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Chapter Outlines

Chapter 20       Plant Viruses

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20.1 History of Plant Viruses Tulipomainia - craze in which bulbs of infected tulips were traded for large sums of money Birth of Virology 1900's Martinus Beijerinck - discovery of a plant virus that infected tobacco Mechanical Transmission Most common technique for experimental infection of plants Inoculate or rub virus-containing preparation into leaves of healthy susceptible plants 1922 Kunkel published the first paper to recognize that insects transmitted some plant viruses 1939 Holmes Handbook of Phytopathogenic Viruses Lists 129 known viral diseases of plants ICTV lists ~900 plant viruses today 1930's to 1950's focus on tobacco mosaic virus (TMV) Virus structure using X-ray crystallography 20.2 Transmission of Plant Viruses Plant viruses do NOT bind to surface receptors A major difference between viral infections of plant and animal viruses Infection of plant cells requires cell wounding Horizontal vs. Vertical Transfer Horizontal Transfer Occurs from plant to plant (within generations) such as when plants are touching each other Vertical Transfer Occurs from parent to offspring such as infected seeds (between generations) Types of Transmission Mechanical means (human and environmental damage) Soil transmission Vegetative propagation (grafting) Piercing and chewing insects and other vectors Seed transmission Pollen transmission Mechanical Transmission Common way to experimental infect plants Wound leaves with virus-containing inoculum Not a very efficient means of transmission At least 500 particles needed to give rise to a viable infections Soil Transmission Plants may be physically damaged by wind Leaves of healthy and infected plants may rub together in the wind, allowing natural transmission to occur Viruses present in the soil may be transmitted to leaves of new plants as wind-blown dust or rain-splashed mud strikes the leaves Soil acts as an abrasive, causing damage that will give the virus access to plant cells Soilborne and waterborne viruses may also infect plants through slightly damaged rootlets Grafting Two young plants are joined together so that the best features (phenotypes) of two different plants combine into one plant A twig or scion is grafted onto a rootstock The scion develops into a new shoot Infection an infected scion is grafted onto a healthy plant's rootstock Vector Transmission Many organisms can act as vectors and spread plant viruses such as Bacteria Fungi Nematodes Parasitic plants Insects (e.g. aphids, leafhoppers, planthoppers, beetles, mites, mealybugs, whiteflies, thrips) Seed and Pollen Grain Transmission If pollen grains are infected, the seedling will grow from that seed or it may infect the plant through the fertilized flower. Plant-to-plant transmission occurs among fruit trees. 20.3 Symptoms of Plant Diseases Caused by Viruses Examples: Dwarfing or stunting of plants Leaf curling Reduced yield Fruit distortion Chlorosis (yellowing) Other color deviations (e.g. variegation of flower petals) “mosaic” patterns on leaves Ring-spots on leaves Wilting and withering Necrosis Bark scaling Many of these symptoms mimic other problems of the plant such as Nutrient deficiencies Toxicities Infections by other types of pathogens (e.g. bacteria, fungi) Viral infections rarely kill the plant Most replicate at the site of infection, resulting in localized symptoms such as necrosis Virus Movement Proteins Virus movement proteins must be compatible with the host in order for the virus to cause a systemic infection Once a plant is infected, it is infected for life 20.4 Diagnosis and Detection of Plant Viruses Most methods to diagnose plant infections are similar to those used in detecting animal and human viral infections: Direct detection of particles by electron microscopy Inclusion bodies in infected cells by light microscopy Infectivity assays Serology (ELISAs) DNA or RNA probes (e.g. PCR) 20.5 Prevention and Control of Plant Virus Diseases Major control methods: Control of insect vectors Removal of alternate hosts (e.g. weeds) Sanitation techniques Use of certified virus-free seed or stock Growing resistant crop varieties Plant quarantines 20.6 Morphology of Plant Viruses Most plant viruses are naked helical rods 3 types of rods Long, helical, flexuous (full of bends or curves) 10 nm X 480-2000 nm in length Rigid helical rods 15 nm X 300 nm in length Short (bacillus-like) rods Morphology of Remaining Plant Viruses Polyhedron-shaped plant viruses do exist 17-60 nm in diameter Multipartite viruses The only plant viruses that contain envelopes are the plant rhabdoviruses and tospoviruses 20.7 Types of Plant Virus Genomes dsDNA is the rarest type of plant virus genome Majority of genomes are +ssRNA (90%), dsRNA and ssDNA Some plant viruses have multipartite genomes (segmented) that are quadra-, tri-, or bipartite /ul> Unlike Animal Viruses, Multipartite Plant Viruses Package Each Nucleic Acid Molecule Into a Separate Virus Particle What are the Advantages Multipartite Genomes of Plant Viruses? i>Reduces the probability of losing the coding capacity of the genome if breakages of the genome occur Removes the requirement for accurate sorting or packaging of genomes into one virus particle What are the Disadvantages Multipartite Genomes of Plant Viruses? All of the individual genome segments must be packaged into a separate virus particle All of these particles must be taken up by a single cell to establish a productive infection or the virus will be defective Large