Role of biotechnology - gene silencing in plant disease control
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An overview of role of biotechnology especially gene silencing approach in plant disease control and success achieved so far and way forward and it's importance in developing countries
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Role of biotechnology - gene silencing in plant disease control
- 2. • Agronomic Traits – Biotic Stress • Insect Resistance – Bt, cystatin • Disease Resistance – Viral- coat protein protection (Papaya ringspot virus) – Bacterial, Fungal, Nematode (Rice blight, rice blast) • Weed- herbicide tolerance (Striga, orobanchia) – ABiotic Stress • Drought, Cold, Heat • Poor soil – Salinity – tomatoes with transport protein – Aluminum Crop Biotechnology
- 3. • Yield • Nitrogen Assimilation – nodulation by rhizobia • Starch Biosynthesis, O2 Assimilation, photosynthesis/Rubisco • Quality Traits – Processing – Post harvest loss reduction – Reproduction: sex barriers, male sterility, seedless fruit – Nutrients (Nutraceuticals) • Macro: Protein (Cassava), Carbohydrates, Fats, Fiber • Micro: vitamins, minerals, phytochemicals • Anti-nutrients: Phytase, Toxin removal • Novel Crop Products – Proteins: nutraceuticals, therapeutics, vaccines • Renewable resources • Biomass conversion, feedstocks, biofuels, phytoremediation
- 5. Prevalence of vitamin A deficiency. Red is most severe (clinical), green least severe. Countries not reporting data are coded blue
- 6. . Rice staple food for 2.4 billion people • Fungal diseases destroy 50 million metric tons of rice per year; varieties being developed resistant to fungi - proteins with anti-fungal properties. • Insects cause a 26 million tons loss of rice per year; insecticidal proteins environmentally friendly control. • Viral diseases devastate 10 million tons of rice per year; Tungro virus genome transgenes defense systems. Cassava Mosaic Virus similar protection system as papaya working in Kenya • Bacterial diseases cause comparable losses - transgenes such as cecropin lytic peptide basis for inbuilt resistance.
- 7. Virus resistance technologies continue to grow Pathogen Derived Resistance Protein Mediated Resistance Coat Protein Mediated Resistance Replicase Mediated Resistance Movement Protein Mediated Resistance RNA-mediated resistance sRNAs that target vRNAs for degradation Non-Pathogen, Protein Mediated Resistance Transcription regulators DNA binding proteins Interferon-like strategies; ds-RNA degrading enzymes Translation initiation factors/co-factors
- 8. • Crop varieties which are resistant against many economically important diseases is a major challenge for plant biotechnologists worldwide. • In this regard, RNA interference (RNAi) has emerged as a powerful modality for battling some of the most notoriously challenging diseases caused by viruses, fungi and bacteria. • This goal can be achieved in a more selective and robust manner with the success of genetic engineering techniques. • Gene silencing can be achieved by introducing a short “antisense oligonucleotide”in to the cell that is complementary to an RNA target. • This experiment was first done by Zamecnik and Stephenson in 1978and continues to be a useful approach, both for laboratory experiments and potentially for clinical applications (antisense therapy).
- 9. The core of RNA silencing: Dicers and Argonautes • RNA silencing uses a set of core reactions in which double-stranded RNA (dsRNA) is processed by Dicer or Dicer-like (DCL) proteins into short RNA duplexes. • These small RNAs subsequently associate with ARGONAUTE (AGO) proteins to confer silencing. DICER AGO Silencing
- 10. Dicer and Dicer-like proteins Dicer’s structure allows it to measure the RNA it is cleaving. Like a cook who “dices” a carrot, Dicer chops RNA into uniformly-sized pieces. In siRNA and miRNA biogenesis, Dicer or Dicer-like (DCL) proteins cleave long dsRNA or foldback (hairpin) RNA into ~ 21 – 25 nt fragments.
- 11. Argonaute proteins ARGONAUTE proteins bind small RNAs and their targets. The Arabidopsis ago1 mutant and the octopus Argonauta argo ARGONAUTE proteins are named after the argonaute1 mutant of Arabidopsis; ago1 has thin radial leaves and was named for the octopus Argonauta which it resembles.
