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Pest, Pathogen resistance: Insects, viruses....
Fichas del conjunto (50)
- 1Need for engineering resistanceCrop losses, 14%, 10 billion on pesticides, conventional pesticides and pollution, growing population and food need...
- 2Biopesticidese.g Bt toxin, from natural sources, easy to degrade, less toxic, specific and not broad spectrum; difficult to identify and develop, effective but limited sources, difficult to apply and maintain, insect resistance can develop
- 3Engineering StrategyEngineer plants to express proteins that are insecticidal or insect repelling
- 4Strategies for engineering and typesSpecific pest-targeted toxin e.g Bt toxin
General antifeedants: alpha amylase inhibitors, proteinase inhibitors, lectins and general toxins - 5Bacillus thuringiensisGram positive, soil dwelling, facultative aerobe, produces crystalline proteins, part of the family of delta endotoxins, insecticidal against lepidoptera(moths and butterflies), coleoptera (beetles) and diptera(flies)
- 6Mechanism of action Bt toxinnative cry protein is inactive protoxin-100KDa; in insect gut, it is cleaved by proteolytic enzymes yielding active 60-80KDa active toxins that bind to glycoprotein receptors on the epithelial brush border membrane, toxin irreversibly inserts into membrane and then epithelial cell lysis ensues followed infiltration of other organisms, gut paralysis and death within 1-3 days.
- 7Cry protein domains and functionsactive protein has 3 domains: domain I: membrane insertion and pore formation
domain II and III: receptor recognition and binding - 8First experimentVaeck et al., 1987: Bt toxin in Tobacco- Nicotiana tabacum through agrobacterium mediated transformation. Full cry protein 1184aa, no protection from tobacco horn worm Manduca sexta, expression of 600-800aa yielded better phenotype
- 9OthersChrysanthemum protected by expression of modified Cry1C gene, against beet amyworm larvae
- 10Initial problems and challengesAmount of Bt toxin was low
Amount of mRNA from transgene was low - 11Modifications (bacterial genes in plants)Cryptic splice sites, Better promoters, Codon usage, Tissue specific promoters
- 12BT transgenicsAs of 2008 BT transgenics comprised 84% of cotton and about 60% of the maize crop in the USA
BT potato has not really taken off (Monsanto's 'New Leaf' potato was taken off the market in 2001 because of lack of interest) - 13Transgenics and organismsCotton: cotton bollworm and pink bollworm as well as tobacco budworm (BollgardTM- Cry1Ac)
Potato: Colorado potato beetles(New LeafTM) Cry3A
Maize: European Cornborer
(Bt-XtraTM Cry1Ab) - 14European cornborerLarval damage is inside the plant and is difficult to see.
Infestations are difficult to predict.
Insecticides used are expensive and have environmental and health implications.
Previous management using insecticides has a poor record of success - 15European cornborer and BtExpression of a single Bt toxin, Cry1A(b), in maize controls attack by the European Corn Borer.
- 16Newer Bt transgenics haveThe latest commercial Bt lines contain several stacked Bt genes active against more than one pathogen
- 17Bt Corn: Commercially availableNovartis- "KnockOut and NatureGard" :Bt toxin gene is CrylAb
Dekalb- "Bt-Xtra" :another CrylAb
Monsanto and Novartis: "YieldGuard"- CrylAb or Cry1A 105
They are produced in different tissues ranging from kernal, stalks, silk, leaves and have different event names e.g. knockout event name is 176 and YieldGard is Mon810(1Ac) - 18Bt Cotton/IndiaBt cotton now comprises >90% of planting in India
- 19Impact of Bt usehigher yield, lower insecticide use and higher net revenue?
- 20Potential Environmental ConcernUse of herbicide markers in transgenesis- herbicide resistance?
use other selectable markers, remove these
Cross toxicity of insecticidal proteins: Monarch butterfly debate?
Unfounded/Compare toxicity to treatment with organophosphorus insecticides.
Development of resistance in pests?
A real problem. Pyramiding and careful introduction and management essential: Gene stacking - 21Monarch Butterfly debate Losey et al., 1999claimed that larvae fed on milkweed leaves artificially dusted with high loads of pollen from Bt Transgenic maize “ate less, grew slower, and suffered a higher death rate than larvae that ate leaves free of corn pollen”.
Only five caterpillars used per experiment.
Amount of pollen applied was very high, over six times as high as the highest level found naturally.
Experiments was conducted as a “no choice” for larvae.
