genetically engineered crops

8/8/2019 Genetically Engineered Crops

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Genetically engineered crops 1

Genetic Engineering

Genetic engineering is a laboratory

technique used by scientists to change the DNA

of living organisms.

DNA is the blueprint for the individuality of anorganism. The organism relies upon the

information stored in its DNA for the

management of every biochemical process. The

life, growth and unique features of the organismdepend on its DNA. The segments of DNAwhich have been associated with specific

features or functions of an organism are called

genes.

Molecular biologists have discovered many

enzymes which change the structure of DNA in

living organisms. Some of these enzymes can

cut and join strands of DNA. Using such

enzymes, scientists learned to cut specific genes

from DNA and to build customized DNA using

these genes. They also learned about vectors,

strands of DNA such as viruses, which caninfect a cell and insert themselves into its DNA.

With this knowledge, scientists started to build

vectors which incorporated genes of their

choosing and used the new vectors to insert

these genes into the DNA of living organisms.

Genetic engineers believe they can improve the

foods we eat by doing this. For example,

tomatoes are sensitive to frost. This shortens

their growing season. Fish, on the other hand,

survive in very cold water. Scientists identified a

particular gene which enables a flounder to resistcold and used the technology of genetic

engineering to insert this 'anti-freeze' gene into a

tomato. This makes it possible to extend the

growing season of the tomato.

Generally, there are four parts to a DNApackage

1. Genes for the desired trait the payload.

An example of such a trait is crop resistance to a

given diseases.

2. Genes for carrying the package into the host

plants DNA. This genetic carrier is called the

vector and is usually taken from a bacterium that

causes tumors in plants, Agrobacteriumtumefaciens. Viruses are also sometimes used as

gene carriers. These bacterial or viral vectorsinfect the new host cells, delivering the

engineered gene into the DNA of the host plant.

3. Genes for ensuring that the genetic package

will express the desired trait persistently (rather

than weakly or not at all). These genes turn onthe desired trait and are called promoters. They

are usually derived from the cauliflower mosaic

virus (CaMV).

4. Genes for helping the biologist find the DNA

segment in which the insertion has beensuccessful. These genes are called markers and

are resistant to antibiotics (usually neomycin or

kanamycin). When the antibiotic is applied to

the new hosts cells, the cells that survive are the

ones carrying the successfully inserted

antibiotic-resistant geneindicating likelihood

that the gene carrying the desired trait has been

successfully inserted as well. Marker genes are

usually derived from bacteria (E. coli).

The methods of Genetic Engineering

Recently, we have begun to learn how to takeevolution into our own hands through genetic

engineering, which involves altering or

manipulating an organism's genome to create a

new and useful result. The methods often used

by genetic engineers are many and varied, but

generally fall under one of three categories: the

plasmid method, the vector method, and the

biolistic method.

The Plasmid Method

The first technique of genetic

engineering, the plasmid method, is the mostfamiliar technique of the three, and is generally

used for altering microorganisms such as

bacteria. In the plasmid method, a small ring of

DNA called a plasmid (generally found in

bacteria) is placed in a container with special

restriction enzymes that cut the DNA at acertain recognizable sequence. The same

enzyme is then used to treat the DNA sequence


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to be engineered into the bacteria; this procedure

creates "sticky ends" that will fuse together if

given the opportunity.

Next, the two separate cut-up DNA sequences

are introduced into the same container, where

the sticky ends allow them to fuse, thus forming

a ring of DNA with additional content. Newenzymes are added to help cement the new

linkages, and the culture is then separated bymolecular weight. Those molecules that weigh

the most have successfully incorporated the new

DNA, and they are to be preserved.

The next step involves adding the newly formed

plasmids to a culture of live bacteria with knowngenomes, some of which will take up the free-

floating plasmids and begin to express them. In

general, the DNA introduced into the plasmid

will include not only instructions for making a

protein, but also antibiotic-resistance genes.These resistance genes can then be used to

separate the bacteria which have taken up the

plasmid from those that have not. The scientist

simply adds the appropriate antibiotic, and the

survivors are virtually guaranteed (barring

spontaneous mutations) to possess the new

genes.

Next, the scientist allows the successfully altered

bacteria to grow and reproduce. They can now

be used in experiments or put to work in

industry. Furthermore, the bacteria can be

allowed to evolve on their own, with a "selectionpressure" provided by the scientist for producing

more protein. Because of the power of natural

selection, the bacteria produced after many

generations will outperform the best of the early

generations.

Many people strongly object to the plasmid

method of genetic engineering because they fear

that the engineered plasmids will be transferred

into other bacteria which would cause problems

if they expressed the gene. Lateral gene transfer

of this type is indeed quite common in bacteria, but in general the bacteria engineered by this

method do not come in contact with natural

bacteria except in controlled laboratory

conditions. Those bacteria that will be used inthe wild - for example, those that could clean up

oil spills - are generally released for a specific

purpose and in a specific area, and they are

carefully supervised by scientists.

The Vector Method

The second method of genetic engineering is

called the vector method. It is similar to the

plasmid method, but its products are inserted

directly into the genome via a viral vector. The

preliminary steps are almost exactly the same:cut the viral DNA and the DNA to be inserted

with the same enzyme, combine the two DNA

sequences, and separate those that fuse

successfully. The only major difference is that

portions of the viral DNA, such as those that

cause its virulence, must first be removed or the

organism to be re-engineered would become ill.

This does yield an advantage - removal of large

portions of the viral genome allows additional

"space" in which to insert new genes.

Once the new viral genomes have been created,

they are allowed to synthesize protein coats andthen reproduce. Then the viruses are released

into the target organism or a specific cellular

subset (for example, they may be released into a

bacterium via a bacteriophage, or into human

lung cells as is hoped can be done for cystic

fibrosis patients). The virus infects the target

cells, inserting its genome - with the newly

engineered portion - into the genome of the

target cell, which then begins to express the new

sequence.

With vectors as well, marker genes such as

genes for antibiotic resistance are often used,giving scientists the ability to test for successful

uptake and expression of the new genes. Once

again, the engineered organisms can then be

used in experiments or in industry. This

technique is also being studied as a possible way

to cure genetic diseases.

Many people object to this type of genetic

engineering as well, citing the unpredictability

of the insertion of the new DNA. This could

interfere with existing genes' function. In

addition, many people are uncomfortable with

the idea of deliberately infecting someone with avirus, even a disabled one.

The Biolistic Method

The biolistic method, also known as the gene-

gun method, is a technique that is most

commonly used in engineering plants - forexample, when trying to add pesticide resistance

to a crop. In this technique, pellets of metal


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(usually tungsten) coated with the desirable

DNA are fired at plant cells. Those cells that

take up the DNA (again, this is confirmed with a

marker gene) are then allowed to grow into new

plants, and may also be cloned to produce more

genetically identical crop. Though this technique

has less finesse than the others, it has provenquite effective in plant engineering.

Objections to this method arise for many of thesame reasons: the DNA could be inserted in a

working gene, and the newly inserted gene

might be transferred to wild plants. Additionally,

this technique is commonly opposed because of

its association with genetically modified foods,which many people dislike. Plants have

elaborate defense mechanisms for dealing with

foreign substances, including foreign DNA. To

overcome these defense mechanisms, genetic

engineers include pathological organisms in thecomplex DNA packages they create to carry the

chosen gene into the host cell.

Transforming Plants through GeneticEngineering Techniques

Cloning of Plant Cells and M anipulation ofPlant Genes

Plant cells exhibit a variety of

characteristics that distinguish them from animalcells. These characteristics include the presence

of a large central vacuole and a cell wall, and theabsence of centrioles, which play a role in

mitosis, meiosis, and cell division. Along with

these physical differences, another factor

distinguishes plant cells from animal cells,

which is of great significance to the scientistinterested in biotechnology: Many varieties of

full-grown adult plants can regenerate from

single, modified plant cells called protoplasts -

plant cells whose cell walls have been removed

by enzymatic digestion. More specifically, whensome species of plant cells are subjected to the

removal of the cell wall by enzymatic treatment,

they respond by synthesizing a new cell wall and

eventually undergoing a series of cell divisions

and developmental processes that result in the

formation of a new adult plant. That adult plant

can be said to have been cloned from a single

cell of a parent plant.

