lecture 13

Question 1

What are plastids?

Answer 1

• Plastids are a major organelle in plant and algal cells
• they are semi-autonomous with their own genetic material
• plastids are capable of differentiating into various types
• some store large amounts of starch (amyloplasts)
• others are photosynthetic (chloroplasts)

Question 2

Where is the second genome of animals cells found? How is this important?

Answer 2

• in the mitochondria

- it means that you could also transform the mitochondria

Question 3

How many genomes do plants have?

Answer 3

3:
- nuclear genome
- mitochondrial genome
- plastid genome (e.g. chloroplast)

Question 4

Why bother putting genes into a plastid?

Answer 4

• plastids are maternally inherited
• some proteins can be toxic in the cytoplasm
• proteins/polymers can accumulate to high levels (it’s what they usually do)
• integration is by homologous recombination

Question 5

What is the plastid genome?

Answer 5

• Ds DNA 120-220 kb circular and linear forms
• up to 10,000 copies per cell
• ~120 genes
• 20-30 kb inverted repeats (IRa and IRb) separate the large and small single copy regions (LSC and SSC)

Question 6

What experiment was performed with unicellular alga?

Answer 6

• Chlamydomonas has a single large chloroplast
• using biolistics, a non-photosynthetic Chlamydomonas mutant was transformed with an ATPase subunit gene
• a high frequency of direct homologous replacement events in the chloroplast was found but there was no integration of plasmid sequences

Question 7

What is homologous recombination in plastids?

Answer 7

• complete replacement of the target gene with the replacement gene
• the only way you can integrate DNA in a plastid

Question 8

What is the standard design of transformation construct?

Answer 8

• the standard design of a transformation construct has two regions of plastid DNA surrounding the gene of interest and a spectinomycin resistance gene (aadA)
• don’t need the shoulder regions

Question 9

Can flowering plant plastids be transformed?

Answer 9

• yes, flowering plant plastids can also be transformed

Question 10

How do you get the transformation construct into the cell?

Answer 10

• put it onto the beads
• load them into the gun
• fire up the gun e.g. helium gun
• accelerates disc which hits stopping plate and small pellets come through and into the tissue

Question 11

What is heteroplasmy?

Answer 11

• a plant cell can have have hundreds of chloroplasts
• for transformation to occur, a DNA-coated particle has to enter a chloroplast
• in a typical experiment, only one or two chloroplasts will have such particles
• this results in a situation called “heteroplasmy”
• this is the presence of two or more different plastid genomes in a cell

Question 12

What is the problem of heteroplasmy?

Answer 12

• heteroplasmic situations are genetically unstable and usually resolve spontaneously (‘homoplasmy’)
• this sorting-out is due to random genome segregation upon plastid division, as well as random plastid segregation upon cell division

1. 50-100 plastids/cell, 50-100 genomes/plastid
2. primary event changes a single plastid genome
3. cell/organelle division under spectinomycin selection — ptDNA is heteroplasmic
4. Regenerated plant reselected on spectinomycin — ptDNA is homoplasmic

• go through rounds of selection with spectinomycin until you eventually have a homoplasmic plant
• can use GFP reporter

Question 13

What is a biotechnology application of the Bt toxin?

Answer 13

• Dipel
• Bt toxins are insecticidal proteins from the Gram positive bacteria Bacillus thuringiensis
• proteins found as crystal inclusions in sporulated bacteria
• potent toxins of specific classes of insects
• • Lepidoptera - butterflies and moths
• • Hymenoptera - ants, bees, wasps, etc.
• • Coleoptera - beetles and weevils
• Early biotech interest in expressing Bt toxins in plants

Question 14

What were some expression problems with the Bt toxin?

Answer 14

• Agrobacterium-mediated transformation used to introduce Bt toxins into plants
• Poor levels of insect resistance because of low protein expression
• Re-engineering the Bt gene increased insect resistance to acceptable levels
• Protein expression increased from barely detectable (WT protein) to <100 ng per 50µg total protein (0.2%)
• preferences for G+C in plants - increased numbers in re-engineered version - changing codons without changing amino acid (redundancy)

Question 15

How were these problems with the Bt toxin overcome?

