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Characteristics of Viral and Viroid Pathogens Svetlana Folimonova Assistant Professor Dept. of Plant Pathology University of Florida Learning Objectives • Introduction to viruses: prevalence, diversity, virus structure, genome composition and organization (with main focus on plant viruses) • Plant virus life cycle • Virus movement • Plant-virus interactions (types of plant response, symptoms) • Detection and identification • Control of plant viruses • Characteristics of viroids Prevalence … Virtually all known life forms have been shown to be infected with viruses Viruses are the major players in the genetic universe 1 cm3 of seawater contains 106-109 virus particles Suttle, C.A. (2005) Nature 437:356 There are millions of diverse bacteriophage species in the water, soil, and gut Edwards and Rohwer (2005) Nat. Rev. Microbiol. 3:504 Viruses dominate biosphere: there are 10-100 viruses per each living cell Slide source: V. Dolja “Introduction to plant viruses” VIRUSES Non-cellular form of life Obligate intracellular parasites The fundamental characteristic is their absolute dependence on a living host organism for their reproduction Exist as inert particles (virions) outside the cell Virions harbor viral genome protected by protein shell Diversity between viruses • There is much biological diversity between viruses: - Host range - Type of disease - Virus particle size (large or small?) - Virus particle structure - Virus genome type (DNA or RNA?) and size - Virus genome replication strategy Virus structure Virus particles (virions) are built of a nucleic acid and a protein shell Icosahedral (spherical) virions (protein subunits are arranged with cubic symmetry) Elongated (helical) virions Slide source: V. Dolja “Introduction to plant viruses” Structure of TMV virion 95% protein, 5% RNA have been reported to retain infectivity at room temp. for at least 50 years 15 x 300 mµ - 2130 protein subunits; - each sununit is associated with 3 nts; -49 nts and 16 1/3 protein subunits per turn - ~ 130 helix turns of protein 8 subunits Virus structure “complex” structure enveloped viruses “simple structure” 10 A. Smallpox virus B. Orf virus C. Rhabdovirus D. Paramyxovirus E. Bacteriophage T2 F. Flexuous-tailed bacteriophage G. Herpes virus H. Adenovirus I. Influenza virus J. Filamentous flexuous virus K. Tobacco mosaic virus L. Polyoma/papilloma virus M. 11 Alflafa mosaic virus N. poliovirus O. Bacteriophage phiX174. Virions under EM Tobacco mosaic virus rigid rods Small pox virus – enveloped Tobacco vein mottling virus – flexuous rods a geminivirus – twinned icosahedra Vesicular stomatitis virus - enveloped Genome composition and organization 13 Viruses infecting plants Genome No. % dsDNA 30 3 ssDNA 166 17 dsRNA 45 4.6 (-) ssRNA 100 10.2 (+) ssRNA 635 65 Total plant viruses: 976 Source: Matthew’s Plant Virology by R. Hull Functions of viral gene products - structural proteins - proteins involved in nucleic acid synthesis (RdRp, RdDp or reverse transcriptase, methyl transferase, helicase) - proteases - proteins involved in virus movement and transmission by vectors Most of the ss (+)-sense RNA genomes code for about 4 to 7 proteins (CTV – 12) 15 Genome organization of Tobacco mosaic virus Genomic RNA 6.4 kb “leaky’ UAG 5’cap 69 ntsNTR MT HEL MT HEL 126 kDa 183 kDa 3’ NTR tRNA-like structure RdRp CP MP 17.6kDa, CP 30 kDa, MP CTV genome (19.3 kb) Replication Movement 1a L1 L2 MT p33 IDR HEL HSP70h CPm p18 p20 1b RdRp p6 p61 CP p13 p23 Virion assembly RNA silencing suppressors Virus Life Cycle 1 Invasion Invasion through leaves: Cytoplasm Cell Wall vectoring insects; mechanical damage Invasion through roots: vectoring nematodes or fungi; mechanical damage Exception: seed and pollen transmission Slide source: V. Dolja “Introduction to plant viruses” Virus Life Cycle 2 Genome uncoating, expression and replication uncoating translation replication Slide source: V. Dolja “Introduction to plant viruses” Virus Life Cycle 3 Particle (virion) assembly Slide source: V. Dolja “Introduction to plant viruses” Virus Life Cycle 4 Cell-to-cell movement Cell-to-cell movement – from the initially infected cell to the vascular bundle. Exception: phloem-limited viruses are usually injected directly into the phloem by their vector. Slide source: V. Dolja “Introduction to plant viruses” Virus Life Cycle 5 Systemic transport through phloem QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Long-distance movement – through the phloem. Further cell-to-cell movement establishes systemic infection in the young leaves. Slide source: V. Dolja “Introduction to plant viruses” Virus movement pathway From Carrington et al. (1996) Plant Cell Vol. 8 (10):1669-1681 Cell walls between different types of cells represent significant barriers for the virus during its cell-to-cell movement, loading into phloem and further unloading out of vascular tissue into parenchyma cells in systemic leaf. Structure of plasmodesmata and comparison to viral particles Viral encoded “movement proteins (MPs)” facilitate these steps. Most MPs are multifunctional. 