PL PA 601 Plant Defenses and Viral Counterstrategies 30 March & 1 April, 2004 S. Lazarowitz

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1 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. p 1

2 PL PA 601 Plant Defenses and Viral Counterstrategies Spring 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: Baulcombe, D. C., and MacFarlane, S. A. (1999). Gene silencing: RNA makes RNA makes no protein. Curr Biol 9: R 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: Chen, C., and Z. Chen Isolation and characterization of two pathogen- and salicylic acid- induced genes encoding WRKY DNA-binding proteins from tobacco. Plant Mol Biol 42: 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: 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: 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: Erickson, F. L., S. Holzberg, A. Calderon-Urrea, V. Handley, M. Axtell, C. Corr, and B. Baker The helicase domain of the TMV replicase proteins induces the N- mediated defence response in tobacco. Plant J 18: Hamilton, A. J., and Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: Kasschau, K. D., and Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95: 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: Llave, C., Kasschau, K.D., Rector, M.A. and Carrington, J.C. (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 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: p 2

3 PL PA 601 Plant Defenses and Viral Counterstrategies Spring 2004 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: Lindenbach, B.D. and Rice, C.M. (2002) RNAi targeting an animal virus: news from the front. Mol Cell 9: 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: 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: Saenz, P., Salvador, B., Simon-Mateo, C., Kasschau, K.D., Carrington, J.C. and Garcia, J.A. (2002) Hostspecific involvement of the HC protein in the long-distance movement of potyviruses. J Virol 76: Tang, G., Reinhart, B.J., Bartel, D.P. and Zamore, P.D. (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17: Vaucheret, H., C. Beclin, T. Elmayan, et al. (1998). Transgene-induced gene silencing in plants. Plant J 16: 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: 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: 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: 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: Recent Reviews: Baulcombe, D. (2001). RNA silencing. Diced defence. Nature 409: Carthew, R. W. (2001). Gene silencing by double-stranded RNA. Curr Opin Cell Biol 13: Dangl, J 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: [For updated model of TMV movement see: Gillespie, T. et al. (2002). Plant Cell 14: ] McDowell, J. M., and J. L. Dangl Signal transduction in the plant immune response. Trends Biochem Sci 25: Hannon, G.J. (2002). RNA interference. Nature 418: 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: 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: Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15: Fire, A., et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 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: 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: 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: p 3

4 PL PA 601 Plant Defenses and Viral Counterstrategies Spring 2004 Kennerdell, J. R., and R. W. Carthew. (2000). Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol 18: 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: Montgomery, M. K., and A. Fire. (1998). Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends Genet 14: 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: Nishikura, K. (2001). A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107: Zamore, P.D., Tuschi, T. Sharp, P. and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATPdependent cleavage of mrna at 21 to 23 nucleotide intervals. Cell 101: p 4

5 Plant Defenses and Viral Counterstrategies Overview of Virus Life Cycle Factors in Pathogenesis Host Defenses Against Plant Virus Infection Recessive resistance Dominant (gene-for-gene) resistance (R-Avr) RNAi

6 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

7 Virus Life Cycle Extracellular (metabolically inert) Attachment* Intracellular (replicating) Entry/Penetration* mrna proteins genome uncoated replication (RNA = cytoplasm DNA = nucleus) Synthesis & Replication progeny virion Assembly Virus particle (virion) 1. Genome (DNA or RNA) 2. Protective protein coat (capsid) with or without envelope 3. Transport, attachment, facilitate penetration Release*

8 Plant Virus Life Cycle: Adaptations to the Cell Wall at Entry and Exit entry/penetration* genome uncoated Attachment/Penetration: Mechanical introduction initially. NO evidence attach to specific receptor sites on cell wall mrna proteins replication (RNA = cytoplasm DNA = nucleus) Transmission: plant viruses: insects, fungi, nematodes, abrasion, seeds, pollen, vegetative propagation progeny virion assembly release * Release: plant viruses channel through wall (MPs) without lysis

9 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

10 Plant Viruses use Movement Proteins to Move Cell to Cell without an Extracellular Phage and without Lysis CELL 1 CELL 2 PD MP Move through plasmodesmata (PD) that have been altered by a virus-encoded movement protein (MP) Encapsidated virus particle (virion) not essential Genome may be associated with MP or with CP CP functions for insect transmission

11 Stages in Pathogenesis Virus infection proceeds Series of obstacles Complete all stages = DISEASE Failure at any stage = Abortive or Nonproductive Infection

12 How a pathogen produces disease in its host Pathogenesis Combined effects of pathogen replication strategy, host defense responses, and the target tissues/organs of the host Viral Factors route of entry (mechanical, vectored by insects most common) 1 dose (number of infecting virions) 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 1 Noted are specific details for plant viruses

13 Viral genes that affect virulence may Alteration of viral virulence 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

14 Plant Virus Infection: The Host Side Immune (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 movement Host 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)

15 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 Neither movement within or between leaves limited What is usually economically important

16 Host Contributions to Pathogenesis: Genetic Approaches Resistance or decreased susceptibility to virus infection Lesion Mimics Genetic 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)

17 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 production Movement 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 production Animals: fever, edema, rash (pox) Plants: Hypersensitive Response (HR) (Avr-R interaction), RNAi

18 Plant Defenses Against Virus Infection Host Defense (Obstacle) Recessive resistance Mutation of a host gene needed for infection of a susceptible host Dominant gene-for gene resistance R gene # product recognizes a viral avirulence (avr) protein to set off active defense response Virus Counterstrategy Compensating mutation allows viral protein to interact with mutated host protein Mutate R gene target (Avr) Reactive Oxygen species # Nitric oxide # Pathogen Response (PR) Genes Salicyclic Acid, SAR response Apoptosis # 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

