biotechnology ammeriolation for viral diseases
TRANSCRIPT
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. HusainiAssistant 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
componentsMembrane All phases of the Only enveloped
life cycle viruses when outside the cell
Translational All types of cells None of the virusesmachinery
Genome dsDNA ds or ss DNA or RNA
Virion Structure
Nucleic Acid
Spike Projections
ProteinCapsid
Lipid Envelope
VirionAssociatedPolymerase
Basic Plant Virus Structures
Helix (rod) e.g., TMV
Icosahedron(sphere)e.g., BMV
Virus Replication1 Virus attachment
and entry
1 2 Uncoating of virion
2
3 Migration ofgenome nucleicacid to nucleus
3
4 Transcription5 Genome replication
4
5
6 Translation of virusmRNAs
6
7 Virion assembly78 Release of new
virus particles
8
Acute Virus InfectionA
mou
nt o
f viru
s
Time
Symptoms
Virus
Virus-induced transformationNormal cells Transformed cells
• Important viral diseases of crops in IndiaCrop Disease Yield loss
(%)Virus Virus group
Cassava Mosaic 18–25 Indian cassavamosaic virus
Begomovirus
Cotton Leaf curl 68–71 Cotton leaf curl virus Begomovirus
Mungbean, Blackgram & Soybean Yellowmosaic
21–70 Mungbean yellowmosaic virus
Begomovirus
Potato Mosaic 85 Potato virus Y Potyvirus
Prevalent plant virus diseases
Crop Virus diseaseRice 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:
Virus ?
Current status of major virus diseases in vegetable crops:
TomatoTLCV
– 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 virusesPotato 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
Blight
lStem pitting – usually results in Loss of woody perennials
Ringspots, oakleaf
Deformities
Phyllody – tissue destined to develop flower parts instead develops leaves
Wilt
Necrotic roots
Mosaics – very common
Tomato mosaic
Alfalfa mosaic
Mosaics on monocots are streaks or stripes
Maize streak Maize mosaic
Necrotic lesionsSystemicLocal
“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
TAXONOMY AND NOMENCLATURE
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)
COMPOSITION AND STRUCTURE
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 naked0 5 9 12 9 14 2 50 100 200 300 200 600 10 300
FamiliesSpecies
Host typeVertebrateInvertebratePlantFungusBacteria
- + ++ ++ ++ ++ - ++ - + ++ - ++ ++ - ++ - ++ - + + +++ - + - - - + + + + +++ - + + +++ - + + -
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
GENOME ORGANIZATIONS
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)*– Dianthoviruses (Red clover necrotic mosaic virus,
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)*– Dianthoviruses (Red clover necrotic mosaic virus,
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
INFECTION CYCLE
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 usinghost machinery
3. Replication in cytoplasm
4b. New virus particlesare assembled
1. Virus particle enters first cell through healable wound
4a. Infectious TMV RNA is shuttled to adjacent cell through plasmodesmata, by virus-codedmovement 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
• Virus Life Cycle 3 Cell-to-cell movement
VIRION
PLASMODESMA
cw
• Virus Life Cycle 4 Systemic transport through phloem
• Virus Life Cycle 5 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 ofwhole 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 relationshipsTerms 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
Types of vector relationships
• Persistent transmission– virus acquired slowly, retained long period
(weeks), transmitted slowly– circulative or propagative transmission– virus acquired and transmitted during feeding
probes to phloem
Types of vector relationships
• Semi-persistent transmission– virus acquired fairly quickly, retained moderate
period (days), transmitted fairly quickly– virus acquired and transmitted during exploratory
probes
Viroids
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 anddetails 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 attendantRNA 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 withina double sphere)
Tobacco mosaic virus(ssRNA within ahelical rod)
Transgenic virus resistant papaya: Main hope for controlling papaya ringspot virus in Hawaii
Biotechnology In
Agriculture
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. SwaminathanWorld 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 ResistanceProtein Mediated Resistance
Coat Protein Mediated ResistanceReplicase Mediated ResistanceMovement Protein Mediated Resistance
RNA-mediated resistancesRNAs that target vRNAs for degradation
Non-Pathogen, Protein Mediated ResistanceTranscription regulators: TFs, and sZFPsDNA binding proteinsInterferon-like strategies; ds-RNA degrading enzymesTranslation 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
AgriBioInstituteUniversity of Sofia, Bulgaria
Alternative: insecticides to control insect vector
Resistance to Bean Golden Mosaic Geminivirus in Brazil; EMBRAPA, Aragio et alDisease 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 activeGene hypermethylated in coding regionPurpose - Viral immunity?
S. Grant (1999)
Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS)
This has recently been termed “RNAi”
Promoters silencedGenes 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
RNAi can be induced by:
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,infection of suspension cell lines)
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 transcriptionin 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)
ArgonauteRNA 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 polymerasesRDR2 - RNA dep.RNA polymeraseDCL3 – dicer-like protein
AGO4 – ARGONAUTEDRM2 – de novo metyltransferaseDRD1 – chromatin remodelling proteinSHH1 – 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
Paramutationinteraction (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
Paramutationinteraction (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 RDR2MOP2 = subunit of Pol IV, V(mediator of paramutation)
mediator of paramutation
RNAi pathways in plants - overviewNatural 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 expressionin 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 proteinse.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 byknock-out of a MIR gene
Phenotypic changes caused byexpression 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 imprintingMammals: 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.
DICER
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
DCL
AGO
AGO
RNA Pol
AGOsiRNA-mediated silencing via post-transcriptional and transcriptional gene silencing
AAAn
DCLMIR gene
RNA Pol
AGORNA Pol
miRNA -mediated slicing of mRNA and translational repression
mRNAAGO
AGOAAAn
AAAn
AAAnAAAn
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.
DCLViral ssRNAViral 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 genemRNA
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-typemRNA
mRNA
Protein translated
Endogenous gene
Sense RNA
Sense construct
Co-suppressed transgenic
PRO ORF
Co-suppression
PRO ORFEndogenous gene
mRNA
siRNA produced
AGO
AGO AAAn
AGO AAAn
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
DCLMIR gene
RNA Pol
AGORNA Pol
mRNAAGO
AGOAAAn
AAAn
AAAnAAAn
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
DCL
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 petuniaCaMV - 35S promoter from Cauliflower Mosaic VirusDFR 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 RNAiBy “Dicer”
21-23 nt RNAs
Fig. 16.39, 3rd Ed.
ATP-dependentHelicase 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 regionSoybean 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, Solanumspecies,
Tobaccorattle 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).