1999)
Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS)
This has recently been termed “RNAi”
Promoters silenced
Genes hypermethylated
in promoter region
Purpose - Viral
immunity?

Other names of post-transcriptional gene
silencing (PTGS) :
– gene silencing
– RNA silencing
– RNA interference
– In certain fungi: quelling
RNAi can spread throughout certain organisms
(C. elegans, plants).

Short history of post-transcriptional gene silencing
Definition: the ability of exogenous double-stranded
RNA (dsRNA) to suppress the expression of the gene
which corresponds to the dsRNA sequence.
1990 Jorgensen :
Introduction of transgenes homologous to
endogenous genes often resulted in plants with both
genes suppressed!
Called Co-suppression
Resulted in degradation of the endogenous and the
transgene mRNA

1995 Guo and Kemphues:
-injection of either antisense or sense RNAs in the
germline of C. elegans was equally effective at
silencing homologous target genes
1998 Mello and Fire:
-extension of above experiments, combination of
sense and antisense RNA (= dsRNA) was 10
times more effective than single strand RNA
Contd….

What is RNA interference /PTGS?
dsRNA needs to be directed against an exon, not an
intron in order to be effective
homology of the dsRNA and the target gene/mRNA is
required
targeted mRNA is lost (degraded) after RNAi
the effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
ssRNA does not work as well as dsRNA

double-stranded RNAs are produced by:
– transcription of inverted repeats
– viral replication
– transcription of RNA by RNA-dependent RNA-
polymerases (RdRP)
double-stranded RNA triggers cleavage of
homologous mRNA
PTGS-defective plants are more sensitive to infection
by RNA viruses
in RNAi defective nematodes, transposons are much
more active

Dicer
Double-stranded RNA triggers processed into siRNAs
by enzyme RNAseIII family, specifically the Dicer family
Processive enzyme - no larger intermediates.
Dicer family proteins are ATP-dependent nucleases.
These proteins contain an amino-terminal helicase
domain, dual RNAseIII domains in the carboxy-
terminal segment, and dsRNA-binding motifs.

Contd…..
They can also contain a PAZ domain, which is thought
to be important for protein-protein interaction.
Dicer homologs exist in many organisms including
C. elegans, Drosphila, yeast and humans
Loss of dicer: loss of silencing, processing in vitro
Developmental consequence in Drosophila and
C. elegans

RISC complex
RISC is a large (~500-kDa) RNA-multiprotein complex, which
triggers mRNA degradation in response to siRNA
some components have been defined by genetics, but function
is unknown, e.g.
– unwinding of double-stranded siRNA (Helicase !?)
– ribonuclease component cleaves mRNA (Nuclease !?)
– amplification of silencing signal (RNA-dependent RNA
polymerase !?)
cleaved mRNA is degraded by cellular exonucleases

Different classes of small RNA
molecules
During dsRNA cleavage, different RNA classes
are produced:
– siRNA
– miRNA
– stRNA

siRNAs
Small interfering RNAs that have an integral role in
the phenomenon of RNA interference(RNAi),
a form of post-transcriptional gene silencing
RNAi: 21-25 nt fragments, which bind to the
complementary portion of the target mRNA
and tag it for degradation
A single base pair difference between the siRNA
template and the target mRNA is enough to block
the process.

miRNAs/stRNAs
micro/small temporal RNAs
derive from ~70 nt ssRNA (single-stranded RNA),
which forms a stemloop; processed to 22nt RNAs
found in:
– Drosophila, C. elegans, HeLa cells
genes
– Lin-4, Let-7
stRNAs do not trigger mRNA degradation

Contd….
role: the temporal regulation of C. elegans
development, preventing translation of their target
mRNAs by binding to the target’s complementary 3’
untranslated regions(UTRs)
conservation: 15% of these miRNAs were conserved
with 1-2 mismatches across worm, fly, and
mammalian genomes
expression pattern: varies; some are expressed in all
cells and at all developmental stages and others have
a more restricted spatial and temporal expression
pattern

Overview of small RNA molecules

Why is PTGS important?
Most widely held view is that RNAi evolved to
protect the genome from viruses (or other
invading DNAs or RNAs)
Recently, very small (micro) RNAs have been
discovered in several eukaryotes that regulate
developmentally other large RNAs
–May be a new use for the RNAi mechanism
besides defense

Recent applications of RNAi
Modulation of HIV-1 replication by RNA interference.
Hannon(2002).
Potent and specific inhibition of human immunodeficiency
virus type 1 replication by RNA interference.
An et al.(1999)
Selective silencing of viral gene expression in HPV-positive
human cervical carcinoma cells treated with siRNA, a primer
of RNA interference.
Jung et al. 2002.
RNA interference in adult mice.
Mccaffrey et al.2002
Successful inactivation of endogenous Oct-3/4 and c-mos
genes in mouse pre implantation embryos and oocytes using
short interfering RNAs.
Le Bon et al.2002

Possible future improvements of RNAi
applications
Already developed:
in vitro synthesis of siRNAs using T7 RNA Polymerase
U6 RNA promoter based plasmids
Digestion of longer dsRNA by E. coli Rnase III
Potentially useful:
creation of siRNA vectors with resistances cassettes
establishment of an inducible siRNA system
establishment of retroviral siRNA vectors (higher efficiencies,

Conclusions
begun in worms, flies, and plants - as an accidental
observation.
general applications in mammalian cells.
probably much more common than appreciated
before:
– it was recently discovered that small RNAs
correspond to centromer heterochromatin repeats
– RNAi regulates heterochromatic silencing
Faster identification of gene function

Powerful for analyzing unknown genes in
sequence genomes.
 efforts are being undertaken to target every
human gene via miRNAs
Gene therapy: down-regulation of certain
genes/mutated alleles
Cancer treatments
– knock-out of genes required for cell proliferation
– knock-out of genes encoding key structural
proteins
Agriculture
Contd…..

• Nucleic acid-mediated resistance (NAMR)
• Pathogen-derived resistance has also been achieved
through the expression of virus sequences, the
acquisition of resistance being dependent on the
transcribed RNA. This RNA-mediated virus resistance
can be considered to be an example of post-
transcriptional gene silencing (PTGS) in plants (Prins et
al, 2008). Napoli et al (1990) firstly reported PTGS
in Petunia hybrida transgenically expressing the
chalcone synthase gene. They observed a co-ordinated
and reciprocal inactivation of the host gene and the
transgene encoding the same RNA. This process has
been called RNA silencing or RNA interference (RNAi)
and occurs in a variety of eukaryotic organisms
(Mlotshwa et al, 2008). The silencing process involves
the cleavage of a dsRNA precursor into short (21-26
nucleotides) (nt) RNAs by an enzyme, Dicer, that has
RNase III domains

• Proteins involved in plant RNA silencing
• Several silencing-associated protein factors have been identified in the
plants. To-date, Dicer-like (DCL) proteins, Argonaute (AGO) proteins and
RNA-dependent RNA polymerases (RdRP) have been reported to play key
roles in RNA silencing (Xi and Qi, 2008). However, RNA helicase
(Kobayashi et al, 2007) and other proteins such as HEN1 (Lózsa et al, 2008)
and HYL1 (Baulcombe, 2004; Dong et al, 2008) are also involved. RdRPs
are particularly important in plant silencing in that they copy target RNA
sequences to generate dsRNA and that they are also required for RNA-
directed DNA methylation (He et al, 2009). Until present, six RdRPs were
reported in Arabdopsis (Brodersen and Voinnet, 2006).
• Arabidopsis thaliana and rice encode for four DCL (DCL1, DCL2, DCL3
and DCL4) proteins with distinct functions. Although DCL1, together with
HEN1 (Xie et al, 2004) and HYL1 has been previously shown to be
involved in miRNA biogenesis (Han et al, 2004), the protein represses
antiviral RNA silencing through negatively regulating the expression of
DCL4 and DCL3 (Qu et al, 2008). It appears to function in the nucleus,
processing both miRNA primary transcripts and precursors (Papp et al,
2003). Purified DCL1 from A. thaliana extracts was shown to be involved in
the production of 21 nt siRNAs (Qi et al, 2005a).

