Plant Viruses

1. Plant VirusesBradley HillmanDept. of Plant Biology and Pathology339 Foran Hall, Cook932-9375 X 334hillman@aesop.rutgers.eduComparative Virology course website: http://www.rci.rutgers.edu/~bhillman/comparative_virology/Index.htm

2. Plant Viruses • Introduction and history • Why study plant viruses? • How do they relate to animal viruses? • How has their study impacted virology? • Symptoms • Composition and structure • Taxonomy and nomenclature • Four families contain both plant and animal viruses • Seven families contain only plant viruses • Many plant viruses belong to genera without family affiliation

3. Plant Viruses (cont’d) • Survey of some major plant viruses • Positive strand RNA • RdRp supergroup 3 (Sindbis-related) • Tobacco mosaic virus (Tobamovirus) • Brome mosaic virus (Bromoviridae) • RdRp supergroup 2 (Flavivirus-related) • Turnip crinkle virus (Tombusviridae) • Red clover necrotic mosaic virus (Dianthovirus) • RdRp supergroup 1(Picornavirus related) • Tobacco etch virus (Potyviridae) • Cowpea mosaic virus (Comovirus) • Negative strand RNA • Sonchus yellow net virus (Rhabdoviridae) • Ambisense RNA • Tomato spotted wilt virus (Bunyaviridae)

4. Plant Viruses (cont’d) • Survey of some major plant viruses (cont’d) • Double-stranded RNA • Wound tumor virus (Reoviridae) • Single-stranded DNA • Bean golden mosaic virus (Geminiviridae) • Double-stranded DNA (pararetroviruses) • Cauliflower mosaic virus (Caulimoviridae) • Expression strategies of + strand RNA viruses • Plant virus infection cycle • Cell-to-cell movement and movement within plants • Plant-to-plant transmission • Brome mosaic virus, a well-studied plant virus • Satellites, defective-interfering RNAs, viroids • Plant defense response to virus infection • Plant viruses and biotechnology

5. Host Systems: Plants 1 • Eukaryotic, but fundamentally different from animals • Plants don’t move, so vectors are very important for moving viruses from one plant to another • Plants are autotrophic and easy to grow in quantity– great bioreactors • Plants have rigid cell walls and very small cell-to-cell connections (plasmodesmata) • Synchronous infection of many cells can be achieved using plant protoplasts (primary cell cultures with cell walls removed)

6. Host Systems: Plants 2 • Developed plant cells are totipotent • Virus in one part of a plant moves to another slowly by cell-to-cell connections; more rapidly through vascular system, mostly phloem • Plant defense response system exists, but is less specific than vertebrate or invertebrate systems • Plants are developmentally complex; viruses may be excluded from some tissues

7. CHARACTERISTICS OF PLANT PATHOGENIC VIRUSES • Loss due to plant viruses is often difficult to quantify, but they are often of great importance as plant pathogens (fungi are most economically important ) • Viruses do not usually kill plants, and symptoms on plants are often subtle • Virus diseases of plants are not subject to chemical control – no effective cure in individual plants • Symptoms, serological, electron microscopic, or molecular methods are used to identify plant viruses • Plant virus disease cycles often are dependent on vectors or alternate hosts

8. Selected highlights of plant virology research

9. Tulipomania – late 16th century Before it was known to be caused by a virus, tulips with color breaking symptoms were prized and traded for large sums of goods – this led to “tulipomania” in the late 1500’s Traded for 1 Viceroy tulip bulb: 4 tons of wheat 8 tons of rye 4 fat oxen 8 fat pigs 12 fat sheep 2 hogsheads of wine 4 barrels of beer 2 barrels of butter 1000 lbs of cheese 1 bed with accessories 1 full dress suit 1 silver goblet

10. Adolf Mayer –1886 – showed that Tobacco mosaic virus was transmissible, could not find bacteria or fungi associated with disease TMV

11. Dmitri Ivanowski - 1892– showed that Tobacco mosaic virus was not retained by filters that retained all bacteria known at that time

