viruses of plants: into the battle between viruses and their host...spread of plant viruses through...

Viruses of Plants:

Into the battle between

viruses and their host

Richard Kormelink

Laboratory of Virology

Wageningen University

Virology Course Rotterdam 2018

Laboratory of Virology research themes


Plant virus research

● Tomato spotted wilt virus-bunyavirus & Geminiviruspathology

● RNAi, virus resistance and viral counter-defence

● Intercellular movement

● Virus imaging (WEMC)

Insect virus research

● Invertebrate DNA virus genetics and evolution

● Baculovirus and insect behavior

● Protein expression and gene therapy systems

● Mechanism of oral infection

Arbovirus research

● Molecular arbovirus-mosquito interactions (WN, CHIKV)

● Viral vaccines

http://upload.wikimedia.org/wikipedia/commons/8/83/Aedes_aegypti_during_blood_meal.jpg

Why study plant viruses?


Food

Why study plant viruses?


• Cause serious losses in food and fiber production

• Excellent models for molecular biology

• Good probes for studying cellular processes

• Vectors for gene expression and synthesis of proteins of commercial

and medical interest in plants.

• Virus-induced gene silencing (VIGS): functional genomics

Impact of Plant virus diseases

~20 plant viruses cause annual

crop-losses of > $ 20 billion

(Rybicki , 2015)

Cassava

Rice

Tomato

Pepper

Viruses of plants


Introduction:

− Plant viruses and diseases

Genetic make up of plant viruses:

− Genetic maps and translation

strategies

− Examples

The plant viral replication cycle

− Differences with viruses in animal

hosts

Plant defense against viruses

− RNA interference

− Resistance genes

− Engineered resistance

− VIGS Abutilon mosaic virus

Examples of plant virus diseases: Tulip

breaking virus


Potyvirus; 750 nm - Ø12 nm

Vector: Aphid

Tulipmania

By 1635, a sale of 40 bulbs for 100,000 florins (70.000

US dollars) was recorded.

A record was the sale of the most famous bulb, the

Semper Augustus, for 6,000 florins (± 4200 US dollars)

in Haarlem

By way of comparison, a ton of butter cost around 100

florins and "eight fat swine" 240 florins.

http://upload.wikimedia.org/wikipedia/commons/c/cc/Semper_Augustus_Tulip_17th_century.jpg

Examples of plant virus diseases :

Potato leafroll virus (PLRV)


Isometric (+RNA) 30 nm

Aphid

Beet necrotic yellow vein virus


Fungus: Polymyxa betaeRoot cells with sporesRhizomania

Rod-shaped (+RNA; segmented): 390, 265, 100, 70nm 20nm

Tobacco rattle virus


Rod-shaped (+RNA; segmented):

185-196nm, 50-115nm, 70nm

23nm

Nematode: Paratrichodorus,

Trichodorus

Potato

Tomato spotted wilt virus (TSWV)

Tomato

PepperAlstroemeria

Iris

Membrane bound (ambisense RNA)

Ø80 - 100 nm

BunyaviridaeTospovirusØ80 - 100 nm

Thrips

Morphology of plant virus particles


Means of spread of some representative

plant viruses


Ng and Falk (2006). Annual Review of Phytopathology Vol. 44: 183-212

Different living organisms can act as vectors to spread viruses

Means of Spread Rel. frequency Examples of genera

Humans Rare Tobamovirus, Potexvirus

Fungi Rare Carmovirus, Tombusvirus, Ophiovirus, Furovirus, Bymovirus

Nematodes Few Nepovirus, Tobravirus

Mites Few Rymovirus, Pigeaon pea sterility mosaic virus

Thrips Few Tospovirus

Beetles Few Comovirus, Tymovirus

Whiteflies Many Begomovirus, Crinivirus

Leafhoppers Many Phytoreovirus, Mastrevirus, Tenuivirus

Aphids Most (65%) Potyvirus, Luteovirus, Cucumovirus, Clostreovirus

Viruses of plants


Introduction:

− History




strategies

− Examples



hosts





− VIGS

More than 1000 different plant viruses

have been described ……………


…… of which 90% has an RNA genome

Virology Course Rotterdam 2018 Baltimore (1971)

e.g. Geminiviruses (4%)

e.g. Caulimoviruses (2%)

e.g. Reoviruses (4%)

Most plant viruses (76%)

Pseudoviridae (2%)

e.g. Plant-infecting

bunyaviruses (TSWV) (14%)

- RNA is directly infectious- RNA can be directly translated

Genetic maps of plant viruses


..or embedded in

the other genes

Viral genome (RNA or DNA)

Virion

Replication

Vector transport

Movement

RNAisuppressor

Polymerase

Coatprotein

To become a successful plant pathogen; a POL gene, one or more CP genes, and 3 additional genes

Functions encoded by (RNA) viruses


1. The coat protein(s) cp protect the viral genome

2. RNA-dependent RNA polymerase RdRP replicate the (RNA) genome

subunits: core polymerase pol to synthesize RNA

helicase hel to unfold secondary structure

methyltransferase mt to add cap structure

3. Proteases (some viruses) pro polyprotein processing

4. Terminal protein (some viruses) VPg priming RNA synthesis

5. Movement protein (plant viruses only!) mp for cell-to-cell transport

6. RNAi suppressor protein (plant only?) various counteracting RNAi defence

7. Vector transmission protein various insect transmission

8. Miscellaneous proteins (not conserved among viruses)

In case of enveloped viruses:

9. Envelope glycoproteins G cell attachment

(Plant) RNA viruses: strategies to

express downstream cistrons


Strategies to circumvent this problem:

1. Segmentation of the genome (i.e. minimization of downstream cistrons)

2. Translation of subgenomic mRNAs (subgenomic promoter)

3. Read-through translation (leaky stop codon)

4. Polyprotein processing (viral protease)

5. Frame-shifting

General problem of eukaryotic RNA viruses:

the eukaryotic ribosome translates only monocistronic mRNAs, while a viral

RNA genome contains at least 3 genes.

Genetic map of Brome Mosaic Virus (BMV)


m7GRNAs

ProteinsCP

1 (3234 nt) 2 (2885 nt) 3 (2117 nt)

4 (876 nt)

109 kDa 94 kDa 33 kDa 20 kDa

m7G m7G

m7G

Tyr Tyr Tyr

Tyr

MPpolhelmt

L M H

RNA 1 RNA 3 + 4 RNA 2

Translation strategies:• genome segmentation• subgenomic mRNA

Multi-partite virus

Advantages of a divided genome


1. Rapid recombination by RNA reassortment (in mixed infections)

2. Increase of genome size (separate encapsidation: multipartite virus)

3. Reduction of lethality (reduction target size)

4. Regulation of gene expression

5. Facilitation of spreading through plants and/or vectors (smaller entities)

Disadvantage: low efficiency of transmission

Genetic map of Tobacco Mosaic Virus (TMV)


69

Amber UAG

3417 4917 6396

4903 5707

5712 6189

m7G

126 kDa

183 kDa

30 kDa

17.6 kDa

CP

Proteins

RNA His

30K - mRNA

CP - mRNAHis

Hism7G

m7G

MP

polhel

helmt

mt

Translation strategies:• Read-through translation (through “leaky”stop codon)• Sub-genomic mRNA

(Two 3' co-terminal subgenomic mRNAs to express MP and CP)-

MP: the cell-to-cell movement protein

Genetic map of Potato Virus Y (PVY)


AAAA 3’

35kDa 52kDa 50kDa 71kDa 6kDa21kDa 27kDa 58kDa 30kDa

AI CI NIa NIb

N-Pro HC-PRO ?? HelicaseATPase

? VPg Protease Polymerase CP

?