• Use of RNA silencing to biotechnological control of virus disease
• Enhanced resistance of transgenic plants to viruses has been shown to have been brought
about by expression of sequences able to trigger RNA silencing (Pruss et al, 2004; Andika et
al, 2005). However, the possible environmental risks (see below) and the difficulties of
transforming some species are obstacles to the application of this technology. Strategies that
confer RNA silencing, such as dsRNA molecules of viral origin, could result in undesired
consequences in hosts with unmodified genomes. Thus RNAi was synthesized in C.
elegans when incubated together with E. coli expressing a dsRNA corresponding to a specific
gene (Timmons and Fire, 1998). An alternative method for the production of resistance in
transgenic plants is the use of Agrobacterium tumefaciens to express dsRNA molecules
(Johansen and Carrington, 2001). Thus expression of a dsRNA coding for green fluorescent
protein (GFP) in N. benthamiana tissues that also had the GFP gene present resulted in
inhibition of GFP production. GFP synthesis was not inhibited when the N.
benthamiana strains used either carried plasmids coding for GFP-specific dsRNA molecules
or for viral suppressors of RNA silencing.
• Strategies using exogenously supplied dsRNA have already been use to combat virus
infestation in plants. E. coliwas used to produce large amounts of dsRNA coding for partial
sequences of two different viruses, Pepper mild mottle virus (PMMoV) and Plum pox
virus (PPV) (Tenllado et al, 2003). Simultaneous injection of dsRNA together with purified
virus particles resulted in the inhibition of both viruses. Interestingly, resistance to infection
was also observed when the crude bacterial preparations were sprayed onto the N.
benthamiana leaves. These data suggest a simple, economic and effective application of RNA
silencing technology. In the near future, we believe other such simple approaches to induce
and enhance the efficiency of RNA silencing will emerge, leading to large scale applications
of this sophisticated molecular pathway.
• Risks related to genetically engineered plants

• The main risks associated with genetically engineered plants (Tepfer, 2002; Keese, 2008) are the transgenic
expression of viral genes in a compatible host, which can directly interfere with the life cycle of other viruses. A
normal transgenic protein, for example those related to cell-to-cell and long-distance movement proteins, may
complement defective viral proteins. Similarly, heterologous encapsidation using viral coat proteins expressed
in the host represent a possible alteration in the process of transmission and host specificity that can
contribute to infection. The natural process of gene flow between crop plants and their wild relatives can
potentially alter the plant genome. Two possible problems are the fixation of crop genes in small populations of
wild plants leading to a loss of biodiversity and consequent population extinction, and increased "weediness"
of wild relatives of the crop plant brought about by gene introgression resulting in plant growth in undesirable
locations. This, however, would only occur if the transgene conferred an advantage that overcame a population
size limiting factor, which would result in increased gene prevalence in the wild population. If the transgene
were to confer resistance to conditions established by human activities, resultant problems could be controlled.
If the transgene were to confer resistance to viruses, other pathogens or climatic conditions, the problems are
far more complex as the selection pressure cannot be controlled.
• Recombination, a covalent joining of nucleic acids that were not previously adjacent (Keese, 2008) might also
allow the flow of plant genes to the virus genome. Recombination is seen to occur by a copy-choice mechanism
during virus replication, involving one or more changes of template while the replicase complex synthesizes
RNA complementary to the template molecule. Different types of recombination occur in the viral RNA
genome, i.e. between identical sequences at equivalent sites (homologous recombination) or between
unrelated sites that lack appreciable sequence identity (nonhomologous recombination). Reports have
identified the incorporation of chloroplast tRNA and cellular mRNA coding for an hsp70 homolog in the virus
genome (Mayo and Jolly, 1991; Masuta et al, 1992). The advantages of recombination to the virus include
elimination of deleterious alleles and creation of new variants. Indeed, sequence comparison has suggested
that recombination might play a key role in viral evolution (Miller et al, 1997). The susceptibility of virus
resistant transgenic plants to recombination and the resultant emergence of new virus diseases is therefore of
particular importance to the genetic engineer. It must be pointed out that recombination can also introduce
point mutations and others errors into the viral genome, leading to a loss of viral fitness.

Epigenetics
- RNA interference

Cenorhabditis
elegans
Discovery of RNA interference
(1998)
- silencing of gene expression with dsRNA

Antisense RNA (= RNA komplementární k mRNA)
can silence gene expression
(již počátek 80. let 20. století)
- direct introduction of antisense RNA (or transcription
in reverse orientation)
- interaction of mRNA and antisense RNA,
formation of dsRNA

What is the mechanism behind?
Original (!) hypotheses:
- antisense RNA mechanically prevents translation
- dsRNA is degraded (RNases)

Cosuppression in Petunia
Result: loss of pigmentation in flower segments
Napoli et al. 1990 Plant Cell 2:279–289
Aim: increase expression of pigment-synthetizing enzym

Expression of antisense RNA was less efficient!!!
Cosuppression in Petunia
Napoli et al. 1990 Plant Cell 2:279–289
Mechanism see later!

What they get the Nobel prize for?
- making proper controls pays off!
- Introduction of even very small amount of dsRNA induce specific silencing (antisence RNA
is less efficient!)
dsRNA has to be a signal!
- for sequence specific silencing
Andrew J. Hamilton, David C. Baulcombe*(1999):
A Species of Small Antisense RNA in Posttran-
scriptional Gene Silencing in Plants, Science 286 (5441): 950-952

RNA interference (RNAi)
= silencing of gene expression mediated by
small RNAs (small RNA, sRNA)
in plants predominantly - 21-24 nt
The precise role of 25-nt RNA in PTGS remains to be
determined. However, because they are long enough to
convey sequence specificity yet small enough to move
through plasmodesmata, it is possible that they are
components of the systemic signal and specificity
determinants of PTGS (Hamilton and Baulcombe, 1999).

