Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Gene silencing assaf's paper


Published on

Published in: Technology

Gene silencing assaf's paper

  1. 1. Identification of Tomato Yellow Leaf Curl Virus (TYLCV) Host Resistance Genes Amer Wazwaz Aug 30th 2012
  2. 2. Large numbers of plant genes with NO defined functions Powerful in silico techniques have been developed for the analysis of genome sequence information But gene function must always be verified in vivo using genetic analysis
  3. 3. Reverse Genetics a powerful tool that establishes a direct link between the biochemical function of a gene product and its role in vivo RNA-MEDIATED INTERFERENCE CHEMICAL MUTAGENESIS VIRUS-INDUCED GENE SILENCING INSERTIONAL MUTAGENESIS FAST NEURON MUTAGENESIS
  4. 4. VIRUS-INDUCED GENE SILENCING Cloning a 200–1300 bp cDNA fragment from a plant gene of interest into a DNA copy of the genome of an RNA-virus Transfecting the plant with this construct using Agrobacterium Double-stranded RNA from the viral genome, including sequence from the gene of interest, is formed during viral replication The double-stranded RNA molecules are degraded into siRNA molecules by the plant Dicer-like enzymes Limited only by the host range of the virus used, TRV and ALSV are the most common vectors
  5. 5. RNA-MEDIATED INTERFERENCE similar to VIGS, but it is heritable DNA construct that produces either s.s or d.s RNA complementary to the gene of interest is introduced into a cell It activates the RNA silencing pathway and degrades some or all of the transcripts from the gene of interest Through amiRNA using promoters that are temporally or spatially specific or inducible by exogenous factor Partial loss of function can be achieved
  6. 6. INSERTIONAL MUTAGENESIS Disruptions in target genes of interest through genome(wide insertions (insertional knockouts Plants carrying an insertion in a gene of interest can be identified by screening the population with PCR using one gene-specific primer and one insertion based primer Result in a total loss of function and the insertion can be easily followed using PCR Phenotypes may not be obvious if the gene function is redundant Insertions in essential genes will typically result in lethality
  7. 7. FAST NEURON MUTAGENESIS Bombardment is used to generate deletions & chromosome rearrangements of various sizes randomly in the genome Seeds are mutagenised with fast neutron radiation and deletions are identified by PCR using primer sequences that flank the gene of interest Laborious because of the number of plants that must be screened Has limitations in terms of the sizes of deletions that can be recovered
  8. 8. CHEMICAL MUTAGENESIS Chemical mutagenesis induced point mutations in DNA in all species in which it has been tested Mutations induced using these mutagens are distributed in the genome randomly As point mutations are less damaging, a high degree of saturation can be achieved in a mutant population facilitating examining of gene function on a genomic level Unlike other reverse genetics techniques, chemical mutagenesis can result in either loss-of-function or gainof-function mutations
  9. 9. Plant resistance to viruses is the outcome of interconnecting gene networks and signaling pathways leading to inhibition of virus replication and/or movement These gene and protein networks have been revealed by largescale microarray analyses and by protein-protein interaction studies, usually using the yeast two-hybrid system ~70 different genes preferentially expressed in R plants
  10. 10. Two inbred tomato lines (line 902 is resistant to the virus (R (line 906-4 is susceptible (S R and S can be distinguished by a single nucleotide polymorphism (SNP) found in hsp70 gene cDNA libraries from the S and R genotypes were prepared and screened for genes preferentially (expressed in R tomato as described (Eybishtz et al. 2009
  11. 11. Silencing of the Hexose transporter LeHT1 gene using a VIGS vector cDNA encoding a fragment of the hexose transporter gene (LeHT1 (518 to 1038 Fragment was T/A cloned into pDrive vector then excised and ligated to TRVII vector using XbaI & KpnI Plasmid was introduced into Agrobacterium by electroporation Agrobacterium cells containing TRV-Hex and TRVI were cultured in YEB medium for 48 h at 28C Agroinoculated into 30 R and 30 S tomato seedlings at the 4-6 leaf stage
  12. 12. Inoculation of tomato plants with different viruses Five days after LeHT1 silencing silenced and 20 non-silenced plants were inoculated 20 with TYLCV Caging plants with viruliferous whiteflies for 3 days (~ 30 insect /plant), kept at 24–27C Plants were grown in a greenhouse at 18–24C, 16 h light Then, plants were mechanically inoculated with Bean Dwarf Mosaic Virus (BDMV), Cucumber Mosaic Virus ((CMV) and with Tobacco Mosaic Virus (TMV
  13. 13. PCR and semi-quantitative PCR • Semi-quantitative PCR analyses of LeHT1 expression in notsilenced susceptible (So:0) and resistant (Ro:0) plants, and silenced resistant plants (Ro:TRV-Hex) 14 days after TRVHex treatment β-actin was used as an internal control in the same samples
  14. 14. Detection of small RNA related to silencing the hexose transporter gene LeHT1 Northern blot-based detection of a 24-nucleotide-long RNA present in LeHT1-silenced plants (Ro:TRV-Hex), but not in not silenced R (Ro:0) plants C 21-nucleotide-long primer used to label the LeHT1 probe, utilized as size marker
  15. 15. TYLCV amounts and appearance of TYLCV inoculated in virus-infected LeHT1-silenced (Ri:TRV-LeHT1) and not-silenced (Ri:0) R plants Comparison of TYLCV amounts estimated by semi-quantitative PCR, 21 days after inoculation
  16. 16. TYLCV amounts and appearance of TYLCV inoculated in virus-infected LeHT1-silenced (Ri:TRV-LeHT1) and not-silenced (Ri:0) R plants Growth inhibition and mild symptoms of Ri:TRV- LeHT1 plants compared with infected not silenced R (Ri:0) and S (Si:0) plants
  17. 17. Visualization of TYLCV in infected leaves by in situ hybridization d.a.i, using a tetramethyl rhodamine labeled viral DNA probe 21
  18. 18. PCD-induced necrosis in TYLCV infected LeHT1silenced R plants Necrosis on stem and petiole of TYLCV-infected LeHT1-silenced R plants (Ri:TRV-Hex); no necrosis on not-silenced infected R (Ri:0) and S (Si:0) plants, and on infected LeHT1-silenced S plants
  19. 19. Detection of H2O2 Oxidative burstrelated damages detected with DAB Plants of the same age stained and not stained with DAB Release of H2O2 is visualized as a reddish brown precipitate
  20. 20. Necrosis in LeHT1silenced R plants (Ri:TRV-Hex) infected with Bean dwarf mosaic virus (BDMV), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) Ri:0 are virusinfected not silenced plants
  21. 21. Conclusion The amounts of LeHT1 transcripts in R tomato was about 120 times higher than in the S plants The expression of the gene was reduced by approximately 60-folds The infected silenced plants contained approximately 30,000 times more virus than the untreated Ri plants
  22. 22. Conclusion Many of the 70 genes were annotated as membranal, suggesting that the mechanism of resistance might involve: virus entry in the cell and/or cell-to-cell trafficking A second line of resistance was uncovered in the silenced R plants in form of programmed cell death ((PCD Not only upon inoculation of TYLCV, but also after inoculation of additional DNA (Bean dwarf mosaic virus) and RNA viruses (Cucumber mosaic virus and Tobacco (mosaic virus