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Lecture 1&2

Lecture 1&2. Agrobacterium mediated plant transformation. A.Tumefaciens gall is not a tiny thing. Biology of A. tumefaciens. Well known to induce crown gall tumor . A.tumefaciens lives around root surfaces (in rhizosphere) where it using nutrients that leak from the root tissues.

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Lecture 1&2

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  1. Lecture 1&2 Agrobacterium mediated plant transformation

  2. A.Tumefaciens gall is not a tiny thing

  3. Biology of A. tumefaciens Well known to induce crown gall tumor A.tumefaciens lives around root surfaces (in rhizosphere) where it using nutrients that leak from the root tissues infects only through wound sites and actively chemotactic to them www-genvagar.slu.se/teknik/ djup/plasm.htm Bacterial T-plasmid produces receptors for acetosyringone Plant wound produces acetosyringone

  4. The basis of Agrobacterium-mediated genetic engineering • T-DNA of A. tumefaciens is excised and integrates into the plant genome as part of the natural infection process. • Any foreign DNA inserted into the T-DNA will also be integrated.

  5. Important genes encoded by Ti plasmid • 1. Cytokinins • (plant hormone for cell plant division and tumorous growth) • 2. Enzymes for indoleacetic acid(auxin) synthesis • Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and preventing aging) • 3. Enzymes forsynthesis and release ofnovel plant metabolites: • theopines (uniques amino acid derivatives) • the agrocinopines (phosphorylated sugar derivatives) . • Opines and agrocinopines are NUTRIENTS for A.tumefacies. • They can not be used by other bacterial species • It provides unique niche for A.tumefaciens Nopaline

  6. Cytokinins are plant hormones that are derivatives of the purine adenine. Zeatin is one of cytokinines which synthesis may be encoded by Ti plamid isolated from corn (Zea mays). Cell specific expression of cytokinin in the A.tumefaciens infected cell

  7. Opines are nutrients that are also for quorum sensing The plant cells start to secrete the opines from transferred bacterial T DNA opine diffuses into the surrounding cells and serves as a signal molecules for the conjugation of the agrobacterium (Quorum sensing)

  8. Ti Plasmid T-DNA region DNA between L and R borders is transferred to plant as ssDNA; T-DNA encoded genes can be substituted by target genes Tumor- producing genes Opine catabolism Virulence region ORI

  9. Agrobacterium A unique bacterial species Plant-Fungal-Animal Transformation

  10. Agrobacterium tumefaciens 1. Soil bacterium closely related to Rhizobium. 2. Causes crown gall disease in plants (dicots).

  11. 3. Infects at root crown or just below the soil line. 4. Can survive independent of plant host in the soil. 5. Infects plants through breaks or wounds. 6. Common disease of woody shrubs, herbaceous plants, particularly problamatic with many members of the rose family. 7. Galls are spherical wart-like structures similar to tumors.

  12. Only known natural example of DNA transport between Kingdoms 1. (Virulent) strains of A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid 2. Bacteria transfer a portion of the plasmid DNA into the plant host (T-DNA). T-DNA 

  13. The T-DNA is transferred from the Bacteria into the Nucleus of the Plant 1. Stably integrates (randomly) into the plant genome. 2. Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell. 3.  Synthesis of plant growth hormones (auxins and cytokinins)  neoplastic growth (tumor formation)

  14. Opine Biosynthesis 1. Within tumor tissues, the synthesis of various unusual amino acid-like compounds are directed by genes encoded on the integrated plasmid. 2. The type of opine produced is specified by the bacterial T-DNA 3. Opines are used by the bacteria as a carbon (nutrient) source for growth. 4. Opine catabolism within bacteria is mediated by genes encoded on the Ti plasmid.

  15. Overview of the Infection Process

  16. How is the signal recognition (acetosyringone and other plant phenolics) converted to gene activation and other cellular responses?

  17. Bacterial 2-Component Signal Transduction Systems 1. Component 1 : Sensor kinase i) Substrate receptor, signal recognition domain, input domain (periplasmic) ii) Signal transduction domain, membrane spanning region iii) Autokinase domain, phosphorylation domain (cytoplasmic) a) ATP binding (sub) domain b) phosphorylation-phosphotransfer (sub)-domain

  18. 2. Component 2 : Response regulator i) Phosphorylation domain ii) DNA binding domain Simplest case: transcriptional activator when phosphorylated First component is typically (auto)-phosphorylated on a His residue and transfers to a Asp group on the response regulator (second component).