RNA interference (RNAi)
gene silencing at
• transcriptional level (TGS)
(transcriptional gene silencing)
- induction of DNA methylation (mRNA not formed)
• posttranscriptional level (PTGS)
(posttranscriptional gene silencing)
- transcript cleavage
- block of translation

Basic mechanism of RNAi
dsRNA in cell is cleaved by
RNase DICER into short
dsRNA fragments – sRNA
Argonaute with a single
strand (from sRNA)
mediates recognision of
complementary sequences,
which should be silenced
(TGS, PTGS)

Small RNA in plants
- 3’ end of sRNA methylated (HEN1) - protection
• miRNA (micro) – from transcipts of RNA Pol II (pre-miRNA)
– hunderds MIR genes (in trans)
• siRNA (small interfering) – from dsRNA of various origin (both internal and external
– thousands types (both in cis and in trans)
….. (+ piRNA in animals)
pre-miRNA
Wang et al. 2004

Dicer-like
- cleavage of dsRNA (21-24nt)
- in Arabidopsis 4 paralogues
(different functions)
DCL1 – 21 nt miRNA (pre-miRNA)
DCL3 – 24 nt siRNA (TGS - RdDM)
DCL2, 4 – 21-22 nt siRNA
(antiviral defence, secondary siRNAs)

Argonaute
RNA binding protein (20-26 nt RNA)
- strand selection (5’ nt, participation of
HSP90)
- 10 genes in Arabidopsis
- main component of RISC
(RNA induced silencing complex)
- block of translation or slicer
(RNAse H-like endonuclease
- PIWI doména)
- role in TGS (RdDM)
(RNA directed DNA methylation)

Mechanism of small RNA action - overview
PTGS (21-22 nt): - specific cleavage of transcript
- - block of translation
TGS (24 nt): - methylation of promoter, heterochromatin formation
- preventing interaction of transcription factors
Pol V

sRNA mode of action also depends
on complementarity
- incomplementarity in cleavage site prevents RNase activity

dsRNA formation
(MIR genes)
?
• RdRP = RNA-dependent RNA Polymerase – synthesis of compl. RNA strand
templates: - transcripts cleaved by RISC
- impaired mRNAs (without polyA or cap)
- transcripts of RNA polymerase IV
+ secondary structures of viral RNAs (!)
mRNA fragment after Ago
cleavage (secondarysiRNA )

RNA polymerases in RNAi
(in plants)
RNA-dependent RNA polymerases (RdRP, RDR)
- form the majority siRNAs in plants
RDR6:
- dsRNA from impaired (not by Ago) RNAs (lacking polyA or cap)
= primary siRNAs
- dsRNA from products of Ago = secondary siRNAs
- RDR1 – close paralogue involved in antiviral defense
RDR2:
- dsRNA from products of RNA polymerase IV (DNA-dependent)

RNA polymerases in RNAi
(in plants)
DNA dependent RNA polymerase (Pol IV and Pol V)
- related to RNA polymerase II (share common subunits)
- specific for plants
RNA polymerase IV
- transcibes chromatin with H3K9me2 and non-met H3K4 (SHH1)
- short transcripts (hundreds nt), with cap, without polyA
- automatically used by RDR2 ( dsRNA  DCL3  siRNA)
RNA polymerase V
- necessary for RNA-directed DNA methylation (RdDM)
- direct interaction with Ago4 – identification of target seq.
RNA polymeráza II – unclear, but important role

RNA-directed DNA methylation (in detail)
- methylation (by DRM2) occurs only under interaction of AGO4(6,9)-siRNA
with RNA Pol V (large subunit C-term domain) and RNA-RNA complementarity
Pol IV a V – RNA polymerases
RDR2 - RNA dep.RNA polymerase
DCL3 – dicer-like protein
AGO4 – ARGONAUTE
DRM2 – de novo metyltransferase
DRD1 – chromatin remodelling protein
SHH1 – dual histon-code reading
(H3K9me2, H3K4)
Saze et al. 2012
SHH1
SHH1
Zhang et al. 2013

RNA-directed DNA methylation
– why so complicated and energy consuming?
- de novo methylation of TE in new insertion sites
- transmission of info from histons (CMT2/3) less reliable (less dense nucleosomes)
- PTGS – TGS transition
SHH1
SHH1
Zhang et al. 2013
Zemach et al. 2013

Secondary siRNA formation
- target RNA (mRNA, TAS transcripts)
- cleaved by Ago + primary sRNA
(miRNA or siRNA)
- RDR6 – complementary strand synthesis:
dsRNA  DCL2(4)  secondary siRNA
Function of secondary siRNA
- signal amplification
- formation of siRNA from neighbor seq.
(transitivity – new targets)
ta-siRNAs (miRNA na TAS)
(trans-acting siRNA – widening of miRNA targets)

- overexpression of pigment gene
(enzyme for pigment synthesis)
caused loss of pigmentation in flower
sectors
Cosupression in Petunia
- occurrence of aberrant transcripts due to overexpression
- formation of dsRNA from aberrant transcripts by RdRP (RDR6)
- formation of siRNAs that silence both transgene and internal gene
Use in functional genomics – overexpression and knock-out
possible with a single gene construct
dsRNA

Paramutation
interaction (in trans) between homologous alleles (epialleles), resulting in heritable
change in gene expression of one allele
(= epimutation)
paramutagenic and
paramutated allele
Mechanism?
mediator of
paramutation

Paramutation
interaction (in trans) between homologous alleles (epialleles), resulting in heritable
change in gene expression of one allele (= epimutation)
- 1. methylated, inactive allele
transcribed by Pol IV
- siRNA formation
- 2. induction of RdDM of
complementary sequences
1.Paramutagenic allele
2. Paramutable allele
MOP1 = ortholog RDR2
MOP2 = subunit of Pol IV, V
(mediator of paramutation)
mediator of
paramutation

RNAi pathways in plants - overview
Natural antisense+ Inverted repeat
Molnár et al. 2011
(DNA methylation)
a b c d
+ secondary siRNAs (theoretically from any RNA fragment primarily cleaved by Ago)

Antiviral systemic
resistance
- newly developed leaves resistant
against infection (1928)
Mechanism?

siRNA are transported
- via plasmodesmata
- through phloem
- passive – diffusion or in direction of stream

Spreading of silencing
- GFP silencing with siRNAs (from vasculature)
green = GFP fluorescence, red = chlorophyll autofluorescence

Function of RNAi
and systemic spreading of sRNA
- antiviral defense – preventing of spreading of infection and new infection
- TE defense – keeping genome integrity, heterochromatin structure
- regulation of development – practically all phases
- stress response (even heritable changes – see bellow)
- regulation of nutrient uptake – e.g. phosphorus
- epigenetic modulation of genetic information in
meristems (heritable changes) - environmental adaptations

Epigenetic regulation of gene expression
in plant development
- regulation mainly with miRNA (21 nt)
(hundreds of MIR genes)
- sometimes mediated with ta-siRNA
(non-coding TAS transcript cleaved by RISC with
miRNA, RDR6, …)
- frequently transported between neighbour cells
(non-cell autonomous)
- targets – genes for regulatory proteins
e.g. transcription factors, components of ubiquitination
pathway, …
(limited reliability of simple promoter fusion experiments with such genes!)

Example: roles of miRNAs and ta-siRNAs in
Arabidopsis leaf development
Pulido A , Laufs P J. Exp. Bot. 2010;61:1277-1291

Modulation of RNAi regulation
of gene expression in plant development
Phenotypic changes caused by
knock-out of a MIR gene
Phenotypic changes caused by
expression of miRNA-resistant variants
of target genes (same protein encoded
by different nucleotide sequence non-
homologous to miRNA)

Parental imprinting
- varying epigenetic modification of alleles inherited from father and mather (parental
imprint) – it results in differencial expression of these alleles in zygote
= alleles of some genes are methylated in one or the other gamet
- evolved in mammals and angiospermous plants
- hemizygote“ state can serve to ensure reasonable feeding of embryo (in
mather body) – prevents evolution of paternal alleles causing excessive
embryo feeding
(in angiosperms – determination of endosperm size)
- in plants – main function – repression of TE activity!