Plants that can be cloned with relative ease

include carrots, tomatoes, potatoes, petunias, and

cabbage, to name only a few. The capability to

grow a whole plant from a single cell means that

researchers can engage in the genetic

manipulation of the cell, let the cell develop into

a completely mature plant, and examine thewhole spectrum of physical and growth effects

of the genetic manipulation within a relativelyshort period of time. Such a process is far more

straightforward than the parallel process in

animal cells, which cannot be cloned into full-

grown adults. Therefore, the results of any

genetic manipulation are usually easier toexamine in plants than in animals.

A Cloning Vector that Works with PlantCells

Not all aspects of the geneticmanipulation of plant cells are readily

accomplished. Not only do plants usually have a

great deal of chromosomal material and grow

relatively slowly as compared with single cells

grown in the laboratory, but few cloning vectors

can successfully function in plant cells. While

researchers working with animal cells can

choose among a wide variety of cloning vectors

to find just the right one, plant cell researchers

are currently limited to just a few basic types of

vectors.

Perhaps the most commonly used plant cloningvector is the "Ti" plasmid, or tumor-inducing

plasmid. This plasmid is found in cells of the

bacterium known as Agrobacterium

tumefaciens, which normally lives in soil. The

bacterium has the ability to infect plants and

cause a crown gall, or tumorous lump, to form at

the site of infection. The tumor-inducing

capacity of this bacterium results from the

presence of the Ti plasmid. The Ti plasmiditself, a large, circular, double-stranded DNA

molecule, can replicate independently of the A.

tumefaciens genome. When these bacteria infecta plant cell, a 30,000 base-pair segment of the Ti

plasmid - called T DNA - separates from the

plasmid and incorporates into the host cell

genome. This aspect of Ti plasmid function has

made it useful as a plant cloning vector.

The Ti plasmid can be used to shuttle exogenous

genes into host plant cells. This type of gene

transfer requires two steps: 1) the endogenous,


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tumor-causing genes of the T DNA must be

inactivated and, 2) foreign genes must be

inserted into the same region of the Ti plasmid.

The resulting recombinant plasmid, carrying up

to approximately 40,000 base pairs of inserted

DNA and including the appropriate plant

regulatory sequences, can then be placed backinto the A. tumefaciens cell. That cell can be

introduced into plant cell protoplasts either bythe process of infection or by direct insertion.

Once in the protoplast, the foreign DNA,

consisting of both T DNA and the inserted gene,

incorporates into the host plant genome. The

engineered protoplast - containing therecombinant T DNA - regenerates into a whole

plant, each cell of which contains the inserted

gene. Once a plant incorporates the T DNA with

its inserted gene, it passes it on to future

generations of the plant with a normal pattern ofMendelian inheritance.

One of the earliest experiments that involved the

transport of a foreign gene by the Ti plasmid

involved the insertion of a gene isolated from a

bean plant into a host tobacco plant. Although

this experiment served no commercially useful

purpose, it successfully established the ability of

the Ti plasmid to carry genes into plant host

cells, where they could be incorporated and

expressed.

A. Tumefaciens Infects a Limited Variety

of Plant Types

The fact that only certain types of plants

were naturally susceptible to infection with the

host bacterial organism initially limited the

usefulness of the Ti plasmid as a cloning vector.

In nature, A. tumefaciens infects only

dicotyledons or "dicots" - plants with two

embryonic leaves. Dicotyledenous plants,

divided into approximately 170,000 differentspecies, include such plants as roses, apples,

soybeans, potatoes, pears, and tobacco.

Unfortunately, many important crop plants,including corn, rice, and wheat, are

monocotyledons - plants with only one

embryonic leaf - and thus could not be easily

transfected using this bacterium.

Overcoming the Limited Range of A.Tumefaciens Infection

Research efforts in the past few years

have reduced the limitations of A. tumefaciens.

Scientists discovered that by using the processes

of microinjection, electroporation, and particle

bombardment, naked DNA molecules can be

introduced into plant cell types that are not

susceptible to A. tumefaciens transfection.

Microinjection involves the direct injection ofmaterial into a host cell using a finely drawn

micropipette needle.

Electroporation uses brief pulses of high

voltage electricity to induce the formation of

transient pores in the membrane of the host cell.A jolt of electricity is used to puncture self-

repairing holes in protoplasts (i.e., the cell

without the cell wall), and DNA can get in

through these holes. Such pores appear to act as

passageways through which the naked DNA canenter the host cell. Particle bombardment

actually shoots DNA-coated microscopic pellets

through a plant cell wall.

These developments, important in the

commercial application of plant genetic

engineering, render the valuable food crops of

corn, rice, and wheat susceptible to a variety of

manipulations by the techniques of recombinant

DNA and biotechnology.


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Genetic Engineering of Grapevines forImproved Disease Resistance

Grapevine diseases cause growers to

invest millions of dollars and numerous hours on

various techniques to reduce losses. Grapevines

with improved disease resistance would be

welcomed, especially if other traits were not

altered. Reduction of pesticide sprays by even

one or two per year would cut the cost of

production and may benefit the environment.

The grapevine breeding program at the New

York State Agricultural Experiment Station in

Geneva is using both traditional breeding

methods as well as biotechnology to develop

disease resistant vines. This article focuses on

the use of genetic engineering in which genes

that code for desired traits are inserted into a

plant. The major advantage of genetic

engineering techniques is the ability to direct

improvement of important cultivars without

altering their essential features.

Gene transfer technology became routine in the

mid 1980's for easily manipulated non-woody

plants such as tobacco. However, it has only

been in the last few years that genetically

transformed grapevines have been produced.

This technology is now progressing rapidly.

Genes that may confer disease resistance

Genes that may confer disease resistance to

plants are now available from a variety of

sources. We have already mentioned the testing

of virus resistance genes in grapevines. These

genes come from a part of the virus itself.Resistance is based on the observation that once

a plant becomes infected with certain viruses, it

is resistant to future attacks. Thus, insertion of a

non-infectious viral gene into a plant provides a

sort of protective vaccine. Genes for resistance

to fungi and bacteria work in different ways.

Some genes that have been isolated from plants

and higher fungi, code for enzymes (such as

chitinase) that degrade a major component of the

outer protective walls of certain fungi. Other

genes act by creating holes in the membrane of

fungal and bacterial cells. These membrane-

active genes have been taken from a variety of

organisms such as plants, mammals,amphibians, and insects. They have also been

synthesized in the laboratory. Much work isneeded to test the numerous genes against

grapevine pathogens.

Basic requirements to engineer diseaseresistance genes into plants

To successfully engineer disease resistance

genes into plants, the following are needed:

1) Recipient cells those are capable of growing

into whole plants,2) A method to transfer the genes into the cells,

3) Proper expression of the genes by the

transformed plant cells,

4) A method to select the transformed cells from

the non-transformed cells,

5) Regeneration of whole plants, and

6) Evaluation of disease resistance.

Success in grapevine transformation came only

when researchers started using what are termed

embryogenic cultures. These cultures are grown

in the laboratory under sterile conditions in anartificial growth medium. The cultures consist of

tiny clumps of cells that are capable of growing

into embryos that can germinate into plants. The

cells originate from the body of the plant

(somatic cells) and not the egg or sperm cells, so

that each embryo is a clone (exact replicate) of

the original plant.

To insert genes into embryogenic

cultures, most researchers working on grapetransformation rely upon modified strains of

Agrobacterium (the bacterium responsible for

crown gall), which transfers genes into plants as part of its normal life cycle. In our Geneva

laboratory, we have taken a different approach.We rely upon the biolistic process whereby

DNA-coated particles of extremely minute size

are used to carry foreign genes into grapevine

cells. DNA coding for the genes of interest is

coated onto the minute tungsten-micro

projectiles. These are accelerated at extremely


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high speeds into the cultured cells using a

biolistic device, also known as the "gene gun"

(Figure 1). There are usually several genes

transferred into each cell penetrated by a

microprojectile. One of these genes might be the

gene of interest coding for a desired trait.

Another gene is used to help separate thetransformed cells from the remaining normal

cells. This is important because usually less than5% of the cells receive and maintain the genes

long-term. We use a gene which confers

resistance to the antibiotic, kanamycin. Selection

for the transformed cells takes place in a

medium containing kanamycin, on which thetransformed cells are able to grow and develop

into embryos. Normal cells without the newly

inserted gene will die on medium with

kanamycin, so that the only growth observed

should originate from cells with the newlyinserted genes.

