gene silencing-for disease managment

gene silencing-for disease managment

1. “Learning is a life long process”

2. GENE SILENCING Submitted by : Rathod Balaji Ulhas ita G. 1 st semester Ph.D. Plant Molecular Biology & Biotechnology Dept. of Biotechnology, J.A.U. An Assignment presentation on Submitted to Dr. F. L. Akbari Head andAssociate Professor, Dept. of plant pathology, College of Agriculture, J.A.U. PL PATH 604 MOLECULAR BASIS OF HOST PATHOGEN INTERACTION

3. Contents 1) Introduction 2) Types of Gene Silencing 3) Transcriptional gene silencing 4) Post-Transcriptional gene silencing 5) Application 6) Significance 9) Advantages and Disadvantages 9) Conclusion

4. INTRODUCTION Gene silencing is a technique that aims to reduce or eliminate the production of a protein from it’s corresponding gene . It generally describe the “switching off ” of a gene by a mechanism other than genetic modification. That is, a gene which would be expressed (“turned on”) under normal circumstances is switched off by machinery in the cell. It occurs when RNA is unable to make a protein during translation . Gene silencing is same as gene knock down but is totally different from gene knock out .

5. 1987 - antisense RNA suppression– - early form of RNA suppression - introduction of the antisense strand of RNA into the cell that corresponds to the target mRNA, the transcript intended to silence Prelude… 1980 - Before RNAi – to generate loss-of-function mutations. - insertion of TDNA elements, transposons and treatment with mutagens or irradiation

6. AIM: Upregulate activity of chsA Expected : More pigments Observed : White sectors Napoli, C., Lemieux, C. and Jorgensen, R., 1990. Introduction of a chimeric chalcone synthase gene into petunia Plant Cell 2:279–89 Loss of mRNAs of both endo-and transgene COSUPPRESION

7. Andrew Z. Fire Craig C. Mello 7

8. Sense RNA Antisense RNA Double stranded RNA Wild type Wild type Twitcher C.elegans UNC -22 null type phenotype

9. Types of Gene Silencing Transcriptional Genomic Imprinting Paramutation Transposon silencing (or Histone Modifications) Position effect DNA methylation Post-transcriptional RNA interference RNA silencing Nonsense mediated decay

10. Transcriptional gene silencing Genomic imprinting: Paramutation :

11. Position effect: RNA Directed DNA Methylation

12. Transposon silencing: Transgene silencing:

13. Post transcriptional gene silencing

14. What is Antisense Technology ?

15. Comparison of different Antisense strategies

16. RNA INTERFERENCE : What about it? During RNAi double stranded RNA molecules (dsRNA)act as inducers or activators of this process. Cleavage of the inducer molecules into smaller pieces first and eventually desruption of the cellular or viral cognate mRNA molecules (target). RNAi seems to be a natural function to protect the genome against invasion by mobile genetic elements (viruses, transposons)

17. Different Classes of RNA Non-genetic RNA RNAs actively participate in RNAi Messenger RNA Ribosomal RNA Transfer RNA Hairpin RNA Small interfering RNA Double stranded RNA Micro RNA

18. COMPONENTS OF RNAi ( MAJOR ACTORS IN RNAi) Dicer Small interfering RNA ( siRNA ) RNA-Induced Silencing Complex (RISC)

19. RISC RNA-induced silencing complex (RISC): a protein complex in which the antisense strand of siRNA duplexes guides endonucleolytic cleavage of target RNAs

20. MECHANISM OF RNA INTERFERENCE STEP 1 : Initiation step Involves binding of the RNA nuclease (Dicer) to a large dsRNA (30-200 nt) and its cleavage into discrete 21 to 25 nucleotide RNA fragments (siRNA) STEP 2 : Effector step siRNAs join a multinuclease complex, RISC, which degrades the homologus single-stranded mRNAs

21. Initiation Step ATP ATP ADP + ppi ADP + ppi DICER KINASE RdRP ss RNA ds RNA Viral RNA/as RNA/ hpRNA 21

22. Effector Step siRNA binding siRNA unwinding RISC activation 22

23. RNAi: How to use? Insertion of double-stranded or small-interfering RNA into a cell Agrobacterium -viral mediated dsRNAtransfer or by infiltration ( Sijen and Kooter 2000). non- Agrobacterium -mediated methods particle bombardment was used to show that RNAi worked at the single cell level in cereals ( Schweizer et al . 2000), direct uptake of dsRNA by fern spores during imbibition (Klink & Wolniak,2000; Stout et al. 2003) Most of these methods utilize an RNAi vector to produce stable or transient dsRNA in vivo .

