Genetic manipulation of plants

What can DNA or RNA of interest be loaded into to insert into a cell?

Plasmid or a ribonucleoprotein

Once you have loaded your DNA what are the delivery techniques you can use?

Biolistic bombardment, nanoparticles, agrobacterium, Poly-ethyl glycol (permeabilises the membrane) and viral vectors

How many countries in the world allow GM?

30/197

Why do we use back crossing for conventional breeding?

It limits the genome content from the line you don’t want

What is back crossing?

a breeding technique where a hybrid offspring is crossed back with one of its parents to produce offspring with genetic characteristics closer to the elite parent

How is conventional breeding monitored?

Uses gene markers

Limitations of conventional breeding?

• VERY TIME CONSUMING

• Genes are linked - desired gene may be tightly linked to undesirable gene

• Polygenic traits - would need multiple unique markers

• Available gene pool is limited

Advantages of conventional breeding?

Can generate many individuals in a relatively short space of time.

You can phenotype relatively easily.

What are genotyping databases?

Digital repositories that store, curate, and share information on genetic variants (DNA variations) and their associated phenotypic traits

What are cultivar lines?

Specifically selected, genetically uniform plant populations maintained through controlled reproduction (self-pollination or asexual propagation) to retain desirable traits

What causes crown gall disease?

Caused by a gram negative bacterium (Agrobacterium tumefaciens), galls produce their own auxins and cytokinins

Agrobacterium tumefaciens mechanism of HGT:

conjugation - highly successful + evolved to be very accurate

Why is HGT by Agrobacterium tumefaciens special?

They can carry out inter-kingdom gene transfer (e.g. from bacteria to plants)

What is the plasmid in Agrobacterium tumefaciens that is responsible for gene transfer?

The Ti plasmid (Tumour inducing)

Condition required for the Ti plasmid to be inserted into the plant?

Plants defences must be reduced

What is the DNA that is transferred from the bacteria to the target called?

T-DNA - target DNA

What does the T-DNA in the Ti plasmid encode for?

encodes specific enzymes that reprogram plant cells to produce plant hormones and food for the bacteria, resulting in crown gall disease

What is a signal that induces virulence genes in A. tumefaciens?

Acetosyringone - given off by wounded plants - accidentally informs A. tumefaciens

When A. tumefaciens recognises Acetosyringone what happens?

It is detected by receptors and it activates Vir-A which upregulates Vir-G

These are essential proteins in Agrobacterium tumefaciens that form a system to detect wounded plant signals

What is the Ti plasmid adapted for?

To be recognised by plant incorporation machinery

What built in alterations does the T-DNA in the Ti plasmid have to overcome the fact that it is a prokaryotic gene being transferred to a eukaryotic gene?

Eukaryotic compatible genetic codon

Poly-A tail

How is the A. tumefaciens vector in research different to the one found in the wild?

Removal of the tumour inducing gene - T-DNA

How the Ti plasmid is used for gene cloning:

• Plasmid is removed from the bacterium

• T-DNA is cut out by a restriction enzyme

• Foreign DNA of interest is cut by same enzyme

• Foreign DNA is inserted into the T-DNA of the plasmid

• The plasmid is reinserted into a lab strain of A. tumefaciens

• T-DNA is inserted into plant cell via conjugation

• Plant cells are grown in culture and a plant is generated from a cell clone

• ALL OF ITS DNA CONTAINS TRANSFERRED GENE

What to include in the molecular design of the inserted gene?

The gene of interest + a selectable marker + left and right boarder + appropriate promoter + poly-A tail + origin of replication

What would you used to design an T-DNA insert for gene cloning?

Requires a restriction endonuclease cloning kit e.g., Golden Gateway - allows you to put 3 genes in at 1 time

Ways to optimise using the Ti plasmid for gene cloning?

• Plasmids cannot be too big - use multiple - one with gene of interest and Vir gene on the other, this allows for co-integration.

• Different optimized plasmids for different plant species - arabadopsis has a bank of plasmids that work well for it.

• Positioning of genes when assembling the construct

What are the left and right boarders?

They are on either end of the transferred sequence

They define exactly which piece of DNA gets cut out of the bacterial plasmid and pasted into the plant's genome

Arrangement of components of the inserted gene - in terms of left and right boarder:

Since the transfer starts at the RB, the DNA closest to the Right Border is the most likely to make it into the plant safely.

Always put the selectable marker near the Left Border - ensures that only fully transformed plant cells are selected

Which way around does transfer work in the Ti plasmid:

Starts at the RB to the LB

What is the binary vector system used for cloning using A.tumefaciens?