In lab, the biolistic process is initially

tested for grapevine transformation using

embryogenic cell suspensions. The embryogenic

cell suspensions were bombarded with "marker

genes" that enable us to track transformation and

gene expression. Using common techniques in

molecular biology, we have been able to extract

DNA from the transformed vines and prove the

presence of foreign DNA supplied via the

biolistic process.

Current work focuses on the use of achitinase-producing gene to confer disease

resistance upon important grapevine cultivars. In

the laboratory, the chitinase enzyme attacks

fungal cell walls and has been shown to inhibit

the growth of pathogens that cause Botrytis

bunch rot and powdery mildew of grapes.

Recent results indicate that the chitinase gene is

expressed in 'Chancellor' and 'Merlot', but it is

too early to judge whether the level of disease

resistance has been increased. Further

experiments are required to obtain plants from

these cultures and to judge the effect of this geneon disease resistance.

In the future, it is likely that multiple

genes for disease resistance will be inserted

simultaneously into important cultivars. There isconcern that the product of a single gene will be

more readily overcome by a pathogen, and that

by pyramiding multiple genes, the resistance

will be stable and long lasting. New genes are

being sought from grapevines and other

organisms. Attempts to create genetic maps of

grapevine chromosomes at Cornell University

should lead to the isolation of important genes.

Finally, any genetically altered vines will have

to undergo stringent field testing to assure that,

not only is the resistance stable, but that theessential features of the vine and the fruit

produced are not altered.

The final challenge will be to assure a

skeptical public of the value of a transgenic

grapevine. This technology will help to reduce

reliance on pesticides, reduce the cost of

production, and permit continued productivity invineyards hit with harmful virus diseases. In 5-

10 years, when transgenic vines and rootstocks

become commercially available, the public

should have become more accustomed to the

consumption and use of transgenic fruits,vegetables, and food and fiber crops. There are

already transgenic tomatoes, squash, potatoes

and cotton on the market.

Figure 1. Diagram of the "gene gun" which is used to

deliver genes into plant cells. The device is driven by highpressure helium gas. When the rupture disk at the end ofthe gas acceleration tube is burst, a strong shock wave of

gas is released, which in turn launches the micro carriers(minute tungsten particles coated with the desired genes).The micro carriers penetrate the plant cells and the genesare released within. When conditions are optimized, cellinjury is minimal and the new genes are maintained by the

plant cells long-term. The gene gun was invented by JohnSanford, Ed Wolf, and Nelson Allen at Cornell University.The device is being used worldwide to genetically engineera variety of organisms, including plants, animals, and

microorganisms. Medical applications such as gene therapyare also being tested.


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Genetic engineering for virus resistance

Plant virus diseases cause severe

constraints on the productivity of a wide range

of economically important crops worldwide. In

India the Green Revolution ushered in intensive

agricultural practices and reduced varietal

diversity, resulting in the emergence of viral

diseases at an alarming pace in the cultivated

crops. Some such diseases, which are especially

relevant, along with their yield losses, are listed

in Table 1. Strategies for the management of

viral diseases normally include control of vector

population using insecticides, use of virus-free

propagating material, appropriate cultural

practices and use of resistant cultivars. However,

each of the above methods has its own

drawback. Rapid advances in the techniques of

molecular biology have resulted in the cloning

and sequence analysis of the genomic

components of a number of plant viruses. A

majority of plant viruses have a single-stranded

positive sense RNA as the genome. However,

some of the most important viruses in tropical

countries have single-stranded and double-

stranded DNA genomes and RNA genomes of

ambience polarity, i.e. genes oriented in both

directions. Genome organization, electron-

microscopic structures and symptoms caused by

some of the viruses, referred to in this review,

are briefly illustrated in Figure 1. on page 8.

Concomitantly, tremendous advances have taken

place in our understanding of plantvirusinteraction in the process of pathogenesis and

resistance. This, along with associated advances

in the genetic transformation of a number ofcrop plants, has opened up the possibility of an

entirely new approach of genetic engineering

towards controlling plant virus diseases. There

are mainly two approaches for developing

genetically engineered resistance depending on

the source of the genes used.

The genes can be either from the pathogenic

virus itself or from any other source. The former

approach is based on the concept ofpathogen-

derived resistance (PDR) 2, 3. For PDR, a part,

or a complete viral gene is introduced into the plant, which, subsequently, interferes with one

or more essential steps in the life cycle of thevirus. Coat protein (CP) of tobacco mosaic virus

(TMV) was inserted into tobacco and observed

TMV resistance in the transgenic plants. The

concept of PDR has generated lot of interest and

today there are several hostvirus systems inwhich it has been fully established. Non-

pathogen-derived resistance, on the other hand,

is based on utilizing host resistance genes and

other genes responsible for adaptive host

processes, elicited in response to pathogenattack, to obtain transgenics resistant to the

virus.

The use of non-PDR type of resistance, even

though reported much less in the literature in

comparison to PDR-based approaches, holds a

better promise to achieve durable resistance.

Various aspects of the above topics have been

reviewed extensively59.

Transgenics with pathogen-derivedresistance

In a number of crops, transgenics resistant to aninfective virus have been developed by

introducing a sequence of the viral genome in

the target crop by genetic transformation. Virus-

resistant transgenics have been developed in

many crops by introducing either viral CP or

replicase gene encoding sequences. Resistance

obtained by using CP is conventionally called

CPMR. Replicas mediated resistance has been

pursued in a number of laboratories and in mostof these cases, resistance has been shown to be

due to an inherent plant response, known as

post-transcriptional gene silencing (PTGS),which is described in more detail later in this

article. Because of the essential nature of the

viral movement protein for intercellular

movement of plant viruses, movement problem

sequence has also been used for achieving viral

resistance. Other pathogen-derived approaches

described in the literature, include the use of


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satellite RNA and defective-interfering viral

genomic components.

Coat protein

The use of viral CP as a transgene for producing

virus resistant plants is one of the most

spectacular successes achieved in plant

biotechnology. Numerous crops have been

transformed to express viral CP and have beenreported to show high levels of resistance in

Powell-Abel et al.4 first reported resistance

against TMV in transgenic tobacco expressing

the TMV CP gene.

The resistance was manifested as delayed

appearance of symptoms as well as a reduced

titre of virus in the infected transgenic plants, as

compared to the controls. The resistance against

TMV using TMV CP in tobacco was also

reported to be effective against other tobacco

viruses whose CP was closely related to that of

TMV but not effective against viruses whichwere distantly related to TMV. Transgenic

potato, expressing the CP of potato virus X

(PVX) also showed resistance against PVX.

However, in marked contrast to TMV, this

resistance was not broken down when PVX

RNA was used as the inoculum, thus indicating

several possible mechanisms of CPMR.

The stage of the viral life cycle at which the

CPMR is effective has been shown to vary. InTMV, it is at the virus disassembly and in the

long-distance transport stage. In the case of

alfalfa mosaic virus (AMV), it is only at the

disassembly stage, whereas in PVX, it is at

multiple stages, including replication, cell-to-

cell and systemic movement stages. In

tospoviruses, the stage affected is believed to be

replication. These mechanistic aspects have been


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dealt with in greater detail elsewhere. Recently,

considerable efforts have been made towards

understanding the molecular basis of the CPMR

especially in tobaccoviruses. These studies may

lead to more rational design of CP-derived

transgenes.

Substantial yield increase observed in field trialsof transgenic papaya and squash (Table 3) has

established CPMR as the most favoured strategyto engineer resistance against many viruses. The

success of CPMR has prompted the production

of transgenic plants expressing multiple CP

genes from more than one virus. Several

important crops have been engineered for virusresistance using CPMR approach and released

for commercial cultivation. These include

tomato resistant to TMV, tomato mosaic virus

implications for further effective control of viral

diseases.Movement protein

Movement proteins (MP) are essential for cell-

to-cell movement of plant viruses. These

proteins have been shown to modify the gating

function of plasmodesmata, thereby allowing the

virus particles or their nucleoprotein derivatives

to spread to adjacent cells. This phenomenon

was first used to engineer resistance against

TMV in tobacco by producing modified MP

which is partially active as a transgene. The

conferred resistance is believed to be based onthe competition between wild-type viruses

encoded MP and the preformed dysfunctional

MP to bind to the plasmodesmatal sites. The

above resistance was moreover seen to be

effective against distantly related or unrelated

viruses, for example resistance against TMV

could be achieved in tobacco using the MP

derived from brome mosaic virus, suggesting

functional conservation of this protein among

several viruses. In contrast to the single MP gene

in tobamoviruses, viral movement is mediated

by a set of three overlapping genes, known asthe triple-gene-block (TGB) in potex-, carla- and

hordeiviruses. However, resistance was

overcome when inoculated with viruses lacking

a TGB, like PVY. This indicated that the

resistance depended upon the interaction of theviral derived and the transgene-derived MPs.

