M1.1 Plant breeding & transformation (focus on Maize)

Question 1

2 methods of domesticating plants?

Answer 1

• refining existing diversity through breeding / sexual crossing (the green revolution)
• introducing new diversity using Agrobacterium-mediated plant transformation

Question 2

What is the likely progenitor of maize?

Answer 2

Teosinte, a subspecies of Zea mays endemic to Mesoamerica

Maize began as a mexican weed and is now a crop produced globally (most in US & China), >1 gigatonne per year (more than wheat/rice)

Question 3

Describe the features of early maize.

Answer 3

Early maize resembles teosinte:
* Fewer kernels
* More spiky
* Highly branched
* Multiple male&female inflorescences
* Hard integument covering the seed which had to be broken open

Question 4

Describe the features of modern maize.

Answer 4

Modern maize has been selected ~9 kya. Domestication saw modification of traits related to agronomy (crop production), growth, yield, floral architecture:
* taller
* more apical dominance (main stem is dominant over side stems)
* arranged in cobs, with greater diameter, and with more kernels per inflorescence
* integument has been selected out, allowing easier access to the seed

Question 5

~_% of differences between maize & teosinte are traceable to less than _ loci

Answer 5

~90%, 10

Question 6

Describe the domestication syndrome for wheat.

Answer 6

• Many were dwarf varieties (wheat used to reach over waist height), allowing more plant resources to be directed to seed production
• Better response to inorganic fertilisers
• Disease resistance (via cyclical outcrossing then backcrossing)

Question 7

Why does Agrobacterium tumefaciens transform plant cells?

Answer 7

Agrobacterium transforms its host in a manner which promotes the host to grow and produce bacterial nutrients. This allows Agrobacterium to become parasitic

Question 8

Name the system used by Agrobacterium to transform susceptible plant cells (often those which are wounded).

Answer 8

a plasmid-borne bacterial conjugation system (multi-gene Type IV secretion system T4SS)

Question 9

What is the name of the type of plasmids which Agrobacterium has?

Answer 9

large Ti (tumour-inducing) megaplasmids

Question 10

Agrobacterium large Ti (tumour-inducing) megaplasmids have a modular structure containing operons (grouped sets of coregulated genes) with different functions. List the operons it contains.

Answer 10

• A Rep (replication) region
• A Vir (virulence) region
• At least one T-DNA (transfer-DNA) region

Question 11

List the 3 phases of Agrobacterium transformation.

Answer 11

• First, Agrobacterium detects a wounded plant cell and prepares to transfer its T-DNA
• Then Agrobacterium performs a specific replicative transfer of its T-DNA into the plant cell
• Then the T-DNA integrates into the plant genome and is transcribed

Question 12

Describe the structure of Agrobacterium megaplasmids.

Answer 12

The megaplasmids have a modular structure containing operons (grouped sets of coregulated genes) with different functions:
* A Rep (replication) region (blue), encoding the proteins & origin required for the plasmid to replicate when inside the bacterial host
* A Vir (virulence) region (red), encoding the proteins required for sensing wounded plant tissue, activating the vir operon, and then transferring the T-DNA to the host (the means of doing this include the secretion system)
* At least one T-DNA (transfer-DNA) region (green). This is a specific sequence which is to be inserted into the plant genome. It’s also delimited, i.e. flanked by specific 25bp sequences (a left border and a right border), which means it’s marked in a way that signals that it’s meant to go into the plant genome

Question 13

Describe how Agrobacterium detects a wounded plant cell and prepares to transfer its T-DNA.

Answer 13

• Agrobacterium binds a susceptible/wounded plant cell, then forms a conjugation complex with it
• Wounded cells release phenolic compounds as a damage response. Bacterial membrane receptors detect the compounds & activate the signal transduction pathway, which induces expression of the bacterial Vir operon
• Bacterial Vir proteins recognise the 25bp left and right borders flanking the T-DNA segment in the Ti plasmid.
• The proteins make a single-stranded cut on either side of the T-DNA, thus cutting out a ssT-DNA fragment
• Other Vir proteins package the T-DNA into a protein-coated, ssDNA complex

Question 14

Describe how Agrobacterium performs a specific replicative transfer of its T-DNA into a wounded plant cell.

