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Websites to brush up on viral diseases Barley Yellow Dwarf http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/BarleyYelDwarf.aspx Papaya Ringspot Virus http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/PapayaRingspotvirus.aspx Tobacco Mosaic Virus http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/TobaccoMosaic.aspx Lab final is in two weeks! –Thursday, November 29 Study guide on web site – Lab manual is ultimate study guide! *If you want an update on your points on exams and assignments, please email me* RNA genome of TMV: ~6,400 nucleotides, three genes, and three major functions RNA REPLICATION RNA ENCAPSIDATION CELL-TO-CELL MOVEMENT QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Virus Life Cycle 2 Genome uncoating, expression and replication Replicase (RNA Viral genome uncoating of virion Host ribosomes translate (express) viral genome translation polymerase) enzyme (gene product) New protein coat subunit (gene product) Cell to cell movement protein (gene product) Gene Products: replication Replicase enzyme makes new copies of virus’ genome 1 Replicase (RNA polymerase) enzyme Host ribosomes New protein coat subunits Cell to cell movement protein The replication cycle of Tobacco mosaic virus (TMV). TMV enters a wounded plant cell to begin the replication cycle [1]. As the cost protein (CP) molecules are stripped away from the RNA [2], host ribosomes begin to translate the two replicase-associated proteins. The replicase proteins (RP) are used to generate a negative-sense (- sense) RNA template from the virus RNA [3]. This - sense RNA is, in turn, used to generate both full-length positive-sense (+ sense) TMV RNA [4] and the + sense subgenomic RNAs (sgRNAs) [5] that are used to express the movement protein (MP) and CP. The + sense TMV RNA is either encapsidated by the CP to form new TMV particles [6] or wrapped with MP [7] to allow it to move to an adjacent cell for another round of replication. Cell-to-cell movement of Tobacco mosaic virus (TMV). In this model, the movement protein (MP) binds to the viral RNA [1]. Host proteins and/or other virus-encoded proteins may be included in the MP-complex [2]. The MPcomplex then moves from cell-to-cell through the plasmodesmata [3]. When the complex is localized to a new cell, the MP (and any host proteins) are presumably released from the TMV RNA [4], allowing for translation of the genomic RNA to express the replicase proteins and to initiate a new round of replication [5], as shown in previous slide. How much would you pay for a diseased plant? ‘Tulip Mania’ The Netherlands 1630 to 1660 Symptoms of tulip breaking virus 2 Viruses http://www.apsnet.org/edcenter/intropp/PathogenGroups/Pages/PlantViruses.aspx ~2500 named viruses - many more undiscovered Obligate, intracellular parasites of virtually all cellular organisms 25% of known viruses are parasites of plants Like other pathogen groups, some viruses infect a broad range of host, whereas others infect a single plant species only Discovery of viruses as agents of disease: Transmission: 1886 sap, 1904 graft, 1922 insect vectors 1892 – sap transmission after passage through bacteria proof porcelain filters Visualization: 1940 electron micrograph Purification: 1950 Genomics: 1980’s 1st virus genome completely sequenced Viruses have the following properties: Very small genome - 104-105 nucleotides – this typically codes for 4-7 proteins Viruses contain a single type of nucleic acid, either RNA (most plant viruses) or DNA When completely assembled (the virion), the nucleic acid is protected by a protein coat (the capsid) consisting of individual protein subunits Viruses multiply inside living cells by hijacking the biosynthetic machinery (ribosomes) of the host Examples of virions (intact particles) of common plant viruses Diversity of plant viruses Genomes of most plant viruses are comprised of ‘single-stranded RNA’ with the virion shaped as a sphere, a rod, or a filament. Typically, the complete genome is contained in a single particle (monopartite), but some viruses require multiple particles (bipartite, tripartite). The other major classifications are double-stranded RNA, singlestranded DNA, double-stranded DNA. 3 Plant viruses and disease Viruses are obligate, biotrophic pathogens – unlike other pathogen groups, many viruses can infect and replicate