- 12. What are small RNAs? •Small RNAs are a pool of 21 to 24 nt RNAs that generally function in gene silencing •Small RNAs contribute to post- transcriptional gene silencing by affecting mRNA stability or translation •Small RNAs contribute to transcriptional gene silencing through epigenetic modifications to chromatin AAAAA RNA Pol Histone modification, DNA methylation
- 13. Mechanism of silencing RNAi is a mechanism for RNA-guided regulation of gene expression in which double-stranded ribonucleic acid (dsRNA) inhibits the expression of genes with complementary nucleotide sequences.
- 14. Sense RNA Antise nse RNA Sense construct: PRO ORF Endogenous gene mRNA Transgene mRNA Protein translated mRNA mRNA Extra protein translated Antisense construct: Transgene Sense-antisense duplex forms and prohibits translation Hypothesis: sense RNA production enhances pigmentation and antisense RNA production blocks pigmentation
- 15. siRNAs – Genomic Defenders siRNAs protect the genome by • Suppressing invading viruses • Silencing sources of aberrant transcripts • Silencing transposons and repetitive elements siRNAs also maintain some genes in an epigenetically silent state
- 16. Cullen Nature Genetics 38:S25, 2006
- 17. Source: ISAAA •Biotech Crops 2008: 125 million hectares (310 million acres up 9.4%) •25 countries 12% increase over 2007, 13.3 M farmers (12 M 2007) •90% resource-poor farmers (12.3 M - 11.0 M 2007) most Bt cotton •New: Egypt, Burkina Faso, Paraguay , Uruguay. •India Bt cotton up to 7.4 M Ha.
- 18. Number 2 in GM production. 17% of the global area of GM plants. In 2007, 98% of soybean in Argentina was GM. Yield have reached over 6 tonns per hectare In 1994-95 production costs were 182 dolars/Ha; in 2007 are 117 dolars/Ha In 1994-95 farmers spent 78 dolars/Ha in herbicides; today they spend 37 dolars/Ha and insecticide use has decrease 90%. Economical benefits of GM soybean USD$ 20 billion + 1 million jobs Problems, yes. Due to the economic success of GM soybean and maize, President Cristina Fernádez de Kirchne created a new tax on Gm soybean exports that producers oppose GM crops an engine of economical development
- 19. Between 2001-05 the chinese government spent 15 billion US dólars on AgBiotech projects; 2006-10 a 400% increase has been projected National Biotechnology program work on the development of over 130 varieties of GM rice and 55 varieties of GM cotton 10 GM products have been aproved for human consumption (rice, maize, soybean and potato). Bt and disease resistent rice is commercially planted in China. GM cotton was used by 7.1 million small farmers in 3.8 million hectares in 2007 with an economical benefit of USD$ 817 million in 2006. China is awakening
- 20. Viruses PlantAb protected mice against genital herpes similar physical props to MCC remained stable in human exhibited no diff in affinity for binding, neutralizing HSV Genetic Engineering Technology Allows Production of Novel Products Metabolic Pathways Active Vaccines Transmissible gastroenteritis virus Antigen Cholera/Hep B/banana Acetylenic &Vernolic Acid Containing Chemical feedstocks Passive Vaccines Ab enteric bacteria E.coli O157:H7 meat foodborne path Polyhydroxybuterate biodegradable plastic
- 21. Alfalfa Plasma Proteins, Foot-and-mouth disease vaccine Maize Anti- HIV and Anti herpes Simplex Antibodies Microbiocides for pulmonary infection Mabs for cancer, autoimmune disease RA, Vaccines hepatitis B, Norwalk virus (Travelers disease), Vaccines & Mabs for animal, Aprotinin for blood loss, Gastric lipase cystic fibrosis, Lettuce Vaccines for Hepatitis B Moss Factor IX for hemophilia B Rice Lactoferrin Lysozyme for GI health, Alternatives to abs in poultry diets, Topical infections, inflammations, B-cell lymphoma vaccine Safflower Therapeutics and oil-based products for oral/dermal delivery Spinach Protective antigen for vaccine against Bacillus anthracis Soybean Tobacco extensin signal peptide - Anti-HSV-2 (IgG) Tobacco Non-Hodgkins B-cell lymphoma, TGF-b glucocerebrosidase for Gauchers Syndrome , Alpha galactosidase for enzyme replacement therapy, IgGs for prevention of dental decay, common cold, GAD 7 cytokines for type 1 Diabetes, Colon cancer surface antigen – Fabrazyme fat-storage disorder Tomato , Potato Banana (someday!) Edible vaccines: Enterotoxigenic E. coli, Norwalk virus, Hepatitis B, Vibrio cholera, Rabies virus-intact Glycoprotein Antimicrobe peptides, Wheat Carcinoembryonic antigen - Murine IgG signal peptide Potato Polyhydroxybuterate biodegradable plastic
- 23. CONCLUDING REMARKS • RNA silencing has become a hot topic of biological research in the last few years. • Gene silencing reveals an entirely new level of post-transcriptional gene regulation. • An extremely useful technique for molecular biology and very powerful biotechnological tool. • The topic is of vital importance for the students interested in biotechnology and plant pathology. • However, a better and comprehensive understanding of RNAi would allow the researchers to work effectively and efficiently in order to improve crop plants nutritionally and manage various mascotous intruders of crop plants.
