Insect Pest resistance - A Biotechnology Approach

Mr. Abhirup Ganguli
Asst. Professor,
Dept. of Biotechnology
Swami Vivekananda
Institute of Modern
Sciences

Where the story began
 For centuries, humans have searched for crop plants
that can survive and produce in spite of insect pests.
 Knowingly or unknowingly, ancient farmers selected
for pest resistance genes in their crops, sometimes by
actions as simple as collecting seed from only the
highest-yielding plants in their fields.
 With the advent of genetic engineering, genes for
insect resistance now can be moved into plants more
quickly and deliberately.
 Bt technology is only one example of ways genetic
engineering may be used to develop insect-resistant
crops now and in the future.

The Bt chapter
 Bacillus thuringiensis, commonly known as Bt, is a
bacterium that occurs naturally in the soil.
 For years, bacteriologists have known that some strains
of Bt produce proteins that kill certain insects with
alkaline digestive tracts.
 When these insects ingest the protein produced by Bt,
the function of their digestive systems is disrupted,
producing slow growth and, ultimately, death.
 Bt is very selective — different strains of the bacterium
kill different insects and only those insects.
 Strains of Bt are effective against European corn borers
and cotton bollworms (Lepidoptera), Colorado potato
beetles (Coleoptera), and certain flies and mosquitoes
(Diptera).

 Bt is not harmful to humans, other mammals, birds,
fish or beneficial insects.
 Bt was first identified in 1911 when it was discovered
that it killed the larvae of flour moths.
 Bt was registered as a biopesticide in the U.S. in 1961.
 Today it is used in insecticide sprays sold to home
gardeners and others worldwide.
 In 1983, the World Health Organization used Bt in
West Africa to control disease-carrying blackflies.
 In the U.S., various strains of Bt are used to control
spruce budworms and gypsy moths in forests,
cabbage worms in broccoli and cauliflower, loopers
or budworms in cotton and tobacco, and leaf rollers
in fruits.

 However, less than one percent of all pesticides
used in the U.S. each year contain Bt (Monsanto).
 As an ingredient of commercial sprays, Bt is
relatively expensive and has some drawbacks.
 Although some pesticides kill on contact, Bt must
be eaten by insects to be effective.
 Sunlight breaks down Bt, and rain washes it from
the plants.
 Therefore, Bt must be applied exactly where and
when the target insects are feeding, and they must
consume it quickly before it disappears.

Bt and biotechnology
 Today, plants can be genetically engineered to produce
their own Bt.
 Genetic (recombinant DNA) engineering is the
modification of DNA molecules to produce changes in
plants, animals or other organisms.
 DNA (deoxyribonucleic acid) is a double-stranded
molecule that is present in every cell of an organism
and contains the hereditary information that passes
from parents to offspring.
 This hereditary information is contained in individual
units or sections of DNA called genes.
 The genes that are passed from parent to offspring
determine the traits that the offspring will have.

 In the last twenty years, scientists made a
surprising discovery — DNA is interchangeable
among animals, plants, bacteria ... any organism!
 In addition to using traditional breeding methods
of improving plants and animals through years of
cross-breeding and selection, scientists can now
isolate the gene or genes for the traits they want in
one animal or plant and move them into another.
 Of course, when a trait is controlled by several
genes, the transfer process is more difficult.
 The plants or animals modified in this way are
called transgenic.

 Bacillus thuringiensis form insecticidal proteins
which are crystalline in nature during sporulation.
 The total dry weight of crystal protein occupies as
much as 30% of the spore dry weight.
 The crystal protein consists of one or more
protoxins and their mass reached upto 160,000
daltons.
 Cleaving of protoxins by proteolysis result in
peptides of 55,000 to 70,000 that are specifically
toxin to lepidopteran and Dipteran insects.
 Bacillus thuringiensis parasporal crystal consist of
one or more 8-endotoxin or crystal (cry) protein of
130 kDa.

