Engineering the plastid

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The presentation describes the advantages of plastid transformation over 'conventional' nuclear transformation, hurdles to plastid transformation, its advantages. The presentation also covers some successful plastid engineering and its potential.

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Engineering the plastid

  1. 1. For a greener future Sachin S Rawat School of Biotech, GGS IP University
  2. 2. A Look at the Plastid
  3. 3. A Plastid is a….  Major organelle of plant and algal cells  Site of manufacture and storage of important chemical compounds  Has circular, dsDNA copies  Replicates autonomously of the cell  Thought to have been originated from endosymbiotic bacteria  Plastid genes show maternal inheritance
  4. 4. Derived from proplastids in meristem
  5. 5. Have diverse functions  Chloroplasts – green plastids – for photosynthesis  Chromoplasts – coloured plastids – for pigment synthesis and storage  Gerontoplasts – control dismantling of photosynthetic apparatus during senescence  Leucoplasts – colourless plastids – monoterpene synthesis  Leucoplasts include amyloplasts (starch), elaioplasts (fats), proteinoplasts (proteins) and tannosomes (tannins)
  6. 6. 120-130 plastid genes Are densely packed and fall into 2 categories:  Photosynthesis-related genes  Genetic system genes - genes for rRNAs, tRNAs, ribosomal proteins and RNA polymerase subunits
  7. 7. A Fresher Look at the Plastid
  8. 8. Why plastid transformation?  High protein expression levels  Absence of epigenetic effects  Uniparental inheritance is commercially favoured  Easy transgene stacking in operons  Increased biosafety – Since plastids are maternally inherited, they aren’t transmitted by pollen
  9. 9. Hurdles to ‘transplastomic’ plants  Difficulty in delivering foreign DNA through double membrane of the plastid  The enormous copy number (polyploidy) of the plastid genome  The desired genetic modification must be in each copy of plastid genome in each cell  Failure to achieve homoplasmy results in rapid somatic segregation and genetic instability  Repeated rounds of selection and regeneration are required
  10. 10. DNA delivery into plastids  2 successful methods include biolistics and polyethylene glycol-mediated transfer  Biolistics is preferred as it is less time-consuming and demanding  Integration of foreign DNA into plastid genome occurs via homologous recombination  Homologous recombination operates in plastids at a high efficiency
  11. 11. Biolistic chloroplast transformation and transgene integration into the plastid genome via homologous recombination
  12. 12. Recent success  Expression of Bt toxin gene from the tobacco plastid genome  High accumulation levels of Bt toxin protein (3-5 % of TSP)  Plants with high-level resistance to herbivorous insects  Co-expression with upstream ORFs further increased Bt toxin accumulation and even resulted in its crystallization in chloroplast  Production of somatotropin (7% TSP) in tobacco plastids
  13. 13. Case Study I – Lactuca sativa
  14. 14. Protoplast isolation  Lettuce seeds were sterilized and sown on MS medium with 2% sucrose  Shoot tips from leaves obtained were transferred to MS medium with 3% sucrose  The leaves were cut into pieces and incubated in PG solution, followed by enzyme solution consisting of 1% cellulase and .25% macerozyme  Protoplast suspension was filtered through nylon mesh  Protoplasts were collected at surface after centrifugation at 70g for 8min
  15. 15. Transformation and culture  10µl transforming DNA and 0.6ml PEG solution was added to protoplast suspension and incubated at 25ºC for 10min  Protoplasts were mixed with 1:1 solution of B5 and 2% agarose to a density of 3.6 X 104 protoplasts per ml  The suspension was plated onto Petri dishes and cultured at 25ºC in the dark  Selection was initiated on the 7th day by fresh medium containing spectinomycin dihydrochloride
  16. 16. Analyses  PCR – specific primers were used to assess the presence of aadA gene in resistant cell lines  Immunoblot analysis – using HRP-conjugated secondary antibodies  Southern and Northern blots were performed to look for target genes and their transcripts  After 2 weeks, non-transformants were yellow while spectinomycin-resistant seedlings were green and growing vigorously
  17. 17.  100% of spectinomycin-resistant lettuce cell lines were true plastid transformants  A limitation was the high frequency of polyploid cell lines
  18. 18. Production of human therapeutic proteins Why lettuce is favoured over tobacco?  Most of the plant is leaf tissue and this tissue contains the greatest number of plastids per cell  Unlike tobacco, lettuce has no toxic alkaloids that need to be removed - low purification and downstream processing costs  Lettuce is a relevant human foodstuff that can be consumed without cooking
  19. 19. Case Study II – Petunia hybrida
  20. 20. Plastid transformation  Leaf pieces were placed on MS medium supplemented with 1 mg/l 6-benzylaminopurine, 0.1 mg/l IAA, 30 g/l sucrose and 0.8% agar (MSB30)  Leaves were bombarded with 1µm, vector-coated gold particles from distance of 6cm  Incubated in dim light for 48h at 25ºC  Leaves were transferred to MSB30 medium with 200mg/l each of streptomycin sulfate and spectinomycin dihydrochloride pentahydrate  Resistant shoots first appeared after 8 weeks
  21. 21. Vector design
  22. 22. Analyses  DNA blot – gene specific primers were used  GUS assay – 5-Bromo-4-chloro-3-indolylbeta-D- glucuronic acid was used to compare the protein expression levels between the wild type and the transformants by detecting fluorescence  Selection on two antibiotics overcomes the problem of spontaneous resistant mutants associated with using spectinomycin alone
  23. 23. Comparing plastid transformants with non-transformants
  24. 24. Good model to study plastid biology  N. tabacum is amphi-diploid  A. thaliana doesn’t give rise to fertile transplastomes These limitations are overcome in Petunia as:  P. hybrida is diploid  Suitable for mutation screening to identify nuclear loci affecting the maintenance and expression of plastid transgenes
  25. 25. A Look at the Future
  26. 26. Metabolic pathways into plastids?  Cost-effective production platform for pharmaceuticals and nutraceuticals  Production of trehalose in tobacco chloroplasts  Tryptophan overproduction by feedback- insensitive synthesis of α-subunit of anthranilate synthase  Simplifying technology, extending crop range
  27. 27. Can we engineer photosynthesis?  Efficiency of photosynthesis  The most abundant protein in the world  Its CO2:O2 specificity that matters  Getting a better RubisCO for your plant  Equally precise tools for nuclear genome required
  28. 28. Plastids for Synthetic Biology  A compact, minimal genome  High transgene expression and low cost ideal for synthetic biology  Designing totally new plastids
  29. 29. References  Bock and Khan; Taming plastids for a green future; Trends in Biotechnology  Lelivelt et al.; Stable plastid transformation in lettuce; Plant Molecular Biology  Zuilen et al.; Stable transformation of Petunia plastids; Transgenic Research
  30. 30. Thank you 

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