Satell ite RN A

Besides using the genomic components of an

infectious virus, a strategy exploiting the use of

satellite RNA associated with certain viruses

received great attention. Some strains of CMV

encapsidate satellite RNA (sat RNA) in addition

to the tripartite messenger sense, single-stranded

RNA genome. CMV sat RNA depends on itshelper virus (HV) CMV for replication,

movement within the plant, encapsidation andtransmission. The presence of sat-RNA

modulates the symptoms induced by the HV and

often depresses HV accumulation in different

host species. Thus, transgenic tobacco plants

expressing multiple or partial copies of CMVsat-RNA showed attenuated symptoms when

challenged with CMV. In addition, tobacco

plants transformed with anti-sense sat-RNA also

showed delayed symptom development with the

cognate virus.Sat-RNA was tested as a bio-control agent in

field trials in many countries with considerable

success. Tomato, containing non-necrogenic sat-

RNA sequences developed only faint symptoms

following CMV infection. The timing of fruit set

and fruit yield in transgenic plants was

comparable with healthy plants. Thus, high-level

of tolerance to CMV conferred by sat-RNA in

tomato was demonstrated. This was further

improved by combining sat-RNA and CMV CP.

The mechanism behind sat-RNA mediated

resistance may be attributed to the reduction inaccumulation of the HV and its long distance

movement and down-regulation of replication.

However, as sat-RNA spreads epidemically,

sufficient caution will have to be exercised in

adopting this technology.

Defective-interfering viral nucleic acids

In several viruses, truncated genomic

components are often detectable in infected

tissues, which interfere with the replication of

the genomic components. These species of DNA

are also called defective interfering (DI) DNAand expression of delayed disease symptoms and

recovery, coupled with increased resistance

upon repeated inoculation have been observed in

plants engineered with DI DNA. For example,

incorporation of subgenomic DNA B thatinterferes with the replication of full length

genomic DNA A and B confers resistance to

ACMV in N. benthamiana. Self-cleaving RNA


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(ribozymes), seen in viroids and some sat-RNA,

were also used with high expectations. There are

a few reports like targeting PLRV CP and

replicase and 5region of TMV RNA and citrus

exocortis viroid. In most of the cases, ribozyme

sequences were ineffective and the resistant

phenotypes observed were use to antisenseRNA.

Transgenics with non-pathogen derivedresistance

The following section describes the non- pathogen-derived strategies, i.e. those utilizing

genes derived from either the host plant or any

other non-pathogenic source. A new

phenomenon called post-transcriptional gene

silencing (PTGS) has recently been shown to be

responsible for the inherent ability of many

plants to specifically degrade nucleic acids in asequence-specific manner, including those of

viruses. Thus, this strategy can be very effective

in engineering virus resistance.

Post-transcriptional gene silencing

Post-transcriptional gene silencing (PTGS) is a

specific RNA degradation mechanism of any

organism that takes care of aberrant, unwanted

excess or foreign RNA intracellularly in a

homology-dependent manner. This activity

could be present constitutively to help normal

development or induced in response to cellulardefense against pathogens. In this mechanism,

the elicitor double-stranded RNA (ds RNA),

commonly produced during viral infection, is

degraded to 2125 nucleotides, termed as small

interfering RNA (siRNA), with the help of a

variety of factors that have already been or are

being identified. A complex of cellular factors,

namely RNA-dependent RNA polymerase

(RdRp), RNA-helicase, translation elongation

factor, RNAse, etc. along with the small 2125

nt RNA (of the elicitor RNA) acting as the guide

RNA. This degradation process, initiating from aconcerned cell having the elicitor RNA, spreads

later within the entire organism in a systemic

fashion. This process is generally regarded to

have evolved as a plant defense mechanism

against invading viruses containing either RNA

or DNA genomes. When the viral RNA is either

the elicitor or target of PTGS, the degradation

mechanism is known as virus induced gene

silencing (VIGS). VIGS comes into play when

plants recover from initial viral infection (viral

recovery) or plants resist super infection of

viruses with genomes bearing homology with

those of the viruses used as primary inoculum.

A similar resistance involving PTGS applies not

only to RNA viruses but also to DNA viruses.Viruses can also induce silencing of host

endogens and transgenes that are similar insequence to the inoculated virus. Silencing can

be achieved when the silenced gene is present in

either sense or antisense orientation. During

silencing, not only the target host gene

transcripts but also the viral RNA forms aredegraded. Thus it is easily conceivable that the

infecting viruses could be inactivated by PTGS

mechanisms if the host carries the transgene(s)

of the same or similar virus.

Many viruses have evolved mechanisms tosuppress host PTGS activity. The balance of the

pro-PTGS and anti- PTGS activities probably

determines the outcome of virus plant

interaction. Table 4 shows the known plant and

viral genes inducing or repressing PTGS. PVX

does not encode for any strong anti-PTGS

activity by itself. Hence PVX-based

recombinant viral vectors containing test genes

from various viruses have been used for

infecting silenced GFP-transgenic plants to

screen for PTGS suppressing activity of the

viruses. None of the genes shown in Table 4 hasbeen used yet for plant transformation studies to

develop or modulate viral resistance.

Essential considerations for developingvirus-resistant transgenics

Variability

Viral genes show high levels of variability. This

may be due to lack of proof reading function of

viral replicases and the high recombination rates

of viral genomes during the progress of

infection. Symptomatic variants or strains ofviruses, as well as geographically distinct

isolates, not showing such variations in

symptoms, have been nevertheless, documented

to contain significant variability in their genes.

Under field conditions, most of the viruses are believed to exist as collection of variants, or

quasispecies, as documented in cassava-

infecting geminiviruses in Uganda and rice


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tungro bacilliform virus, a double-stranded DNA

virus. As with naturally occurring virus

resistance genes, when considering virus

resistance under field conditions, strain

specificity and breadth of protection are

important questions. There is often a general

correlation between the extent of protection andthe relatedness between the challenge virus and

virus from which the transgene was derived. It isclear from the case of transgenic papaya that the

level of resistance is dependent upon the homo

logy between the prevalent viral isolate and the

transgene. It is imperative that in any viral

transgene strategy, sequence of the aggressive prevalent strain of the virus in that region is

used. Sufficient information on the degree of

diversity amongst the biologically

indistinguishable viral strains needs to be

collected before designing the transgene. It isespecially true of whitefly transmitted

geminiviruses, where the evolution of the virus

is rapid. A wide variety of virus genotypes may

be present, either maintained in different

cultivated hosts or on endogenous weed species.

Depending upon change in the vector behaviour,

e.g. feeding on to a new host more frequently

than it was doing earlier and vector population

build-up, viruses of different populations may

start infecting new hosts leading to further

changes in their genotype.

The success of any transgenic strategy isdependent upon the level of resistance to

multiple inoculation of the same or related

strains, by vector transmission. In recent years,

efforts have been made to identify the variants

and to assess the genetic relatedness between

them.

Biological risks

The concept of using pathogen-derived genes to

induce transgenic resistance has no doubt raised

a number of ecological concerns. Risk

perceptions boil down to two major items, (i)recombination between viral-derived transgene

and non target virus111, (ii) transmission/vector

host range changes brought about by

heteroencapsidation, i.e. encapsidation of the

genome of non-target virus with thetransgenically expressed CP. Field trials

conducted so far with transgenics have not

indicated that expression of viral transgenes

leads to the emergence of new super strain or

change in transmission behaviour of common

viral pathogens. However, sufficient care should

be taken to avoid any risks due to

heteroencapsidation while designing the

constructs. The strong linkages shown by CP

with insect transmission of viruses, have made possible hetero encapsidation, an important

factor to be considered while designing CPbased transgenes. Coat protein genes have been

designed from PPV, such that a DAG motif in

the CP, believed to play an important role in

vector transmission, was deleted to prevent any

further insect transmission of heteroencapsidatedvirions. The use of these constructs in producing

transgenic plants has shown that

heteroencapsidation of ZYMV was significantly

reduced without compromising virus resistance

of the plants. Similar results have also beenreported recently in transgenic N. benthamiana

expressing mutated PPV CP, which were not

only resistant to PPV, but were also suppressed

in heteroencapsidation, when infected with chilli

vein mottle virus and PVY.

