PL PA 601 Plant Defenses and Viral Counterstrategies

30 March & 1 April, 2004 S. Lazarowitz

A. Virus Definitions and Basic Principles (Lazarowitz 2001). 1. Unique properties of viruses

a. Obligate intracellular parasites that do not undergo binary fission. b. Parasitic genomes related to plasmids that can exist in 2 forms: (1) extracellular metabolically

inert form (the virion); and (2) intracellular replicating form. 2. Structure of viral genomes: DNA or RNA, single (ss) or double (ds) strand.

a. Virion (virus particle): Genome surrounded by protective protein coat (the capsid). Can further be surrounded by lipid-containing membrane (the envelope).

b. All virion proteins are viral-encoded. Lipid membrane (in enveloped viruses) is encoded by the host: acquired from host cell membranes (plasma membrane, Golgi, ER, nuclear envelope) during virion assembly.

c. CP: coat protein, makes up the capsid. Rep: replicase, viral replication protein.

i. Required to replicate viral genome. ii. Recognizes viral genome structure: the origin of replication (ori).

MP: movement protein, required for cell-to-cell and systemic virus spread. i. Uniquely encoded by plant viruses. ii. Overcome barrier of plant cell wall.

B. Virus multiplication: Viruses do it from within. 1. Stages of viral life cycle: (1) attachment; (2) penetration and uncoating; (3) synthesis and

replication; (4) progeny virus assembly; (5) release. 2. Based originally on studies of animal viruses and bacteriophage 3. Getting In and Out: Consequences of the cell wall, plant viruses differ at stages (1) attachment,

and (5) release. a. No evidence for specific receptors on plant cell walls. b. Plant viruses encode movement proteins that allow them to move cell-to-cell without

going through an extracellular phase (Lazarowitz and Beachy 1999). C. The pathogenic process as a competition between virus and host.

1. Nonhost: not permissive for virus replication 2. Host: non-specific or specific defenses

a. Specific plant defenses: (i)recessive resistance; (ii)dominant R genes (HR); (iii) RNAi

b. Virus counterstrategy i. Mutate viral protein that interacts with essential host factor. ii. Mutate Avr recognized by plant R gene product. iii. Encode suppressor of RNAi defense.

c. Viral Avr can be CP, Rep or MP D. Recessive resistance is important as a strategy against plant virus infection.

1. Hypothesized to result from mutation of a host factor essential for virus infection. 2. Recent studies on recessive resistance against potyvirus infection support this hypothesis.

a. Potyvirus VPg found to interact with host cap-binding protein eIF4E (Leonard et al., 2000). b. Arabidopsis plants that lack eIF4E(iso) are specifically resistant to potyvirus infection

(Duprat et al., 2002; Lellis et al., 2002). c. A candidate gene approach maps recessive resistance against potyvirus infection in

pepper to a mutant allele of eIF4E (Ruffel et al., 2002). E. Gene-for-gene resistance occurs, but is not the most important plant defense against viruses.

1. Hypersensitive cell death is commonly associated with gene-for-gene resistance and may strengthen the induction of host responses.

2. Cell death and resistance are separable events. 3. A detailed study of N gene resistance and the development of HR against TMV in tobacco (Wright

et al., 2000) shows that cell death precedes tissue collapse and that PD remain functional up until the stage of cell collapse. This suggests that closing down PD is not part of the development of the HR. Xylem transport is also transiently disrupted in the developing TMV necrotic lesion.