Uses 2 separate plasmids:

Binary vector (small)- containing the T-DNA with the gene of interest

Helper Ti plasmid (large) - which lacks T-DNA containing the gene of interest but carries the Vir genes

Preferred method for transformation of monocots:

Biolistics: gene delivery system (informally called a gene gun)

Process of gene delivery by biolistics:

• Start with micro-particles of gold or tungsten

• Chemically "precipitate" your plasmid DNA onto the surface of these beads

• Helium fills the chamber and pressure builds against the rupture disk

• The pressure eventually causes the rupture disk to break, and the resulting burst of helium propels the DNA/gold-coated macro-carrier into the stopping screen.

• When the macro-carrier hits the stopping screen, the DNA-coated gold particles are propelled through the screen and into the target cells.

What is protoplast fusion?

A technique that merges two distinct plant or microbial cells (protoplasts) lacking cell walls to create a hybrid cell with combined genetic material

Benefits of protoplast fusion?

Allows for the combining of species that are sexually incompatible to transfer desirable traits like disease resistance - can even be done in species with different numbers of chromosomes

What sort of media does protoplast fusion use and why?

Uses mannitol media to stabilize osmosis so that cells don’t lyse

What does it mean that plants have a highly plastic development programme?

Means that a single genotype can produce a wide range of phenotypes in response to varying environmental conditions.

They are able to revert to a pluripotent and totipotent state if the right chemicals, phytohormones and conditions are added

As plants are sessile organisms this developmental plasticity is essential for their survival

Essential components of plant tissue media:

• Aseptic technique.

• Essential elements:

  • Macro: N, P, K, Mg, Ca, S

  • Micro: Mn, Cu, Mb, Fe, Zn

• Organic supplement – vitamins and amino acids

• Carbon source – sucrose

• Plant growth regulators (phytohormones)

• Agar

How a plant grows from a collection of cells on media:

Unorganised mass of cells forms first, then it starts expressing chloroplast and photosynthesis takes place and everything kicks in for growth

How to confirm transformation of you gene of interest has taken place:

• Phenotypic assay – growth of plant on selective medium/treatment

• Could use a reporter gene - to express a certain colour

• PCR: Is the gene construct there?

• RT-PCR: Is the transgene transcribed?

• Enzyme assay: Is the gene product active?

• In situ hybridisation: Where is the gene expressed? (C)

How is A.tumefaciens actually integrated into the host in the lab?

Section healthy plant tissue to expose cell surfaces.

Add a surfactant to the bacterial suspension to break surface tension and allow the liquid to enter the leaf's stomata.

Submerge tissue in the transformed bacteria culture and apply a vacuum to pull air out of the leaf, then release it to force the bacteria into the intercellular spaces

This allows the conjugation to take place

New selection marker used for transgenic plants:

RUBY construct for selection

This produces all the genes needed for enzymatic anabolism of making betalain

When under the control of a promoter it is strongly expressed and the tissue is turned red

Why would we want to develop genes and put them into elite cultivar lines?

To increase crop yield

Abiotic factors that cause crop losses?

Exposure to radiation, water, temperature, nutrients

2 methods to improve crop yield:

1. Good agronomic practices: subsistence farming: no slash and burn, appropriate + balanced use of fertilizers, appropriate irrigation practices

2. Genetic modification

What are the two main traits that are engineered into cultivar lines to improve crop yield?

1. Herbicide tolerance (HT/HR)

2. Insect resistance (IR)

Increasingly, these two traits are stacked together

Greatest challenges facing maintained crop yield in the future?

• Resistant to GM herbicides

• Mitigating losses due to biotic and abiotic factors

How do herbicides work and when are they applied?

Usually interfere with aspects of plant metabolism not shared with animals

Careful timing of application required to kill weeds and prevent harming crops - primarily when they are at a young stage

How are herbicides classified?

By their mode of action (MOA)

Some different modes of action of different herbicides?

• Fatty acid synthesis

• Amino acid synthesis - MUST BE CAREFUL AS HUMANS SHARE THE SAME AMINO ACIDS

• Photosynthesis

• Cell wall synthesis

• Hormone transport

How to breed or genetically incorporate resistance into crops (so that the weed is killed but not the crop)? - 3 ways

1. Source from another species or develop a mutation in the herbicide target protein

2. Cause over-expression of herbicide target protein/enzyme, so that plant has resistance

3. Insert a gene that expresses for a protein that is able to catabolize (detoxify) the herbicide

2 main herbicides used in agriculture? + their positives and negatives

Atrazine

• Good: disrupts photosynthesis

• Good: some plant selectivity (different sensitivity in different plants)

• Bad: Long life in soil + water

So, it was banned in EU, 2004

Glyphosate

• Good: disrupts aromatic amino acid biosynthesis - BUT by a pathway that we don’t have

• Bad: indiscriminate (broad spectrum) in terms of plants

• Good: but readily broken down by soil microorganisms

What does glyphosate target?