Parental imprinting
Mammals:
imprint establishment connected with meiosis
- extensive demethylation, specific methylation of some genes
Plants:
imprint establishes during development of gametophyte
(haploid phase in plants: (polen) polen tube + embryo sac)
demethylation in a part of gametophyte – genetically terminal
lines - central cell of embryo sac (DME - DEMETER)
- vegetative nucleus of polen tube (block of DDM1, DME)

Epigenetic changes in microgametophyte
Primary function:
induction of proper TE methylation in
generative cells (sperm cells):
Demethylation – reactivation of TE – siRNA
formation – transport to spermatic cells –
induction of proper TE methylation
Inhibition of methylation (repression DDM1) + actively (DME)
In vegetative nucleus

Primary function:
- block of TE and endosperm specific genes
in egg cell
- regulation of endosperm size
- block of gametophytic developmental program?
Epigenetic changes in megagametophyte

- repression state kept primarily by di-(tri-)methylation of
histon H3K27
- in vegetative Arabidopsis tissue – thousands genes!
- Maintenance of H3K27me2 mediated by gene specific
Polycomb repressive complex (PRC2)
- keep histon methylation after replication
Repression of gene expression independent
on methylation and siRNA:
PcG (POLYCOMB GROUP) proteins

Example:
vernalization
VIN3 – deacethylation of histons, VRN2 – induction of H3K27 methylation
after vernalization VRN2 (VERNALIZATION) PcG protein binds with
other proteins to promoter of FLC gene (repression by H3K27me2),
expression of FLC blocks flowering
-
Repression of FLC
allows formation
of generative
organs

Vernalization – primary signal?
VIN3 chromatin:
- permanently H3K27me3
repressive mark (PRC2)
- transcription induces activation
mark H3K36me3 and acethylation
(? Hypothetically low temperature induce
disassembly of nucleosome in TSS?)
- bivalent chromatin labeling
( animal stem cells)
- quantitative response

• Mechanism of RNAi
• ‘RNA interference’ refers collectively to diverse RNA-based
processes that all result in sequence-specific inhibition of gene
expression at the transcription, mRNA stability or translational
levels. It has most likely evolved as a mechanism for cells to
eliminate foreign genes. The unifying features of this phenomena
are the production of small RNAs (21-26 nucleotides (nt) that act
as specific determinants for down-regulating gene expression
[17,19,20] and the requirement for one or more members of the
Argonaute family of proteins [21]. RNAi operates by triggering the
action of dsRNA intermediates, which are processed into RNA
duplexes of 21-24 nucleotides by a ribonuclease III-like enzyme
called Dicer [22-24]. Once produced, these small RNA molecules or
short interfering RNAs (siRNAs) are incorporated in a multi-subunit
complex called RNA induced silencing complex (RISC) [21,25]. RISC
is formed by a siRNA and an endonuclease among other
components. The siRNAs within RISC acts as a guide to target the
degradation of complementary messenger RNAs (mRNAs) [21,25].
The host genome codifies for small RNAs called miRNAs that are
responsible for endogenous gene silencing.

• 3. Methods to Induce RNAi in Plants
• One of the biggest challenges in RNAi research is
the delivery of the active molecules that will
trigger the RNAi pathway in plants. In this system,
a number of methods for delivery of dsRNA or
siRNA into different cells and tissue include
transformation with dsRNA forming vectors for
selected gene(s) by an Agrobacterium mediated
transformations [19,35], delivery cognate dsRNA of
uidA GUS (β-glucuronidase) and TaGLP2a: GFP
(green fluorescent protein) reporter genes into
single epidermal cells of maize, barley and wheat
by particle bombardment [36], introducing a
Tobacco rattle virus (TRV)-based vector in tomato
plants by infiltration [37],

The Small RNA World
www.plantcell.org/cgi/doi/10.1105/tpc.110.tt0210

What are small RNAs?
•Small RNAs are a pool of 21 to 24 nt
RNAs that generally function in gene
silencing
•Small RNAs contribute to post-
transcriptional gene silencing by
affecting mRNA stability or translation
•Small RNAs contribute to
transcriptional gene silencing through
epigenetic modifications to chromatin
AAAAA
RNA Pol
Histone modification, DNA methylation

The core of RNA silencing:
Dicers and Argonautes
RNA silencing uses a set of core
reactions in which double-stranded
RNA (dsRNA) is processed by Dicer or
Dicer-like (DCL) proteins into short
RNA duplexes.
These small RNAs subsequently
associate with ARGONAUTE (AGO)
proteins to confer silencing.
AGO
Silencing

Dicer and Dicer-like proteins
From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006) Structural basis for
double-stranded RNA processing by Dicer. Science 311: 195 -198. Reprinted with permission from AAAS. Photo credit: Heidi
Dicer’s structure allows it to measure the RNA it
is cleaving. Like a cook who “dices” a carrot,
Dicer chops RNA into uniformly-sized pieces.
In siRNA and miRNA biogenesis, Dicer or
Dicer-like (DCL) proteins cleave long dsRNA
or foldback (hairpin) RNA into ~ 21 – 25 nt
fragments.

Argonaute proteins
Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998) AGO1 defines
a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith, S.K., Hannon, G.J., and Joshua-Tor,
L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – 1437. with permission of AAAS.
ARGONAUTE proteins bind
small RNAs and their targets.
The Arabidopsis ago1 mutant and
the octopus Argonauta argo
ARGONAUTE proteins are
named after the argonaute1
mutant of Arabidopsis; ago1
has thin radial leaves and was
named for the octopus
Argonauta which it resembles.

RNA silencing - overview
AGO
RNA Pol
AGOsiRNA-mediated
silencing via post-
transcriptional and
transcriptional gene
silencing
MIR gene
RNA Pol
AGO
RNA Pol
miRNA -
mediated slicing of
mRNA and
translational
repression
mRNA
AGO
AGO
AAAn

siRNAs – Genomic Defenders
• siRNAs protect the genome by
• Suppressing invading viruses
• Silencing sources of aberrant
transcripts
• Silencing transposons and
repetitive elements
•siRNAs also maintain some genes
in an epigenetically silent state
•
Reprinted by permission from Macmillan Publishers, Ltd: Nature. Lam, E., Kato, N., and Lawton, M. (2001)
Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411: 848-853. Copyright 2001.

Viral induced gene silencing (VIGS)
- overview
AGO
Most plant viruses are
RNA viruses that
replicate through a
double-stranded RNA
intermediate.
Viral ssRNA
Viral dsRNA
Virus-encoded
RNA-dependent
RNA polymerase
(RdRP)
AGO
Double-stranded RNA
is cleaved by DCL to
produce siRNA which
associates with AGO
to silence virus
replication and
expression.