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PL PA 601 Plant Defenses and Viral Counterstrategies Spring 2004

4. Initiation of HR is at least a two stage process: Single TMV-infected cell is not sufficient. F. Homology-dependent gene silencing (now known as RNAi) appears to be an innate defense response to

virus infection in plants (Baulcombe and MacFarlane, 1999). 1. Potyvirus synergism led to the discovery of a virus-encoded suppressor of posttranscriptional gene

silencing (PTGS): TEV and PVY HC-Pro suppresses PTGS. 2. CMV 2b also shown to suppress PTGS (Anandalakshmi et al., 1998; Baulcombe and MacFarlane,

1999; Brigneti et al., 1998; Kasschau and Carrington, 1998). 3. Survey of viruses from diverse families suggest that suppression of PTGS may be widely used as a

counterstrategy by RNA and DNA viruses (Voinnet et al., 1999). a. Differences in spatial pattern and degree of suppression suggest different viral-encoded

suppressors may have distinct modes of action. 4. A small (25-nt) antisense RNA appears to be the specificity determinant of PTGS (Hamilton and

Baulcombe, 1999; Elbashir et al., 2001; Zamore et al., 2000). 5. A viral MP can suppress the RNAi systemic signal (Voinnet et al., 2000).

G. RNAi is highly conserved among eukaryotes. 1. Biochemical and/or genetic studies in Drosophila, C. elegans and Arabidopsis are defining the

pathway (Baulcombe, 2001; Bernstein et al., 2001; Caplen et al., 2000; Dalmay et al., 2000b; Dalmay et al., 2001; Hammond et al., 2001; Ketting et al., 2001; Montgomery et al., 1998; Nishikura, 2001; Tang et al, 2003).

2. Recent studies suggest a role in defense against animal virus infection (Lindenbach and Rice, 2002) 3. Studies in C. elegans and Arabdiopsis suggest a role in regulating development (Kasschau et al.,

2003; Kettering et al., 2001; Llave et al., 2002). References Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., and Vance, V. B. (1998). A

viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95: 13079-84. Baulcombe, D. C., and MacFarlane, S. A. (1999). Gene silencing: RNA makes RNA makes no protein. Curr

Biol 9: R599-601. Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S. W., and Baulcombe, D. C. (1998). Viral pathogenicity

determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17: 6739-46. Chen, C., and Z. Chen. 2000. Isolation and characterization of two pathogen- and salicylic acid- induced genes

encoding WRKY DNA-binding proteins from tobacco. Plant Mol Biol 42: 387-96. Dalmay, T., Hamilton, A., Rudd, S., Angell, S., and Baulcombe, D. C. (2000). An RNA-dependent RNA

polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101: 543-553.

Dalmay, T., Horsefield, R., Braunstein, T. H., and Baulcombe, D. C. (2001). SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. EMBO J 20: 2069-2078.

Duprat, A., Caranta, C., Revers, F., Menand, B., Browning, K.S. and Robaglia, C. (2002) The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses. Plant J 32: 927-934.

Erickson, F. L., S. Holzberg, A. Calderon-Urrea, V. Handley, M. Axtell, C. Corr, and B. Baker. 1999. The helicase domain of the TMV replicase proteins induces the N- mediated defence response in tobacco. Plant J 18: 67-75.

Hamilton, A. J., and Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: 950-2.

Kasschau, K. D., and Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95: 461-70.

Kasschau, K.D., Xie, Z., Allen, E., Llave, C., Chapman, E.J., Krizan, K.A. and Carrington, J.C. (2003) P1/HC-Pro, a Viral Suppressor of RNA Silencing, Interferes with Arabidopsis Development and miRNA Function. Dev Cell 4: 205-217.

Llave, C., Kasschau, K.D., Rector, M.A. and Carrington, J.C. (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605-1619.

Lazarowitz, S. G. (2001). Plant Viruses. Virology. D. M. Knipe and P. M. Howley, eds. Philadelphia, Lippincott, Williams and Wilkins: Chapter 14.

Lellis, A.D., Kasschau, K.D., Whitham, S.A. and Carrington, J.C. (2002) Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection. Curr Biol 12: 1046-1051.

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Leonard, S., Plante, D., Wittmann, S., Daigneault, N., Fortin, M.G. and Laliberte, J.F. (2000) Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol 74: 7730-7737.

Lindenbach, B.D. and Rice, C.M. (2002) RNAi targeting an animal virus: news from the front. Mol Cell 9: 925-927.

Pruss, B., Ge., X., Shi., S.M., Carrington, J.C. and Vance, V.B. (1997). Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9: 859-868.