 The δ-endotoxins are dissolved in the alkaline
conditions or the insect midgut and release proteins of
Molecular Mass 65,000 – 160,000 to become active
forms of the endotoxins, which are then processed by
proteases to yield smaller toxin fragments by binding
to midgut epithelial cells and causes osmotic lysis
through pore formation in the cell membrane.
 The crystal protein of Bt is of single polypeptide of 130
kD of which toxin peptide is half that size.
 The genes encoding crystal proteins are situated in
plasmid rather than in chromosome of bacteria for
instance, in HD-1 strain, there are two plasmids in
bacteria contains insecticidal toxin gene.

 B. thuringiensis toxins are highly specific in such way
that they are non-toxic to other organs.
 Having these unique properties, they can be used as
safe insecticides and also an effective alternative to
chemical insecticides.
Mode of Action:
 Insecticidal crystals are composed of large proteins
that are essentially inactive when a insect ingest some
of the insecticide crystal proteins.
 The alkaline environment of the insect midgut (pH 7.5
to 8.0) causes crystal to dissolve and release their
constituent protoxin.

 At this stage, crystal protein is inactive, but the presence of
specific protease within gut region of the insects trimmed
to an N-terminal 65-70 kDa truncated form and convert
inactive protein to become active protein immediately (Fig.
20.6).

 The processed active protein then binds to a specific
receptor on the border membrane of the cells lining
the midgut and insect itself into the cell membranes.
 When about eight of these aggregate together, this
forms a pore or channel through the membrane,
resulted process is the leakage of cell content
continuously causing the death of cells and
eventually killed by colloid or osmotic lysis.
 These systems are indispensable for nutrient
absorption.
 Once damage occurs, insects stop feeding
immediately and finally starve to death possibly
within 24 hrs.

Isolation of Bt Gene:
 The exact location of Bt gene either in the plasmid or
chromosomal DNA is indispensable prior to isolation.
 The conformation of toxin gene can be had by conjugating
Bt strain with other strain devoid of insecticidal activity.
 Bt strains are followed by separation of plasmid and
chromosomal DNA fractions by separation of plasmid and
chromosomal DNA.
 If toxin gene is encoded in plasmid, it is then subjected for
sucrose density centrifugation and fractionation is followed
for plasmid DNA.
 The media and larger plasmid are treated with
endonucleases and targeted into pBR322 plasmid.
 The cloned banks were transformed into E coli. and
screened by immunological method.

Classification of Crystal Protein:
 A large number of Bt insecticidal σ endotoxin protein
gene have also been cloned and sequenced.
 Till date more than 130 genes have been identified.
 Bt insecticidal proteins are commonly known as cry
genes, based on amino acid sequence in their protein.
 The protein encoded by cry I genes are toxic only to
caterpillar.
 The cry II genes encoded proteins are toxic to
lepidopteran or dipteran (flies of mosquito), while the
cry IV proteins are active only against diptera.
 However, cry III gene produces proteins that can be
weaponised against beetle i.e., coleoptera larvae (Table
20.2)

First Generation of Transgenic (Bt)Plants:
 In some of the earliest experiments, both full-length
and truncated cry genes were successfully introduced
into model plants such as tobacco and potato by
designing plant expression vector such as modified
Agrobacterium tumeifaciens, which contains
constitutive promoter, full length genes, truncated
genes and terminated signal, provide some good
protection against insect challenge.
 Later investigation has clearly revealed that expression
of unmodified cryA genes was too low to provide
complete protection.
 In the Bt odessey, modification of toxin was enforced
to enhance resistance against insect.

 The cloning of the Bt 2 gene from B-thuringenesis
and characterization of the polypeptide expressed in
bacterial E. coli system was analysed.
 The characterized Bt is 1,155 amino acid long and is a
potent toxin to several lepidoptera larvae such as
tobacco pest (Maduca sexta), tomato pest (Heliothis
vivescence and Helicoverpa).
 Bt2 is a protoxin generate smallest fragment that still
possess full toxic mapped in the NH2-terminal half
of the protein between amino acid position 29 and
607.
 Insecticidal activity in transgenic plants was
evidenced by feeding leaves of transgenic plants on
M. sexta larvae and confirms 75-100% mortality of
the larvae.