Viral-induced gene silencing
summary
• RNA-mediated gene silencing is an important
tool in plant defense against pathogens
• siRNAs interfere with viral replication
• siRNAs act systemically to aid in host plant
recovery and resistance
• Most viruses produce suppressor proteins
that target components of the plant’s siRNA
defense pathway; these proteins are
important tools for dissecting RNA silencing
pathways

Silencing of transgenes
• Transgenes introduced into plants are
frequently silenced by the siRNA pathway
• Silencing can be triggered by:
• Very high levels of gene expression
• dsRNA derived from transgenes
• Aberrant RNAs encoded by transgenes
• Transgenes are silenced post-
transcriptionally and transcriptionally

Example: Manipulation of gene
expression to modify pigmentation
Wild-type petunia
producing purple
anthocyanin
pigments
Chalcone synthase (CHS)
is the enzyme at the start
of the biosynthetic pathway
for anthocyanins
Photo credit Richard Jorgensen; Aksamit-Stachurska et al. (2008) BMC Biotechnology 8: 25.
Anthocyanins
Chalcone synthase
(CHS)

Sense RNA
Antisense
RNA
Sense construct:
PRO ORF
Endogenous gene
mRNA
Transgene
PRO ORF
mRNA
Protein translated
mRNA
mRNA
Extra protein translated
Antisense construct:
PRO
ORF
Transgene
Sense-antisense duplex forms
and prohibits translation
Hypothesis: sense RNA production enhances
pigmentation and antisense RNA production
blocks pigmentation

Surprisingly, both antisense and
sense gene constructs can inhibit
pigment production
Photo credit Richard Jorgensen
Plants carrying CHS transgene
CaMV 35S pro : CHS CaMV 35S pro : CHS
Sense Antisense
OR

Silenced tissues do not express
endogenous or introduced CHS
Napoli, C., Lemieux, C., and Jorgensen, R. (1990) Introduction of a chimeric chalcone synthase gene into
petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289.
Transgene RNA
Endogenous
gene RNA
Purple
flowers
White
flowers
This phenomenon, in which both the
introduced gene and the
endogenous gene are silenced, has
been called “co-suppression”.

Co-suppression is a consequence
of siRNA production
De Paoli, E., Dorantes-Acosta, A., Zhai, J., Accerbi, M., Jeong, D.-H., Park, S., Meyers, B.C., Jorgensen, R.A., and
Green, P.J. (2009). Distinct extremely abundant siRNAs associated with cosuppression in petunia. RNA 15: 1965–1970.
PRO ORF
Wild-type
mRNA
mRNA
Protein translated
Endogenous gene
Sense RNA
Sense construct
Co-suppressed transgenic
PRO ORF
Co-suppression
PRO ORF
Endogenous gene
mRNA
siRNA
produced

Most siRNAs are produced from
transposons and repetitive DNA
Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., Givan, S.A., and Carrington, J.C.
(2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57.
Most of the cellular siRNAs are derived from transposons and other repetitive sequences. In
Arabidopsis, as shown above, there is a high density of these repeats in the pericentromeric
regions of the chromosome.
Abundance of
small RNAs
Abundance of
transposon/
retrotransposons
Chromosome
Centromere

microRNAs - miRNAS
• miRNAs are thought to have evolved from siRNAs,
and are produced and processed somewhat similarly
• Plants have a small number of highly conserved
miRNAs, and a large number of non-conserved
miRNAs
• miRNAs are encoded by specific MIR genes but act on
other genes – they are trans-acting regulatory factors
• miRNAs in plants regulate developmental and
physiological events

microRNAs - miRNAS
MIR gene
RNA Pol
AGO
RNA Pol
mRNA
AGO
AGO
AAAn
microRNAs slice mRNAs or
interfere with their translation

MIR genes are transcribed into
long RNAs that are processed to
miRNAs
•miRNAs are encoded by MIR genes
•The primary miRNA (pri-miRNA)
transcript folds back into a double-
stranded structure, which is processed
by DCL1
•The miRNA* strand is degraded 3'
5' miRNA
miRNA*
3'
5' pri-miRNA
miRNA
MIR gene
mRNA target

miRNAs and vegetative phase change
Germination
zygote
JUVENILE
PHASE
Vegetative phase
change
Vegetative phase change is the transition
from juvenile to adult growth in plants.
ADULT
PHASE
REPRODUCTIVE
PHASE
EMBRYONIC
PHASE

Phase change may involve a
temporal cascade of miRNAs and
transcription factors
miR156 SPL
miR172 AP2-like
In Arabidopsis, SPL9
directly activates
transcription of
MIR172b

Low Med High
miRNAs contribute to
developmental patterning
miRNA distribution patterns
can spatially restrict the
activity of their targets
miRNAs can move between
cells to spatially restrict
activity of their targets
miRNA
concentration
gradient
Activity of targetLow Med High
HIGH MED LOW
HIGH MED LOW In some cases the
movement of miRNAs
from cell to cell
establishes a gradient

In roots, miR165/6 moves from
endodermis into vascular cylinder
Reprinted by permission from Macmillan Publshers Ltd. Scheres, B. (2010). Developmental biology: Roots respond to an inner calling. Nature 465: 299-300;
Carlsbecker, A. et al., (2010) Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate. Nature 465: 316-321.
miR165/6 PHB
Movement of miR165/6
inwards from the endodermis
in which it is produced helps to
establish the radial pattern of
the root

Conclusions
Small RNAs contribute to the regulation and defense
of the genome, and confer silencing specificity
through base-pairing
siRNA targets include repetitive-rich heterochromatin,
transposons, viruses or other pathogens
miRNAs and tasiRNAs targets include regulatory
genes affecting developmental timing or patterning,
nutrient homeostasis and stress responses

Post-Transcriptional Gene Silencing
(PTGS)
• Also called RNA interference or RNAi
• Process results in down-regulation of a
gene at the RNA level (i.e., after
transcription)
• There is also gene silencing at the
transcriptional level (TGS)
– Examples: transposons, retroviral
genes, heterochromatin

• PTGS is heritable, although it can be modified
in subsequent cell divisions or generations
–Ergo, it is an epigenetic phenomenon
Epigenetics - refers to heritable changes in
phenotype or gene expression caused by
mechanisms other than changes in the
underlying DNA sequence.

Antisense Technology
• Used from ~1980 on, to repress specific genes
– Alternative to gene knock-outs, which were/are very
difficult to do in higher plants and animals
• Theory: by introducing an antisense gene (or asRNA) into
cells, the asRNA would “zip up” the complementary mRNA
into a dsRNA that would not be translated
• The “antisense effect” was highly variable, and in light of the
discovery of RNAi, asRNA probably inhibited its target by inducing
RNAi rather than inhibiting translation.

Discovery of PTGS
• First discovered in plants
– (R. Jorgensen, 1990)
• When Jorgensen introduced a re-engineered gene into petunia
that had a lot of homology with an endogenous petunia gene,
both genes became suppressed!
– Also called Co-suppression
– Suppression was mostly due to increased degradation of
the mRNAs (from the endogenous and introduced genes)

Discovery of PTGS (cont.)
• Involved attempts to manipulate pigment
synthesis genes in petunia
• Genes were enzymes of the flavonoid/
anthocyanin pathway:
– CHS: chalcone synthase
– DFR: dihydroflavonol reductase
• When these genes were introduced into petunia
using a strong viral promoter, mRNA levels
dropped and so did pigment levels in many
transgenics.