Answer 14

• The T-DNA is transmitted via the Type IV secretion system. The transfer is made efficient using a “hypodermic needle”:
  
  • 2 different Type IV secretion systems:
    
    • Trb for Ti plasmid transfer between bacteria (plasmid conjugation)
    • virB for T-DNA transfer to plant cells (translocation)
  • These type IV secretion systems are encoded by multigene operons. Highly homologous & conserved across bacterial species (similar machinery in T4SS involved in conjugative transfer between bacteria, or between bacteria and plants)
  • The elements of the secretion system assemble into a “hypodermic needle” on the surface of the bacterium — for Agrobacterium, this allows effective transfer of DNA into recipient plant cells

Question 15

Describe how Agrobacterium T-DNA, once transferred into the plant cell, integrates into the plant genome and is transcribed.

Answer 15

• Once the T-DNA is introduced, it is integrated randomly into the host plant genome as a dsDNA segment
• The parasitic part: the T-DNA segment contains genes encoding enzymes for the synthesis of:
  
  • Enzymes which provide opines (bacterial nutrient which the plant cannot metabolise) → allows bacterium to grow
  • Plant growth hormones (auxins, cytokinin) → these promote unregulated plant growth (ie tumours). More plant tissue means the plant produces more opines

Question 16

How can we use Agrobacterium to insert foreign T-DNA into a random section of the plant genome?

Answer 16

• We first transform Agrobacterium with a binary plasmid system
• We use this system to simplify the Agrobacterium system. You don’t want to engineer anything but this T-DNA into the genome – Vir region is unnecessary clutter
• Then we let Agrobacterium infect & transform the plant
• Each of the 2 plasmids has its own different bacterial selection marker, and you test for both, so that you can isolate only the bacteria which have successfully incorporated both plasmids. The vector plasmid destined to go into the plant also contains a plant selection marker

Question 17

Why do we use 2 plasmids in the binary plasmid system, rather than just editing the one Agrobacterium megaplasmid?

Answer 17

• 2 different plasmid are easier to work with than one really big one
• Separating the material into 2 also means that the Vir regulon doesn’t end up in the transformed plant

Question 18

Name the 2 types of plasmid in the Agrobacterium binary plasmid system and state the function of each.

Answer 18

• The Vir helper plasmid / disarmed Ti plasmid (smaller) contains the functions required for T-DNA transfer
• T-DNA binary vector plasmid (larger) contains the T-DNA to be inserted into the plant genome

Question 19

The Vir helper plasmid / disarmed Ti plasmid (smaller) contains the functions required for T-DNA transfer. What does it contain?

Answer 19

• Compatible replication origins
• The Vir regulon (which is used to activate the larger plasmid)
• A bacterial selection marker (different from that used in the larger plasmid) which serves to confirm that this plasmid has been successfully incorporated into the bacterium

Question 20

The T-DNA binary vector plasmid (larger) contains the T-DNA to be inserted into the plant genome. What does it contain?

Answer 20

• A customised & disarmed T-DNA segment, flanked by a LB and RB, usually including:
  
  • The gene(s) of interest
  • A plant selection marker allowing the rescue of transformed plants only (the rest of it can be arbitrary sequences)
  • NOT the genes encoding tumourigenesis & opine production (the modularity of the Agrobacterium megaplasmid means it can be disassembled and these genes are removed)
• A bacterial selection marker (different from that used in the smaller plasmid) which serves to confirm that this plasmid has been successfully incorporated into the bacterium
• A replicative origin

Question 21

Describe the procedure of Agrobacterium-mediated plant transformation.

Answer 21

• Agrobacterium is transformed with the 2 plasmids (successful transformants are isolated by screening for the 2 different bacterial selective markers)
• Plant material is co-cultivated with the engineered Agrobacterium strain
• Agrobacterium is cured via microbial/antibiotic treatment
• Plantlets from transformed cells are regenerated via plant-specific antibiotic selection (eg the engineered T-DNA could contain kanamycin)
• Regenerated plants are rescued, grown, and the transgenic seed harvested (and can then be bred as normal)

Question 22

Biolistic transformation is another method of producing transgenic plants. When would you use this method as opposed to Agrobacterium-mediated transformation?

Answer 22

When you want to do transient expression of transgenes (preferable for transforming organelles)

Question 23

Give 2 advantages of biolistic transformation over Agrobacterium transformation (given you only want transient expression).

Answer 23

• Compatible for a greater range of plants, eg ferns & monocots (also algae)
• Quicker & easier

Question 24

Summarise the process of biolistic transformation and explain why it is transient.

Answer 24

• High-velocity, DNA-coated microparticles (often gold/tungsten) are used to bombard a cell
• Results in plasmids being introduced into cells (sometimes more than one) – these do transform the cell, but may not be passed to offspring, hence the transience