within their hosts without causing obvious symptoms Consequently, why symptoms (disease) develop is poorly understood. Increasingly, it appears that many viruses suppress normal recycling of messenger RNA (this is called RNA silencing). Thus, symptoms may result from prolonged, over expression of the host’s own genes. In nature, viruses that cause severe symptoms in their principal host are likely to go extinct. Ring spot symptom Foliar symptoms of virus diseases Typically, viruses cause chlorosis or color breaks in mottled or mosaic patterns Corn mosaic Calico mosaic of potato Tulip break Lettuce big vein Bean yellows mosaic Internal symptoms of virus diseases Stem pitting Net necrosis of of potato leaf roll Black line symptom hypersensitive reaction at the graft union 4 Local lesion symptom of a virus disease Hypersensitive reaction (HR) observed after artificial inoculation of infected plant sap onto a local lesion or ’indicator’ host. Indicator hosts were developed to aid viral disease diagnosis. Virus movement within plants First leaf infected After infection, viruses move systemically throughout a plant via the phloem transport system. First down, then up, then throughout. Black represents areas of the plant where the virus can be found Virus transmission between plants Vegetative propagation Grafting High rates of transmission for nearly all viruses Mechanical transfer of sap (rubbing, cutting, handling, etc. - relatively uncommon in plant production but important in the lab ) Seed ~100 viruses – efficiency (% of seed with virus) usually low Pollen 9 viruses – usually a fruit crop (blueberry, caneberry, cherry) Mite, Nematode & Fungal vectors – a few dozen viruses INSECTS !! aphids, leafhoppers, whiteflies, others – this is the most common and economically important means of virus transmission 5 Important insect vectors of plant viruses Aster leafhopper Green peach aphid Alate (winged) adult Apterous (wingless) adult Silver leaf whitefly Mother with children Non-persistent and Persistent types of vector transmission Feeding behavior of aphids anti-bruising lifters and conveyers ‘Test-probe’ into ‘Feeding-probe’ – often epidermal cell into phloem tissue This type of feeding behavior is associated with ‘non-persistent’ or ‘stylet-born’ virus transmission (~230 viruses) Test-probes are frequent and of short duration (seconds) This type of feeding behavior is associated with ‘persistent’ or ‘circulative’ virus transmission. (~80 viruses) Feeding-probes are infrequent and of long duration (min to hr) 6 Persistent (circulative) virus transmission After uptake, virus particles move through the insect’s digestive system – In the hind gut, the virus is transferred to the hemocoel (blood), then concentrates in the salivary gland. The virus is transmitted in saliva during feeding probes. Characterization of types of vectored transmission Persistent: Non-Persistent: (circulative / propagative) (stylet-born) Minutes to hours Acquisition time seconds yes Incubation period in insect no yes Insect infective after molt no Minutes to hours Transmission time seconds no Virus inoculated mechanically yes yes Disease spread reduced with insecticides NO! Vector control and non-persistent transmission 100% Incidence of virus infected plants (%) Watermelon mosaic virus potyvirus, non-persistent aphid vector Treatments: untreated insecticide (can accelerate test probe behavior) light mineral oil (clogs stylet) reflective foil mulch (winged aphid repelled by reflected sky-color Disease progress curves reveal treatment differences 0 Time (days) 70 7 Viral diagnostics • Pathogenicity -- Bioassays using indicator hosts • Transmissibility – Vector transmission assays • Architecture of the virus particle -- Electron microscopy • Antigenic properties of the viral protein coat - ELISA (enzyme-linked immunosorbent assay) • Characterization of viral nucleic acid – – Polymerase chain reaction amplification (PCR) and agarose gel electrophoresis – Detection of double-stranded RNA in host tissue – Genome sequencing Enzyme-linked immunosorbent assay (ELISA) is the most practical method for testing large numbers of plants for virus infection Antibody is made in a rabbit or mouse WASH WASH Purified antibody Is attached To plate ELISA plate – colored wells are positives Cartoon of strip-type ELISA: www.whfreeman.com/catalog/static/whf/kuby/content/anm/kb07an01.htm 8