12. Martinus Beijerinck - 1898– repeated demonstration that Tobacco mosaic virus was not retained by filters that retained all bacteria known at that time • Believed results • Did extensive dilution experiments • Showed diffusion of infectious agent through agar • Named “contagium vivum fluidum”, later virus

13. Wendell Stanley – 1935 • At Rockefeller Foundation in Princeton • Crystallized TMV, thought it was only protein TMV Stanley Hall, U.C. Berkeley

14. Bawden and Pirie - 1936 • Crystallized Tomato bushy stunt virus (TBSV); find that it and TMV contain phosphorous – conclude that it is not a protein, but is a nucleoprotein TMV TBSV

15. Markham and Smith - 1949 • Two classes of particles in purified Turnip yellow mosaic virus preparations: • light ones containing only protein, which were not infectious • heavy ones containing protein+nucleic acid, which were infectious empty full

16. Myron Brakke - 1951 • Development of density gradient centrifugation • Isopycnic: particles reach position of equal density in gradient • Rate-zonal: Particles sediment differentially through medium as a function of size, shape, and density • Equilibrium zonal: Combination of the above

17. Virus Aa: RNA A capsid a Fraenkel-Conrat 1955-56 protein • Complete, infectious TMV particles can be reconstituted in vitro from the RNA and protein components • RNA alone is infectious • RNA can be “transcapsidated” in protein from closely related virus; resulting virus has properties of RNA strain RNA reconstitute in vitro inoculate plants symptoms (A) extract virus virus Aa

18. Fraenkel-Conrat – 1955-56 Virus Aa: RNA A capsid a Virus Ab: RNA A capsid a Virus Bb: RNA B capsid b Transcapsidation RNA RNA RNA protein protein protein inoculate plants inoculate plants inoculate plants no symptoms no symptoms no symptoms symptoms (A) symptoms (B) symptoms (A) extract virus extract virus extract virus virus Aa no virus virus Bb no virus virus Aa no virus

19. Crick and Watson – 1956 • TMV virions are composed of one nucleic acid and many identical protein subunits: RNA does not have the coding capacity to make many different subunits

20. Casper and Klug – 1962 • Structure of Tomato bushy stunt virus solved by X-ray crystallography, the first icosahedral virus so determined

21. Heinz Sanger – 1978 • Complete sequence of Potato spindle tuber viroid • First pathogen sequence to be determined • Yielded relatively little information that was immediately useful 1 cggaactaaa ctcgtggttc ctgtggttca cacctgacct cctgagcaaa aaagaaaaaa gataggcggc tcggaggagc gcttcaggga tccccgggga aacctggagc gaactggcaa aaaaggacgg tggggagtgc ccagcggccg acaggagtaa ttcccgccga aacagggttt tcacccttcc tttcttcggg tgtccttcct cgcgcccgca ggaccacccc tcgccccctt tgcgctgtcg cttcggctac tacccggtgg aaacaactga agctcccgag aaccgctttt tctctatctt cttgcttccg gggcgagggt gtttagccct tggaaccgca gttggttcct 359

22. Paul Ahlquist – 1984 • Infectious viral RNA transcribed in vitro from cDNA clones • Done with Brome mosaic virus – with 3 RNAs • Brought reverse genetics to RNA viruses RNA Inoculate plants RNA cDNA

23. Roger Beachy – 1986 • Transgenic plants expressing TMV coat protein are resistant to virus infection • First example of “pathogen-mediated resistance”

24. Bill Dougherty – 1991 • RNA was critical component in resistance in pathogen-mediated resistance • All of the hallmarks that later came to be associated with PTGS and RNAi were first observed with Tobacco etch virus (TEV) (1993 Lindbo et al., Plant Cell 5:1749-1759)

25. SYMPTOMS

26. Plant Virus Symptoms • Viruses rarely kill plants • Most severe disease usually in least well-adapted host/pathogen systems • Levels of tissue specificity differ among plant viruses • Some infect all or most tissues • Most cause symptoms only in aerial portions • Some accumulate only in roots • Symptoms in fruit or flowers may be most harmful • Systemic symptoms only in developing tissue • Local necrotic lesions undetectable in natural infections