Ruffel, S., Dussault, M.H., Palloix, A., Moury, B., Bendahmane, A., Robaglia, C. and Caranta, C. (2002) A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E). Plant J 32: 1067-1075.

Saenz, P., Salvador, B., Simon-Mateo, C., Kasschau, K.D., Carrington, J.C. and Garcia, J.A. (2002) Host-specific involvement of the HC protein in the long-distance movement of potyviruses. J Virol 76: 1922-1931.

Tang, G., Reinhart, B.J., Bartel, D.P. and Zamore, P.D. (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17: 49-63.

Vaucheret, H., C. Beclin, T. Elmayan, et al. (1998). Transgene-induced gene silencing in plants. Plant J 16: 651-659.

Voinnet, O., and Baulcombe, D. C. (1997). Systemic signaling in gene silencing. Nature 389: 553. Voinnet, O., Vain, P., Angell, S., and Baulcombe, D. C. (1998). Systemic spread of sequence-specific

transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95: 177-87.

Voinnet, O., Pinto, Y. M., and Baulcombe, D. C. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96: 14147-52.

Voinnet, O., Lederer, C., and Baulcombe, D. C. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103: 157-167.

Wright, K.M., Duncan, G.J., Pradel, K.S., Carr, F., Wood, S., Oparka, K.J., and Santa-Cruz, S. (2000). Analysis of the N gene hypersensitive response induced by a fluorescently tagged tobacco mosaic virus. Plant Physiol 123: 1375-1385.

Recent Reviews: Baulcombe, D. (2001). RNA silencing. Diced defence. Nature 409: 295-296. Carthew, R. W. (2001). Gene silencing by double-stranded RNA. Curr Opin Cell Biol 13: 244-248. Dangl, J. 1998. Innate immunity. Plants just say NO to pathogens. Nature 394: 525, 527. Lazarowitz, S. G. and R. N. Beachy (1999). Viral movement proteins as probes for investigating intracellular

and intercellular trafficking in plants. Plant Cell 11: 535-548. [For updated model of TMV movement see: Gillespie, T. et al. (2002). Plant Cell 14: 1207-1222.]

McDowell, J. M., and J. L. Dangl. 2000. Signal transduction in the plant immune response. Trends Biochem Sci 25: 79-82.

Hannon, G.J. (2002). RNA interference. Nature 418: 244-251. RNAi in C. elegans and Drosophila (where most of the genetics and biochemistry has been done): Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. (2001). Role for a bidentate ribonuclease in the

initiation step of RNA interference. Nature 409: 262-366. Caplen, N. J., Fleenor, J., Fire, A., and Morgan, R. A. (2000). dsRNA-mediated gene silencing in cultured

Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 252: 95-105. Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide

RNAs. Genes Dev 15: 188-200. Fire, A., et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis

elegans. Nature 391: 806-11. Hammond, S.M., Bernstein, E., Beach, D. and Hannon, G.J. (2000). An RNA-directed nuclease mediates

posttranscriptional gene silencing in Drosophila cells. Nature 404: 293-296. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R., and Hannon, G. J. (2001). Argonaute2, a link

between genetic and biochemical analyses of RNAi. Science 293: 1146-1150. Kelly, W. G., Xu, S., Montgomery, M. K., and Fire, A. (1997). Distinct requirements for somatic and germline

expression of a generally expressed Caernorhabditis elegans gene. Genetics 146: 227-238.

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Kennerdell, J. R., and R. W. Carthew. (2000). Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol 18: 896-898.

Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J., and Plasterk, R. H. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15: 2654-2659.

Montgomery, M. K., and A. Fire. (1998). Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends Genet 14: 255-8.

Montgomery, M. K., Xu, S., and Fire, A. (1998). RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci U S A 95: 15502-7.

Nishikura, K. (2001). A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107: 415-418.

Zamore, P.D., Tuschi, T. Sharp, P. and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25-33.