RNA

PROTEINS

145 9306

POLYPROTEIN

Proteolytic cleavage

VPg

Translation strategy:polyprotein processing

Recombinative nature of potyviruses


Evolved by recombinationCore modules

A very successful group of plant pathogenic viruses

Genetic Map of TSWV


vcRNA

vRNA

Gn/Gc (glycoproteins)

3’

5’

M RNA

5’

3’

NSm (movement protein)

(-) (+)

3’

5’3’

5’

N (nucleoprotein)

S RNA

vcRNA

vRNA

NSs (suppressor of silencing)

(-) (+)

L RNA

L (RNA-dependent RNA polymerase)

vcRNA 5’

vRNA 3’ 5’

3’

(-)

Membrane bound Ø80 - 100 nm

Family BunyaviridaeGenus TospovirusNegative & Ambisense RNA

Translation strategies:• Segmented RNA genome• Subgenomic mRNAs• Processing precursor proteins (GP)

Ambisense

RNA segments

Comparison of an animal- and plant-

infecting Bunyavirus: NSm is the major

genetic difference


BUNYAVIRUS HOSTS VECTORSOrthobunya-/Nairo-/Phlebovirus Mammals, Birds Moquitoes, sandflies, ticksHantavirus Rodents Rodent-borneTomato Spotted Wilt Virus Plants Thrips

NSm is the plant viral movement protein Bunyaviruses replicate in their insect vector !!

NSs 32 kDa)

N (28 kDa)

Phlebovirus

RdRp (241 kDa)

Gn-Gc (139 kDa)

Orthobunyavirus/Hantavirus/Nairovirus

RdRp (247-459 kDa)

L RNAv

vc

M RNAGc-Gn (108-160 kDa)

vvc

S RNA

NSs (11-13 kDa)

N (26-50 kDa)

vvc

RdRp (331.5 kDa)

NSm (33.6 kDa)

Gn-Gc (127.4 kDa)

NSs (52.4 kDa)

N (28.8 kDa)

Tospovirus

Viruses of plants


Introduction:

− History




strategies

− Examples



hosts





− VIGS

Infection cycle of plant viruses


Penetration into plant cell

Uncoating/translation

Intracellular multiplication

Transport: cell-to-cell

long-distance

Vector

-arthropods

-nematodes

-fungi

Acquisition

Inoculation

Plant host

Nonpersistent transmission

Persistent transmission

Plant virus infection: Initial interactions


• No receptor-mediated uptake

plasma membrane covered by rigid cell wall

• Penetration not by endocytosis

but by transient wounding of cell wall/membrane

• Uncoating occurs by ribosomes

“co-translational disassembly”

TMV

Ribosomes

Ontmanteling en vroege translatie

cell wall

co-translationaldisassembly

early translation

Uncoating andtranslation

Entry

Replication cycle of TMV as function of time


5’ 3’

126 kDa 183 kDa read through CP

MP3419

4919 5709

6191

OAS

0 min parental virus

5’ - 3’ disassembly(cotranslational)

2-3 min

3-5 min

20-30 min

25-35 min all coat protein subunits removed

10 min 3’ - 5’ disassembly (coreplicational?)

45 min

30-40 min initiation of assembly

Replication of a single stranded

(+) RNA virus


5’ 3’(+)

5’3’(-)

(-)

RIReplicative Intermediate

5’ 3’(+)

3’

5’

RI

5’3’(-)

(+)5’

3’

RF(+)

(-)viral polymerase(RdRp)

helicase

Disassembly and early translation(synthesis of viral polymerase)

?

Difference between a (+)-sense and (-)-sense RNA virus


Plus-strand RNA virus

[ RNA is directly infectious[ RNA is directly translatable

Minus-strand RNA virus

[ RNA is not infectious[ RNA is not directly translatable

5’3’(-)

lipid membranepolymerase

5’3’

(+)

5’

3’

(-)

ORFtranslation

(more polymerase etc.)

glycoproteins

nucleocapsid

polymerase

Virus particles must carry some molecules of the viral polymerase

RNA viruses replicate in the cytoplasm ……


Nucleusgeminivirusrhabdovirus

Chloroplasttymovirus

Vacuolecucumovirusalfamovirus

GolgiTospovirus

Endoplasmic reticulumcomovirusbromovirus

Cytoplasm/viroplasmpotyvirus (pinwheels?)potexvirustobamovirus (X-bodies)tospovirus

