1. Which two classes of plant hormones are synthesized in crown gall tissues? What is the function of the “oncogenes” encoded on the T-DNA?
Plantgrowth hormons: auxin & cytokinin
“oncogenes” are tumor inducing genes that lead to uncontrolled cell growths
2. Which unusual compounds are found in many crown galls? Name the two most common examples. What is the purpose of these compounds?
Octopine and Nopaline : (are carbon and nitrogen source for the agrobacterium)
Opine synthesis genes are encoded on the T-DNA
Opines induce expression of genes required for opine uptake and catabolism
Carbon, nitrogen and energy source for Agrobacterium that cannot be catabolized by the plant
3. What is the name of the phenolic metabolite that is released by wounded plant cells and activates Agrobacterium vir gene expression?
Acetosyringone is perceived by the transmembrane VirA sensor protein encoded on the Ti plasmid.
It is a Lignin component which is sensed the wounding of the plant
4. What is a “two-component system”? Which two-component system functions as a major activator of Ti plasmid encoded vir genes. What is the function of the two components?
a two-component system recognizes signals —> allows organisms to sense and responds to changes
one part recognizes, the other part responds (signal mostly phosphorylation)
VirA: is a transmembrane sensor protein, that recognises Acetosyringone
VirA phosphorylates VirG in cytoplasm
VirG binds to vir promotors and activates transcripiton of virulence genes
Summarize the recognition and virulence activation in Agrobacterium upon finding a host plant.
5. Briefly describe the key features (genes, regions) of Ti plasmids. Which part of the Ti plasmid is transferred into host cells, how is this part confined and which genes does it contain?
Part transfered into host cells: Between T-RNA border (Oncogenes and opine synthesis genes)
6. What is the function of VirD2?
1. VirD2 is an endonuclease that nicks the Ti plasmid at 25 bp direct repeat border sequences that flank the TDNA.
2. The helicase VirD1 is required for unwinding the Ti-plasmid
3. VirD2 remains covalently bound to the 5’-end of the single stranded T-DNA
7. Which type of secretion system does Agrobacterium employ for delivery of the T-complex into host cells?
Type 4 secretion system, encoded on VirB operon
“needle" pilius inserts into plant cell to transfer T-DNA
How long does it take agrobacteria to reliably transfer genes into the plant host?
2 days, heat experiments
8. In plant genetic engineering, how can you detect transformed plants?
Add selective marker (eg. herbicide resistance)
Add fluorescence marker (eg GFP)
9. What are binary vector systems?
Create an overview, what VirA - VirG functions are
What does VirE2 do? Design an experiment that proves that VirE acts inside the plant cell, not the bacteria
Experiment:
Delete VirE on bacterial plasmid
Express VirE in transgenic plants
Infect wt and transgenic plants with VirE and observe suceptibility of plants
Describe the protocoll for flora dipping of Arabidopsis flowers in Agrobacteria culture
Dip female flowering Arabidopsis plant in Agrobacterium with selectable marker (herbicide resistance)
Wait for seed production
Plate seeds on herbicide media, only transformed seeds with herbicide resistance will grow
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