Flavonoid/anthocyanin pathway in plants
Strongly pigmented compounds

DFR construct introduced into petunia
CaMV - 35S promoter from
Cauliflower Mosaic Virus
DFR cDNA – cDNA copy of the DFR
mRNA (intronless DFR gene)
T Nos - 3’ processing signal from the
Nopaline synthase gene
Flowers from 3 different transgenic petunia plants carrying copies of
the chimeric DFR gene above. The flowers had low DFR mRNA levels
in the non-pigmented areas, but gene was still being transcribed.

• RNAi discovered in C. elegans (first animal) while
attempting to use antisense RNA in vivo
Craig Mello Andrew Fire (2006 Nobel Prize in
Physiology & Medicine)
– Control “sense” RNAs also produced suppression of target
gene!
– sense RNAs were contaminated with dsRNA.
– dsRNA was the suppressing agent.

Double-stranded RNA (dsRNA) induced
interference of the Mex-3 mRNA in the nematode
C. elegans.
Antisense RNA (c) or
dsRNA (d) for the mex-
3 (mRNA) was injected
into C. elegans
ovaries, and then mex-
3 mRNA was detected
in embryos by in situ
hybridization with a
mex-3 probe.
(a) control embryo
(b) control embryo hyb.
with mex-3 probe
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense
mRNA. (2) the suppressing signal moved from cell to cell.
Fig. 16.29

PTGS (RNAi) occurs in wide variety of
Eukaryotes:
– Angiosperms
– C. elegans (nematode)
– Drosophila
– Mammalian cells
– Chlamydomonas (unicellular
– Neurospora, but not in Yeast!

Mechanism of RNAi: Role of Dicer
1. Cells (plants and animals) undergoing RNAi contained
small fragments (~25 nt) of the RNA being suppressed.
2. A nuclease (Dicer) was purified from Drosophila embryos
that still had small RNA fragments associated with it, both
sense and antisense.
3. The Dicer gene is found in all organisms that exhibit RNAi,
and mutating it inhibits the RNAi effect.
Conclusion: Dicer is the endonuclease that degrades dsRNA into
21-24 nt fragments, and in higher eukaryotes also pulls the
strands apart via intrinsic helicase activity.

Generation of 21-23 nt fragments of target RNA in a
RNAi-competent Droso. embryo lysate/extract.
32P-labeled ds
luciferase (luc) RNAs,
either Pp or Rr, were
added to reactions 2-10
in the presence or
absence of the
corresponding mRNA.
The dsRNAs were
labeled on the sense
(s), antisense (a) or
both (a/s) strands.
Lanes 11, 12 contained
32P-labeled, capped,
antisense Rr-luc RNA.
Fig. 16.30

The dsRNA that is added
dictates where the destabilized
mRNA is cleaved.
The dsRNAs A, B, or C were
added to the Drosophila extract
together with a Rr-luc mRNA that
is 32P-labeled at the 5’ end. The
RNA was then analyzed on a
polyacrylamide gel and
autoradiographed.
Results: the products of Rr-luc mRNA
degradation triggered by dsRNA B are
~100nt longer than those triggered by
dsRNA C (and ~100 nt longer again for
dsRNA A-induced degradation).
Fig. 16.31

Model for RNAi
By “Dicer”
21-23 nt RNAs
Fig. 16.39, 3rd Ed.
ATP-dependent
Helicase or Dicer
Active
siRNA
complexes
= RISC
- contain
Argonaute
instead of
Dicer
Very efficient process
because many small
interfering RNAs
(siRNAs) generated
from a larger dsRNA.

In plants, fungi, C. elegans & Drosophila, a RNA-
dependent RNA polymerase (RDR) is involved in the
initiation (b) or amplification (c) of silencing (RNAi).
CBP and PABP block access for RDR.
PABP missing.
D. Baulcombe 2004 Nature 431:356

Why RNAi silencing?
• Most widely held view is that RNAi evolved to
protect the genome from viruses (and
perhaps transposons or mobile DNAs).
• Some viruses have proteins that suppress
silencing:
1. HCPro - first one identified, found in plant
potyviruses (V. Vance)
2. P19 - tomato bushy stunt virus, binds to
siRNAs and prevents RISC formation (D.
Baulcombe).
3. Tat - RNA-binding protein from HIV

Micro RNAs (MiRNAs)
• Recently, very small (micro) MiRNAs have
been discovered in plants and animals.
• They resemble siRNAs, and they regulate
specific mRNAs by promoting their
degradation or repressing their
translation.
• New use for the RNAi mechanism besides
defense.

DCL1 mutant
Comparison of Mechanisms of MiRNA Biogenesis and Action
Better complementarity of MiRNAs and targets in plants.

Summary of differences between plant and animal MiRNA systems
Plants Animals
# of miRNA genes: 100-200 100-500
Location in genome: intergenic regions Intergenic regions, introns
Clusters of miRNAs: Uncommon Common
MiRNA biosynthesis: Dicer-like Drosha, Dicer
Mechanism of repression mRNA cleavage Translational repression
Location of miRNA
target in a gene: Predominantly Predominantly the 3′-UTR
the open-reading frame
# of miRNA binding
sites in a target gene: Generally one Generally multiple
Functions of known
target genes: Regulatory genes Regulatory genes—crucial
crucial for development, for development, structural
enzymes proteins, enzymes

• 3.1. Agroinfiltration
• Agroinfiltration is a powerful method to study processes
connected with RNAi. The injection of Agrobacterium carrying
similar DNA constructs into the intracellular spaces of leaves for
triggering RNA silencing is known as agroinoculation or
agroinfiltration [41]. In most cases agroinfiltration is used to
initiate systemic silencing or to monitor the effect of suppressor
genes. In plants, cytoplasmic RNAi can be induced efficiently by
agroinfiltration, similar to a strategy for transient expression of T-
DNA vectors after delivery by Agrobacterium tumefaciens. The
transiently expressed DNA encodes either an ss- or dsRNA, which
is typically a hairpin (hp) RNA. The infiltration of hairpin constructs
are especially effective, because their dsRNA can be processed
directly to siRNAs, while the constructs expressing ssRNA can also
be useful to induce silencing [42-45] and for dissecting the
mechanism of gene silencing, especially concerned with its
suppressors, systemic silencing signal and also for simple protein
purification [42-45]. Besides, they provide a rapid, versatile and
convenient way for achieving a very high level of gene expression
in a distinct and defined zone

• Micro-Bombardment
• In this method, a linear or circular template is transferred into
the nucleus by micro-bombardment. Synthetic siRNAs are
delivered into plants by biolistic pressure to cause silencing of
GFP expression. Bombarding cells with particles coated with
dsRNA, siRNA or DNA that encode hairpin constructs as well
as sense or antisense RNA, activate the RNAi pathway. The
silencing effect of RNAi is occasionally detected as early as a
day after bombardment, and it continues up to 3 to 4 days of
post bombardment. Systemic spread of the silencing occurred
2 weeks later to manifest in the vascular tissues of the non-
bombarded leaves of Nicotiana benthamiana that were
closest to the bombarded ones. After one month or so, the
loss of GFP expression was seen in non-vascular tissues as
well. RNA blot hybridization with systemic leaves indicated
that the biolistically delivered siRNAs induced due to de novo
formation of siRNAs, which accumulated to cause systemic
silencing [29].