Mitochondriontombusvirus

DNA/RNAvirus

.…… at cellular membranes


Chloroplast

Cytoplasm

Cytoplasm

Vacuole

Cowpea mosaic virus replication at the ER membranes

Cucumber mosaic virus replication at the vacuole membraneTurnip yellow

mosaic virus

replication at the

chloroplast

membrane

uninfected infected

After multiplication in the primary cell,

the virus spreads


Primary infection

vector

Early processesdisassemblymultiplication in the first cell

Active transport(viral function)

Defence response(tobacco: N gene)

Passive transportPhloem (xylem)

Systemic infection

AND NEXT OVER LONG DISTANCEUSING THE VASCULAR SYSTEM (Fast)

… MOVE FROM CELL-TO-CELL (Slow)

Local lesion

Long- distance transport: viruses follow

the route of assimilates


sink

source

sink

Most plant viruses move over long distances through the phloem (sieve elements).

Vascular transport in a plant:

Xylem: water and minerals

Phloem: metabolites

During systemic infection plant viruses

have to pass the thick cell wall


Plant cell

Chloroplast

Plasmodesma

Cell wall

PMCW

ER

ER

Spread of plant viruses through

plasmodesma: a complex pore


Rod shaped viruses Ø10-20 nm

Icosahedral virusesØ20-80 nm

Cell 1

Cell 2

ER

Viral RNAØ10 nm

2 nm

plasmodesmain cell wall

The problem!

Physical pore size 2 nm

Size exclusion limit ~1 kDa

Plant-infecting viruses must modify the PD

For this they have movement proteins

Modification of plasmodesmata


Plasmodesma modified

by Tobamovirus

Plasmodesma modified

by Comovirus/Tospovirus

Cowpea mosaic virus: movement of

mature virions


Cell wallCell 1

Cell 2

Transport tubule (MP) with virions

MP structurally modifies the plasmodesma and formsa virion-containing tubule

Electron tomogram of an isolated tubule

PM

Tubule

The TMV MP enlarges the diffusion limits

of plasmodesmata


LYCH-10 kDa dextran

Wildtypeplant

MP transgenicplant

T=30’T= 0

Micro-injected probe LYCH (450 Da)

To remember


• Most plant viruses are transmitted by insects (but do not replicate)

• No receptor-mediated entry, but entry through wounding (insect’s

stylet)

• Plant viruses move through plasmodesmata that are modified by a

viral movement protein. Move as genome or virion.

• Long-distance transport follows the route of metabolites (through the

phloem)

Viruses of plants


Introduction:

− History




strategies

− Examples



hosts





− VIGS

Defense in plants


Against Viruses

First line of defence:

RNA interference (RNAi)

(RNA silencing)

Second line of defence:

Resistance (R)-genes

multigenic (hard to breed, but durable)

monogenic (easy to breed, but less durable)

Principle of RNAi


dsRNA

Dicer/DCL

I.

II. 21-24 nt siRNAs

III. Activation of RISC AGO

IV. Translational inhibition Cleavage

AGO AGO

AAA40S

60SAAA

40S

60S

mRNAstarget

mRNAstarget

21 nt siRNAs

Translational arrest or

cleavage or target RNAs

22 nt miRNAs (host encoded)

Translational arrest or

cleavage or target RNAs

24 nt siRNAs

DNA and histone methylation

(epigenetics)

• Gene regulation

• Development and regulation

of chromosome dynamics

Defense against molecular parasites (viruses and transposable elements)

Antiviral RNAi


Replication

dsRNA intermediates

AAA

Viral mRNAs

Dicer/DCL

I.

II. 21 nt siRNAs

III. Antiviral RISC AGO


AGO AGO

AAA40S

60SAAA

40S

60S

mRNAsViral

mRNAsViral

RNA virus

(-)RI

5’ 3’(+)

3’

5’

Trigger is dsRNA

RF (+)

(-)

21-25 nt siRNA

Hamilton et al., 1999

Antiviral RNAi


Replication

dsRNA intermediates

AAA

Viral mRNAs

Dicer/DCL

I.

