Gene transfer in plants

Why gene transfer? Crop improvement

disease resistance stress tolerance improved performance value-added traits

Basic studies
gene expression reverse genetics - understanding functioning of unknown genes biochemistry and metabolism

Gene transfer strategies

Systems and vectors
Agrobacterium Direct DNA uptake Virus-based vectors

How do we find transformed plant cells? selectable markers

in yeast and Chlamydomonas, one uses nutritional markers to complement auxotrophic phenotypes of laboratory strains (leu, trp, his, ura) in E. coli, one usually uses antibiotic resistance markers (kan, tet, chl, str, amp) in plants, one uses antibiotic or herbicide resistance markers (kan or other aminoglycosides, one of several herbicides) selectable markers used in plants are foreign genes - derived from bacteria, usually; they must be specifically tailored for expression in plant cells

Making gene transfer work: expression vectors

as with selectable markers, foreign genes must usually be modified so that they can be expressed in plants most variation in expression vectors is seen in promoters constitutive or regulated promoters regulation may be natural - promoters derived from plant genes with desirable expression characteristics deliberately designed - constitutive or basal promoters may be specifically altered to produce a desired expression characteristic a variation on this theme - plant promoters can be modified to contain sequences recognized by bacterial or eucaryotic transcription factors tet repressor (tetracycline-inducible) lac repressor (galactoside-inducible) ACE1 activator (copper-dependent) glucocorticoid activator (glucocorticoid-inducible) these systems can be turned on with the application of a specific inducer (usually benign) in order for these to work, the plant must also contain the corresponding transcription factor Other factors to consider introns may increase expression of some genes, and in some plants inadvertent RNA processing signals must be removed from AUrich coding regions subcellular localization information may be incorporated into expression systems

Agrobacterium-mediated gene transfer

the keys
- to make a segment of DNA that contains a selectable marker and a gene of interest to look like a T-DNA - to get this T-DNA into an Agrobacterium cell so that it can be mobilized by the vir genes - to produce and find transformed plant cells that can be regenerated into normal, fertile plants

requirements
- a transfer cassette bounded by functioning borders - ways to get this cassette into Agrobacterium - disarmed Ti plasmids that retain functional vir genes

Advantages and disadvantages of Agrobacterium as a gene transfer tool

advantages - technically simple - yields relatively uncomplicated insertion events (low copy number, minimal rearrangements) - unlimited size of foreign DNA - efficient (for most plants) - adaptable to different cell types, culture procedures (protoplasts, tissue sections, non-culture methods) - transformants are mitotically and meiotically stable Disadvantages - host range is limited: not all plants may be susceptible to Agrobacterium - with susceptible plants, accessible culture/regeneration systems must be adaptable to Agrobacterium-mediated gene transfer

Direct DNA uptake

Electroporation
the observation: passing a brief, intense electrical pulse through a suspension of (wall-less) cells in a solution of DNA results in the introduction of significant quantities of DNA into the cell mechanism: poorly understood disadvantages: - can be only used with protoplasts (requires successful regeneration of plants from protoplasts to be used for making transgenic plants) - frequency of stable transformation is low (0.001 or less) - in stable transformants, the integrated DNA is extensively rearranged (high copy number, extensive recombination and other alterations); this can lead to some mitotic and meiotic instability advantages: - specialized vectors are not needed (expression cassettes can be made in standard E. coli vectors) - useful for high-efficiency transient expression of foreign genes in plants (for this, no selectable marker is needed)

Coprecipitation
the observation: Ca/DNA coprecipitates, when formed at cell surfaces, can be taken up by cells in plants, wall-less cells are amenable to coprecipitation; the mechanism of uptake is poorly understood advantages and disadvantages - as with electroporation

