lecture 11

Question 1

Why is studying the pathogen of an organism helpful to studying the biology of that organism?

Answer 1

• 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

Question 2

What/how many GM plants are different countries growing?

Answer 2

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

Question 3

What is one of the fastest adopted agricultural technologies in history?

Answer 3

• 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

Question 4

What are the current crops that are being transformed and grown at a large scale?

Answer 4

• soybean
• maize
• cotton
• canola

Question 5

Are transgenic animals as widely used?

Answer 5

• no

- GM crops very important while GM animals generally only used in research

Question 6

What is a plant cancer?

Answer 6

• 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)

Question 7

What is a gall?

Answer 7

• 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

Question 8

Why do we get different manifestations of gall?

Answer 8

• different strains of the bacteria that cause the disease

Question 9

What did Erwin Smith show?

Answer 9

• 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”

Question 10

What is Bacterium tumefaciens?

Answer 10

gram negative bacterium found commonly in the soil

Agrobacterium

Question 11

What has since been shown about crown gall disease?

Answer 11

• 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’)

Question 12

What are opines?

Answer 12

• 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

Question 13

What happened in 1971?

Answer 13

• US president Richard Nixon declared war on cancer

- stimulated research on crown gall as a model for human cancer

Question 14

Was crown gall a good model for studying human cancer?

Answer 14

no

Question 15

What was the ‘tumour inducing principle’?

Answer 15

• 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

Question 16

What is the biology of crown gall?

Answer 16

• 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

Question 17

How does Agrobacterium infection occur?

Answer 17

1. sensing and chemotaxis: wound signals, flagellated bacteria
2. attachment, intimate contact
3. T-DNA transfer and integration

Question 18

How was the infection process of Agrobacterium investigated?

Answer 18

• 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

Question 19

How is the Ti plasmid involved in tumour formation?

Answer 19

• 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

Question 20

What is the role of the Vir region in the agrobacterium Ti Plasmid?

Answer 20

• 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

Question 21

What are the different genes and their functions in the Vir region?

Answer 21

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

Question 22

What is the T-DNA region?

Answer 22

• 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

Question 23

What basically happens in the transfer of a T-DNA strand?

Answer 23

• 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

Question 24

What are the major proteins involved in the process that produces a T-strand?

Answer 24

• 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

Question 25

What is involved in the transfer of the T-complex?

Answer 25

- 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)

Question 26

How do we get T-complex integration?

Answer 26

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

Question 27

What does VirF do?

Answer 27

- 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

Question 28

What happens once the T-DNA is integrated into the host chromosome?

Answer 28

- 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