II. 21 nt siRNAs

III. Antiviral RISC AGO


AGO AGO

AAA40S

60SAAA

40S

60S

mRNAsViral

mRNAsViral

RNA virus

Post transcriptional gene silencing

(PTGS)

Virus-induced antiviral RNAi


PVX

TMV – “PVX”

STOP

STOP

21-24 nt (siRNA)

Hamilton et al., 1999

Antiviral RNAi in plants, insects….


AND mammals

Plant host defence against viral invasion

AAA

Dicing by DCL4 (2)

21 nt siRNAs

AGO1

AGO1

AAA40S

60S

AGO1

AAA40S

60S

Cell-to-cell spread

AGO1

AGO1

Cell-to-cell spread

Immunization

Immunization

Amplification

RDRs

DCL4

Secondary siRNAs

RDR (1,2,6): host-encoded RNA-dependent RNA polymerase

Aberrant/cleaved Viral RNA

Cytoplasm

Amplification is essential for a strong

antiviral response

AAA

Dicing by DCL4 (2)

21 nt siRNAs

AGO1

AGO1

AAA40S

60S

AGO1

AAA40S

60S

Cell-to-cell spread

AGO1

AGO1

Cell-to-cell spread

Immunization

Immunization

Amplification

RDRs

DCL4

Secondary siRNAs

Plants become highly susceptible

to RNA viruses


Cytoplasm

How come viruses still achieve a

successful infection?


Wild type PVY reverses GFP silencing ........and so does CMV

Viral counter-defence!!

Genetic maps of plant viruses


..or embedded in

the other genes

Viral genome (RNA or DNA)

Virion

Replication

Vector transport

Movement

RNAisuppressor

Polymerase

Coatprotein

To become a successful plant pathogen; a POL gene, one or more CP genes, and 3 additional genes

P0

Viral counter-defence by RNA viruses

AAA

Dicing by DCL4 (2)

21 nt siRNAs

AGO1

AGO1

AAA40S

60S

AGO1

AAA40S

60S

Cell-to-cell spread

AGO1

AGO1

Cell-to-cell spread

Immunization

Immunization

Amplification

RDRs

DCL4

Secondary siRNAs

P19

NSs

2b

NSs

P38

P19

2b

NSs

2b

HC-Pro

P38

SPCSV RNaseIIIHC-Pro

P1


Cytoplasm

Side effects?


RNAi suppressors that sequester siRNAs often bind the structurally resembling miRNAs as well

Interference on the miRNA pathway

Plant Virus RNA silencing suppressors

(RSS)


RNAi suppressor activityembedded in viral geneswith diverse functions

(polymerase, movement protein, capsid protein…

Also animal/human virus RSS!


Mutant plant viruses

lacking a RSS....


Wang et al. (2010)

.....are less virulent, and viral titersin the host are lower

......unless the host’s RNAi system is compromised as well

Mutant animal-infecting

viruses lacking a RSS....


HIV Tat is a suppressor of RNAi, anda mutant lacking Tat replicates tolower titers.......

........but replicates to wild type levelsagain when trans-complemented withEbola VP35 (RSS) or Rice hoja blanca virus NS3 (RSS).

RNAi suppressor proteins act cross-kingdom!!

Schnettler et al. (2009)

Complication: Animal-virus RSS often

are IFN-anatagonists as well.


Cullen et al. (2014)

Antiviral RNAi against DNA viruses

AAA

Dicing by DCL4 (2)

21 nt siRNAs

AGO1

AGO1

AAA40S

60S

AGO1

AAA40S

60S

Cell-to-cell spread

siRNAs

AGO1

Cell-to-cell spread

Immunization

RDRs

DCL4


Nucleus

Caulimovirus

Geminivirus

Transcription

Dicing by DCL3/4/2

21 nt siRNAs

24 nt siRNAs

Antiviral RITS

AGO4

Cytoplasm

DN

A/h

isto

ne

meth

yla

tion

An epigenetic antiviral defence mechanism

(Transcriptional Gene Silencing)