Targets the shikimic acid pathway

Particularly the enzyme: EPSP synthase

How does the glyphosate target EPSP synthase?

By competitive inhibition - tighter binding by glyphosate than by PEP. Both have a similar chemical structure.

What does glyphosate prevent the production of?

Chorismate - blocking the production of key precursors needed for protein synthesis among other things

CAUSES PLANT TO DIE

Do humans have the Shikimate pathway?

No - only present in plants and microbes

Safe for humans

Example of an agrobacterium strain that is naturally immune to glyphosate and how it was overcome?

CP4 EPSPS binds glyphosate in a non-inhibitory conformation (meaning it doesn’t have a herbicide effect) because of negative interactions between the Ala-100 side chain and the oxygen atoms of the phosphonate group of the glyphosate.

THIS IS IDEAL AS THE PLANT IS RESISTANT

A single point mutation of Ala-100-Gly will re-establish the extended inhibitory binding of glyphosate.

How do we ensure that the crop is resistant to the herbicide?

Need to have a mutant version of this enzyme to create selective resistance in the crop- so that it won’t bind the chemical as strongly

The Goal: Find a "Goldilocks" mutation where the enzyme rejects the herbicide but still accepts the natural molecules it needs to process to keep the plant alive.

2 most common ways scientists insert a mutant gene that is resistant to glyphosate inhibition into the target plant?

Transformation via A. tumefaciens

Biolistics - gene gun

How are crop plants made resistant to glyphosate?

Transfer of mutant EPSP synthase genes from microorganism into plants

If we take the glyphosate resistance mutant and transform it into plants we need to be aware of certain things:

Moving from a bacterial host to a plant - many changes are necessary

• Must check ORF - bacteria and plants have different ORFs

• Must change G:C ratio - for chemical stability

• Need a good promoter with strong constitutive expression

• We need to supply the poly-A tail at the end of the gene

• We need a left and right boarder made of 24 nucleotide sequence

Once you have found the perfect EPSP enzyme that rejects glyphosate what is the next hurdle you have to face?

The Shikimate pathway happens inside the chloroplast, but your transgenic DNA is inserted into the nucleus.

We need chloroplast transit signal

How do we get out transformed gene targeted to the chloroplast?

The Chloroplast Transit Peptide (CTP) is a short sequence of amino acids ligased to the front of your protein so that it could be post-translationally targeted to chloroplast

For a plant to be resistant to glyphosate what must the Ti plasmid contain?

The glyphosate resistant ESPS gene + the chloroplast targeting sequence

Use of glyphosate resistant plants?

In US majority of soybean and cotton is GM (glyphosate resistant)

• This increases yield

Environmental concern of making plants resistant to glyphosate?

Problem of herbicide resistance arising in non-target species

As time goes on a greater population of weeds also become resistant to glyphosate

Cause of transgene escape (e.g. escape of resistance to glyphosate) + what it effects?

When pollen escapes to weed relatives and is introduced to their DNA

Via pollen to “weedy relatives” producing “super weeds” is possible because of heritability of nuclear DNA in pollen gametes

Or glyphosate not so readily broken down in the soil as assumed - Microbes and fungi share the shikimate pathway and can be affected

Conclusion of glyphosate as a herbicide?

Glyphosate looked like the ideal herbicide: it killed all plants, was non-toxic to animals, and it was broken down in soils

Recent studies show that, especially in colder climates, that glyphosate persists longer in the soil that was originally thought, is transported in the soil and may affect non-target organisms.

Advantages of chloroplast transformation:

• Chloroplasts are inherited maternally meaning the chances of escape of transformed genes via pollen are eliminated - reduces transgene escape

• There will be a high expression level of the transformed gene due to the high copy number of genomes per cell (5k - 10k)

• Chloroplast transcription and translation are prokaryote-like - therefore, less modification of prokaryote genes required transformation

• Less chance of gene silencing and regulatory positional effects than if it were nuclear targeted

Other approaches for resistance to glyphosate?