No attempt to duplicate real conditions e.g. rain wash.
The Bt corn used was a line expressing 40X more Bt toxin than the most commonly planted line - 22Bt transgenics/ indirect benefitsLevels of other insect infestation in and around Bt cornfields. Aphid levels reduced since decreased reliance on broad spectrum pesticides meant more beneficial aphid predators and as such less aphids around the area
- 23Plant derived antifeedantscowpea trypsin inhibitor CpTi- first one: conferred tolerance to tomato moth on potato
GNA conferring tolerance to aphid on potato
Proteinase inhibitor conferring resistance to tobacco budworm on tobacco - 24antifeedants arebroad spectrum conferring resistance to fungi (e.g. serine protease inhibitors), nematodes (e.g. lectins) and sometimes mammals (some serine protease inhibitors)
lectins e.g Mannose effective against fungi, nematode, coleoptera, diptera, hemiptera but not mammals
antifeedants can be used in the pyramiding scheme - 25Agglutinins: GNA (Galanthus nivalis agglutinin)Wu et al., 2002
- 26Agglutinins: ASAL- Allium sativum (onion) leaf agglutinin- resistance against sap-sucking insects Yarasi et al., 2008read
- 27Anderson et al., 2004 Emerging diseases proportionsVirus: 47% Bacteria:16% Fungi:30%
- 28Mayse and Price, 1978Highly complex ecosystemic interactions
- 29Pathogen Derived ResistanceEngineering resistance in host using genetic elements from pathogen's own genome
- 30Cassava mosaic disease: important in developing countriesnew strain EACMV-UG (East African Cassava Mosaic
Begomovirus), recombinant btwn ACMV and EACMV; highly virulent; spread by whiteflies (sap sucking bugs) or planting of infected shoot; cause yield loss - 31Examples of Viral diseasesPotato virus Y: yield loss
Potato Leaf roll virus: spread by aphid, symptoms in next growing season
Banana bunchy top: grower can identify disease, yield loss
Bract mosaic virus (banana): not easily detectable, yield slowly declines
Barley Yellow dwarf: of small grains including wheat; was very important in the States %35 loss - 32Losses, viral diseasesViruses are a major cause of economic losses, and are particularly serious economic pathogens in Africa and parts of Asia
For example, virus diseases of cassava and sweet potato (staple crops in large areas of East and West Africa) result in yield losses of 25-50%. - 33OthersTobacco mosaic virus, Cassava mosaic geminivirus, Pepper ringspot virus
- 34Engineering resistanceResistance genes: Dominant and Recessive
Pathogen-Derived Resistance: Protein-mediated resistance e.g coat proteins, movement proteins, replicase
RNA-mediated resistance
Antibody-mediated resistance
Others: Ribosome-inactivating proteins, Protease inhibitors - 35Resistance genes-Dominant R genes conferring resistance against pathogens and inherited in a mendelian manner
(Innate immunity) - 36Resistance genesresistance to TMV was introgressed into Nicotiana tabacum from N. samsun
Many potato plants have been bred to express Resistance genes Rx, Ry
Usually NBS LRR proteins; They cause localized hypersensitivity response and systemic acquired resistance; necrosis of the virus
Problem with R genes: Pathogen drift that causes loss of resistance
Conventional breeding takes time
Ry for Potato
virus Y (PVY) in potato and Sw5 for Tomato spotted wilt virus (TSWV) in
tomato - 37Resistance genes transferAccelerated plant breeding
Transfer single or multiple resistance genes across from wild cultivars to agronomic cultivars
R genes transferred between more distantly related species, e.g. between tobacco and Arabidopsis, may not function properly - 38Examples of natural transfer of R genesExamples include the introduction of the tobacco N gene (resistance to TMV) into tomato, potato Rx and Nx genes (potato virus X) into tobacco, and transfer of resistance genes against bacteria (Pseudomonas syringae) and fungi (downy mildew) from resistant to susceptible Arabidopsis ecotypes
- 39Recessive resistance genesNot NBS LRR genes
They are genes in the host that encode proteins that the virus needs in its life cycle, i.e. they render the host susceptible to the virus - 40Recessive Resistance: knocking out susceptibility genesMost plant viruses contain RNA genomes (CaMV doesn't).
e.g. Turnip mosaic virus (part of potyvirus)- 9kb genome, the whole virus is an ORF, gives polypeptide of 300KDa, it encodes a protease that cleaves it.