27. Plant Virus Symptoms • Stunting - common • Mosaics & mottles - common • Ringspots - common • Abnormal growth/tumors/enations - rare • Blights - rare • Wilts – rare • Systemic necrotic lesions – relatively rare

28. Blight

30. Stem pitting – usually results in loss of woody perennials

31. Ringspots, oakleaf

32. Phyllody – tissue destined to develop flower parts instead develops leaves Deformities

33. Wilt Necrotic roots

34. Mosaics – very common Tomato mosaic Alfalfa mosaic

35. Mosaics on monocots are streaks or stripes Maize streak Maize mosaic

36. Necrotic lesions Local Systemic “Local lesion” or “hypersensitive” response is an apoptotic response. Cells within a short distance of the initially inoculated cell begin to undergo programmed cell death in advance of virus invasion, preventing further virus spread.

37. May be viral coat protein or another viral gene product Hypersensitive response – an apoptotic reaction to infection

38. TAXONOMY AND NOMENCLATURE

39. Virus taxonomy and nomenclature • Modified binomial is used • Taxonomy depends on particle properties, nucleic acid properties and especially sequence • Family is the highest taxonomic level that is commonly used; ends in viridae, e.g., Bromoviridae • Genus ends in suffix virus, e.g., Bromomovirus • Species is usually the commonly used virus name; it is italicized in formal usage, e.g., Brome mosaic virus • Small genome sizes, gene shuffling make broad taxonomic schemes difficult (above Family level)

40. COMPOSITION AND STRUCTURE

41. Virus properties: Plant viruses are often simpler than animal viruses • Genome sizes 0.3 - 300 kb; plant viruses 0.3-30 kb • May have single-stranded or double-stranded RNA or DNA genome; most plant viruses ssRNA • If RNA, may be + or – sense; most plant viruses + sense ssRNA • May have one or many proteins in particles; most plant viruses have 1-2 • May or may not have lipid envelope; most plant viruses do not

42. Types of plant virus genomes • double-stranded (ds) DNA (rare) • single-stranded (ss) DNA (rare) • ssRNA, negative sense (rare) • ssRNA, positive sense (common) • dsRNA (rare)

44. Virus types, by nucleic acid DNA RNA ss ds ss ds env naked env naked env naked env naked Families Species 0 5 9 12 9 14 2 5 0 100 200 300 200 600 10 300 Host type Vertebrate Invertebrate Plant Fungus Bacteria - + ++ ++ ++ ++ - ++ - + ++ - ++ ++ - ++ - ++ - + + +++ - + - - - + + + + +++ - + + +++ - + + -

45. Plant viruses are diverse, but not as diverse as animal viruses – probably because of size constraints imposed by requirement to move cell-to-cell through plasmodesmata of host plants Plant viruses often contain divided genomes spread among several particles

46. Basic Plant Virus Structures Helix (rod) e.g., TMV Icosahedron (sphere) e.g., BMV

47. Helical symmetry • Tobacco mosaic virusis typical, well-studied example • Each particle contains only a single molecule of RNA (6395 nucleotide residues) and 2130 copies of the coat protein subunit (158 amino acid residues; 17.3 kilodaltons) • 3 nt/subunit • 16.33 subunits/turn • 49 subunits/3 turns • TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion • Self-assembly also occurs in the absence of RNA TMV rod is 18 nanometers (nm) X 300 nm

48. TBSV icosahedron is 35.4 nm in diameter Cubic (icosahedral) symmetry • Tomato bushy stunt virusis typical, well-studied example • Each particle contains only a single molecule of RNA (4800 nt) and 180 copies of the coat protein subunit (387 aa; 41 kd) • Viruses similar to TBSV will self-assemble in vitro from protein subunits + nucleic acid in an energy-independent fashion T= 3 Lattice C N Protein Subunits Capsomeres

49. GENOME ORGANIZATIONS

50. Plant virus genome organizations • Very compact • Most are +sense RNA viruses, so translation regulation very important • Use various strategies for genome expression • Only a few genes absolutely required: • Replicase • Coat protein • Cell-to-cell movement protein • Other genes present in some viruses