1) MPs are required for movement 2) MPs bind to virus genomes 3) MPs interact with plant cytoskeleton 4) MPs localize to plasmodesmata 5) MPs alter and gate plasmodesmata, increase their size exclusion limit Virus genome may be associated with MP or coat protein during cell-to-cell movement 26 Genome organization of Tobacco mosaic virus Genomic RNA 6.4 kb “leaky’ UAG 5’cap 69 ntsNTR MT HEL MT HEL 126 kDa 183 kDa 3’ NTR tRNA-like structure RdRp CP MP 17.6kDa, CP 30 kDa, MP Functional characteristics from the pioneer research on TMV 30-kDa MP: •It binds single stranded RNA in a sequence nonspecific manner to form a stable RNP complex •It interact with the host PD machinery to induce an increase in the SEL(size exclusion limit) from 1kDa ~ 30kDa •It gates PD at the leading edge of TMV infection. •It is present within the PD structure •It mediates the cell-to-cell trafficking of itself and viral progeny RNA, leading to the infection of neighboring cells •It associates with microtubules and microfilaments as well as with ER 28 Movement proteins are a general feature of many plant viruses Lucas. 2006. Virology. 344:169-184 Viral proteins involved in systemic transport Genus Virus Viral factors Genus Virus Viral factors 30 Systemic movement of TMV expressing GFP in Nicotiana benthamiana 31 Virus Life Cycle 6 Plant-to-plant transmission . Aphids Whiteflies Mealybugs Leafhoppers Treehoppers Beetles Mites Thrips Nematodes Slide source: V. Dolja “Introduction to plant viruses” Plant-virus interactions 33 Types of plant response to inoculation with a virus Plant is immune: virus does not replicate in protoplasts nor in cells of the intact plant Infection is limited to initially infected cells: replication, but no cell-to-cell movement (could be due to an ineffectual virus movement protein) Infection is limited to initial leaf: replication, cell-to-cell movement, but no long distance movement (inability to enter phloem or due to a host resistance gene-mediated response, i.e. hypersensitive response when the plant resistance gene product recognizes some viral product, local lesions formation) 34 Hypersensitive response Examples: Tobamoviruses tobacco N gene recognizes the replicase protein tobacco N’ gene recognizes the coat protein tomato Tm2 gene recognizes the movement protein pepper L gene recognizes the coat protein eggplant recognizes the coat protein Potexviruses potato Nx recognizes the coat protein potato Nb recognizes the replicase protein 35 Types of plant response to inoculation with a virus (cont.) Plant is immune: virus does not replicate in protoplasts nor in cells of the intact plant Infection is limited to initially infected cells: replication, but no cell-to-cell movement (could be due to an ineffectual virus movement protein) Infection is limited to initial leaf: replication, cell-to-cell movement, but no long distance movement (inability to enter phloem or due to a host resistance gene-mediated response, i.e. hypersensitive response when the plant resistance gene product recognizes some viral product, local lesions formation) Systemic infection 36 Types of plant response to inoculation with a virus (cont.) Does systemic infection equal disease? 37 Papaya ringspot virus Types of plant response to inoculation with a virus (cont.) Does systemic infection equal disease? Papaya ringspot virus Tolerance (latent infection: little or no obvious symptoms) Disease (severe symptoms, death) Recovery (due to RNA silencing; virus replicates and causes symptoms in new leaves but with time and further plant growth symptoms disappear) Resistance (SAR: active host response to infection by diverse pathogens that cause necrotic cell death, resulting in diminished susceptibility to later pathogen attack; VIGS: active response by the host induced by infection with a virus that leads to specific38 degradation of viral RNAs) Symptoms Systemic symptoms General: Abnormal growth and developmental malformations (reduced growth, dwarfing, stunting of plants, etc.) Reduced life span of plants, death Foliage symptoms: Mosaics Yellows Ring spots Other: Symptoms on stems, fruit, roots Local symptoms Necrotic lesions None 39 Some Examples: A potyvirus in tobacco Peanut stunt virus in white clover Cowpea chlorotic mottle virus in cowpea Barley stripe mosaic virus in barley Tobacco mosaic virus in tobacco Necrotic local lesions on N. tabacum Glurk leaf, demonstrating Holmes’ Ngene resistance following inoculation with TMV. Photo: K.-B. G. Scholthof. 