Comparison of anti-viral strategies

The success of transgenic approach varies for

any specific host/virus combination. A range of

phenotypes is observed amongst the virus-

resistant transgenic plants. While CPMR confers

broad-spectrum, less complete resistance, Rep-mediated resistance produces immunity against

the virus, but to a limited spectrum of strains.

Similarly, in RNA-mediated resistance,

antisense RNA targeting mRNA of DNA viruses

has more potential than against positive stranded

RNA virus. Any antisense RNA/ribozyme

strategy should bear in mind the

association/dissociation parameters of the

molecules. Pyramiding of different transgenes or

combination of transgenes with natural

resistance targeting different events in viral life

cycle will increase the confidence level in themanagement of viral diseases and will ensure

stability of resistance at the field level.

Durability, broad-spectrum character of the

transgene- derived resistance coupled with

enhanced crop yield of the transgenics viv--vishealthy, untransfomed plants, etc. are some of

the essential parameters, which any important

strategy must incorporate.


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Genetic engineering of plants to enhanceresistance to fungal pathogens

Recent applications of techniques in

plant molecular biology and biotechnology to

the study of host pathogen interactions have

resulted in the identification and cloning of

numerous genes involved in the defense

responses of plants following pathogen

infection. These include: genes that express

proteins, peptides, or antimicrobial compounds

that are directly toxic to pathogens or that reduce

their growth; gene products that directly inhibit

pathogen virulence products or enhance plant

structural defense genes, that directly or

indirectly activate general plant defense

responses; and resistance genes involved in the

hypersensitive response and in the interactions

with avirulence factors. The introduction and

expression of these genes, as well as of

antimicrobial genes from non plant sources, in a

range of transgenic plant species have shown

that the development of fungal pathogens can be

significantly reduced. The extent of disease

reduction varies with the strategy employed as

well as with the characteristics of the fungal

pathogen, and disease control has never been

complete. Manipulation of salicylic acid,

ethylene, and cytokinin levels in transgenic

plants have provided some interesting results

with regard to enhanced disease tolerance or

susceptibility. The complex interactions among

the expressed gene product, plant species, and

fungal pathogen indicate that the response oftransgenic plants cannot be readily predicted.

Combinations of defense gene products have

shown considerably more promise in reducing

disease than single-transgene introductions. The

use of tissue-specific or pathogen-inducible

promoters and the engineered expression of

resistance genes, synthetic antimicrobial

peptides, and elicitor molecules that induce

defense responses have the potential to provide

commercially useful broad-spectrum disease

resistance in the not-too-distant future. The

issues and challenges that will need to be

addressed prior to the widespread utilization of

these transgenic plants are highlighted.Introduction

One of the challenges facing breeders during the

development of improved crop cultivars for

agricultural use is the incorporation of resistance

to diseases. Since domestication of plants for

human use began, diseases have caused major

yield losses and have impacted the well-being of

humans worldwide (Agrios 1997). Virtually all

agricultural crop cultivars in use today have

some form of genetic resistance incorporated,

generally against a number of diseases. Thismay involve single or multiple genes that are

characterized as having recessive or dominant

effects. Without the incorporation of these

resistance genes, crop productivity and yield

would be substantially reduced (Agrios 1997).

With the beginning of the molecular era of plant

biology in the early 1980s, a major area of

research has been to identify, clone, and

characterize various genes involved in disease

resistance. As a result, many intriguing

mechanisms, which plants have evolved to

respond to pathogen infection. The approachesthat have been taken by researchers can be

grouped into five general categories: (1) The

expression of gene products that are directly

toxic to pathogens or that reduce their growth.

These include pathogenesis-related proteins (PR

proteins) such ashydrolytic enzymes (chitinases,

glucanases), antifungal proteins (osmotin- and

thaumatin-like), antimicrobial peptides (thionins,

defensins, lectin), ribosomeinactivating proteins

(RIP), and phytoalexins. (2) The expression of

gene products that destroy or neutralize a

component of the pathogen arsenal such as polygalacturonase, oxalic acid, and lipase. (3)

The expression of gene products that can

potentially enhance the structural defenses in the

plant. These include elevated levels of

peroxidase and lignin. (4) The expression of

gene products releasing signals that can regulate

plant defenses. This includes the production of

specific elicitors, hydrogen peroxide (H2O2),


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salicylic acid (SA), and ethylene (C2H4). (5)

The expression of resistance gene (R) products

involved in the hypersensitive response (HR)

and in interactions with avirulence (Avr) factors.

The selection of genes to genetically engineer

into plants to protect against fungal diseases has

been based, in part, on evaluation of the toxicityof the gene product to fungal growth or

development in vitro, and to the prominence ofthe particular gene(s) in a disease resistance

response pathway. In other instances, enhanced

levels of protein expression were reasoned to

provide a greater inhibitory effect on fungal

development than lower naturally occurring orinduced levels in the plant. Other genes were

selected in genetic engineering efforts for their

ability to induce an array of naturally occurring

defense mechanisms in the plant.

Hydrolytic enzymes

The most widely used approach has been to

overexpress chitinases and glucanases, which

belong to the group of PR proteins and have

been shown to exhibit antifungal activity in

vitro. Since chitins and glucans comprise major

components of the cell wall in many groups of

fungi, the overexpression of these enzymes in

plant cells is postulated to cause the hyphae to

lyse and thereby reduce fungal growth. The

specific roles of these hydrolases in resistance to

disease have been difficult to prove innontransgenic plants, since the enzymes are

frequently encountered in both resistant and

susceptible tissues, and their expression can also

be induced by environmental triggers and plant

senescence. However, following expression of

different types of chitinases in a range of

transgenic plant species, the rate of lesion

development and the overall size and number of

lesions were reduced upon challenge with many

fungal pathogens, including those with a broad

host range, such as Botrytis cinerea and

Rhizoctonia solani. However, chitinaseexpression was ineffective against other

pathogens, such as Cercospora nicotianae,

Colletotrichum lagenarium, and Pythium spp.,

indicating that differences exist in sensitivity of

fungi to chitinase. The characteristics ofchitinases from different sources can vary, e.g.

in substrate binding specificity, pH optimum,

and localization in the cell, and this can lead to

differences in antifungal activity, highlighting

the importance of appropriate selection of the

gene to be used against a targeted pathogen or

group of pathogens. While the results from these

efforts have not been spectacular in terms of the

level of disease control, they demonstrate that

the rate of disease progress and overall diseaseseverity can be significantly reduced. There are

fewer examples of the expression of glucanasesin transgenic plants but the results have

generally been similar to that for chitinase

expression. The combined expression of

chitinase and glucanase in transgenic carrot,

tomato, and tobacco was much more effective in preventing development of disease due to a

number of pathogens than either one alone,

confirming the synergistic activity of these two

enzymes. As a general rule, the deployment of

genetic engineering approaches that involve theexpression of two or more antifungal gene

products in a specific crop should provide more

effective and broad-spectrum disease control.

Pathogenesis-related proteins

Other PR proteins that exhibit antifungal

activity, including osmotin- and thaumatin-like

proteins (TLP), and some uncharacterized PR

proteins have been engineered into crop plants.

Osmotin is a basic 24-kDa protein belonging to

the PR-5 family whose members have a high

degree of homology to the sweet-tasting proteinthaumatin from Thaumatococcus danielli and

are produced in plants under different stress

conditions. The PR-5 proteins induce fungal cell

leakiness, presumably through a specific

interaction with the plasma membrane that

results in the formation of transmembrane pores.

Osmotin has been shown to have antifungal

activity in vitro and, when tested in combination

with chitinase and -1,3-glucanase, showed

enhanced lytic activity. When expressed in

transgenic potato, osmotin was shown to delay

expression of disease symptoms caused byPhytophthora infestans. Thaumatin- likeproteins is also expressed in plants in response

to a range of stress conditions and was

demonstrated to have antifungal activity in vitro.