Biolistics
the introduction of DNA into cells using microprojectiles literally, DNA-coated particles are shot into target cells targets may be cells, tissues, whole plants can be used for stable transformation, transient expression advantages: - specialized vectors are not needed (expression cassettes can be made in standard E. coli vectors) - useful for high-efficiency transient expression of foreign genes in plants (for this, no selectable marker is needed) - theoretically unlimited host range (applicable to all plants) - may be used with methods that obviate the need for tissue culture/regeneration disadvantages: - in stable transformants, the integrated DNA is extensively rearranged (high copy number, extensive recombination and other alterations); this can lead to some mitotic and meiotic instability - does not completely eliminate the need for tractable tissue culture/regeneration systems

double-stranded plasmid

T-DNA

homologous recombination, concatamer formation, double-strand breakage, nuclease trimming

Micromanipulation
a single-cell technique conceptually analogous to biolistics - delivery of DNA into individual cells an excellent tool for specific transient expression studies of little use for stable transformation of plants [note that this is the technique of choice for transgenic animal production]

Virus-based vectors
Requires the ability to modify plant viruses to express foreign genes Potentially very useful for transient and whole-plant expression studies, and for obtaining high levels of expression in plants Not practical for producing stably transformed plants Host-range limitations are not a factor For specialized studies (production of foreign proteins in plants, expression of potentially lethal genes, etc.), virus-based vectors and systems may be superior

Chloroplast transformation
Issues Expression levels Selectable markers siting Multi-gene expression bio-containment

Expression levels Chloroplast localization of proteins encoded by foreign genes has been observed to increase steady-state levels of the proteins Glyphosate resistance Bt toxins PHA biosynthesis

Selectable markers Gene expression signals Antibiotic resistance Metabolic selections

Siting getting proteins where they belong Nuclear genes uses nuclear gene expression signals Agrobacterium-friendly requires protein translocation information transit peptides susceptible to gene silencing Chloroplast genes uses chloroplast gene expression signals RNA processing?? NEP, PEP, bifunctional promoters? RNA stability?? translation controls? gene silencing?

Multigene expression using nuclear genes using chloroplast genes

A.

B. x + x + +

x + +

using caulimovirus translation strategies

AAAAAA

+
VI

Caulimoviruses

type member - cauliflower mosaic virus ds DNA viruses circular genome of about 8000 bp DNA strands have gaps or interruptions DNA encodes seven open reading frames all are encoded by the same DNA strand six of the seven open reading frames are "tightly packed" in the genome - 1-3 bp overlaps, or separated by <5 bp DNA genome encodes just two transcripts - "19S", "35S"

Questions: how are seven proteins made from just two mRNAs? how does the ds DNA genome replicate? how does the virus move from cell to cell, from infected to uninfected plant?

Gene expression two promoters one poly(A) site (the larger mRNA is terminally redundant - the poly(A) signal is recognized in the larger, but not the smaller, transcript) the larger mRNA encodes the products of genes I, II, III, IV, and V the smaller mRNA encodes the gene VI product translation of the larger mRNA requires a trans factor (the gene VI product)

Genome replication larger mRNA (the 35S RNA) serves as the template for the synthesis of new DNA, via reverse transcription a specific tRNA is the primer for reverse transcription, and template switching is needed for procustion of a full-length ss DNA oligoribonucleotides that are resistant to RNAse H action serve as primers for second strand synthesis (presumably catalyzed by DNA repair machinery)

VII I

II III

IV

VI

CAT VII I II III IV V VI

CAT?? +++

CAT VII I II III IV V

CAT VII I II III IV V

VI

++

A.
P1 * HC-pro P3 * P1 * HC-pro CI NIa NIb CP

P3

CI

NIa

NIb

CP

P3

CI

NIb

CP

B.

NIa

NIa

BT expression in chloroplasts

single gene construct

protein accumulation

Kota et al., PNAS 96, 18401845, 1999

multiple gene construct

protein accumulation

De Cosa et al., Nature Biotechnology 19, 7174, 2001

Biocontainment Nuclear genes can be transmitted by pollen, seed, vegetative means Mendelian inheritance Chloroplast genes maternally-inherited (usually) -> not transmitted by pollen

Glyphosate resistance in plants with chloroplast-localized EPSP synthase genes constructs

Expression: protein

mRNA

Maternal inheritance

Ye et al., Plant J. 25, 261-270, 2001