Agrobacterium Flashcards
what are the two general types of genes present on the pTi (tumor inducing plasmid)?
T-DNA
vir genes
what are vir genes?
- Encodes all of the functions required for the transfer of the T-strand to the plant
- function is to make a tumor to pump out opines
- The vir genes of the Ti plasmid are located outside the transferred region and required for the translation process
- induced by phenolic compounds released by plants in response to wounding
virulence region
produces all of the structures and produce required to pump out and ship to the plant
- encodes all the functions required for the transfer of the T-strand to the plant
what is the mechanism behind T-DNA transfer from agrobacterium to the plant?
- Vir A is embedded in the membrane and receives the signal from acetosyringone to activate
- VirA is then phosphorylated
- VirG is phosphorylated and can bind to target promoters
- T-dna is generated when a nick at the right boundary creates a primer for synthesis of a new DNA strand
- in order to not copy the whole genome, two oriTs are used and virC ensures that the right border is cut first and then the left
- Overdrive is the binding site for overdrive, and Vir C has an affinity for VirD1 and VirD2.
- VirD1&2 nick the strand
- Synthesis of the new strand displaces the old strand
- Helicases unwind the T strand which is then coated by the Vir E protein
- One T strand produced per bacterial cell
- transferred to plant nucleus via Vir E, D1, and D2 via T4SS channel
- exact T-DNA integration is unknown
How is transfer of T-DNA similar to bacterial conjugation?
The t-dna is transferred as a complex of single stranded dna with the vir e2 single stand binding protein
- The single stranded t-dna is converted into ds dna and integrated into the plant genome
What is the function of ChvE and where is the gene found?
- found on the chromosome
Which two genes of the vir region are constitutively expressed?
Vir A and G
G is inducible
What is acetosyringone and what is its role?
- Produced by wounded plant cells (phelic compound)
- Agrobacterium infection succeeds only on wooded plants
- Phenolic compound - crosslinks to proteins to inactivate them (Used to make proteins to make them non digestible )
- Is one of the phenolic signals that signals it has been wounded
- Perceived by agrobacterium to initiate transfer
- Cellulose makes it hard to get through the cellulose wall
What role does arabinose play in the activation of the vir region?
- Arabinose = building blocks for cellulose synthesis
- Agrobacterium can sense the cell is wounded and still alive because it makes new cell wall (arabinose)
- Chve binds arabinose and has an affinity to the periplasmic domain for vir A - making it 100x more sensitive to binding to acetyl
Which two proteins of the vir region are related to other bacterial two-component system?
the virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria
What role does phosphorylation play in the activation of the vir region?
- Scheme is called phosphohistidine relay
- Vir A is autophosphorylated due to it sensing acetosyringone
- The phosphates are transferred to the effector
Vir G is phosphorylated and activates transcription - Opens the DNA binding domain
Which proteins of the vir region travel to the plant nucleus?
Vir E, virD2, and vir D1
Which proteins contain nuclear localization sequences (NLSs)?
E and D
Why does the VirD1/VirD2 complex nick at the right border first?
Nicks at the right border first due to overdrive; overdrive recruits vir C
In the VirD1/VirD2 complex, which one plays a role similar to TraI?
VirD2
How much of the T-DNA is actually transferred to the plant?
just the T strand
What components are usually found on the mini-Ti plasmid?
oriT, kanamycin resistance to select for transformation in the plant, the right and left border sequences with the engineered gene(s) in between.
What is a “bom” (or “mob”) site?
- Basis of mobilization
- oriT that works with tra
- Will cut the miniTi
- oriT of the mobilization plasmid
What is the function of the modified Ti plasmid?
Provides the vir functions to the vector
How does kanamycin resistance allow transformed plant cells to survive on Kan plates?
Kanamycin is toxic for bacterial ribosomes. Since the chloroplast is recently derived from cyanobacteria, its ribosomes are prokaryotic and therefore sensitive to kanamycin. The kan resistance gene breaks down the kanamycin in the selection plates, which allows the transformed plant cells to survive.
What does the helper plasmid supply in the mobilization of the vector plasmid into Agrobacterium?
- two component system
- Clone small plasmids to engineer with our genes and make it able to be transported to agrobacterium then agrobacterium into a plant
- makes use of a binary vector
- vir genes on the mobilization plasmid is required and are transferred to the plant residue on a modified pTi
- first, delete the T-DNA on the helper plasmid to remove the left and right borders and not transfer any helper DNA
- now there are only left and right borders on the mob plasmid
- the mob plasmid consists of the right and left border sequences, a selectable marker (kanamycin resistance) and a polylinker for insertion of a foreign gene
- may or may not have an oriT in the helper plasmid
T-DNA
- Part that is transferred from the plasmid
- 23 kb region that is transferred to the plant (only the t strand is actually transferred)
- Integrates into the chromosome
- used to generate opine synthesis and plant hormone synthesis
pTi
- Transforms a portion of the large plasma and transfers to a eukaryote and has a promoter that can be read by a eukaryote
- About 200 kb
opines
- Genetically engineers to plant to make this
- Conjugates of basic amino acids with alpha ketoglutarate and pyruvic
- Unnatural combination not found in nature often - Most systems cannot break it down
- Agrobacterium can break it down as a food source
- Engineers plant to produce this
- 7% of dry mass can be in opines
- Allows the bacterium to use octopine and nopaline nutrient (most other soil bacteria lack these genes)