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16/26


Antimicrobial proteins, peptides, and othercompounds

Defensins and thionins are low molecular mass

(around 5 kDa) cysteine-rich peptides (4554

amino acids in length) found in

monocotyledonous and dicotyledonous plantspecies, which were initially derived from seeds

and have antimicrobial activity. Viscotoxin from

mistletoe (Viscum album) is a thionin. It was

proposed that these peptides play a role in

protecting seeds from infection by pathogens.Defensins are also found in insects and

mammals, where they play an important role in

curtailing or limiting microbial attack. These

peptides may exert antifungal activity by

altering fungal membrane permeability and (or)

inhibiting macromolecule biosynthesis, and

thionins may be toxic to plant and animal cellcultures as well. The overexpression of

defensins and thionins in transgenic plants was

demonstrated to reduce development of several

different pathogens, including Alternaria,

Fusarium, and Plasmodiophora, and provided

resistance to Verticillium on potato under field

conditions. Chitin-binding peptides (hevein- and

knottin-types) are 3640 residues in length and

have been recovered from the seeds of some

plant species. They contain cysteine residues and

were demonstrated to have antifungal activity in

vitro. It was postulated that the presence ofcations, particularly Ca2+, may have inhibited

the activity of these peptides in vivo. It has been

demonstrated that combined expression of

chitinase and RIP in transgenic tobacco had a

more inhibitory effect on Rhizoctonia solani

development than the individual proteins.

Therefore, dissolution of the fungal cell wall by

hydrolytic enzymes should enhance the efficacy

of antifungal proteins and peptides in transgenic plants. Human lysozyme has lytic activity

against fungi and bacteria, and when expressed

in transgenic carrot and tobacco, enhancedresistance to several pathogens, including

Erysiphe andAlternaria.Antimicrobial peptides have been

synthesized in the laboratory to produce smaller

(1020 amino acids in length) molecules that

have enhanced potency against fungi

Enhancement of the specific activities of

antifungal enzymes or the creation of variants

with broad activity using directed molecular

evolution (DNA shuffling) has also been

proposed as a method to enhance the efficacy of

transgenic plants in the future.

Phytoalexins

These are low molecular mass secondarymetabolites produced in a broad range of plant

species, which were demonstrated to have

antimicrobial activity and are induced by

pathogen infection and elicitors. Phytoalexins

are synthesized through complex biochemical

pathways, such as the shikimic acid pathway,

and genetic manipulation of these pathways to

suppressor enhance phytoalexin production has been difficult to achieve. Similar to the

hydrolytic enzymes, it has not been easy to

conclusively demonstrate the role played by

phytoalexins in enhancing resistance to diseasein many hostpathogen interactions. A mutant of

Arabidopsis deficient in the production of the

indole-type phytoalexin camalexin was shown to

be more susceptible to infection by Alternaria

brassicicola but not toBotrytis cinerea. Using

transgenic plants, it has been possible to also

show that the overexpression of genes encoding

certain phytoalexins, resulted in delayed

development of disease and symptom production

by a number of pathogens on several plant

species.

Inhib ition of pathogen virulence products

The plant cell wall acts as a barrier to

penetration by fungal pathogens and numerous

strategies have evolved among plant pathogens

to overcome this. These include secretion of a

range of plant cell wall degrading enzymes

(depolymerases) and the production of toxins

such as oxalic acid by fungal pathogens. Several

strategies to engineer resistance against fungalinfection have targeted the inactivation of these

pathogen virulence products.

Polygalacturonase inhibiting proteins (PGIP) areglycoproteins present in the cell wall of many

plants and that can inhibit the activity of fungal

endopolygalacturonases. The expression of

PGIP in transgenic plants led to contrasting

results: in transgenic tomato expressing a bean

PGIP, resistance to Fusarium, Botrytis, or

Alternaria was not enhanced while in transgenic

tomato expressing a pear PGIP, colonization of


17/26


leaves and fruits by Botrytis was reduced. As

with the PR proteins and antifungal compounds,

disease development was reduced by PGIPs but

not totally prevented in the transgenic plants.

Another developed strategy that could have

potential to reduce pathogen infection is

immunomodulation, the expression of genesencoding antibodies or antibody fragments in

plants (plantibodies) that could bind to pathogenvirulence products. The antibodies can be

expressed inter- or extracellularly and can bind

to and inactivate enzymes, toxins, or other

pathogen factors involved in disease

development. Currently, there are no publishedreports on the expression of antifungal

antibodies in transgenic plants that have led to a

reduction in disease. However, it has been

demonstrated that antilipase antibodies inhibited

infection of tomato by Botrytis cinerea, whenmixed with spore inoculum, by preventing

fungal penetration through the cuticle. Similarly,

infection by Colletotrichum gloeosporioides on

various fruits was inhibited using polyclonal

antibodies that bound to fungal pectate lyase.

Genetic engineering of antibody expression in

plants is extremely challenging technically and

the applications to fungal disease control

(immunization) have yet to be determined.

Alteration of structural components

Lignifications of plant cells around sites ofinfection or lesions have been reported to be a

defense response of plants that can potentially

slow down pathogen spread. The enzyme

peroxidase is required for the final

polymerization of phenolic derivatives into

lignin and may also be involved in suberization

or wound healing. A decrease in polyphenolic

compounds, such as lignin, in potato tubers by

redirection of tryptophan in transgenic plants

through expression of tryptophan decarboxylase

rendered tissues more susceptible to

Phytophthora infestans, illustrating the role of phenolic compounds in defense. Reduction of

phenylpropanoid metabolism through inhibition

of phenylalanine ammonialyase activity in

transgenic tobacco also rendered tissues more

susceptible to Cercospora nicotianae. Overexpression of a cucumber peroxidase gene in

transgenic potato, however, did not increase

resistance of tissues to infection by Fusarium or

Phytophthora. Overexpression of a tobacco

anionic peroxidase gene in tomato did enhance

lignin levels but resistance to fungal pathogens

was not enhanced.

Activation of plant defense responses

One activator of host defense responses iselicitor molecules from an invading pathogen.

These can trigger a network of signalling

pathways that coordinate the defense responses

of the plant, including HR, PR protein, and

phytoalexin production. A gene encoding the

elicitor cryptogein (a small basic protein, 98

amino acidsin lengths) from the pathogen

Phytophthora cryptogea was cloned and

expressed in transgenic tobacco under control of

a pathogen-inducible promoter. Challenge

inoculation with a range of fungi induced the

HR as well as several defense genes, and growthof the pathogens was concomitantly restricted.

Resistance to the pathogens was not complete,

possibly because of the time needed for

production of the transgenic elicitor following

initial infection. Another elicitor, INF1, was

shown to act as an Avr factor in the tobacco

Phytophthora infestans interaction and triggered

the onset of the HR. Expression of the gene

encoding the AVR9 peptide elicitor from

Cladosporium fulvum in transgenic tomatoes

containing the Cf-9 gene resulted in a necrotic

defense response. The development of lesionsresembling the HR induced through expression

of a bacterial proton pump gene (bacterio-opsin)

from Halobacterium halobium activated

multiple defense systems in transgenic tobacco

plants in the absence of pathogen challenge. In

transgenic potato, expression of bacterioopsin

enhanced resistance to some pathogens but had

no effect on others (Abad et al. 1997), while in

poplar, there was no effect on disease

development. Expression of elevated levels of

H2O2 in transgenic cotton, tobacco, and potato

reduced disease development due to a number ofdifferent fungi, including Rhizoctonia,

Verticillium, Phytophthora, and AlternariaOther activators of plant defense responses

include signaling molecules such as SA,

ethylene, and jasmonic acid. The roles of SA asa signal molecule for the activation of plant

defense responses to pathogen infection and as

an inducer of systemic acquired resistance


18/26


(SAR) have been extensively studied. Altered

auxin or cytokinin expression has the potential

to also affect mycorrhizal colonization of plant

roots.Resistance genes

Resistance gene products may serve as receptorsfor pathogen Avr factors or recognize the Avr

factor indirectly through a coreceptor. This gene

forgene interaction triggers one or more signal

transduction pathways that in turn activates

defense responses in the plant to prevent

pathogen growth. These defense responses

include the development of the HR, expression

of PR proteins, and accumulation of SA and can

lead to the development of SAR. Ethylene and

jasmonic acid may also be involved in signalling

the defense responses in the gene-forgene

interaction. Efforts to clone an array of R genesinvolved in fungal disease resistance have met

with some success. The R-gene products that

have been cloned from tomato, tobacco, rice,

flax,Arabidopsis, and several other plant species

shared one or more similar motifs: a serine or

threonine kinase domain, a nucleotide binding

site, a leucine zipper, or a leucine-rich repeat

region, all of which may contribute to

recognition specificity. There are several

examples of the expression of R genes in

transgenic plants. The overexpression of the

HRT gene, which controls the HR to turnipcrinkle virus in Arabidopsis, did not confer

enhanced resistance to Peronospora tabacina.