Antiviral RNAi against DNA viruses

AAA

Dicing by DCL4 (2)

21 nt siRNAs

AGO1

AGO1

AAA40S

60S

AGO1

AAA40S

60S

Cell-to-cell spread

siRNAs

AGO1

Cell-to-cell spread

Immunization

RDRs

DCL4


Nucleus

Caulimovirus

Geminivirus

Transcription

Dicing by DCL3/4/2

21 nt siRNAs

24 nt siRNAs

Antiviral RITS

AGO4

Cytoplasm

DN

A/h

isto

ne

meth

yla

tion

V2

P6

C2

βC1

V2

Transcriptional gene silencing (TGS) of

geminiviruses

Rodriguez-Negrete et al. (2009)

Involving siRNA-dependent DNA methylation (RdDM) of corresponding viral DNA sequences.


Second line of defence:

Dominant Resistance (R) genes-

sensors of innate immunity


HRN CCC NB-ARC LRR

Breeding companies (Rijk Zwaan, Enza Seeds, Bejo Seeds etc.):

single dominant R genes

Innate immune sensors in plants and

mammals


(Plants: intracellular NLRs = dominant R genes)

The Bunyaviridae: Genome organization


NSs 32 kDa)

N (28 kDa)

Phlebovirus

RdRp (241 kDa)

Gn-Gc (139 kDa)

Orthobunyavirus/Hantavirus/Nairovirus

RdRp (247-459 kDa)

L RNAv

vc

M RNAGc-Gn (108-160 kDa)

vvc

S RNA

NSs (11-13 kDa)

N (26-50 kDa)

vvc

RdRp (331.5 kDa)

NSm (33.6 kDa)

Gn-Gc (127.4 kDa)

NSs (52.4 kDa)

N (28.8 kDa)

Tospovirus

NSs: viral defence against host innate immune responses

NSm: facilitates movement of viral entity between plant cells

Kormelink et al. (2011), Vir. Research

Kormelink (2011) in “ The Bunyaviridae” (Elliott & Plyusnin, eds.)

• The TSWV NSs is an RNAi suppressor

• The vertebrate-infecting bunyavirus NSs proteins acts

as IFN antagonists

• The TSWV tospovirus non-structural proteins NSs and

NSm trigger the resistance genes Tsw- and Sw-5

Exploiting RNAi:

Engineering multiple virus resistance


Transgene cassette with

target gene sequences from serveral viruses

Transcription

dsRNADICER

Viral specific siRNAs

TYRV-t WSMoV TCSV TSWV GRSV

Transgenic line

Wild type

High efficiency of resistance (66-73%) due to dsRNA

constructs

Virus-induced gene silencing (VIGS)


Defining gene functions using VIGS

pTRV2LB RB

MCS2x35S CP Rz NOSt

pTRV1RBLB

2x35S NOStRzRdRp

MP

16K

pTRV2LB RB

PDS2x35S CP Rz NOSt

+ PDS

pTRV1

+

pTRV2

At different time points:

Observe virus symptoms and

silencing effect

T=0

Inject A. tumefaciens suspensions

with TRV1 and TRV2 expression

cassette to N. benthamiana plants.

•pTRV1+ pTRV2-PDS• Plant viruses

Control

PDS silenced

Discovery and Identification of (new)

viruses by deep-sequencing of siRNAs


Li et al. (2012)Deep Sequencing of Small RNAs in Tomato for Virus and Viroid Identification and Strain Differentiation

To remember


• Plants have different mechanisms of defense against virus

infections (RNA interference, resistance genes)

• Plant viruses express factors that trigger these defense

mechanisms

• But also express factors that counteract (suppress) these

defense mechanisms

• Plant viruses can effectively be stopped by inducing RNAi

(transgenic resistance; hairpin constructs)

• VIGS is a powerful tool to identify gene functions

• sRNA sequencing is a powerful tool for virus discovery

viruses of plants: into the battle between viruses and their host...spread of plant viruses through...

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