lecture 11 Flashcards
Bacterial transformation of plants - overview of GM agriculture - crown gall disease, a plant 'cancer' - the biology of Agrobacterium (white collar criminals) - Vir genes and T-strands - DNA transfer and events within the plant - biotechnology - Describe the roles of VirA and VirG from Agrobacterium in T-DNA transfer
1
Q
Why is studying the pathogen of an organism helpful to studying the biology of that organism?
A
- pathogens, particularly those that are really well adapted to an organism, understand the organisms biology exquisitely
- e.g. “alleyway muggers” vs “white collar criminals” such as Agrobacterium
- studying human biology by studying its pathogens/plant biology by its pathogens, is a great way to learn how an organism functions
- it is the pathogens that know the organism better than anything else
2
Q
What/how many GM plants are different countries growing?
A
2012: over 5
- Australia: <1 million hectares; cotton, canola
- US: 70 million hectares; papaya (hawaii)
- Canada: 10.8 million hectares
- South america (e.g. Brazil and Argentina): soy beans
- China
- India
- Some european countries e.g. portugal and spain
3
Q
What is one of the fastest adopted agricultural technologies in history?
A
- GM
- initially industrial countries were the first to adopt it while developing countries lagged
- 2012 developing countries overtook industrial in terms of GM crops being grown
- a lot of that by small farmers (couple of hectares or less)
- china and india etc
- 1%/small changes in agricultural production feed millions of people — don’t need to make huge changes to make a difference as in other areas of medical research
4
Q
What are the current crops that are being transformed and grown at a large scale?
A
- soybean
- maize
- cotton
- canola
5
Q
Are transgenic animals as widely used?
A
- no
- GM crops very important while GM animals generally only used in research
6
Q
What is a plant cancer?
A
- called ‘crown gall disease’ because tumours typically form at the junction between root and stem (the crown)
- huge mass of undifferentiated cells
- major problem for grapes, (almonds, cherries and so on) and fruit trees; galls can form on a wide range of dicots and some monocots (e.g., asparagus)
7
Q
What is a gall?
A
- large mass of undifferentiated cells
- can also have ones where there is organisation
- ‘shooty’ gall - leaf-like identity of gall cells
- ‘rooty’ gall
- all caused by the same thing but different manifestations
8
Q
Why do we get different manifestations of gall?
A
- different strains of the bacteria that cause the disease
9
Q
What did Erwin Smith show?
A
- Erwin Smith showed that a bacterium ‘Bacterium tumefaciens’ caused galls on many plants
- “when minced galls are buried in the earth near the roots of sound trees, the latter develops galls. The disease is therefore a communicable one”
10
Q
What is Bacterium tumefaciens?
A
gram negative bacterium found commonly in the soil
Agrobacterium
11
Q
What has since been shown about crown gall disease?
A
- galls can be removed from the plant and grown in culture without added hormones (auxin and cytokinin)
- different Agrobacterium strains produce galls that look different (rooty vs shooty)
- galls make low molecular weight compounds — opines. Different strains produce galls that make different opines.
- Each Agrobacterium strain can grow on its own opine
- Galls do not need bacteria after initial infection (‘tumour inducing principle’)
12
Q
What are opines?
A
- there are many different opines
- the two most studied are:
- octopine — carbonyl compound is pyruvic acid
- nopaline — carbonyl compound is alpha-keto glutaric acid
- combination of an amino acid (typically arginine) and a carbonyl compound (or in some cases a sugar)
- carbonyl compound typically keto acids
13
Q
What happened in 1971?
A
- US president Richard Nixon declared war on cancer
- stimulated research on crown gall as a model for human cancer
14
Q
Was crown gall a good model for studying human cancer?
A
no
15
Q
What was the ‘tumour inducing principle’?
A
- transfer of the DNA sitting in the agrobacterium into a plant cell
- that DNA got called the transfer DNA/T-DNA
- bacterium that had worked out how to transform a plant by introducing DNA into the plant
16
Q
What is the biology of crown gall?
A
- two basic types of agrobacterium sitting in the soil: those which are infectious and those which aren’t
- the only difference between them is whether they have the Ti Plasmid
- within that plasmid is a region of DNA called the T-DNA
- senses wounded plants and migrates to hunker very close to them
- closely related to E. coli
- transfer T-DNA region from the bacterial cell to the plant cell - becomes incorporated in the plants DNA
- causes a dysregulation of hormone production
- starts to produce hormones cytokines and auxin which are necessary for cell division
- cells reenter the cell cycle
- also synthesising opines in large amounts
- this is used as a food by agrobacterium (can only live on its own type of opine)
- Ti Plasmid is the site where opines are metabolised back into food
- also conjugation of bacteria - transfer of plasmid
17
Q
How does Agrobacterium infection occur?
A
- sensing and chemotaxis: wound signals, flagellated bacteria
- attachment, intimate contact
- T-DNA transfer and integration
18
Q
How was the infection process of Agrobacterium investigated?