inoculums are necessary for infection 20.8 Plant Virus Life Cycles Plants contain an impermeable cell wall Viruses enter through a break in the cell wall or channels called plasmodesmata Viruses have evolved specialized movement proteins to allow them through the plasmodesmata from one infected cell to neighboring cells Functions of Movement Proteins Associate with RNA or DNA viral genomes to form nucleoprotein complexes These complexes allow the plasmodesmata microchannels to dilate, delivering the complex to the cytoplasm of the neighboring cell Form a tubular structure that is inserted into a plasmodesmata pore, allowing the passage of virus particles from one cell to another Appear to be derived from host plant genes that encode for chaperonins and plasmodesmata-related proteins Systematic plant infections may occur if the viruses can be transported longer distances through the vascular system (the phloem) Plant viruses “channel” through the plant cell wall via movement proteins without causing cellular lysis Once viral particles have reached the cytoplasm, the genome is uncoated Low Ca++ concentrations may facilitate uncoating 90% of plant viruses contain +ssRNA genomes Use the host cell mRNA processing and translational machinery 5' ends of viral mRNAs will take one of several forms: A 5' phosphate group A 5' cap A virus-encoded VPg covalently attached to the 1st nucleotide of the RNA 5' Untranslated Regions (UTRs) of Viral RNAs Differs significantly from cellular mRNAs by length and secondary structure May contain internal ribosomal entry site (IRES) Allows for cap-independent translation 3' end of Viral +ssRNAs May take 1 of the following forms: Poly(A) tail tRNA-like structure 3' hydroxyl (OH) group Plant Virus +ssRNA Genome Replication These viruses encode their own RNA-dependent RNA polymerases for genome replication Utilize host factors for the formation of the replicase complex The +ssRNA is copied into a -ssRNA intermediate that serves as a template for the production of genomic +ssRNAs. These genomic RNAs are used for Translation Replication Genomic RNAs in new virions Most Plant Viruses Have at Least Three Genes 1 or more involved with the replication of the viral genome 1 or more concerned with cell-to-cell movement of the virus 1 or more encoding a structural protein that makes up the coat or capsid protein of the virus Role(s) of Host Factors in the Plant Virus Life Cycle Not clear Researchers are investigating this aspect of plant virology Plant Virus Exit Viruses exit through the modified plasmodesmata without causing cell lysis This differs from animal and human viruses that lyse cells or bud through the plasma membrane of cells Families and Genera of Viruses Infecting Plants. See Figure 20-9. 20.9 Plant Satellite Viruses and Satellite Nucleic Acid Satellite viruses encode their own coat protein but lack the genes that would encode the enzymes necessary for replication Satellite viruses are completely dependent upon helper viruses for replication Satellite RNAs or DNAs become packaged in a protein capsid form the helper virus Satellite Viruses and Nucleic Acids Share the Following Properties Genetic material is 200-1500 nucleotides in length (has little sequence similarity to the helper virus genome) Replication requires a helper virus May have a dramatic affect on disease symptoms of the plant Replication of the satellite virus interferes with replication of the helper virus Satellites are replicated on their own nucleic acid template 20.10 Plants and RNA Silencing: Plants Possess an Immune System of Their Genomes Plants are not defenseless toward foreign invaders. Nonspecific barriers Bark Saponins Bioflavenoids (possess antiviral and antibacterial properties) Some plants accumulate heavy metals or other toxic substances from soil, making them more resistant to microbial infection Plant Defenses Plants do not possess an active immune system like animals (e.g. antibodies, complement, phagocytes, interferon) Plants possess and RNA interference or RNAi or RNA silencing phenomena RNAi A form of genomic immunity that responds to invading nucleic acid It is a sequence-specific antiviral defense mechanism in plants 2002 Science "breakthrough" of the year Craig C. Mello and Andrew Z. Fire shared the Nobel Prize in Physiology or Medicine for their discovery of the mechanism of RNAi in C. elegans in 2006 The RNAi defense mechanism is triggered by dsRNA molecules 90% of plant viruses are +ssRNA viruses (create dsRNA intermediates during genome replication) Viral dsRNA replication intermediates activate a host cell RNase III-like dicer that chops the dsRNA into 21-24 nucleotide dsRNAs called short-interfering RNAs (siRNAs) 20.11 Tobacco Mosaic Virus (TMV) Plant viruses as a general rule are named after the first plant on which they are found. TMV infects tobacco Most studied plant virus Most plant viruses infect a wide variety of hosts Allows them to be well characterized in model plants such as tobacco or Arabidopsis TMV History Martinus Beijerinck (1851-1931) is credited for his TMV experiments and coining the term “virus” Rosalind Franklin hypothesized that the TMV particle was hollow and that its RNA genome was single-stranded TMV Structure Helical shaped 300 nm length X 18 nm diameter One of the most stable viruses (resistant to chemical and physical agents that inactivate other viruses) Capsid composed of 2130 identical subunits +ssRNA genome 6400-6600 nucleotides in length Purified TMV RNA is 95% RNA and 5% coat protein TMV particle self-assembles Host Range and Transmission Broad Over 550 species of flowering plants can be infected