In this article we will discuss about the insect resistance in plants.
Insect control is serious and greatest challenge for agricultural crops. The global scenario of crop damage inflicted by insects is a matter of serious concern. Modern agriculture provides novel solutions to age old problems. Despite the use of wide array of insecticides, extent of damage seems to be far reaching implications.
Pest management and chemical control of insects reaches more than $12 billion annually and total loss accountable to 25-30% of the total production. In addition, pest resistance insect infestans may pose possible devastation of the crop. Besides, environmental problems are also associated with the indiscriminate use of insecticide.
In this exorbitant exercise some notable exceptions are the utility of biological methods of insect control i.e., insect toxins produced by Bacillus thuringiensis. Although usage of B. thuringiensis in the control of the insects appears novel approach but in reality it is age old practice because these have been used for more than 40 years as a biological insecticide.
Genetic engineering for insect’s resistance offers an attractive approach and can supersede all conventional method of control. Apart from Bt crystal proteins, several other biotechnological approaches such as protease inhibitor,α-amylase inhibitors, chitinase and cholesterol oxidase provided reasonable success in the insect control strategy by transgenic crops.
Several commercial genetically engineered insect resistant crops have been released are transgenic corn, cotton potato which expresses Bt toxins. In North America these crops are already growing in vast area and many more transgenic plants are in the pipeline to release in other countries.
Bacillus Thuringiensis — A Weapon for Insect Control:
Bacillus thuringiensis is spore-forming gram-positive bacteria exist in many locations such as soil, plant surface, dust and grain storage. These are used as bioinsecticide for four decades. The bacteria were first discovered in 1901 from diseased silk worm larvae. Dr. Berliner isolated these killer bacteria from diseased flour moth larvae. They exhibit specific toxicity to mediterranean flour moth (Ephestia kuhnella) larvae and not to meal worm larvae.
Bacillus species by the presence of B, thuringiensis can be distinguished from related parasporal crystals that are formed during sporulation. Indiscriminate use and environmental concern with chemical pesticide mooted the commercialization of first Bt strain in 1960.
Thuricide, a trade name for Bt was marked to control lepidopteron insects. Later several other strains were replaced by thuricide completely and were sprayed directly on plants to prove their insecticide activity.
Until 1977, it was belived that Bt could act only against lepidoptera pest. This view was reoriented when Goldberg isolated the strain Bacillus thuringiensis Israelensis from pond in which mosquito breed extensively. Further studies revealed its potential insecticide role to other insects like elm leaf beetle and Colorado potato beetle larvae. In the odessey of Bt, several hundred strains have been isolated and successfully characterized.
Different strains of B. thuriengensis differ in their mode of insecticidal activity. Most of the Bt are active against lepidoptera and some strains are specific to Diptora and coleoptera have also been observed. Bt crystal protein is killer protein.
Spraying of Bt on plants leads to rapid degradation of crystal protein by UV light and loosening their activity. These problems are effectively addressed by producing transgenic plants that express crystal toxic protein continuously and protected against degradation.
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 osmolytic 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 e 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 I 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).
Different cry group are further divided into sub-families, for example, cry I group was divided into cry IA, lb, Ic and similarly five sub-families were made for cry IA.
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 is 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 group of researcher continued their work and conducted field trials with transgenic plants expressing Bt proteins. The outcome of their study was found to be display of 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 of 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.
Chitinase:
The enzyme chitinase have been explored as insecticide protein. Transgenic plants expressing chitinases in tobacco provides protection against tobacco bud worm after insects starts feeding the tissues. Chitinase enzyme target chitin structure known as peritrophic membrane present in the insects midgut lumen. For effective control, large amount of chitinase has to be produced in the plant as peritrophic membrane tends to regenerate continuously in the 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 numatode 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 encode 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.