  19. The EnvZ/OmpR System of E. coli Senses changes in extracellular osmolarity

  20. Agrobacterium tumafaciens senses acetosyringone via a 2-component-like system 3 components: ChvE, VirA, & VirG 1. ChvE • periplasmic protein binds to sugars, arabinose, glucose • binds to VirA periplasmic domain  amplifies the signal

  21. 2. VirA : Receptor kinase 1. Membrane protein five functional domains: a) Periplasmic binds ChvE-sugar complex does NOT bind acetosyringone b) Transmembrane domain c) Linker region BINDS acetosyringone NOTE this is on the cytoplasmic side! d) Transmitter domain (His) auto- phosphorylates and then transfers to the response regulator protein VirG e) Inhibitory domain  in absence of analyte will bleed off the phosphate from the His in the transmitter domain (to an Asp)

  22. 3. VirG : Response Regulator a) Receiver domain that is phosphorylated on an Asp residue by the His on the transmitter domain of VirA b) Activates the DNA binding domain to promote transcription from Vir-box continaing promoter sequences (on the Ti plasmid)

  23. sugars VirA Periplasmic domain ChvE receiver acetosyringone VirG Transmitter DNA-binding Inhibitory domain

  24. Crown gall tumors a natural example of genetic engineering.

  25. Agrobacterium/plant interactions Agrobacterium at wound site transfers T-DNA to plant cell. opines Agrobacterium in soil use opines as nutrients.

  26. Genes required to breakdown opines for use as a nutrient source are harbored on the Ti plasmid in addition to virgenes essential for the excision and transport of the T-DNA to the wounded plant cell. 23 kb T-DNA tra bacterial conjugation pTi ~200 kb vir genes for transfer to the plant opine catabolism

  27. Ti plasmids can be classified according to the opines produced 1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas). 2. Octopine plasmids: carry genes(3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate, but remain as callus tissue.

  28. H2N CNH(CH2)2CHCO2H HN NH HO2C(CH2)2CHCO2H 3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out. (Nopaline)

  29. Ti plasmids and the bacterial chromosome act in concert to transform the plant 1. Agrobacterium tumefacienschromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface. 2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

  30. 3. T-DNA encodes genes for opine synthesis and for tumor production. 4. occ(opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient.

  31. Agrobacterium chromosomal DNA pscA chvA chvB T-DNA-inserts into plant genome tra bacterial conjugation for transfer to the plant pTi vir genes opine catabolism oriV

  32. Generation of the T-strand Left Border Right Border T-DNA overdrive 5’ virD/virC VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

  33. Generation of the T-strand Left border Right border T-DNA gap filled in virE T-strand D virD/virC 1. Helicases unwind the T-strand which is then coated by the virE protein. 2. ~one T-strand produced per cell.

  34. Left border Right border T-DNA D T-strand coated with virE virD nicks at Left Border sequence 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome

  35. The vir region is responsible for the transfer of T-DNA to the wounded plant cell. virA is the sensor. activated virG membrane constitutive virA virG Note: activated virG causes its own promoter to have a new start point with increased activity. positive regulator for other vir genes receptor for acetyl-syringone

  36. P P virA is the sensor. Asg 1 2 Asg virA triggers auto-phosphorylation of virA bacterial membrane Acetylsyringone is produced by wounded plant cells (phenolic compound). virG activates transcription from other vir promoters. 3 VirA phosphorylates virG which causes virG to become activated. virG virG is the effector.

  37. The vir region is responsible for the transfer of T-DNA to the wounded plant cell. effector sensor virE virG virA virD virB virC ssDNA binding protein. Binds T-strand. membrane protein; ATP-binding endo- nuclease nicks T- DNA Binds overdrive DNA. Note: The virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria.

  38. Generation of the T-strand Left Border Right Border T-DNA overdrive 5’ virD/virC VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

  39. Generation of the T-strand Left border Right border T-DNA gap filled in virE T-strand D virD/virC 1. Helicases unwind the T-strand which is then coated by the virE protein. 2. ~one T-strand produced per cell.

  40. Left border Right border T-DNA D T-strand coated with virE virD nicks at Left Border sequence 1. Transfer to plant cell. 2. Second strand synthesis 3. Integration into plant chromosome

  41. VirB1 may have local lytic activity that allows assembly of the transporter at specific sites in the cell envelope. • The processed VirB1* peptide is secreted through the outer membrane by an unknown mechanism. • The structural components of the pilus are VirB2 and VirB5. • Complexes of VirB7/9, formed by disulfide bridges, may initiate assembly of the VirB channel. • The exact role of VirB3, 4, 6, 8, 10 and 11, and VirD4 in the transporter apparatus is unknown. Assembly of the Agrobacterium T-Complex Transport Apparatus

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