Plant Defenses and Viral Counterstrategies

Overview of Virus Life Cycle

Factors in Pathogenesis

Host Defenses Against Plant Virus InfectionRecessive resistanceDominant (gene-for-gene) resistance (R-Avr)RNAi

Viruses Defined by Unique Properties

Obligate intracellular parasites that do not undergo binary fission

distinguish from bacterial pathogens and other parasites

Parasitic genomes related to plasmids that can exist in two forms

distinguish from other life forms

Virus Life CycleExtracellular (metabolically inert) Intracellular (replicating)

genome uncoated

proteins

mRNA replication(RNA = cytoplasmDNA = nucleus)

Synthesis &

Replication

progeny virion Assembly

Attachment*Entry/Penetration*

Release*Virus particle (virion)1. Genome (DNA or RNA)2. Protective protein coat (capsid)

with or without envelope3. Transport, attachment,

facilitate penetration

Plant Virus Life Cycle: Adaptations to the Cell Wall at Entry and Exit

entry/penetration*

genome uncoated

mRNA replication

proteins

progeny virion assembly

“release” *

(RNA = cytoplasmDNA = nucleus)

Release:plant viruses “channel” through wall (MPs) without lysis

Transmission:plant viruses: insects, fungi, nematodes, abrasion, seeds, pollen, vegetative propagation

Attachment/Penetration:Mechanical introduction initially. NO evidence attach to specific receptor sites on cell wall

Virus Intercellular Transport The goal is for progeny viruses to infect new host cells and repeat the infectious cycle

➧ May be an immediate neighbors or a distant cells

➧ In animals and humans, the circulatory and nervous systems are conduits for systemic infection (dissemination to distant sites)

All Phage and most Animal Viruses go through an extracellular form (released virions) to infect additional cells, even locally

➧ Local infection will occur through interstitial or extracellular fluids (e.g. respiratory viruses will spread through mucus)

All Plant Viruses spread directly cell to cell within a leaf without an extracellular phase (local movement)

➧ An extracellular form is transported leaf to leaf through the phloem (nutrient transport system) for systemic (long distance) infection

➧ For most, but not all, plant viruses the extracellular form appears to

be virus particles

Plant Viruses use Movement Proteins to Move Cell to Cell without an Extracellular Phage and without Lysis

CELL 1 CELL 2

Move through plasmodesmata (PD) that have been altered by a virus-encoded movement protein (MP)

PD

MP

Encapsidated virus particle (virion) not essentialGenome may be associated with MP or with CPCP functions for insect transmission

Stages in Pathogenesis

Virus infection proceeds Series of obstacles

Complete all stages DISEASE=

Failure at any stage Abortive or Nonproductive Infection

=

Pathogenesis

• How a pathogen produces disease in its host

• Combined effects of pathogen replication strategy, host defense responses, and the target tissues/organs of the host

Viral Factors

dose (number of infecting virions)

route of entry (mechanical, vectored by insects most common)1

path to susceptible cells (Local: cell-to-cell. Systemic: phloem, although a few spread via xylem)1

rates of viral multiplication and spread

effects of virus on cell function

intracellular state of viral genome 1Noted are specific details for plant viruses

Alteration of viral virulence

• Viral genes that affect virulence may

1) Affect the ability of the virus to replicate (CP, Replicase)

2) Enable the virus to spread in the host or between hosts (MP, CP)

3) Defeat the host’s defense mechanism (suppress RNAi:e.g. HC-Pro)

4) Produce gene products that are directly toxic• Demonstrated for animal viruses

• No example for plant viruses

Plant Virus Infection: The Host SideImmune (non-host)

No virus replication in plant (even in initially inoculated cell) or in protoplasts from that plant

Failure: Incompatible interactions with host factors required for viral uncoating, transcription, and/or replication

Infectible (host)Subliminal infection: multiplication limited to inoculated cells

Failure: MP not function in cell-to-cell movementHost RNAi response

Hypersensitivity: infection limited to localized zone of cells by induced host responses ( R-Avr interaction)