A
- using the tools of bacterial mutagenesis e.g. transposon mutagenesis
- integration into the bacterial genome of a transposon that affects one of the three steps of infection
- Tn5 inserted into a DNA region of a plasmid inserted into E. Coli
- Plasmid carries a certain antibiotic resistance (kanamycin)
- agrobacterium has rifamycin resistance
- try to get Tn5 to migrate from E. coli to Agrobacterium
- at the end of the experiment select for cells that have Kanamycin resistance (meaning they have Tn5) and Rif resistance (meaning they have the agrobacterium chromosomes)
- take this bacterium and see if it can infect a plant
- if not it means one of these processes has been affected
- test all Kan^r/Rif^r bacteria for ability to form a crown gall
- select all that are avirulent
- test for site of Tn5 insertion — most are in the bacterial chromosome and affect attachment to plant surface
- the cel locus codes for a gene involved in cellulose synthesis (coat made of cellulose that enables close attachment)
- the att locus encodes cell surface proteins involved in attachment
19
Q
How is the Ti plasmid involved in tumour formation?
A
- 200-250 kb in size
- two regions are associated with tumour formation
- the T-DNA region (tumour inducing principle)
- the Vir region (in opine synthesis region)
- approximately half the plasmid is involved in opine synthesis and half opine degradation
20
Q
What is the role of the Vir region in the agrobacterium Ti Plasmid?
A
- A wounded plant releases chemicals: sugars from damaged cells and a compound called acetosyringone (phenolic)
- trying to block the wound site
- these sorts of compounds are sensed by a protein on the bacterial membrane: a chemosensor
- also a kinase on the inner surface
- substrate for the kinase is a protein called VirG (a gene regulator/transcription factor)
- activated once phosphorylated
- now capable of going and binding to promoter regions for all the other genes in the Vir region and activating them
21
Q
What are the different genes and their functions in the Vir region?
A
Locus/no. of genes/function:
- A / 1 / environmental sensory
- G / 1 / transcription factor
- C / 2 / T-DNA processing
- D / 5 / T-DNA processing
- E / 2 / T-DNA processing
- B / 11 / transmembrane pore
- F / 1 / F-box protein
- overall focus is to get T-DNA into a plant cell
- produce only one T-DNA strand
22
Q
What is the T-DNA region?
A
- T-DNA has no genes involved with excision, transfer or integration into host DNA
- left and right borders - imperfect 25 bp repeats that define the transferred segment
- relies entirely upon the Vir region for its own transfer
- integration into the host DNA is done by proteins/genes found in the host cell
23
Q
What basically happens in the transfer of a T-DNA strand?
A
- single-stranded nicking of T-DNA borders
- endogenous repair mechanisms will come along and try to repair the strand causes displacement of T-strand — unwinding of T-strand, DNA synthesis
- T-strand is ss-DNA
- Formation of a pore in the bacterial membrane
- Unknown how the T-strand gets across the plant cell wall/membrane/nuclear membrane
- also transfers a protein across called VirF
24
Q
What are the major proteins involved in the process that produces a T-strand?
A
- VirD1 binds to borders and recruits VirD2 ‘relaxase’
- VirD2 ‘nicks at borders and covalently attaches to 5’ end
- other bacterial enzymes responsible for DNA synthesis etc
- VirE2 is a ssDNA binding protein and coats/protects the ss T-strand
- pore formed by VirB
- nuclear localisation signals on VirB target T-strand to nuclear membrane of plant cell/take it into the nucleus
25
Q
What is involved in the transfer of the T-complex?
A
- T-strand covered with VirE2 and with VirD2 covalently attached to its 5’ end
- Transfer by a type IV secretion pathway that translocates T-DNA and associated proteins across the cell envelope
- Requires at least one host protein (particularly for attachment)
- Type IV secretion is often associated with pathogenesis (e.g. Helicobacter pylori)
- ‘Injectosome’
- pore complex formed from VirB1-VirB11
- VirD4 involved in recruiting DNA-protein complexes for translocation
- either acting as a syringe or as a pore (unsure)
26
Q
How do we get T-complex integration?
A
T-strand covered with VirE2 and VirD2 covalently attached to its 5’ end
- protection and transport through cytoplasm to nucleus (nuclear localisation signals - NLS)
- subsequent steps largely depend on host cell proteins and processes
- integration into genome shows a preference for insertion into transcribed regions
27
Q
What does VirF do?
A
- T-strand along VirF injected into plant cytoplasm
- T-strand and VirF move into nucleus
- Inside nucleus VirF associates with host cell proteins to produce an E3 ligase that selectively adds ubiquitin to VirE2
- this step is necessary for chromosomal integration of the T-strand
- i.e. gives the proteins a means for getting rid of the proteins which are now unnecessary/inhibitory
28
Q
What happens once the T-DNA is integrated into the host chromosome?
A
- T-DNA genes behave like eukaryotic genes
- oncogenic genes stimulate cell division
- Tms promotes auxin synthesis
- Tmr promotes cytokinin synthesis
- Tml determines tumour size
- opine synthesis genes
