Plants Fight it out Intrinsic defence mechanism The magic world of Gene silencing
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1 I LOVE YOU
2 Plants Fight it out Intrinsic defence mechanism The magic world of Gene silencing Over expression of Chalcone synthase gene to get Purple Petunias Napoli, Lemieux & Jorgensen,1990 Desired Effect Gene expression Outcome Gene Silencing Post-transcriptional gene silencing (PTGS)
3 Since its discovery in plants, gene silencing has been discovered in many other things. like. Fungi Worms Flies Mice One phenomenon many terms Post-transcriptional gene silencing Co-suppression Quelling RNA interference (RNAi) RNA silencing
4 Expression Silencing DICER(DCL) double-stranded RNA sirnas(21-25nt) RISC RNA-Induced Silencing Complex mrna AAAAA AAAAA
5 Small interfering RNA pathway Virus Sense transgene RdRp Hairpin transgene dsrna sirna (21-24 nt) DCL4 DCL2 DCL3 21/22 nt 24 nt Degradation of mrna
6 RNA silencing for host delivered resistance dsrna Pathogen Sequence Nucleus Dicer Fungi Nematodes Insects Transgenic mrna si RNAs RNA-Induced Silencing Complex Degraded pathogen RNA Invading pathogen No disease
7 sirna based resistance to PVY resistant PVY PVY-NIa 35 S PVY-NIa ocs nos NPT II nos spacer N. tabacum W38 single copy line #16 T1 progeny
8 microrna Pathway DNA Pol II A A A A pre-mirna DCL1 Double-stranded fold-back RNA mirna-mirna* Ago mirna duplex Programmed RISC High homology A A A A mrna binding Partial homology A A A A mrna degradation mrna translation inhibition
9 Artificial microrna mediated resistance to Tomato spotted wilt virus Neena Mitter, Keith Chua, Roger Mitchell, Sudeep Bag, Keri Druffel, and Hanu Pappu.
10 Artificial MicroRNA Technology Biogenesis of mirna is not affected even if the mature mirna is changed. Artificial mirna:mirna* Construct Endogenous Plant pre-mirna Artificial mirna mirna* mirna 21-nucleotide Endogenous mirna precursor amirna Sense Antisense Min. 100-nucleotide Linker shrna
11 Artificial mirnas for virus resistance 21 nt from Target virus Plant microrna Express in plants mirna-mirna* Virus specific Ago Programmed RISC High homology Viral mrna degradation A A A A
12 Artificial MicroRNA Technology Virus Gene Targeted mirna Backbone Reference Cucumber mosaic virus 3 UTR mir159a Duan et al, 2008 Cucumber mosaic virus 2b silencing suppressor mir171a Qu et al, 2007 Turnip yellow mosaic virus Turnip mosaic virus P69 silencing suppressor HC-Pro silencing suppressor mir159a mir159a Niu et al, 2006
13 Tospoviruses No. of tospovirus reported Absent Little/No info Melon yellow spot virus Groundnut bud necrosis virus Capsicum chlorosis virus Melon severe mosaic virus Impatiens necrotic spot virus Iris Yellow Spot Virus Tomato Spotted Wilt Virus Peanut chlorotic fanspot virus Groundnut ringspot virus Calla lily chlorotic spot virus Watermelon silver mottle virus Tomato zonate spot virus Peanut yellow spot virus Polygonum ringspot virus Tomato yellow fruit ring virus Zucchini lethal chlorosis virus Chrysanthemum stem necrosis virus Tomato chlorotic spot virus Watermelon bud necrosis virus
14 Tomato spotted wilt virus (TSWV) Background - Type species of the genus Tospovirus - Account for losses exceeding US$1 billion/year globally Tomato spotted wilt virus Pappu et al (2009) Susceptible Hosts Tomato Spotted Wilt Disease Insect Vectors Environment > 900 species of plants Warm season Thrips species (Family Thripidae)
15 Tomato spotted wilt virus (TSWV) Structural Proteins Nucleoprotein (N) (attach to genome) Tripartite Genome Organisation S Segment Glycoprotein (G n and G c ) Polymerase (L) Envelope Spherical M Segment L Segment Genome Gene Function Segment S Nucleoprotein (N) Coat Protein Non-structural Protein (NSs) Silencing Suppressor M Glycoprotein (G c /G n ) Insect Transmission Non-structural Protein (NSm) Movement Protein L Polymerase (L) Viral Replication
16 amirna Construct Design -TSWV
17 amirna construct design Engineered 6 N gene targeting amirna constructs and 6 NSs gene targeting amirna constructs Sequence Position Used only 173 nucleotides of mir159a instead of 273 nucleotides used previously Introduced a T at 5 end of mature microrna Designed constructs with and without wobble in the mir*
18 Transient Assay for amirna expression level Method Agroinfiltration N. benthamiana leaf 3 dpi 6 dpi amirna expression 3 dpi 6 dpi
19 Transient Expression Assay Agroinfiltrate with tospo amirna constructs targeting N gene Challenge with virus 3 days post agroinfiltration Monitor for symptoms ELISA for N gene expression
20 Transformants Generation Transformants Screening amirna Expression Analysis TSWV Resistance Analysis Conserved sequences among TSWV Conserved sequences among Tospoviruses Multiple sequences for durable and broad range resistance Targeting genes involved in insect transmission TSWV Inoculation DAS-ELISA Absorbance Value Symptom Monitoring Seeds Harvested
21 It is a small world after all..
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