The authors proposed that multiple factors may

be involved in determining the resistance

response, or that the resistance may be HR-

independent. A combination of several

interacting genes, similar to that for the

antifungal proteins, will likely be required. An

enhanced understanding of R-gene structure and

function could, however, make it possible to

modify functional domains in the future to tailor

R genes for use in providing broad-spectrumresistance to diseases in transgenic plants. Other

potential approaches to the use of R genes for

engineering disease resistance in plants.

Scientific challenges

Besides identifying and cloning potentially

useful genes to engineer into plants, the

development of transgenic plants with enhanced

fungal disease resistance faces additional

challenges. Depending on the plant species,

transformation frequencies can be as low as 1

10%, and out of hundreds of confirmed

transgenic lines, only a few may have

appropriate transgene expression levels. Recent

advances in plant transformation should providenew opportunities to overcome some of these

difficulties. The positive relationship of highlevels of PR proteins and antifungal compounds

with enhanced disease resistance in plants has

been documented in many but not all cases.

However, as indicated previously, there are a

number of examples where transgene productsexpressed at high levels induced plant cell

damage or had other undesirable effects.

Wound-inducible and pathogen inducible

promoters, which have advantages for

engineering specific disease resistance againstfungal pathogens by expressing antifungal

compounds only at sites of infection or wounds,

have also been described. Targeting of the

engineered protein to the apoplastic space or to

the vacuole has been achieved and may enhance

the antifungal activity, depending on the mode

of infection of the pathogen. Future research will

require the fine tuning of engineered gene

expression and establishment of the optimal

expression levels and target site in the cell

needed to prevent pathogen infection.

Commercial development

Despite close to 100 published scientific reports

, of which about 30% are on tobacco used as a

model system, only a few of the transgenic crops

have been field-tested, and wide-scale

deployment may not yet be realized for another

510 years. Development of transgenic plants

with enhanced disease resistance is also being

actively pursued in the private sector, Therefore,

genetic engineering of novel disease resistance

traits in crop plants has the potential to provide

control of devastating pathogens with reducedfungicide applications. Expression of an

antifungal trait throughout the growing season,

from seed to harvest, under prolonged disease

conducive conditions, can also provide

significant advantages for disease managementusing this technology.


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Resistance to bacterial diseases throughgenetic engineering

The livelihoods of millions of farmers

have been threatened by current outbreak of a

banana bacterial wilt disease caused by

Xanthomonas campestris pv. musacearum,

which is very destructive and rapidly spreading

in Uganda. Bananas are the highest value staple

food and source of income for millions of peoplein this region. Economic impact of the disease is

clear as a result of widespread destruction of

banana, pre-harvest rotting of fruits, and a lack

of farmers ability to grow bananas in disease

endemic areas. The transgenic approach shows

potential for the genetic improvement of the

crop using a wide set of transgenes currently

available which may confer bacterial resistance.

Introduction

Banana (Musa sp.) is the fourth most important

global food crop after rice, wheat and maize interms of gross value of production. The

livelihoods of millions of Ugandan farmers have

been threatened by the current outbreak of

bacterial wilt disease caused by the bacterium

Xanthomonas campestris pv. musacearum The

disease, which has been identified as a bacterial

wilt was first reported in Ethiopia, where it

caused only minor problems since banana

production is smallscale and scattered. Xcm wilt

was initially identified in the major banana-

producing appears to be manifesting itself as a

disease threat of potential epiphytoticproportions. X cm infection can result in severe

losses in banana production and affects banana

productivity to early ripening and rotting of

fruits even in the absence of apparent external

signs of the disease, and wilting and death of

banana plants. If unchecked, the disease would

cause massive losses in the Ugandas western

districts, an area of intensive banana cultivation,

and to neighboring countries. The disease

attacks all varieties of banana.

The first symptoms include discoloration at the

tip of the flower and withering of the flower

bracts. Other symptoms include yellowing,

wilting and premature ripening in young plants.

When the banana is cut, a pink-purple colorationconfirms presence of the disease. Even in some

cases where these other symptoms fail to show,the coloration is always seen. The plant dies

within a month from the first appearance of any

of the symptoms.

Use of genetic transformation technologies for

Musa, may provide a timely and cost-effectivemeasure to address the dangers of the spread of

this disease. Even if resistant germplasm sources

are identified, which to date have not; a breeding

cycle for banana germplasm development may

be expected to require 6-20 years utilizingconventional breeding methodologies. More

recently, transgenic plants have been produced

that are resistant to a wide variety of bacterial

diseases. These forms of resistance follow a

number of chemical strategies, including the use

of genes for bacterial toxin tolerance,

antimicrobial peptides, and other defense related

proteins that tend to act as bactericidal

compounds.

Strategies for developing BacterialDiseases resistant plants

One approach to control bacterial disease is to

improve a plants' defense against a particular

pathogen. This has been made possible by

genetic engineering by using genes found in

fungi, insects, animals and other plants.

Antimicrobial proteins, peptides, and lysozymes

that naturally occur in insects, plants, animals,

and humans are now a potential source of plant

resistance.Expression of antimi crobial proteins

Antimicrobial peptides (AMPs) with -helicalstructures are ubiquitous and found in many

organisms. AMPs have been isolated from frogs,

insects, and mammalian phagocytic vacuoles.

AMPs are selective for prokaryotic membranes

over eukaryotic membrane due to the

predominantly negatively charged phospholipids

in the outer leaflet of the prokaryotic membrane.


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Such preference is considered a regulatory

function in target selectivity.Magainins

Magainin is a defense peptide secreted from theskin of the African clawed frog (Xenopus

laevis), first discovered by Zasloff (1987).Magainins and their analogs have been studied

as a broad-spectrum topical agent, a systemic

antibiotic, a wound-healing stimulant, and ananticancer agent. However, only magainin

analogs (MSI-99 and Myp30) have recently

been transferred into plants for used against

bacteria. Li et al. (2001) have reported disease

resistance, to both a fungal and a bacterial pathogen, conferred by expression of a

magainin analog, Myp30, in transgenic tobacco

(Nicotiana tabacum var. Petit Havana). Another

analog MSI-99, when expressed in tobacco viachloroplast transformation conferred both in

vitro and in planta resistance to plant pathogenic

bacteria and fungi.Cecropins

Cecropins are antibacterial lytic peptides native

to the hemolymph ofHyalophora cecropia, the

giant silk moth. These peptides interact with the

outer phospholopid membranes of both Gram-

negative and Gram-positive bacteria and modify

them by forming a large number of transient ion

channels. Native (Cecropin B), mutant (SB37,MB39) and synthetic (Shiva-1,

D4E1) cecropins are active in vitro against a

wide range of plant pathogenic bacteria

including Erwinia carotovora, E. amylovora,

Pseudomonas syringae, Ralstoniasolanacearum

and Xanthomonas campestris whereas they exert

no toxicity at bactericidal concentration to

cultured cells or protoplasts of several plant

species. Therefore, cecropins are considered as

potential candidates to protect plants against

bacterial pathogens. Transgenic tobacco plants

expressing cecropins have increased resistanceto P. syringae pv. tabaci, the cause of tobaccowildfire. Synthetic lytic peptide analogs, Shiva-1

and SB-37, produced from transgenes in potato

plants reduce bacterial infection caused by

Erwinia carotovora subsp. atroseptica in

transgenic potato plants. Transgenic apple

expressing the SB-37 lytic peptide analog

showed increased resistance to E. amylovora,

pathogen for fire blight, in field tests. More

recently, the expression of the D4E1 in poplar

has resulted resistance to Agrobacteriumtumefaciens andXanthomonas populi .