- 24. • References: • Biotechnology and Plant Disease Control-Role of RNA Interference Shabir H. Wani*, Gulzar S. Sanghera, N. B. Singh; American Journal of Plant Sciences, 2010, 1, 55-68 • Plant pathology G.N.Agrios;5th edition • Post transcriptional gene regulation by siRNA and miRNA Pete Burrows;MIC 759,October 26, 2006 • An innovation from the plant cell American plant biologists society;2013 • Modern Ag Biotech Applications Martina Newell-McGloughlin
- 25. • Biotechnology :A new era for plant pathology and plant protection(class notes) Dr.P.M. Bhattacharya • Sense molecular biology WIKIPEDIA,GOOGLE IMAGES • A practical approach to the understanding and teaching of RNA silencing in plants Ariel A. Bazzini ,Vanesa C. Mongelli, H. Esteban Hopp
- 26. Thank you
Editor's Notes
- ANTIBODY-CONTAINING SOYBEANS During the early 1990s, researchers discovered that special vaccination of flocks of chickens caused them to secrete antibodies [against the bacterial strains chosen for vaccination] into the whites of the eggs they laid. Those egg whites are now chopped-up to prepare a commercial piglet feed that removes all E. coli bacteria of specific diarrhea-causing strains from the intestines of piglets. xx That egg-white-based feed product works via each antibody “latching onto” one of the diarrhea-strain-specific E. coli bacteria within the piglet’s digestive system, then the combined pair is excreted by the animal. Because antibodies are pure protein molecules, there are no regulatory issues pertaining to meat residues. Today’s periodic outbreaks of beef-borne E. coli 0157:H7 bacterial disease occur because cattle became tolerant of E. coli 0157:H7 in the 1970s [it had previously killed infected cattle] xxi and humans are now sometimes exposed to that deadly bacteria when the hide or digestive system contents of cattle come into contact with meat (e.g., at slaughterhouses). xxii A study published by USDA in April, 2000 showed that reducing E. coli 0157:H7 in live cattle prior to slaughter greatly increases slaughterplant safety. xxii It is now possible for biotechnology to cause specific antibodies (e.g., specific to E. coli 0157:H7) to be produced in soybeans, so such future soybeans could be fed to livestock for 72 hours prior to slaughter in order to eliminate outbreaks of foodborne diseases such as E. coli 0157:H7, Salmonella spp, etc. 12. “VACCINE-CONTAINING” SOYBEANS AND MAIZE During 2001, a U.S. company will introduce a biotechnology derived maize that produces antigens for the swine disease known as transmissible gastroenteritis virus (TGEV). When that maize is eaten and those antigens in it tough lymph tissues in the swine’ digestive system, the animals’ immune system rapidly produces antibodies that protect it against TGEV. Similar plant vaccines are expected in the future for human diseases such as hepatitis B. Medical Benefits Plants have been a valuable source of pharmaceuticals for centuries. During the past decade, however, intensive research has focused on expanding this source through rDNA biotechnology. The re-search brings closer to reality the pros-pect of commercial production in plants of edible vaccines and therapeutics for preventing and treating animal and hu-man diseases. Possibilities include a wide variety of compounds, ranging from vac-cine antigens against hepatitis B and Nor-walk viruses (Arntzen, 1997; Dixon and Arntzen, 1997; Mason et al., 1992, 1998) and Pseudomonas aeruginosa and Staphy-lococcus aureus (Brennan et al., 1999) to vaccines against cancer and diabetes. In addition, genetically modified strains of probiotic microorganisms are also possi-ble vehicles for successful delivery of vac-cines and digestive aids (e.g., lactase) through the stomach and the small intes-tine. Two seminal papers supported the use of rDNA biotechnology-derived plants for pharmaceutical production (Ma et al., 1995, 1997). These reports were soon followed by one (Ma et al., 1998) describing results of successful hu-man clinical trials with an edible vaccine against a pathogenic strain of E. coli and a monoclonal antibody against cariogenic Streptococcus mutans. Haq et al. (1995) reported the expression in potato plants of a vaccine against E. coli enterotoxin against the toxin in mice. Human clinical trials suggest that oral vaccination against either of the closely related enterotoxins of Vibrio cholerae and E. coli induces pro-duction of antibodies that can neutralize the respective toxins by preventing