Answer 15

• McBride et al (1995) expressed WT Bt toxin in plastids
• Bt toxin represented 3-5% of total leaf protein
• High levels of insect resistance and no adverse effects on plant growth
• Plastid transformation attracts considerable attention for high level production of foreign proteins

Question 16

How can we use viruses to deliver genes?

Answer 16

• viral nucleic acids are infective, can cross cell walls, and can spread from cell-to-cell
• most plant viruses have ssRNA genomes that serve as mRNA without further transcription

Question 17

What is potato virus X?

Answer 17

• potato virus X (PVX) is a widespread virus, with symptoms ranging from mild leaf mottling to a severe mosaic
• however symptoms are often not visible
• the ssRNA genome is 6.4kb long and encodes an RNA-dependent RNA polymerase (RdRp), a coat protein (CP) and 3 movement proteins (MP1-3)

Question 18

How do we take advantage of PVX ?

Answer 18

• cDNA inserts can be placed between MP3 and CP genes
• in vitro transcription of the DNA from a bacterial promoter produces infective RNA, which can be rubbed onto leaves with a little ground glass
• this leads to transient but systemic expression of the cDNA-encoded protein
• size limitations (1-2kb)

why not incorporate PVX into a T-DNA?

• can put the entire virus inside a T-DNA
• enters into the nucleus
• using Agrobacterium to deliver viral T-DNAs increases the number of infected cells and improves the yield of protein
• you want to get translation to occur and harvest the plant before its natural defences occur

Question 19

How can plants act as factories for recombinant protein production?

Answer 19

• Depending on the protein, yields of up to 0.5-5g recombinant protein per kg leaf biomass have been achieved

a) growing plants and bacteria
b) agroinfiltration
c) plant incubation
d) biomass harvest
e) extraction
f) protein purification

Question 20

Why use plants as protein factories?

Answer 20

• technology in hand
• simple, inexpensive means of producing complex proteins
• elimates the risk of contamination with animal pathogens (animal ethics)
• heat stable environment

Question 21

What are some Agrobacterium-mediated transformation research application?

Answer 21

• what Agrobacterium can do
• T-DNA ‘plug-n-play’
• promoter trapping
• GAL4 trapping
• cytokinins and root development

Question 22

What can Agrobacterium do?

Answer 22

• transfer T-DNA to region to a eukaryotic cell
• get T-DNA into the nucleus
• T-DNA genes may be expressed without integration (transient transformation)
• T-DNA may be integrated randomly into genome (stable transformation)

Question 23

What is in the plant molecular biologists’ toolkit?

Answer 23

Using Agrobacterium you can:

• add new functions to an organism (e.g. herbicide resistance to soybeans)
• remove an existing function (e.g. FlavrSavr tomato)
• express proteins transiently in mature organs (e.g. leaf)

Question 24

What does a “typical” gene have?

Answer 24

• three modular parts
• each has its own separate and largely independent function
• promoter: regulates the expression of a nearby gene
• coding region: will be expressed if near a promoter
• 3’ UTR
• Non-Coding DNA isn’t expressed

Question 25

What is T-DNA plug-n-play?

Answer 25

- right and left border - if you want to add a function: promoter driving function of a coding region both of which are inserted in T-DNA, also a stop sequences (3' UTR) - left and right border + promoter --> random integration so if it lands near something it will change the way it is expressed - coding region + stop signal = if it happens to land near promoter plant will express coding region according to that promoter - adding NC DNA may inactivate something

Question 26

What is promoter trapping?

Answer 26

- randomly insert a promoterless Kan^r gene into a plant genome and screen for kanamycin resistant calli - has to be inserted in cells undergoing division - to be expressed, the promoterless Kan^r gene must insert near a promoter that is activate in calli - region around where there is active transcription occurring is a good site for T-DNAs to insert into them - conformation of the T-DNA allows that to happen better than the conformation of DNA in genes that are inactive - T-DNAs seem to preferentially insert next to genes that are being transcribed (evidenced by the fact that inserting a fully functional gene vs a promoterless gene has little impact on the number of cells that are transformed, [5 vs 10 in 10,000])