• Virus Induced Gene Silencing (VIGS)
• Modified viruses as RNA silencing triggers are used as a mean for inducing
RNA in plants. Different RNA and DNA viruses have been modified to serve
as vectors for gene expression [46,47]. Some viruses, such as Tobacco
mosaic virus (TMV), Potato virus X (PVX) and TRV, can be used for both
protein expression and gene silencing . All RNA virus-derived expression
vectors will not be useful as silencing vectors because many have potent
anti-silencing proteins such as TEV (Tobacco etch virus), that directly
interfere with host silencing machinery . Similarly, DNA viruses have not
been used extensively as expression vectors due to their size constraints for
movement [53]. However, a non-mobile Maize streak Virus (MSV)-derived
vector has been successfully used for long-term production of protein in
maize cell cultures [48]. Using viral vectors to silence endogenous plant
genes requires cloning homologous gene fragments into the virus without
compromising viral replication and movement. This was first demonstrated
in RNA viruses by inserting sequences into TMV [54], and then for DNA
viruses by replacing the coat protein gene with a homologous sequence
[53]. These reports used visible markers for gene silencing phytoene
desaturase (PDS) and chalcone synthase (CHS), providing a measure of the
tissue specificity of silencing as these have been involved in carotenoid
metabolic pathway.

• The PDS gene acts on the antenna complex of the thylakoid
membranes, and protects the chlorophyll from photooxidation. By
silencing this gene, a drastic decrease in leaf carotene content
resulted into the appearance of photobleaching symptom [55,56].
Similarly, over expression of CHS gene causes an albino phenotype
instead of producing the anticipated deep orange color [57]. As a
result, their action as a phenotypic marker helps in easy
understanding of the mechanism of gene silencing. shows some
general characteristics for currently available virus-derived gene
silencing vectors. Most viruses are plus-strand RNA viruses or
satellites, whereas Tomato golden mosaic virus (TGMV) and
Cabbage leaf curl virus (CaLCuV) are DNA viruses. Though RNA
viruses replicate in the cytoplasm DNA viruses replicate in plant
nuclei using the host DNA replication machinery. Both types of
viruses induce diffusible, homology- dependent systemic silencing
of endogenous genes. However, the extent of silencing spread and
the severity of viral symptoms can vary significantly in different
host plants and host/virus combinations. With the variety of
viruses and the diversity of infection patterns, transmission
vectors, and plant defenses it is not surprising that viruses differ
with respect to silencing [

• Management of Plant Pathogenic Viruses
• Antiviral RNAi technology has been used for viral
disease management in human cell lines [77-80]. Such
silencing mechanisms (RNAi) can also be exploited to
protect and manage viral infections in plants [19,81].
The effectiveness of the technology in generating virus
resistant plants was first reported to PVY in potato,
harbouring vectors for simultaneous expression of both
sense and antisense transcripts of the helper-
component proteinase (HC-Pro) gene [82]. The P1/HC-
Pro suppressors from the potyvirus inhabited silencing
at a step down stream of dsRNA processing, possibly by
preventing the unwinding of duplex siRNAs, or the
incorporation into RISC or both [83]. The utilization of
RANi technology has resulted in inducing immunity
reaction against several other viruses in different plant-
virus systems .In phyto-pathogenic DNA viruses like
geminiviruses

non-coding intergenic region of Mungbean yellow mosaic India virus
(MYMIV) was expressed as hairpin construct under the control of the
35S promoter and used as biolistically to inoculate MYMIV-infected
black gram plants and showed a complete recovery from infection,
which lasted until senescence [84]. RNAi mediated silencing of
gemini viruses using transient protoplast assay where protoplasts
were co-transferred with a siRNA designed to replicase (Rep)-coding
sequence of African cassava mosaic virus (ACMV) and the genomic
DNA of ACMV resulted in 99% reduction in Rep transcripts and 66%
reduction in viral DNA [85]. It was observed that siRNA was able to
silence a closely related strain of ACMV but not a more distantly
related virus. More than 40 viral suppressors have been identified in
plant viruses [86]. Results from some of the well-studied virus
suppressors indicated that suppressors interfere with systemic
signaling for silencing [44]. During last few years, the p69 encoded by
Turnip yellow mosaic virus has been identified as silencing
suppressors that prevented host RDR-dependent secondary dsRNA
synthesis [87]. P14 protein encoded by aureus viruses suppressed
both virus and transgene-induced silencing by sequestering both long
dsRNA and siRNA without size specificity [88].

• Multiple suppressors have been reported in Citrus tristeza virus
where p20 and coat protein (CP) play important role in
suppression of silencing signal and p23 inhibited intracellular
silencing Multiple viral components, viral RNAs and putative RNA
replicase proteins were reported for a silencing or suppression of
Red clover necrotic mosaic virus [89. These suppressors of gene
silencing are often involved in viral pathogenicity, mediate
synergism among plant viruses and result in the induction of more
severe disease. Simultaneous silencing of such diverse plant
viruses can be achieved by designing hairpin structures that can
target a distinct virus in a single construct. Contrarily, the RNAi
system may cause an increase in the severity of viral pathogenesis
and/or encode proteins, which can inactivate essential genes in
the RNAi machinery [94] that helps them in their replication in the
host genome [17]. Transgenic rice plants expressing DNA encoding
ORF IV of Rice tungro bacilliform virus (RTBV), both in sense and in
anti-sense orientation, resulting in the formation of dsRNA, were
generated. Specific degradation of the transgene transcripts and
the accumulation of small RNA were observed in transgenic plants

• . In RTBV-ODs2 line, RTBV DNA levels gradually rose from an
initial low to almost 60% of that of the control at 40 days
after inoculation [95]. For the effective control of PRSV and
Papaya leaf-distortion mosaic virus (PLDMV), an
untranslatable chimeric construct containing truncated PRSV
YK CP and PLDMV P-TW-WF CP genes has been transferred
into papaya (Carica papaya cv. ‘Thailand’) by Agrobacterium-
mediated transformation via embryogenic tissues derived
from immature zygotic embryos of papaya [96]. Based on
sequence profile of silencing suppressor protein, HcPro, it
was that PRSV-HcPro acts as a suppressor of RNA silencing
through micro RNA binding in a dose dependent manner. In
plants expression of PRSV-HcPro affects developmental
biology of plants, suggesting the interference of suppressor
protein in micro RNA-directed regulatory pathways of plants.
Besides facilitating the establishment of PRSV, it showed
strong positive synergism with other heterologous viruses as
well [97].

