Lecture 13

Genes and Traits of Interest for

Transgenic Plants
Introduction
• The whole purpose of biotechnology is to manipulate the
genome of important plants, typically by adding a few genes at a
time.
• Traits can be manipulated by inserting DNA originating from any
organism with that trait of interest into the target plant.
• Thus far in crop biotechnology, much work has been
accomplished in conferring traits to plants such as the ability to
survive herbicide treatment, insect resistance, disease
resistance, and stress tolerance. However, there is growing
interest in producing drugs and industrial proteins in plants as
well as enhancing the nutrition of plant products.
TRAITS FOR IMPROVED CROP PRODUCTION

• The growth of healthy plants that yield quality products

requires farmers to deal with everchanging environmental
conditions and pests.
• Transgenic approaches to helping farmers with these
challenges are being broadly used today, while additional
products are in the developmental pipeline.
• Plants with improved tolerance to high temperatures, saline
conditions, and drought are likely to find their way into
production in the future.
 Herbicide Resistance
The first transgenic application to be widely adopted in agriculture was
resistance to herbicides. Weeds are generally regarded to be the most
serious problem for farmers and result in reduced yields because they
compete with crop plants for water, light, and nutrients.
 Chemical herbicides are widely used by many farmers because they are
cost-effective and efficient at killing weeds.
• Most effective herbicides for agricultural production must be somewhat
selective, meaning that they should kill the target weeds but not the crop
plant. Using single-gene traits in transgenic plants can provide a very
specific way to protect the crop plant from the effects of a given herbicide.
• Herbicides generally work by targeting metabolic steps that are vital
for plant survival.
• For example, glyphosate kills plants by inhibiting the production of
certain amino acids that the plant requires for survival. Glyphosate is
the active ingredient in the herbicide Round Up TM. Thus, crops such
as soybean and corn that have been engineered to be resistant to
glyphosate were given the name “Round Up Ready.”
• Glyphosate works by binding to and inhibiting the enzyme 5-enol
pyruvylshikimate3-phosphate synthase (EPSPS), which is active in the
shikimate pathway leading to the synthesis of choris mate-derived
metabolites, including the aromatic amino acids (tyrosine,
phenylalanine, and tryptophan)
• To make plants resistant to glyphosate, a form of the EPSPS enzyme
that is functional in plants but is not affected by the herbicide was
used.
• In addition to being present in plants, the EPSPS protein can also be
found in bacteria. So scientists at Monsanto, the inventors of Round
Up, looked for and identified a form of EPSPS from a soil bacterium
that was not sensitive to treatment with glyphosate.
 Figure 1
Resistance to glyphosate in RoundUp ReadyTM plants is engineered
by expressing a form of the 5-enolpyruvylshikimate-3-phosphate
(EPSP) synthase (EPSPS) enzyme that is resistant to the herbicide. In
the absence of this transgenic enzyme, glyphosate inhibits the plant
EPSPS and ultimately blocks the synthesis of chorismate, the
branchpoint precursor to the essential aromatic amino acids:
tryptophan, phenylalanine, and tyrosine. The transgenic EPSPS is
unaffected by glyphosate, and can carry out the synthesis of EPSP
leading to chorismate production.
• An alternative strategy to engineer herbicide resistance is to express
a protein that will inactivate the herbicide if it is sprayed onto plants.
• This is the approach used in resistance against the herbicide
glufosinate, the active ingredient in the product LibertyTM, generating
a trait in crop plants often called “LibertyLink.” Glufosinate kills plants
by inhibiting the plant enzyme glutamine synthetase (GS), which is
responsible for synthesis of the amino acid glutamine.
• As part of the chemical reaction that produces glutamine, GS utilizes
excess plant nitrogen in the form of ammonium that is incorporated
into the amino acid. When GS is inhibited in glufosinate-treated
plants, ammonium concentrations inside the plant rise to toxic levels
• The glufosinate compound is naturally produced in some
Streptomyces bacteria.
• In addition to having phytotoxic activity, glufosinate also servers as an
antibiotic because it is toxic to some other bacteria.
• Bacterial strains that are resistant to glufosinate produce an enzyme,
encoded by the bar gene, called phosphoinothricine acetyltransferase
(PAT)
• The bar gene was isolated from a strain of Streptomyces
hygroscopicus, which degrades glufosinate, and has been transferred
into several crop plants. The LibertyLink trait is currently widely used
in transgenic corn, canola, and cotton varieties.
• Resistance to glufosinate in LibertyLinkTM plants is
engineered by expressing an enzyme that directly targets
and inactivates the herbicide.
• Glufosinate kills plants by inhibiting glutamine synthetase.
This enzyme is responsible for production of the amino acid
glutamine in a reaction that can sequester excess nitrogen by
incorporating ammonia (NH4 þ).
• If this enzyme is inactivated by glufosinate, excess ammonia
accumulates and the plant is killed. An enzyme encoded by
the bacterial bar gene in transgenic plants inactivates
glufosinate.
Insect Resistance
• A number of proteins with negative effects on insects have been
tested as potential weapons for use in engineering insect-resistant
transgenic crops.
• Genes for several proteins have been expressed in transgenic plants
and were shown to inhibit insect growth or cause higher insect death
rates.
• These include genes for protease inhibitors, which interfere with
insect digestion; lectins, which kill insects by binding to specific
glycosylated proteins; and chitinases, enzymes that degrade chitin
found in the cuticle of some insects.
• Although each of these genes has been shown to have some negative
impact when consumed by insects and may have some utility in insect
control, none have been as effective or widely adopted as genes
encoding endotoxins from the bacterium Bacillus thuringiensis (Bt).
 Figure
• The Bt toxin binds to very specific receptor son the epithelial
membrane of the insect gut. The toxin then forms channels in the
membrane that leads to ion leakage and ultimately, death of the
insect. This mode of action explains the specificity of Bt (from the
presence of the necessary receptors) and also shows why the toxin
needs to be eaten by the insect to function.
 Pathogen Resistance
• Plant pathogens such as viruses, fungi, and bacteria are a severe and
constant threat to agricultural crop production. Multiple transgenic
approaches have been used to attempt plant disease control, although
relatively few of these have yet made their way into the field of
production.
• The most effective way to control pathogens in a field setting is to use
plants that are resistant to the problem pathogen.
• Resistance to a particular pathogen can often be conferred by a single
plant gene (an R gene), the product of which is active in recognition of
the presence or activity of a single virulence factor from the pathogen
(encoded by an Avr gene). In plant pathogen systems, this relationship
is known as a gene-for-gene interaction.
• Plant breeders have historically taken advantage of this system,
although it can sometimes take many years to identify a plant line
with the desired resistance and to breed that trait into useful
cultivars.
• Another disadvantage to the breeding approach is that unwanted or
undesirable genes may sometimes be linked to the R gene, and it can
be difficult to separate them from the R gene using traditional
breeding methods. Finally, useful R genes are sometimes not easy to
transfer because of barriers in crossing different species. Therefore,
the ability to clone and transfer a single R gene from one plant variety
or species to another represents an encouraging option to adapt and
speed up the process.
Figure
• Resistance to specific strains of plant pathogens can be
conferred by the protein product of a single resistance (R)
gene. Most plant R genes function by recognizing the activity
or presence of a specific virulence factor from the pathogen.
In addition to the ability to induce basal defenses, these
pathogen “effectors” areal so active in attacking various host
proteins. The protein products of R genes guard against
pathogens via surveillance of specific targeted host proteins.
When these R-gene mediated defenses are triggered, the
plant responds with a hypersensitive response and rapid
activation of defense gene expression.