 Several groups of researcher continued their work
and conducted field trials with transgenic plants
expressing Bt proteins.
 The outcome of their study was found to display
two important factors.
 One is, they demonstrated that Bt gene could be
systematically expressed in transgenic plants.
 Another is expression level of Bt toxin protein.
 They showed that level of insecticidal protein in
transgenic plants with exceptional few was
relatively low, generally not sufficient to provide
protection to the plant against insect challenge.

Engineering of Bt Cry Genes:
 The enigma of low Bt gene expression become the
target for many research groups involved in insect
control and greater attention was given to the
engineering of Bt gene to accelerate its expression, so
that complete protection can be accomplished.
 Bacillus thuringiensis cry genes are typically bacterial
genes. Their DNA sequence has high A/T content than
plant genes in which G/C ratio, higher than A/T.
 The overall value of A/T for bacterial genes is 60-70%
and plant genes with 40-50%.
 As a consequence, GC ratio in cry genes codon usage is
significantly insufficient to express at optimal level.

 Moreover, the A/T rich region may also contain
transcriptional termination sites (AATAAA
polyadenylation), mRNA instability motif (ATTTA)
and cryptic mRNA splicing sites.
 These regions might be recognised by the plant
transcriptional system as destabilising sequence or as
introns.
 Some critical assessment was made in the gene
modification of cry genes like crylAb, and crylAc genes
in transgenic tobacco, tomato and cotton.
 Partial modification in cry IAb involves the removal of
seven out of 18 polyadenylation sites and seven out of
13 ATTTA sequences.

 As a consequence, there was considerable increase in
protection of plants and ten-fold increase in Bt protein
concentration when compared with unmodified genes.
 Further increase in Bt protein production (upto 0.2 to
0.3% of total soluble protein) to 100-fold level have been
contrived by removing remaining poly-adenylation sites
and ATTA sequence and changes to a total of 356 of the 615
codons.
 Apart from modification of Bt gene by removal of some
sequence, resynthesis of the genes contain higher G/C
content, solved one of the major problems of low
expression.
 This allowed the codon usage to be accorded for particular
crop.
 The synthetic modified gene is exactly proportion as native
gene.

 The track record of this Bt gene expression showed
substantial increase in the expression of cry 3A gene in
transgenic potatoes and it was achieved by increasing its
overall G/C content from 36% to 49%, which result in the
fine protection against Colorado potato beetle larvae.
 The performance of transgenic cotton expressing 100-fold
increase in cry 1Ab or cry 1AC were confirmed by effective
control of cotton pests such as cotton boll worm in 1990.
 This high level expression was achieved by using strong 35S
promoter with duplicated enhancers and sequence
modification in certain regions of the gene with predicted
mRNA secondary structure.
 Innacone (1997) reported sequence modification of cry 3B
endotoxin gene resulted in high level of expression.
 When cry 3B native gene was transferred into egg-plant
(Solanum melongena) low expression of toxin protein and
no resistance were recorded.

•The Bt 43 belonging to
the cry 3 class was
partially modified by its
nucleotide sequence by
replacing four target
regions using
reconstructed synthetic
fragments.
•The coding sequence of
Bt 43 wt (wild type) was
partially redesigned and
nine DNA fragments
were identified as target
for substitution
(modification).

 Synthetic Bt genes were designed in such a way that in
their modified region, researchers deliberately avoids
the sequences such as ATTA sequence,
polyadenylation sequence and splicing sites, which
might destabilize the messenger RNA.
 In addition, codon usage from AT to high GC ratio was
improved for better expression.
 The modified gene resulted in four versions (BtE, BtF,
BtH and Btl).
 In the modified version of Btl gene, overall G + C ratio
was increased from 34% of the wild type gene to 45%
(Fig. 20.7).
 Transgenic plants obtained with modified versions
BtH and Btl, were completely resistant to Leptinotorsa
decemli.