Susceptible: systemic movement and replication“sensitive” - disease symptoms“tolerant” - little or highly attenuated disease symptoms“masked symptoms” - temporarily symptomless subject to environmental conditions (e.g. temperature)

Potential Barriers to Cell-to-Cell and Systemic (Long Distance) Movement

Movement out of initial inoculated cells or limited cell-to-cell movement

Local lesion (microscopic or macroscopic)

Movement out of parenchyma cells into vascular tissues in inoculated leaf

Movement out of vascular tissue into parenchyma cells in systemic leaf

What is usually economically important

Neither movement within or between leaves limited

Host Contributions to Pathogenesis: Genetic Approaches

Resistance or decreased susceptibility to virus infection

Lesion MimicsGenetic mutants: Maize (rp1), Barley (mlo)

Transgenic plants: reduced catalase (tobacco)

Inhibit ubiquitin pathway

Halobacterium bacterio-opsin (bO) proton pump

Spontaneous cell death mutants (Arabidopsis: acd, lsd)Suppressors of these mutants (phx)

Enhanced Disease Susceptibility (eds, ndr) mutants (Arabidopsis)

Disease Symptoms Result from Injury to Discrete Cell Populations in the Plant

Injured mesophyll cells: Lack of photosynthesis produces yellowing (chlorotic symptoms).

Hypersensitive response (HR) is characterized by local apoptosis and cell collapse. Usually produces necrotic lesions. (Avr-R

interaction)Injured phloem: Disruption of photoassimilate transport to developing parts of plant produces developmental abnormalities (blistering, or upward or downward leaf curl due to uneven growth of leaf lamina) or wilting, and death of the plant.

Injured meristems/developing cells (RNAi): Developmental abnormalities.

Mutational studies implicate all plant virus genes in symptom productionMovement proteins directly determine pathogenic properties (as tested in transgenic plants). Coat protein and replicase do so indirectly as the result of their effects on the efficiency of virus replication and spread

Host defense responses can contribute to symptom productionAnimals: fever, edema, rash (pox)

Plants: Hypersensitive Response (HR) (Avr-R interaction), RNAi

Plant Defenses Against Virus InfectionHost Defense (Obstacle) Virus Counterstrategy

Recessive resistanceCompensating mutation allows viral protein to interact with mutated host protein

Mutation of a host gene needed for infection of a susceptible host

R gene# product recognizes a viral ‘avirulence’ (avr) protein to set off active defense response

Pathogen Response (PR) Genes

Apoptosis#

Salicyclic Acid, SAR response

Reactive Oxygen species#

Nitric oxide#

Dominant ‘gene-for gene’ resistanceMutate R gene target (Avr)

RNA interference (RNAi)*#

[Induced Gene Silencing]Suppress posttranscriptional gene silencing

# Innate defense (immune) responses common to animals and plants*Homology-dependent gene silencing, posttranscriptional gene silencing

Potyviruses: Recessive Resistance is Due To Failure of an Essential Interaction

Translation of Capped mRNA

m7G-cap

eIF4F complex

pA 3’AUGeIF4E (CBP)

p220

eIF4A

m7G-cap pA 3’AUG

recruits 43S ribosome subunit initiation complex

scan

43S

eIF4F complex

(+factors)

m7G-cap pA 3’

60S subunit

AUGtranslate

pA tail cooperates in recruitment of eIF4F

Potyviruses: Recessive Resistance is Due To Failure of an Essential Interaction

5’AnP1 HC-Pro P3 CI 6 NIa NIb CP

RNA replication module

3’(VPg-Pro)

VPg

Yeast 2-hybrid and in vitro binding assays:TEV or PVY VPg binds Arabidopsis or Tobacco eIF4E or eIF(iso)4E (the cap binding protein)

The ability of TEV or PVY mutants to overcome recessive resistance in pepper or in lettuce maps to VPg

Arabidopsis mutants that do not express eIF(iso)4E are specifically resistant to potyvirus infection (TEV, PVY, turnip mosaic virus)

Using a candidate gene approach, the recessive resistance alleles against PVY and TEV infection in pepper map to eIF4E containing specific missense mutations.