45 Tomato spotted wilt virus in peanut Leaf roll virus on grape leaves. (Courtesy of A. Baudoin) 49 Citrus tristeza virus in citrus Citrus tristeza virus in citrus Symptoms Systemic symptoms General: Abnormal growth and developmental malformations (reduced growth, dwarfing, stunting of plants, etc.) Reduced life span of plants, death Foliage symptoms: Mosaics Depend on virus strain, host, Yellows time, environmental conditions, Ring spots presence of other viruses! Other: Symptoms on stems, fruit, roots Local symptoms Necrotic lesions None 52 Symptoms of the mosaic strain of PVY in potato Symptoms of the necrotic strain of PVY in potato Pictures courtesy of Nan Jing Ko 53 Sweet orange cv. Valencia grafted on sour orange rootstock affected by Citrus tristeza virus (CTV) Stem pitting induced by CTV on a grapefruit trunk. Pictures by L. Navarro IVIA, Valencia 54 Vein-clearing on TMV-infected tobacco. Picture credit W.O. Dawson 55 Effect of temperature on symptom development and Cymbidium ring spot virus spreading of virus-infected N. benthamiana plants. Szittia et al. (2003) EMBO 22:633-640. 56 Recovery phenotype was first described in 1928 (a) Typical symptoms of TRSV in N. benthamiana plants at 10 days p.i. (b) Recovery of a plant from initial infection at 40 days. Siddiqui et al. (2008) J. Gen Virol 89:150208. Virus synergism in mixed infections Tobacco plants infected singly with either potatovirus X (PVX) or potatovirus Y (PVY) show mild symptoms, whereas doubly infected plants develop a severe synergistic disease in systemically infected leaves. 59 Detection and identification… your ideas here?! 60 Detection and identification Virus biology observations: - Host range - Symptoms (possible causes by nutrient deficiencies, toxicities, insects, etc. need to be eliminated and confirmed by transmission to a healthy plant) - Modes of transmission (vegetatively, mechanically, by plant sap passed through bacterial filters, insect vectors) Virus properties: - LM (inclusion bodies) and EM (virus particles) - Virus purification - Serology (only for known viruses for which antisera are available) - dsRNA isolation - PCR, RT-PCR (for known viruses) - Virus genome sequencing - small RNA preparation, sequencing using Next generation sequencing technologies, virus genome seq. assembly - Microarray 61 Control of plant viruses Prevention of infection!!! Control of plant viruses - Keep out of an area through quarantine, inspection, and certification programs that would prevent or limit local (within state, between states) or international (between countries) virus movement and warranty the use of virus-free seed, tubers, budwood - Eradication of diseased plants - Controlling the insect vectors (oil sprays, repellents, insecticides, predators…) - Breeding plants for resistance - Transgenic resistance (natural resistance genes, viral sequences, genes from other sources) Today, 80% of Hawaiian papaya is genetically engineered, and there is still no conventional or organic method to control ringspot virus. 13% of the zucchini grown in the US was genetically modified to resist three viruses. - Systemic acquired resistance - Mild isolate protection (cross-protection) Large-scale utilization of cross-protection Virus system Host plant Country Tobacco mosaic virus tomato Tomato mosaic virus tomato, pepper China Tien and Chang (1983) Cucumber mosaic virus with satellite pepper China Tien and Chang (1983) Cocoa swollen shoot virus cocoa West Africa Zucchini yellow mosaic virus squash, melon, watermelon Israel Apple mosaic virus apple New Zealand Passion fruit woodiness virus passion fruit Australia Papaya ringspot virus papaya Taiwan, Thailand, Mexico, Florida, Hawaii Netherlands, U.K., Japan, France, USA Reference Rast (1972), Fletcher (1975), Oshima (1975), Marrou (1978) Posnette and Todd (1951) Yarden et al. (2000) Chamberlin et al. (1964) Simmonds (1959) Yeh and Gonsalves (1984) Cross-protection by Citrus tristeza virus is one of the best examples of cross-protection of major economic importance A total of 50 million orange trees have been protected with a mild strain in Brazil (Costa and Muller, 1980). Cross-protection was also used to control the disease in South Africa, Australia, Peru and other countries. Viroids - Pathogenic agents in plants - Unencapsidated naked small circular RNA molecules, a few hundred nucleotides long, with high degree of secondary structure - Lack the protein coat of viruses - Do not code for any protein - Replicate in the nuclei of infected cells - Rapidly move from cell to cell and through the phloem - Readily transmitted mechanically in the field, vegetative propagation, some - by pollen and 66 seed Potato spindle tuber viroid was the first to be identified and is the most studied. Viroids in citrus: Citrus exocortis viroid – 375 nts Citrus viroid III – 297 nts Citrus viroid IV – 284 nts Citrus bent leaf viroid – 318 nts 68 Suggested reading material ‘Matthew’s Plant Virology’ by Roger Hull (2002)