Note:
Dotted boxes are indicated by the replacement of nucleotide sequence.

 A modified synthetic cry IA (b) gene was transferred to
cabbage cultivar and their expression resulted in
significant insecticidal activity of transgenic cabbage
plant against the larvae of diamond moth.
 These results also reveal that synthetic gene based on
monocot codon usage can be expressed in
dicotyledons plants for insect challenge.
 Efficient expression of modified Bt gene under the
control of strong CamV 35S is well documented.
 In addition, other promoters such as wound inducible
promoters, chemically inducible promoters and tissue
specific promoters have also been used.
 In certain cases, Bt protein has been made to express
in the chloroplast of tobacco by rubisco small subunit
promoter fused to plastic signal peptide.

 Surprisingly chloroplast transformation can be performed
for better expression of even unmodified Bt protein.
 This shows that transcriptional and translational
machinery of plastids are similar to chloroplast.
 Therefore, modification of crylAc sequence in many cases
was found to be unnecessary.
 Apart from dicotyledons, several members of
monocotyledons were transformed with Bt crystal protein.
 Transformation of maize with truncated cry 1A6 gene by
complete replacement of codons with maize codons
resulted in increased percentage of GC content of the gene
(37% to 65%).
 As a consequence, transgenic maize provided excellent
protection against European corn borer.

 Another case study, among monocot is the
expression of synthetic Cry IA (b) gene in Indica
rice.
 Transgenic rice plants displayed high level
expression in their leaves and result in high
effective control of pest of rice in Asia.
 The yellow stem borer (YSB) and the striped stem
borer (SSB) and feeding inhibition of two leaf
folder species.

Second Generation of Insect Resistant
Transgenic Plants:
 First generation of transgenic insecticidal plants consist
of δ-endotoxins are currently using in large scale in
agriculture.
 Although Bt toxin is a remarkable protein by providing
protection to the several economically important plants
from insects challenge.
 Their overall performance was found to be not efficient
against some economically-important insect’s pest such
as northern and western corn-root worms and also boll
weevil.
 As a consequence, alternative strategies have been
evolved to characterize novel insecticidal proteins.

 The best way is to screen bacterial production of
insecticidal proteins in physiological stages of
bacterial growth other than looking for protein
and sporulation stage, where production of Bt
protein occur normally.
 In addition, screening is done for new sources of
insecticidal protein even in plant sample,
particularly in tropical plants.
 Following are some of the non-Bt insecticidal
proteins providing protection against several pests.

VIP’s Toxin Protein (vegetative insecticidal
protein):
 While searching for novel insecticides effectives alternative
to Bt insecticide protein was discovered that certains
Bacillus species produces novel insecticidal protein during
vegetative stages (lag phase) of growth in culture.
 Their presence was confirmed in supernatant obtained
from bacillus clarified culture.
 Supernatant fluids of Bacillus cereus when tested, exhibit
potent insecticidal activity against corn root worms.
 The insecticidal protein was identified as VIP1 and VIP2.
 In addition, VIP3A a new class of insecticidal protein shows
no sequence homology to known Bt cry proteins and
specifically binds to non-Bt receptors into insect midgut.

 Currently, VIP3 is under review for its efficacy in reducing the
rate of insect resistance development.
 These three vip proteins exhibits number of positively
charged residues followed by a hydrophobic core region.
 The efficacy of vip proteins as insecticide potency shows that
they exhibit acute bioactivity against susceptible insect
(ng/ml of diet).
 Bt toxin protein shows bioactivity with the same
concentration of vip proteins.
 The better insecticidal activity is associated with VIP 2A
protein in particular as it display insecticidal activity against
wide spectrum of lepidopteran insects such as beet army
worm, cut worm and a army worm.
 In the susceptible insects protein vip 3A causes gut paralysis
after specifically binds to gut epithelium followed by complete
lysis of these cells.