Translation initiation factors recognize the cap and translation starts. Arabidopsis isoEIF4E recognizes TuMV Vpg cap. TuMV requires isoEIF4E (EIF4E does not recognize the cap) Mutations within transcription factors EIF4E or isoforms such as isoEIF4E affect the binding activity of the cap (VpG) of the irus for the initiation of transcription. Example; done in Barley, alleles of gene called rym conferred resistance to barley yellow mosaic - 41Coat protein mediated: TMVcotranslational disassembling: ribosomes attach to the accessible cap of TMV during translation moving through the RNA driving off the coat proteins. Produce a lot of transgene coat protein that blocks up the end of the virus and prevents the ribosome from attaching- keep the 5' end well attached to coat protein preventing translation. Engineer coat protein that makes it bind the RNA more strongly: higher level of resistance. the virus can not disassemble itself for translation.
Examples: GM squash (courgette/zucchini) plants expressing the coat protein of Zucchini yellow mosaic virus;
GM papaya plants expressing the coat protein of Papaya ringspot virus
Future: Cassava with resistance to several viruses including Cassava brown streak
Maize with resistance to Maize streak virus (developed in South Africa) - 42coat protein-mediated GM Resistance to Papaya Ringspot Virus in Papaya Dennis Gonsalves 1978coat protein gene of a mild mutant of a PRSV strain from Hawaii was used in biolistic transformation of red-fleshed Sunset cultivar. Transgenic line 55-1 of Sunset was inbred to homozygosity for the single copy coat protein gene and named SunUp. The Rainbow cultivar was developed to create a virus-resistant, transgenic yellow-fleshed papaya to replace virus-susceptible Kapoho. Rainbow is an F1 hybrid from SunUp and nontransgenic Kapoho
- 43Transgenic PRSV-resistant rainbow cultivarsResistance of PRSV held up under strong disease pressure. It is still predominant in Hawaii. Replaced Kapoho which was susceptible to PRSV.
- 44Replicase and Movement ProteinHemingway et al expressed mutant replicase forms (GED, GAD, ADD reping ADD) in potato and got high resistance against potato virus X because transgenic replicase outcompetes wildtype replicase
- 45Maize streak virus-resistant transgenic maize, first for Africa; Shepherd et al., 2007MSV is a geminivirus, it is a DNA virus, spread by aphids.The virus genome consists of a single circular ss-DNA of 2687 bp. The C1 and C2 ORFs encode a protein repA that is essential for virus replication. repA is a signalling protein that starts off replication using host mechanism so that virus can be multiplied. Expressed mutant forms of repA in the plant. Most of the transgenic plants were phenotypically abnormal -reduced yield? (cos expression of a viral signalling protein that interferes with replication affects host division also). However there was improved resistance/partial resistance. some were good.
- 46RNA-mediated Resistance RNAi mediated resistance to Cassava brown streak Uganda virus in Cassava Yadav et al., 2011Trigger defence that leads to the degradation of the RNA genome. Transgenic cassava plants were transformed with a hairpin RNA-i construct derived from the CBSV genome and containing part of the coat protein sequence; partial resistance
- 47Antibody-mediated Resistance Safarnejad et al., 2011Engineer genes for modified monoclonal antibody fragments and express in plants.
E.g. tobacco expressing TMV-specific cytosolic single chain Fv antibody: Better phenotype, reduction of necrosis
Transgenic expression in citrus of single-chain antibody fragments to Citrus tristeza virus conferred resistance - 48Other non-conventional: Ribosomal inactivating proteins and protease inhibitorsRibosome inactivating proteins – Transgenic tobacco expressing Pokeweed antiviral protein (PAP) shows broad-spectrum resistance to several viruses including CMV, PVY and PVX
Protease Inhibitors – Plant viruses belonging to the Picorna-like superfamily express their proteins by proteolytic cleavage of a polyprotein precursor. Tobacco plants expressing rice cystatin show resistance to TEV and PVY but not TMV - 49Engineered virus resistant crops that have potential for release in developing countriesCassava (ACMV), Cereals (Barley yellow mosaic virus), Maize (Maize streak virus), Papaya (PRSV), Potato (Potato virus X, Potato virus Y)
- 50TILLINGTargeting Induced Local Lesions in Genomes : mutagenize with ethyl methanosulfate (EMS) or radiation, then amplify with PCR, you get mismatch heteroduplexes caused by the mutations and Heteroduplexes were incubated with the plant endonuclease CEL I, (cleaves heteroduplex mismatched sites) and the resultant products analyzed and traced back to source plant.