Attacins

Attacins are another group of antibacterial proteins produced by Hyalophora cecropiapupae. The mechanisms of antibacterial activity

of this protein are to inhibit the synthesis of the

outer membrane protein in gram negative

bacteria. Attacin expressed in transgenic potato

enhanced its resistance to bacterial infection by

E. carotovora subsp. atrospetica. Transgenic

pear and apple expressing attacin genes have

significantly enhanced resistance to E.

amylovora in in vitro and greenhouse. In field

tests, reduction of fire blight disease has been

observed in transgenic apples expressing attacingenes. Transgenic apple expressing attacin

targeted to the intercellular space, where E.

amylovora multiplies before infection, has

significantly reduced fire blight, even in apple

plants with low attacin production levels.

Lysozymes

Lysozymes are a ubiquitous family of enzymes

that occur in many tissues and secretions of

humans, animals, as well as in plants, bacteria

and phage. The lysozyme attacks the murein

layer of bacterial peptidoglycan resulting in cellwall weakening and eventually leading to lysis

of both Gramnegative and Gram-positive

bacteria. Hen egg-white lysozyme (HEWL), T4

lysozyme (T4L), T7 lysozyme, human and

bovine lysozyme genes have been cloned and

transferred to enhance plant bacterial or fungal

resistance. The lysozyme genes have been used

to confer resistance against plant pathogenic

bacteria in transgenic tobacco plants. T4L, fromthe T4-bacteriophage, also has been reported to

enhance resistance of transgenic potato against

E.carotovora, which causes bacterial soft rot.Transgenic apple plants with the T4L gene

showed significant resistance to fire blight

infection. Human lysozyme transgenes have

conferred disease resistance in tobacco through

inhibition of fungal and bacterial growth,

suggesting the possible use of the human

lysozyme gene for controlling plant disease.

There is evidence of efficacy of bovine


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lysozyme isozyme c2 (BVLZ) transgene against

a variety ofXanthomonas campestris strains in

both monocotyledon and dicotyledon crops

including tomato, tobacco, rice and potato. Since

this bactericidal transgene has been shown to

function in monocot and has clear efficacy

against at least several strains ofX. campestris,its usefulness as a transgene for resistance to X.

campestris in Musa has a high probability ofsuccess.

Thionins

Thionins are plant antimicrobial proteins which

are able to inhibit a broad range of pathogenic

bacteria in vitro. Carmona et al. (1993) reported

the expression of alpha-thionin gene from barley

in transgenic tobacco confers enhanced

resistance to two pathovars of P. syringae.

Unfortunately, most thionins can be toxic toanimal and plant cells and thus may not be ideal

for developing transgenic plants.Expression of plant defense genes

Plants have their own networks of defense

against plant pathogens that include a vast array

of proteins and other organic molecules

produced prior to infection or during pathogen

attack. Recombinant DNA technology allows

the enhancement of inherent plant responsesagainst a pathogen by either using single

dominant resistance genes not normally presentin the susceptible plant (Keen, 1999) or by

choosing plant genes that intensify or trigger the

expressions of existing defense mechanisms.Plant resistance (R) genes

Pathosystem-specific plant resistance ( R) genes

have been cloned from several plant species.

These includeR genes that mediate resistance to

bacterial, fungal, viral, and nematode pathogens.

Many of these R gene products share structural

motifs, which indicate that disease resistance to

diverse pathogens may operate through similar pathways. TheBs2 resistance gene of pepper

specifically recognizes and confers resistance to

strains of X. campestrispv. vesicatoria that

contain the corresponding bacterial avirulence

gene, avrBs2. Transgenic tomato plants

expressing the pepper Bs2 gene suppress the

growth ofXcv. TheBs2 gene is a member of the

nucleotide binding siteleucine-rich repeat

(NBS-LRR) class of R genes. The Xa1 gene in

rice confers resistance to Japanese race 1 ofX.

oryzae pv. oryzae, the causal pathogen of

bacterial blight.Xa1 is a member of the NBS-

LRR class of plant disease resistance genes. The

rice Xa21 gene confers resistance to X. oryzae

pv. oryzae. Fifty transgenic rice plants carryingthe cloned Xa21 gene display high levels of

resistance to the pathogen. The sequence of the predicted protein carries both a leucine-rich

repeat motif and a serinethreonine kinase-like

domain. The Pto gene is another class of R

genes, encoding a serine/threonine protein

kinase that confers resistance in tomato to P.

syringaepv tomato strains that express the type

III effector protein AvrPto. Overexpression of

Pto in tomato under control of the cauliflower

mosaic virus (CaMV) 35S promoter has been

shown to activate defense responses in theabsence of pathogen inoculation. Pto-

overexpressing plants show resistance not only

to P. syringae pv tomato but also toX.

campestris pv vesicatoria and to the fungal

pathogen Cladosporium fulvum. Therefore, Pto

genes are considered as potential candidates to

protect plants against pathogens.

Current status of M usa transformation

Genetic transformation using microprojectile

bombardment of embryogenic cell suspension isnow routine. An efficient method for direct gene

transfer via particle bombardment of

embryogenic cell suspension has been reported

in cooking banana. The recovery of transgenic

plants of banana obtained by means of

Agrobacterium tumefaciens mediated

transformation has been reported. The protocol

has been developed forAgrobacterium mediated

transformation of embryogenic cell suspensions

of the banana. At present most of the

transformation protocol use cell suspension,

however establishing cell suspension is lengthy process and cultivars dependent. The protocol

has also been established using shoot tips from

various cultivars of Musa. This technique is

applicable to a wide range ofMusa cultivars

irrespective of ploidy or genotype. This process

does not incorporate steps using disorganized

cell cultures but uses micropropagation, which

has the important advantage.


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Resistance to nematode diseases throughgenetic engineering

Nematodes are recognized as severe

production constraints to bananas and plantains,

with losses due to nematodes estimated at about

20% worldwide. Locally however, losses of

40% or greater can frequently occur, particularly

in areas prone to tropical storms due to toppling

as a result of wind damage on affected plants.

Nematode management in bananas and plantains

is mainly based on crop rotation and chemical

control. However, crop rotation is not often

practiced and use of nematicides is often not

practical or affordable.

Nematode resistance in banana

There is evidence that nematode resistance and

tolerance sources, though limited, are present in

the Musa gene pool. Some resistance has been

identified against the most damaging nematode

species, the burrowing nematode (Radopholus

similis), but this needs to be combined with

consumer acceptable traits. However,

Pratylenchus sp. causes more losses than R.

similis. Furthermore, several species of

nematodes are often present together,

necessitating a broad spectrum resistance in

order to reduce these losses significantly. There

are several possible approaches for developing

transgenic plants with improved nematode

resistance. The use of proteinase inhibitors (PIs),

as nematode antifeedants, is an important

element of natural plant defence strategies. This

approach offers prospects for novel plantresistance against nematodes and reduces use of

nematicides. The potential of PIs for transgenic

crop protection is enhanced by a lack of harmful

effects when humans consume them in seeds

such as rice and cowpea. Cysteine PIs (cystatins)

are inhibitors of cysteine proteinases and have

been isolated from seeds of a wide range of crop

plants consumed by man including those of

sunflower, cowpea, soybean, maize and rice.

Cysteine proteinases are not involved in

mammalian digestion. Transgenic expression of

PIs provides effective control of both cyst and

rootknot nematodes. The cystatins Oc-I and an

engineered variant Oc-ID86 was shown tomediate nematode resistance when expressed in

tomato hairy root, Arabidopsis plants, rice and pineapple. The partial resistance (709%) was

conferred in a small-scale potato field trial on a

susceptible cultivar by expressing cystatins

under control of the CaMV35S promoter. There

is no evidence that expression of cystatinsimpairs plant growth or yield in trials. The

enhanced transgenic plant resistance to

nematodes has been demonstrated by using dual

proteinase inhibitor constructs. Full resistance is

achieved by pyramiding a cystatin with naturalresistance genes. Since this nematicidal

transgene not only has been shown to function in

rice, which like Musa is a monocotyledon, and

also has clear efficacy against a wide range of

nematode species and has been consumed for

years from foods such as seeds of rice and maize

by human beings, its usefulness as a transgene

for development of transgenic Musa for

resistance to nematodes can be evaluated as

having a high probability of success. The other

genetically engineered crops

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