Papaya Ringspot Virus. Tobacco Mosaic Virus. RNA World

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1 Websites to brush up on viral diseases Barley Yellow Dwarf Papaya Ringspot Virus Tobacco Mosaic Virus RNA World 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 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. 1

2 Virus Life Cycle 2 Viral genome Genome uncoating, expression and replication uncoating of virion Host ribosomes translate (express) viral genome Gene Products: translation Replicase (RNA polymerase) enzyme (gene product) New protein coat subunit (gene product) Cell to cell movement protein (gene product) Replicase enzyme makes new copies of virus genome replication Replicase (RNA polymerase) enzyme Host ribosomes Cell to cell movement protein New protein coat subunits 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. 2

3 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. GMO is controversial why? religious philosophical (ethical limits?) legal (concentrate power?) technical (safe?) GMO : it is more than one technology trans formation (foreign DNA) cis formation (DNA within species) gene knockouts / gene editing 3

4 Interesting observations on viral plant diseases Sometimes the plant recovers! --- so called Shock diseases Mild strain protects against disease by severe strain! --- Cross-protection : used in Brazil for a citrus virus Date first observed 1950s 1970s Transgenic plants that express viral coat protein gene are resistant! --- Hypothesis of the day: Virus can t disassemble 1980s Plants that express inversions of viral genes are resistant! --- Inverted gene has no product. Hmmmmm? 1990s Recovery from viral diseases Blueberry Shock Disease Beet Necrotic Yellow Vein Blueberry bush without leaves on right is Middle plant is recovering from showing shock for the first time. It will initial symptoms caused by BNYVV show the shock reaction for 1 to 2 years and be symptom-free thereafter, although it will still carry the virus and transmit it through pollen. 4

5 Citrus Tristeza Virus Stem pitting At graft union From Japanese citrus production guide : In areas where it is difficult to find a virus-free field, pre-inoculation with a mild CTV strain protects trees against infection with a severe strain of CTV. Protection of papaya with the coat protein gene of papaya ringspot virus: a success story Inoculation with Papaya mosaic virus Transgenic Non-transgenic Center: healthy, transgenic plants Borders: diseased papaya Transgenic papaya article: Field trials with transgenic papaya in Hawaii 5

6 So, what is the biochemical mechanism that accounts for the interesting observations on virus diseases? RNA Silencing RNA Silencing: 1) Replication of viral genome creates double stranded RNA because the + sense and minus stands are templates for each other e.g., BLUE strand +ss RNA virus Plant and animal cells have a two step enzyme process to recycle RNA virus destroyed See: 2) Enzyme DICER targets double stranded RNA molecules, and chops it into small pieces called silencing inducing RNAs (sirnas, about 20 base pairs in length) 3) sirnas become templates for the RISC enzyme complex. RISC uses the template to guide destruction of the original RNA molecule, now and in the future. 6

7 If RNA silencing is an efficient mechanism for destruction of double stranded RNAs, which are formed during replication of the gemome then how do viruses succeed? a. they outrun it b. they suppress it c. both = Examples of where a virus has been shown to suppress RNA silencing Viral ssrna Viral Replicase Viral dsrna or Viral ssrna dsrna Host RNA polymerase dsrna is formed during replication RISC DICER sirna Host enzyme that prepares ssrna for recognition by DICER - old normal messenger RNA also is recycled with this enzyme Continued progression of viral infection 7

8 Identification of silencing suppressors Intact leaf of N. benthamiana Infiltration with Agrobacterium carrying cassette Infiltration with Agro carrying -cassette and ds- cassette -cassette, ds- cassette, and cassete with silencing suppressor SS dsrna induces silencing of mrna S S Introduction of inverted repeat of results in silencing of P1/HC-Pro is a viral gene that suppresses RNA silencing S S 8

9 How easy is it to engineer plant resistance to a viral disease? Agrobacterium: nature s genetic engineer In nature, genetic instructions to produce a gall are transferred to the plant cell In biotechnology, the gall genes are replaced with a gene of interest e.g. 9

10 Engineering viral resistance 1) Choose virus --- e.g. TMV 2) Choose sequence --- coat protein 3) Make an inverted repeat of target sequence gemome bp Or buy it: e.g.: ttgaaaatca-tgattttcaa ensures mrna will double on itself, which prepares it for DICER 4) Attach promoter and insert into Agrobacterium 35S 5) Create and select transformants Life Technologies catalog: $338 ElectroMAX A. tumefaciens LBA4404 Cells are Agrobacterium cells that have been specially prepared for transformation by electroporation. They transform a variety of plant species at high efficiency

11 Making a transgenic plant = our gene of interest Plant tissue culture medium containing kanamycin T-DNA is that part of the plasmid transferred to the plant s nucleus. our gene of interest 35S plus a gene for kanamycin resistance These plantlets/plant express our gene of interest plus gene conferring kanamycin resistance Advantages of plant transformation over conventional breeding Genetic Transformation: Allows introduction of genes from sexually incompatible species Permits creation of novel types of resistance (viral coat protein) Is much less time consuming than breeding Permits insertion of multiple genes Lacks deleterious effects associated with backcrossing Works especially well for clonally propagated crops (trees, potatoes) Watch INNATE video on JR Simplot Co. website on use of RNAi to improve Russet Burbank potato:. 11