them from binding to gut cells. Ma et al. (1995, 1998) showed that tobacco plants could express secretory antibodies or “planti-bodies” against the cell surface adhesion protein of S. mutans. Used as a bactericid-al mouthwash, the antibodies prevented bacterial colonization by the microorgan-ism and development of dental caries for four months. A similar approach showed that soy-bean- produced antibodies protected mice against infection by genital herpes (Zeit-lin et al., 1998). Compared to antibodies produced in mammalian cell culture, the plantibodies had similar physical proper-ties, remained stable in human reproduc-tive fluids, and exhibited no differences in their affinity for binding and neutralizing herpes simplex virus. Hence, the differ-ence in the glycosylation processes of plants and animals does not appear to af-fect the immune functions of the plant-derived antibodies. Non-Hodgkins B-cell lymphoma, the most widespread cancer of the lymph sys-tem, is difficult to treat because the B-cell tumors are variable and response to treat-ment can vary from person to person. Hence, effective therapy requires “person-alized medicine” tailored to the genetic makeup of each patient’s tumor. Unfortu-nately, conventional treatment methods do not meet the needs for rapid produc-tion of customized antibodies in suffi-cient quantities. Monoclonal antibodies used in conventional treatment also tend to be expensive and unreliable, and those produced in bacteria have solubility and conformation problems. A system using tobacco mosaic virus (TMV) was developed to produce in to-bacco plants (Nicotiana benthamiana) a therapeutic vaccine against non-Hodgkin’s B-cell lymphoma in a mouse model (McCormick et al., 1999). Using cells cloned from malignant B-cells of mice, TMV DNA was modified with a tu-mor- specific sequence from the gene cod-ing for the immunoglobin cell surface marker. Plants were then infected with the modified virus, resulting in expres-sion of cancer-specific antibodies. B-cell proteins were then extracted from the plant leaves for vaccination of the mice. Eighty percent of the mice receiving the while all untreated mice died within three weeks of contracting the dis-ease. A similar approach was used to devel-op a vaccine against insulin-dependent diabetes mellitus (IDDM), an auto-im-mune disease in which insulin-producing cells of the pancreas are destroyed by the cytotoxic T lymphocytes. The “oral toler-ance” method of preventing or delaying autoimmune disease symptoms involves the ingestion of large amounts of immu-nogenic proteins that turn off the auto-immune response. This method of vacci-nation is gaining recognition as a poten-tial alternative to systemic drug therapy, which is often ineffective. Insulin and pancreatic glutamic acid decarboxylase (GAD), which are linked to the onset of IDDM, are candidates for use as oral vac-cines. Blanas et al. (1996) described the development in a mouse model of a po-tato- based insulin vaccine that is almost 100 times more powerful than the exist-ing vaccine in preventing IDDM. Feeding diabetes-prone mice potatoes engineered to produce immunogenic GAD reduced the incidence of disease and immune re-sponse severity. rDNA biotechnology-derived vac-cines are potentially cheap, convenient to distribute, and simple and safe to ad-minister. Production of medically im-portant substances via rDNA biotech-nology engineering of plants and micro-organisms offers multiple advantages. For plants, production can be done vir-tually anywhere and has the potential to address problems associated with deliv-ery of vaccines to people in developing countries. Products from these alterna-tive sources do not require a so-called “cold chain” of refrigerated transport and storage, although they will require segregation from conventional foods to prevent inappropriate consumption. Pharmaceuticals or therapeutics pro-duced via genetic engineering of plants also offer an alternative delivery meth-od, feeding versus injection (Howard, 1999), and an alternative to extraction from animal sources. Furthermore, rDNA biotechnology-derived vaccines may also be safer than many conven-tional vaccines because they consist of pathogen or antibody subunits rather than whole microorganisms. The use of plants can facilitate abundant produc-tion of therapeutic proteins without the risk of contamination by animal patho-gens, and at substantially reduced cost. Biotechnology Report: Benefits C O N T I N U E D