An over-expression gene of EPSPS was detected in petunia - was inserted transgenically into other plant species and can tolerate glyphosate 2-4 times higher than that required to kill wild type weed plants.

Soil microorganisms possess enzymes glyphosate oxidase that converts glyphosate to glyoxylate - the gene was isolated from Ochrobactrum anthropi and was introduced into crop plants

Only way you can transform the chloroplast genome?

Using biolistics: gene gun

A. tumefaciens alters the nuclear genome but the protein produced by the transformation is targeted to the chloroplast

Design of a chloroplast transformation construct for mutant EPSP synthase:

Must design a cassette that the chloroplast genome will accept:

Flanking Regions (trnA and trnI): Act as the "Left and Right Borders" for chloroplasts. They are actual sequences found in the chloroplast genome - plant's machinery sees them and "swaps" your transgene precisely via homologous recombination.

The Selectable Marker (aadA): This gene provides resistance to Spectinomycin - allowing you to kill off any plastids that didn't take up the DNA.

The Cargo (mEPSPS): This is your mutant version of the EPSPS gene

• For both of these you need you ribosomal promoters before and terminator sequences after them

UTRs (Untranslated regions) which interact with regulatory proteins: 5’ UTR (promoter) and 3’ UTR: PolyA tail

How to ensure that all chloroplasts in a population contain the engineered construct?

Do multiple rounds of selection > to achieve a homoplasmic state

Why was the chloroplast targeted construct created?

To try and reduce transgene escape

What is jasmonate signalling paired with?

Circadian rhythm signalling

Why are chloroplast transformations not widely used currently?

A. tumefaciens is set up to targeted to the nuclear DNA

3 types of plant tissue systems:

Dermal tissue

Ground tissue

Vascular tissue

3 methods of primary metabolism in plants:

Photosynthesis, cellular respiration + photorespiration (not actually very beneficial to the plant as no sucrose is made)

What is secondary metabolism in plants?

The specialized metabolic pathways that produce organic compounds not directly essential for basic growth, development, or reproduction, but critical for survival.

These compounds, which are often produced in response to stress, act as ecological defences against herbivores and pathogens, signalling molecules for symbiotic interactions, and protection against UV radiation

What is Bt?

Bacillus thuringiensis (Bt) - this is a natural insecticide

Bacteria that kills lepidopteran (moth) insects.

Benefits of Bt?

• It is naturally occurring, i.e., organic

• It affects specific insects - conserves natural enemies

• It does not persist in the environment (unless in spore form)

• Reduced use of insecticide (environmental and cost benefit)

• Higher yield - increases profits

How has Bt been used via GM?

In 1995 the first GE corn was produced that included Bt genes for resistance against certain insect species

How does the Bt toxin work?

A compound with the same effect as a proteinase inhibitor as it disrupts the gut on an insect

Parasporal crystal is made by the bacteria and solubilises

They get cleaved naturally by chemicals in the system of the animal

This activates the toxin which binds to cadherens on gut lining causing deregulation of the opening of ion channels

Solute pours uncontrollably into the cells causing apoptosis

NOT A PROTEINASE INHIBITOR - IT IS AN ADDITIONAL DEFENCE

In which gene is the Bt toxin produced?

cry genes

How to engineer cry expression in crops:

• Bt cry or toxins encoding genes have higher A/T content than in plants - must change G/C optimisation

• Must be specific for different species.

• Need eukaryotic terminator with poly-A tail

Where do all plastids come from in a zygote?

The mother

Why is stacking traits important in reducing herbicide/insecticide resistance?

If a plant expresses only one toxin, a single mutation in a population might allow them to survive.

However, by stacking multiple toxins that target different biological pathways, it becomes statistically much harder to evolve resistance.

Why is the maternal inheritance of plastids less important in insecticide resistant traits and more important in herbicide resistant traits?

Plants are fertilised by pollen which doesn’t contain any plastids - preventing transfer of the resistant trait

Additional benefit of Bt maize?

Prevents loss of crop via wounding then infection by fungal species (Fusarium)

What has been the issue of introducing the Bt gene into maize?

It has introduced a selective pressure therefore some insects have become resistant to it

How do we protect the effectiveness of Bt?

High dose and refuge strategy:

Works on the assumption that resistance is recessive

Creates a buffer zone of non-Bt maize (normal non-resistant maize)

Then if any insects carry both resistant genes (RR) they won’t survive in the population for long as they will by chance mate with a susceptible insect

Mating of resistant (RR) and susceptible (SS) moths produces heterozygous (RS) moths that die when they feed Bt maize

This dilutes resistance genes