19 Potyviruses: Recessive Resistance is Due To Failure of an Essential Interaction Translation of Capped mrna eif4f complex p220 eif4e eif4a (CBP) m 7 G-cap eif4f complex AUG recruits 43S ribosome subunit initiation complex pa 3 (+factors) 43S m 7 AUG G-cap pa 3 scan 60S subunit m 7 AUG translate G-cap pa 3 pa tail cooperates in recruitment of eif4f

20 Potyviruses: Recessive Resistance is Due To Failure of an Essential Interaction 5 VPg (VPg-Pro) P1 HC-Pro P3 CI 6 NIa NIb CP 3 An RNA replication module 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.

21 Rx resistance to PVX strains in potato Gene-for-Gene Resistance Can Occur Without Cell Death No 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 tobacco Necrotic 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

22 Examples of Virus Genes that Overcome Natural Dominant Resistance Virus Resistance gene Mutants Overcome Resistance* TMV N (tobacco) N (tobacco) Tm-1(tomato) Tm-2 (tomato) replicase CP replicase MP PVX Nx (potato) CP Rx (potato) CP Any viral protein can be an Avr (viruses do it from within) *How identified as Avr

23 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

24 Time post-transfer 32C/20C N Gene-Mediated HR to TMV is Characterized by Cell Collapse and Necrosis 11 hr 15 hr Upper leaf epidermis (13-15 hr) Cell Collapse (visible symptoms) Palisade mesophyll (15-18 hr) Wright et al. (2000) Plant Physiol. 123: 1375

25 Time post-transfer 32C/20C 8h Cell death precedes tissue collapse TMV: N mediated HR But PD remain functional until cells collapse 9h 14h Evans blue (bright field) TMV GFP (confocal) Wright et al. (2000) Plant Physiol. 123: 1375

26 Xylem Transport is Transiently Disrupted in Developing TMV Lesion Single infected cells do not initiate HR 10h 11h At least 1 hr before visible cell collapse 11h Uninf d: vacuum grease (prevent transpiration) TMV.GFP MP (96h at 32C, h at 20C) 20h [Texas Red labeled xylem] Wright et al. (2000) Plant Physiol. 123: 1375

27 Virus-Induced Gene Silencing Gene expression in plants suppressed in a sequencespecific 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 CP Levels of transgene RNA and vrna drastically decreased Several mechanistically related phenomena Homology-dependent Gene Silencing Post-transcriptional gene silencing (PTGS) causing variation in transgene expression Cosuppression of endogenous genes and transgenes Sequence-specific degradation of RNA

28 PTGS Can Be Mediated by a Systemic Signal Stages: initiation systemic spread of silencing signal maintenance Phenomenon of homology-dependent gene silencing also found in C. elegans, fungi, paramecium, Drosophila, humans From Brigneti et al. EMBO J. 17:6739 (1998)

29 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, TMV Mapped to P1/HC-Pro (gene product, not RNA required) HC-Pro: enhances pathogenicity HC-Pro affects step common to broad range of viruses Transactivation of viral replication

30 Expression of HC-Pro Enhances Pathogenicity Mock infected PVX-5 TEV Vance et al. Virol. 206:583 (1995) PVX-NoHC PVX-5 TEV replicase PVX-HC replicase PVX-noHC replicase PVX-HC 25K8k 12K 25K 25K 8kAUG 12K 8k no AUG 12K Pruss et al. Plant Cell 9:859 (1997) P1/HC-Pro HC-Pro HC-Pro CP CP CP

31 PVY, But Not PVX, Suppresses Gene Silencing From Brigneti et al. EMBO J. 17:6739 (1998)

32 HC-Pro Expressed from a PVX Vector Suppresses Gene Silencing From Brigneti et al. EMBO J. 17:6739 (1998)

33 Viral Suppression of PTGS: A Common Counterstrategy of Many Viruses TEV, PVY: HC-Pro CMV (cucumo): 2b CpMV (como):? ACMV (gemini): TrAP (AL2) NMV, NVX, VMV (Potex, not all will!):? RYMV (sobemo): P1 TMV (tobamo):? TRV (tobra):? TBSV (tombus): 19K?=protein unknown 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

34 PVX 25 kd Movement Protein Prevents Spread of Silencing Signal While trying to study if systemic silencing occurs during virus infection (previous studies all Agro-induced silencing in transgenic plants) Voinnet et al. (2000) Cell 103:157

35 PTGS: An Innate Anti-Viral Defense System Operating at the RNA Level Activated when viral RNA or transgene or aberrant RNA is recognized Also induced by dsrna Targeted degradation of RNA in a sequence-specific manner Restricts viral (or transgene) RNA accumulation in infected cells Specificity 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 front Would suppress virus movement as well as RNA accumulation Precise 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

36 Genetic and Biochemical Models for RNAi/PTGS RdRp Dicer-2 eif2c similarity sirna 21-23bp-RNA R2D2 Dicer-2 complex AGO2:23bp RNAs:RNase RdRp R2D2: transition of sirna from nuclease substrate to nuclease specificity factor RISC (RNA-induced Silencing Complex) Dicer-2 Dicer-2:siRNA:R2D2 RdRp extend dsrna RdRp extend dsrna AGO2: 23 bp RNA: RNase?= helicase needed to unwind dsrna: smg-2 (C.elegans), sde3 (Arabidopsis)

37 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)?