Leonard et al. (2000) J Virol, 74, 7730, (2002); Duprat et al. (2002) Plant J 32:927; Ruffel et al. (2002) Plant J 32:1067: Lellis et al (2002) Curr Biol 12, 1046.

Gene-for-Gene Resistance Can Occur Without Cell Death

Rx resistance to PVX strains in potatoNo visible symptoms or necrotic lesions

Rx expressed in N. benthamiana: PVX resistance associated with necrotic local lesions

Cell death and resistance are separate events

N resistance to TMV in tobaccoNecrotic lesions (requires oxygen)Low oxygen: No necrotic lesions, but resistance not compromised

Hypersensitive cell death is commonly associated with gene-for-gene resistance and may strengthen the induction of host responses

Examples of Virus Genes that Overcome Natural Dominant Resistance

TMV

Resistance gene Mutants Overcome Resistance*Virus

N (tobacco) replicase

N’(tobacco) CP

Tm-1(tomato) replicase

Tm-2 (tomato) MP

PVX Nx (potato) CP

Rx (potato) CP*How identified as Avr

Any viral protein can be an Avr (viruses do it from within)

N Gene Resistance to TMV in Tobacco

Best characterized example of a dominant (R gene) gene-for-gene interaction against virus infection

N gene cloned: Encodes an NBS-LRR cytoplasmic protein

Sequence similarity to Toll (Drosophila) and IL-1R. Mutational studies suggest N mediates rapid gene induction and TMV resistance through a Toll-IL-1-like pathway

TMV replicase appears to be the target recognized by the N gene-encoded protein

Single infected cell not sufficient to induce HR response

N Gene-Mediated HR to TMV is Characterized by Cell Collapse and Necrosis

11 hr 15 hr

Upper leaf epidermis(13-15 hr)

Palisade mesophyll(15-18 hr)

Cell Collapse (visible

symptoms)

Wright et al. (2000) Plant Physiol. 123: 1375

Time post-transfer 32C/20C

Cell death precedes tissue collapse

Evans blue (bright field) TMV–GFP (confocal)

8h

9h

14h

Tim

e po

st-tr

ansf

er

32C

/20C TMV: N–mediated HR

But PD remain functional until cells collapse

Wright et al. (2000) Plant Physiol. 123: 1375

Xylem Transport is Transiently Disrupted in Developing TMV Lesion

10h

11h

20h

11h Uninf’d: vacuum grease (prevent transpiration)

TMV.GFP∆MP(96h at 32C, 48-120h at 20C)

Single infected cells do not initiate HR

Wright et al. (2000) Plant Physiol. 123: 1375

At least 1 hr before visible cell collapse

[Texas Red labeled xylem]

Virus-Induced Gene Silencing

Gene expression in plants suppressed in a sequence-specific manner by infection with virus vectors that carry fragments of host genes

First evident in attempts to engineer CP-mediated resistance

Resistance not necessarily dependent on expression of CPLevels of transgene RNA and vRNA drastically decreased

Homology-dependent Gene SilencingSeveral mechanistically related phenomena

Post-transcriptional gene silencing (PTGS) causing variation in transgene expression

Cosuppression of endogenous genes and transgenesSequence-specific degradation of RNA

PTGS Can Be Mediated by a Systemic Signal

initiationsystemic spread of silencing signalmaintenance

Stages:

From Brigneti et al. EMBO J. 17:6739 (1998)

Phenomenon of homology-dependent gene silencing also found in C. elegans, fungi, paramecium, Drosophila, humans

Synergism

Interaction of 2 unrelated viruses in the same host causes dramatic increase in symptoms and accumulation of 1 of the co-infecting viruses

Commonly seen in co-infections with a potyvirus

TEV, PVY: enhance pathogenicity of PVX, CMV, TMVMapped to P1/HC-Pro (gene product, not RNA required)

HC-Pro: enhances pathogenicity

HC-Pro affects step common to broad range of virusesTransactivation of viral replication