24. RNAi Technology and its Implication in Phyto -Nematodes Management Genes responsible early stages of parasitism During the stylet thrusting in the plant tissue, several proteins are injected into the host by nematodes, which are responsible for the development of parasitism . Identification and targeting of these genes can, prevent the entry and establishment of nematodes on its particular hosts. Hg‐syv46 proteins ubiquitin like protein and cysteine proteinase are the few examples of involved in parasitism activities . Genes responsible for phytonematodes development The major development stages of nematodes include the fecundity of female, embryo growth and juvenile advancements. Targeting the genes regulating these stages will lead to disruption of the normal life cycle and thus helps in management by hindering the process of further progeny development . Suppression of genes responsible for chitin synthesis leads to the delayed egg hatching and targeting the genes responsible for moulting normally affects the normal development and due to that one can achieve effective management of phytonematodes

25. RNA Interference: An Optimistic Approach to Enhance Plant Disease Resistance ( Nakayashiki et al., 2005 ).- commercial exploitation of this approach to develop disease-resistant plants. mpg1 (a hydrophobin gene, which acts as a cellular relay for adhesion and trigger for the development of appressorium in M. oryzae ) and polyketide synthase-like genes were silenced in 70 to 90% of the transformed plants . Escobar et al., (2001) had demonstrated RNAi of the iaaM and ipt oncogenes for the management of a crown gall disease. Niu et al., (2006) have silenced viral suppressor genes P69 and HC-Pro of Turnip yellow mosaic virus and Turnip mosaic virus infections, respectively with the help of RNAi

26. Schwind et al., (2009) reported that transgenic tomato plant exhibited resistance to PSTVd infection which are expressing a hairpin RNA construct derived from PSTVd sequences. Hoffman et al.,(2008 ). Downregulating Suc transporter genes of Heterodera schachtii with RNAi found to reduce female nematode development

27. Applications of RNAi in crop improvement

28. Sr. no. Plants Target gene Functions Reference Nutritional value 1 Sweet potato IbSBEII Increase the amylose content Takiko et al. 2006 2 Cassava CYPT 9D 1, CYPT 9D 2 Depletion of cynogenic Glucoside content Jorgensen et al. 2005 3. Canola COMT, C4H, C3H, FSH Reduction in lignin content Bhinu et al. 2008 5. Coffee CaXMT , CaMXMT , CaDXMT Decaffeinated coffee Ogita et al. 2003 7. Peanut Arah 2 Eliminate allergy Dodo et al . 2007 8 Rice BiP 3 Mediate innate immunity in rice Park et al. 2010 Table: Other case studies of application of RNAi in crop plants

29. Disease Resistance 9 Banana BBrMV Resistance to BBrMV Rodoni et al. 1999 10 Cantaloupe PRSV- W Resistance to papaya ring spot virus Krubphachaya et al. 2007 11 Tobacco AC 1 gene Resistance against tomato leaf curl virus Wang et al. 2008 12 Rice OsGA 20 ox 2 Modification of plant height Qiao et al. 2007 13 Rice Tryptophan decarboxylase Delaying senescence & cold stress tolerance in rice Kang et al. 2009 14 Tomato LeETR 4 Early ripening of tomato Kevany et al. 2008 15 Tobacco PMT genes Reduction in nicotine content Wang et al. 2008 16 Strawberry Chalcone synthase Increase cinnamoyl glucose esters Hoffman et al. 2006 17 Tomato Chalcone synthase Parthenocarpic tomatoes Elio 2007 6. Barley BYDV Resistance to barley yellow dwarf virus Wang et al. 2000 Continue..

30. BIOTIC STRESS TOLERANCE

31. Advantages of gene silencing: Downregulation of gene expression simplifies "knockout" analysis. Easier than use of antisense oligonucleotides. Si RNA more effective and sensitive at lower concentration. Cost effective High Specificity middle region 9-14 are most sensitive With Si RNA, the researcher can simultaneously perform experiments in any cell type of interest Can be labelled Ease of transfection by use of vector blocking expression of unwanted genes and undesirable substances. Inducing viral resistance Powerful tool for analysing unknown genes in sequenced genomes. Useful approach in future gene therapy. Oligonucleotides can be manufactured quickly, some within one week; the sequence of the mRNA is all that is needed

32. Disadvantages of gene silencing: ” High pressure injection” and electroporation can cause significant injection damage to the integrity of the normal tissues and organs andthus preclude the utilisation in a clinical set-up. Liposomes/cationic encapsulated Si RNA may also be toxic to the host and may cause severe host immune responses. Other emerging strategies includes chemical modification of Si RNA molecules and encapsulated with different molecules are still in their infancy and need to be thoroughly investigated before used in therapeutic applications .

33. Application of Gene silencing Specific gene silencing using RNA i in cell culture. Cancer treatments RNA interference has been used for applications in biotechnology, particularly in the engineering of food plants that produce lower levels of natural planttoxins . Such techniques take advantage of the stable and heritable RNA i phenotype in plant stocks. For example, cotton seeds. Modulation of HIV-I replication by RNA i . Small RNA and it’s application in andrology and urology. Developing technologies for epigenomic analysis and clinical application of molecular diagnosis. Currently there are at least six oligonucleotide drugs inducing RNA i for illness including cancer.

34. Conclusions RNAi seems to be the best method in this era of exploration for best and effective eco-friendly approach alternative to pesticides for plant disease management. We hope, this technology will find its niche in plant disease management to augment agricultural crop production in near future

35. Bibliography Molecular biology of the cell 5th edition by whatson , baker , bell, gann , levinn , losick . Molecular biology by David p. Clark ELSEVIER. RNA i guide to gene silencing by Gregory J. Hannon. Gene silencing theory, techniques and applications by Anthony J. catalano Wikipedia – gene silencing