Proteinase Inhibitors:
 These are the inhibitor proteins reduces feeding
efficiency of insects by inactivating their digestive
enzyme.
 Thereby deprived insects from having nutrition.
 Transgenic plants expressing proteinase inhibitors act on
insects as growth retardant, when feeds on the plant.
 Several plants, particularly pulses contains substantial
amount of inhibitor proteins.
 Once these proteins enters insects digestive system,
paralyse protein digestive enzymes.
 The gene for these proteins in plants have been
characterised and exploited in transgenic technology for
the production of insect resistant plants.
 One classic example is the cloning of well characterized
trypsin inhibitor gene.
 Transfer of this gene into plants provides considerable
protection against several insects.

Lectins:
 These are haeme agglutinin proteins present in many
plants which are used to control insects due to its effective
insecticidal property.
 Once it was thought, lectins could be an alternative to Bt 6
endotoxin.
 The ability of lectin to bind glycosylated proteins on insect
midgut is well characterized.
 Its insecticidal activity however, confirms only when
insects are exposed to high level in the diet.
 There have been many reports of transgenic plants
expressing lectin Coding genes.
 But their performance as insecticidal protein was found to
be unsatisfactory due to their minimal level of expression.
 However, there have been a number of reports on
transgenic maize expressing wheat germ lutin, jacaline or
rice lectin when tested for European corn borer
demonstrated minimum level of larvae growth.

Cholesterol Oxidase:
 This protein belonging to the member of large family of
acysterol oxidase, where boll weevil larvae fed by a diet
containing cholesterol oxidase (CO) showed structural
alteration in the midgut epithelial cells.
 After exposure to ‘CO’-exhibited cellular attenuation
accompanied by local cytolysis.
 These cytological symptoms suggesting that ‘CO’ alters
the cholesterol incorporated into the membrane.
 Cholesterol is indispensable for the structural integrity
and normal functions of all cell membrane.
 A cholesterol oxidase catalyses oxidation of cholesterol
to produce ketosteroids and hydrogen peroxide.

 Therefore, any interference in the incorporation of
cholesterol into the membrane may Jeopardise the
integrity of cell membrane and eventually cell lysis
and death.
 Transgenic plants expressing active cholesterol
oxidase has been demonstrated in tobacco
protoplast transformed with native cholesterol
oxidase gene.
 Once insect’s starts feeding on transgenic plants,
their midgut epithelium seems to be primary target
to CO and consequently leads to death of insects.

Toxin A (TcdA):
 The expression of the toxin A (TcdA) from
photorabdus luminiscence in transgenic plant
represents an important step in searching for new
novel genes for insect challenge.
 This was happened to be the classic example of
exploiting symbiotic bacteria involved in biocontrol of
insects in nature.
 Photorabdus luminiscence is a bacteria lives
symbiotically within the nematode Heterorhabditis.
 This nematode is parasite to insects and is highly
pathogenic to large number of insects.
 The pathogenecity of insects is mainly due to the
presence of symbiotic bacteria Photorabdus
luminiscence.

 When nematode invades an insect and regurgitates the
bacteria that then produce toxin A that kills the insects.
 Bacterial derived toxin A has excellent activity against
atleast one lepidopteron pest (Manduca sexta)
comparable to those of Bt insecticidal activity.
 It also exhibited some activity against southern corn
root worm an important pest of corn.
 There has been report on the expression of tcdA gene,
encodes 283 kDa protein, toxin A in Arabidopsis
thaliana.
 The tcdA gene consists of 7, 548 bp encoding toxin A is
one of the largest transcripts ever produced in a
transgenic plant.

 Expression level was found to be increased using
high-dose strategy in which addition of 5′ and 3′
untranslated region (UTR) of tobacco osmotin
gene increased toxin A production 10 fold.
 This studies could help to reduce the rate of
resistance development and consequently in
pest-resistance management.

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