Expression of HC-Pro Enhances Pathogenicity

replicase CPP1/HC-Pro25K

12K

8kPVX-5’TEV

replicase CPHC-Pro25K

12K

8kAUG

replicase CPHC-Pro25K

12K

8kno

AUG

PVX-HC

PVX-noHC

Mock infected

PVX-5’TEV

PVX-NoHC PVX-HC Pruss et al. Plant Cell 9:859 (1997)

Vance et al. Virol. 206:583 (1995)

PVY, But Not PVX, Suppresses Gene Silencing

From Brigneti et al. EMBO J. 17:6739 (1998)

HC-Pro Expressed from a PVX Vector Suppresses Gene Silencing

From Brigneti et al. EMBO J. 17:6739 (1998)

Viral Suppression of PTGS: A Common Counterstrategy of Many Viruses

TEV, PVY: HC-Pro

CMV (cucumo): 2b

ACMV (gemini): TrAP (AL2)

NMV, NVX, VMV (Potex, not all will!): ?

RYMV (sobemo): P1

TMV (tobamo): ?

TRV (tobra): ?

TBSV (tombus): 19K

?=protein unknown

CpMV (como): ?

All tested for suppressing PTGS of GFP transgene based on infection with particular virus or expression of viral “pathogenicity factor” from replicating PVX vector

NOT suppress PTGS: ALMV (alfamo), TBRV (nepo), several potex (PVX, FoMV) in THIS study, but…

Voinnet et al. (1999). PNAS 96:14147

PVX 25 kD Movement Protein Prevents Spread of Silencing Signal

While trying to study if systemic silencing occurs during virusinfection (previous studies all Agro-induced silencing in transgenic plants)…

Voinnet et al. (2000) Cell 103:157

PTGS: An Innate Anti-Viral Defense System Operating at the RNA Level

Activated when viral RNA or transgene or ‘aberrant’ RNA is recognizedAlso induced by dsRNA

Targeted degradation of RNA in a sequence-specific mannerRestricts viral (or transgene) RNA accumulation in infected cellsSpecificity mediated by ~23 nt silencing RNAs (siRNAs) that

correspond to the target and accumulate

Not cell autonomous: Sequence-specific signal molecule spreads away from cells where process initiated (“Systemic Signal”)

Activate viral RNA degradation in cells beyond the infection frontWould suppress virus movement as well as RNA accumulationPrecise signal not known, but thought to incorporate a nucleic acid

Viruses suppress this response (PVY, TEV, CMV, PVX), although current evidence suggests the precise mechanisms differ

Virus-induced Gene Silencing: A Method for Rapid Functional Genomics

Genetic and Biochemical Models for RNAi/PTGS

21-23bp-RNA–R2D2–Dicer-2 complex

eIF2C similarity

RdRp

RdRp

Dicer-2

Dicer-2Dicer-2:siRNA:R2D2

AGO2:23 bp RNA:

RNase

RISC (RNA-induced Silencing Complex)

RdRp extend dsRNA

RdRp extend dsRNA

AGO2:23bp RNAs:RNasesiRNA

R2D2: transition of siRNA from nuclease substrate to nuclease specificity factor

?= helicase needed to unwind dsRNA: smg-2 (C.elegans), sde3 (Arabidopsis)

Proposed Roles for PTGS (RNAi)Surveillance system to repress transposon activity (repress

transposition, C. elegans)

Regulate expression of genes needed for normal growth and development

• Endogenous microRNAs (21-24 nts) found in Arabidopsis, C. elegans, Drosophila and human cells

• Arise from structured precursor RNAs, mostly from intergenic regions. Accumulation developmentally regulated.

• Proposed role in range of posttranscriptional and epigenetic events. Negatively regulate developmentally important genes by interacting with mRNAs encoding key regulatory factors.

Anti-viral defense• HC-Pro acts to inhibit miRNA-guided cleavage of mRNAs encoding several transcription factors. Why some viruses cause developmental abnormalities during infection (pathogencity factors)?