• Effects of targeted region of RNAi in various plant
virus systems.
• Host system Virus Targeted region
Soybean Bean pod mottle virus Pds ,Actin
N. benthamiana,
M. esculenta
African cassava mosaic virus pds, su, cyp79d2
Barley, wheat Barley stripe mosaic virus pds
Arabidopsis Cabbage leaf curl virus gfp, CH42, pds
N. benthamiana, N.
tabacum
Tobacco mosaic virus pds, psy
Arabidopsis,
tomato, Solanum
species,
Tobacco
rattle virus
Rar1, EDS1,
NPR1/NIM1,
pds, rbcS, gfp

• CPMP
• coat protein mediated protection In 1986, Beachy and co-workers
demonstrated that the expression of the coat protein gene of TMV in
transgenic tobacco plants could provide a considerable level of protection
against virus infection (reviewed by Beachy et al., 1990; Register and
Nelson, 1992). Since then, CPMP in transgenic dicotyledonous plants has
proven effective for more than 20 plant viruses (Hull and Davies, 1992).
Recently, CPMP has also been extended to monocots, as demonstrated by
expression of the coat protein of rice stripe virus in transgenic rice, where it
provides protection against the homologous virus that is obligately vectored
by viruliferous planthoppers (Hayakawa et al., 1992). These findings open
new avenues for plant protection in the most important agricultura1 crops.
In most instances, CPMP extends only to the virus or to related strains with
substantially similar coat protein, but there are a few instances where the
expression of the viral coat protein of one virus can provide at least some
limited protection of transgenic plants against heterologous virus infections
(Beachy et al., 1990; Gadani et al., 1990; Hull and Davies, 1992; Pang et al.,
1992). In most cases, CPMP acts only against the virion, whereas inoculum
consisting of naked virus RNA is frequently able to elicit infections.
However, exceptions to this general rule exist, as with PVX, a plus-sense
ssRNA virus where CPMP is effective against both RNA and viral inoculum
(Braun and Hemenway, 1992). These different results suggest that multiple
protective mechanisms may be involved in the cross-protection
phenomenon.

• REPLICASE-MEDIATED PROTECTION
• A recent development of considerable importance in pathogen- derived
resistance has been the demonstration by Zaitlin and co-workers that
expression of the 54-kD protein of TMV in transgenic plants offered higher
levels of protection against a TMV infection than CPMP (Carr et al., 1992).
The 54-kD protein is presumed to be derived from a subgenomic RNA of
TMV that contains an open reading frame overlapping the carboxy-terminal
portion of the 183-kD replicase 10 Scholthof et al. Plant Physiol. Vol. 102,
1993 coding region, but this protein has not been detected either in
infected plants or in plants transformed with this open reading frame
(Carr.et al., 1992). Experiments by Carr et al. (1992) show that the 54-kD
protein, rather than the RNA transcript, is responsible for virtual immunity
to TMV challenge in transgenic plants. The authors speculate that variations
of the temporal expression and accumulation of the 54-kD protein in
transgenic plants may disrupt the replication cycle of TMV. Similar
experiments have been performed with PEBV, a bipartite plus-sense ssRNA
virus (MacFarlane and Davies, 1992). As with TMV, expression of this
putative 54-kD replicase-based protein of PEBV in transgenic plants
provided protection against challenge by the homologous virus and two
closely related strains of PEBV. Some of the plants were susceptible, and
nucleotide sequence analyses of the transgene revealed the presence of
mutations that prevented the translation of the PEBV 54-kD protein.

• Thus, although the 54-kD protein was not detected in
protective transgenic plants, the open reading frame
and the putative protein appeared to be essential for
protection. MacFarlane and Davies (1992) detected two
virus variants that overcame the replicase-based
resistance in inoculated plants that were maintained for
a prolonged period of time. This is not unexpected,
since viruses are rapidly replicating entities. Thus, it is
highly likely that strains able to circumvent resistance
will evolve, particularly as such genes become widely
used in crop protection, and it can be expected that
multiple forms of protection strategies will be
necessary to realize the maximum potential for virus-
free transgenic crops when they are subjected to field
conditions.

• In analogous experiments, Braun and Hemenway (1992) expressed full-
length and amino-terminal portions of the replicase gene of PVX in tobacco
and found good resistance to subsequent PVX infection. In a comparison
with plants expressing the coat protein gene of PVX, they observed that
transgenic plants expressing the replicase derivatives provided more
effective protection against virus infection than CPMP. As was the case with
other replicase-based strategies, transcripts but not the predicted protein
were detected in the transgenic plants, even though in vitro experiments
indicated that the transcripts were translationally competent (Braun and
Hemenway, 1992). A related study (Anderson ,et al., 1992) showed that a
defective replicase protein of CMV, a plus-sense ssRNA virus with three
components, protected transgenic plants from virus challenge. The
defective protein may act as a dominant negative mutant that interacts with
wild-type components of the replicase system to inactivate the complex and
therefore interfere with the virus life cycle (Anderson et al., 1992). These
recently developed replicase based strategies offer new possibilities for
protecting plants from the deleterious effects of virus infection, including
yield reduction, and they will also increase our understanding of strategies
utilized by viruses for replication in plant cells.

• cRNA OR ANTISENSE RNA STRATEGIES
• The use of RNA complementary to part of the viral genome
(antisense RNA) is another potential pathogen-derived
resistance strategy that may have some utility for protecting
plants from systemic virus infection (for a review, see
Bejarano and Lichtenstein, 1992). In one case, expression of
an RNA transcript complementary to a replication-associated
portion of the viral genome of tomato golden mosaic virus, a
ssDNA virus that replicates in the nucleus, resulted in a
positive correlation between the accumulation of antisense
RN.4 and reductions in symptom development of virus
inoculated plants (Day et al., 1991). In other experiments,
transgenic potato plants expressing an RNA complementary
to the coat protein gene of PLRV, a phloem-limited plus sense
ssRNA virus, also provided protection from virus infection
comparable to that of transgenic plants expressing PLRV coat-
protein (Kawchuk et al., 1991). Further research is needed to
clarify the mechanism(s) of CPMP, and we may find that this
form of protection is due, at least in part, to complementary
(or antisense) RNA interactions with the virus genome.

• The results of Lindbo and Dougherty (1992) that were
discussed earlier certainly suggest that transcript
accumulation rather than coat protein accumulation could
result in plus "sense" RNA interference with replication of the
minus sense replicative intermediate. Thus, antisense RNA
technology directed to the coding template may be useful for
many viruses and could be particularly effective for those
restricted to particular tissues, such as PLRV, which are
phloem limited and dependent on aphids for transmission.
Antisense RNA technology may also be applicable to viruses
that replicate in the nucleus, such as the ssDNA-containing
geminiviruses, where replication probably occurs in close
proximity to the site where antisense RNA transcripts are
produced. Perhaps the inability of antisense transcripts to be
transported to cytoplasmic replication sites may partly
explain why earlier studies with the coat protein-based
antisense RNAs were unsuccessful against high-titer viruses
such as CMV and PVX.

• satRNAs
• satRNAs are small RNAs that are not infectious by themselves and require
helper viruses for their replication and encapsidation (reviewed in Palukaitis
et al., 1992). In some cases, satRNAs enhance the severity of symptoms in
conjunction with the helper virus infection, and in other cases the
symptoms are ameliorated. The transgenic expression of satRNAs of a
number of viruses has decreased virus symptoms and/or titers in a manner
that appears to mimic a natural system (Palukaitis et al., 1992). A probable
risk of this strategy is that in transgenic plants these satRNAs could mutate
during their amplification and, in conjunction with a virus infection, exhibit
a shift from an attenuating form to a virulent satRNA. Moreover, a virus or
satRNA producing a mild reaction on one host plant could elicit severe
symptoms on another host or in combination with a different strain of
helper virus. However, practical experience with field tests in China have
provided no evidence to support this hypothesis. In an 8-year study on
tomato and pepper plants using mild or attenuating combinations of CMV
and satRNA to mechanically inoculate plants, severe strains of satRNA in
conjunction with CMV infections have not yet emerged (Tien and Wu,
1991).

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