Agrobacterium

Question 1

what are the two general types of genes present on the pTi (tumor inducing plasmid)?

Answer 1

T-DNA
vir genes

Question 2

what are vir genes?

Answer 2

• 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

Question 3

virulence region

Answer 3

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

Question 4

what is the mechanism behind T-DNA transfer from agrobacterium to the plant?

Answer 4

• 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

Question 5

How is transfer of T-DNA similar to bacterial conjugation?

Answer 5

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

Question 6

What is the function of ChvE and where is the gene found?

Answer 6

• found on the chromosome

Question 7

Which two genes of the vir region are constitutively expressed?

Answer 7

Vir A and G
G is inducible

Question 8

What is acetosyringone and what is its role?

Answer 8

• 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

Question 9

What role does arabinose play in the activation of the vir region?

Answer 9

• 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

Question 10

Which two proteins of the vir region are related to other bacterial two-component system?

Answer 10

the virA-virG system is related to the EnzZ-OmpR system that responds to osmolarity in other bacteria

Question 11

What role does phosphorylation play in the activation of the vir region?

Answer 11

• 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

Question 12

Which proteins of the vir region travel to the plant nucleus?

Answer 12

Vir E, virD2, and vir D1

Question 13

Which proteins contain nuclear localization sequences (NLSs)?

Answer 13

E and D

Question 14

Why does the VirD1/VirD2 complex nick at the right border first?

Answer 14

Nicks at the right border first due to overdrive; overdrive recruits vir C

Question 15

In the VirD1/VirD2 complex, which one plays a role similar to TraI?

Answer 15

VirD2

Question 16

How much of the T-DNA is actually transferred to the plant?

Answer 16

just the T strand

Question 17

What components are usually found on the mini-Ti plasmid?

Answer 17

oriT, kanamycin resistance to select for transformation in the plant, the right and left border sequences with the engineered gene(s) in between.

Question 18

What is a “bom” (or “mob”) site?

Answer 18

• Basis of mobilization
• oriT that works with tra
• Will cut the miniTi
• oriT of the mobilization plasmid

Question 19

What is the function of the modified Ti plasmid?

Answer 19

Provides the vir functions to the vector

Question 20

How does kanamycin resistance allow transformed plant cells to survive on Kan plates?

Answer 20

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.

Question 21

What does the helper plasmid supply in the mobilization of the vector plasmid into Agrobacterium?

Answer 21

• 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

Question 22

T-DNA

Answer 22

• 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

Question 23

pTi

Answer 23

• 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

Question 24

opines

Answer 24

• 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)

Question 25

VirG

Answer 25

- analogous to J - positive acting transcription factor; not active unless A

Question 26

VirA

Answer 26

- the membrane embedded sensor - senses the phenolic wound compound

Question 27

VirB

Answer 27

- forms the channel - T4SS; ATP binding

Question 28

VirC

Answer 28

- binds overdrive DNA; - targets the mobilization so the nicking enzyme cuts on the right instead of the left side - When t-dna is being transferred, to not copy the whole genome, two oriTs are used. - Vir C ensures that the right border is cut first and then the left to ensure that the T-DNA is being cut and not the other part of the replicon

Question 29

VirD1 & VirD2

Answer 29

- endo-nuclease nicks T-DNA - similar to tri i and y; 1 = y, 2 = i - 2 does the nicking and covalently binds to the 5’ end

Question 30

VirE2

Answer 30

- produces enough protein to coat the one strand of T DNA - Rolling circle except the circle doesn't go all the way around - Contains a short sequence that has the T-DNA go to the nucleus - Nuclear localization sequence - ssDNA binding protein, binds T-DNA strand

Question 31

ChvE

Answer 31

- Chromosomal location virulence - Receives an additional signal - Gene is encoded on the chromosome - works with arabinose

Question 32

nuclear localization sequence (NLS)

Answer 32

- Vir E2, and VirD has this - Tells t-DNA to go the nucleus

Question 33

overdrive

Answer 33

- Apart of ChvE - The sequence where vir c binds - Cutting site is on the right side because of overdrive; overdrive is binding site for vir C and vir C has an affinity for the vir D1-2 site

Question 34

Disarmed vectors

Answer 34

do not produce tumors; can be used to regenerate normal plants containing the foreign gene

Question 35

what are the components of the Ti plasmid?

Answer 35

- T-DNA - Vir genes - Opine catabolism - Bacterial conjugation

Question 36

What are the two main classes of genes that are transferred to the plant?

Answer 36

- Hormone synthesis (shi - shoot induction and roi - root induction) - Opine synthesis (nos - nopaline synthesis and ocs - octapine synthesis)

Question 37

what bacterium can transform plant cells into tumors?

Answer 37

A. tumefaciens - the infectious agent is a plasmid carried by the bacterium

Question 38

opine catabolism

Answer 38

allows the bacterium to use octopine and nopaline nutrient - most other soil bacteria lack these genes

Question 39

bacterial conjugation

Answer 39

- the vir region is similar to tra in other bacteria - this is for the transfer of the entire pTi between different agrobacterium

Question 40

what is the hypothesis of how T-DNA is integrated into host chromosome?

Answer 40

- probably through nonhomologous recombination that targets AT rich sites - then T-DNA is expressed at its site of integration

Question 41

which components of the tra region are analogous to virD1/virD2?

Answer 41

tray and trai

Question 42

binary vector

Answer 42

the vir genes are required for mobilization and transfer to the plant on a modified pTi

Question 43

explain how miniTi can be placed into the chromosome of the plant

Answer 43

- goes from the miniTi to the agrobacterium to the plant - within E. coli you have the clone miniTi with the genes of interest that you want in the plant - insert the helper plasmid with the tra region that may or may not have an origin - the helper plasmid supplies TraY/TraI to cut the segment of T-DNA that is on the miniTi and is cut at the bom site - T-DNA is transferred where the vir processes continuous and is transferred to the plant