by TMV Important commercial corps infected by TMV: Tobacco Peppers Tomatoes Potatoes Easily transmissible from crop to crop Occurs mainly through mechanical transmission Not known to be spread by insect vector Cigarettes and other tobacco products such as chewing tobacco or snuff can be important sources of TMV and contribute to its spread No smoking should be permitted in the greenhouse Symptoms and Diagnosis of TMV Infection Not distinct Varies depending upon virus strains, plant age, growing conditions TMV almost never kills plants, but infection lowers the quality and quantity of the crop TMV Diagnosis Electron micrograph of particles in plant tissues are required for a positive identification of TMV in infected plants TMV Life Cycle TMV enters the plant cell via mechanical transmission A few CP subunits are removed, the 5' end of the genome is exposed Uncoating step is initiated (may also have a Ca++ requirement) TMV replicates in the cytoplasm of the plant cell The 5' end of the +ssRNA genome contains a terminal cap (m7pppG) followed by an AU-rich UTR of 70 nucleotides The 3' end of the genome contains a UTR that can fold into a tRNA-like structure that can accept histidine in vitro The tRNA-like structure is important for -ssRNA synthesis during genome replication TMV ORFs The first ORF codes for an RNA dependent RNA polymerase (RdRp) that has helicase and methyltransferase activity It is made up of 2 proteins of 126 kd or 183 kd The 183 kd protein is produced as a readthrough of the UAG during translation About 5-10% of the time, the 126 kd protein is produced because the ribosome stops translation at the UAG The 2nd ORF codes for a 30 kd movement protein The 3rd ORF coes for a 17.5 kd CP TMV RNAs The TMV viral RNA is infectious and acts like a mRnA that is translated by the host machinery After RdRP is synthesized, it begins to replicated full-length -ssRNAs or 3 additional shorter -ssRNAs, called subgenomic RNAs which function as monocistronic RNAs. TMV Viral Replication Complexes (VRCs) Does TMV spread quickly from cell to cell via intact VRCs? The VRCs increase in size on the ER in association with the cytoskeleton The VRCs move along microtubules of filamentous actin and myosin and through the plasmodesmata as large bodies Movement through the plasmodesmata is facilitated by action of the viral MPs or VRCs TMV Control Nearly impossible to prevent TMV infection in nature because of the stability of the virus. TMV occurs wherever tobacco is grown On average, it reduces crop yields by 30-35%, reducing the market value The virus is stable in dead plant matter in the soli and on contaminated seeds. It can exist for years in cigars and cigarettes made from TMV-infected leaves. Examples of Control Measures Used to Prevent the Spread of TMV when Maintaining and Transplanting Seedlings in the Greenhouse or Field Use only uncontaminated soils for seedling production Prohibit smoking during work Ask workers to wash hands regularly Destroy and remove all infected plants from nurseries Do not plant tomato or pepper seedlings in the same flower beds Spray plants with skim milk or buttermilk to reduce mechanical spread Rotate crops Plant TMV-resistant varieties 20.12 Citrus Tristeza Virus (CTV): Significance, Transmission, Symptoms, Detection, and Control Significance: CTV occurs wherever citrus is grown It infects nearly all citrus species, especially sweet oranges, mandarins, lemons, limes and grapefruit The word tristeza means sadness in Portuguese and Spanish Farmers coined this word in the 1930s when devastating epidemics of CTV occurred in Brazil and Argentina CTV Transmission Efficiently transferred by aphids and grafting CTV introduced into new geographic areas by importation of infected budwood or fruits that may also carry aphids Aphid feedings as brief as 10 minutes are long enough to bring in the virus from infected plants CTV Symptoms Dieback Defoliation (loss of leaves) Stem pitting Small or poor fruit quality Stunting Decline and death Symptoms are caused by starvation of the roots CTV Detection Infected buds or patches from trees are grafted onto healthy lime seedlings which are grown in temperature-controlled greenhouses Within 2-6 months infected lime seedlings will show symptoms of citrus tristeza Commercial ELISA kits are now available CTV Control Remove infected trees from established orchards Use tristeza-tolerant rootstocks during grafting Quarantine Prohibit the importation of host plants from countries where CTV epidemics occur CTV Virus Structure and Genome Largest known plant virus via electron microscopy Particles are nonenveloped, long flexuous or threadlike helical rods that are 2000 nm by 11 nm in diameter Genome: +ssRNA, ~19,300 nucleotides in length Codes for 17 protein products 2 papain-like proteases Methyltransferase Helicase 20.13 The Next Target: Anti-Crop Bioterrorism Crops would serve as a good target for biological agents Viruses have been responsible for heavy crop losses, including famine and massive economic losses Most crop varieties are genetically identical or nearly identical If one plant is susceptible to disease, all plants in the same field will be susceptible to disease Anti-Crop Bioterrorism Research Research and development programs existed in the UK, France, Germany and Japan before WWI 1946-1973 - U.S. maintained an active biological weapons program that included anti-crop agents The former Soviet Union maintained an active anti-crop program until it dissolved in 1991 The main focus of these programs is on fungal and bacterial pathogens. Few plant viruses listed as important anti-crop pathogens