Engineering resistance to insect pests in plants

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Resistance may be defined as 'a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species'. In terms of insect-resistance, the relationship between the plant and its insect pest is altered (Nicholl, 2008). "Tools of molecular biology and genetic engineering have provided humankind with unprecedented power to manipulate and develop novel crop genotypes towards a safe and sustainable agriculture in the 21st century" (Ranjekar, et al, 2003).

Today, people tend to forget that life would cease to exist without the simple process of photosynthetic fixation of carbon dioxide by plants. Plants are the most vital source of the human food chain. The fact that genetically modified plants have now created a greater variety of foods has been particularly advantageous to our economy, agriculture, and food diversity. (Nicholl, 2008). Plants are cheap to grow compared to the high cost requirements of microbial or mammalian cells, and thus cost reduction make transgenic plants an attractive option for producing high value proteins leading to plant made pharmaceuticals (Nicholl, 2008).

Selective breeding of plants arose long ago. Gregor Medel crossed pea plants to produce certain colors and textures in the 18th century. However, genetic engineering has taken selective breeding to a different level by combining and choosing traits from one plant to express in another species. Potential benefits of crop plant improvement include: increased productivity of crops and reduced losses due to biological agents, increasing growth of crops in areas that we are presently physically unable, increase efficiency of energy conversion due to reduction of photorespiration, and extending shelf life and nutritional value (Nicholl, 2008). Dr. Teetes states benefits include, "Plant resistance to insect pests has advantages over other direct control tactics. For example, plant resistance to insects is compatible with insecticide use, while biological control is not. Plant resistance to insects is not density dependent, whereas biological control is. Plant resistance is specific, only affecting the target pest" (Teetes, 2009).

Insect resistance, with respects to agriculture biotechnology, is a key methodology of improving agricultural productivity. By genetically engineering plants, biotechnologists can make precise changes to specific genes to alter the plant to resist insects and there are several successful ways that insect-resistant crops have been created. These include: regeneration technology via agrobacterium transformation, selection of specific transformants via selectable gene markers, and most importantly modification of Bt endotoxin coding sequences to produce better sequences produced by that plant cell (Tappeser, et al, 1997).

Bt plants, in which a bacterial toxin to confer resistance to caterpillar pests, have been successfully established and are grown commercially in many countries. Several major pests, including the tobacco budworm, Colorado potato beetle, Indianmeal moth, and diamondback moth, have demonstrated the ability to adapt to Bt in the laboratory. It has been reported that the diamondback moth evolved high levels of resistance in the field as a result of repeated use of Bt (McGaughey and Whalon). As Bt use increases on more acres, some scientists predict that insect resistance to Bt will be a major problem. Considerable controversy exists about how Bt should be managed to prolong its usefulness (Daniel, et. al 2008).

Table : Countries and their respective transgenic plants in 2008, also showing their scale in Mha

Insects and nematodes cause massive destruction to crops around the world (Committee et al., 2000). It is estimated that the loss due to insects is at 10-20% per year (Ferry, et al. 2005). Because of these high figures, these losses can cause detrimental socio-economic problems for farmers and consumers around the world. The introduction of engineering transgenic plants has been one of the biggest uses of this technology (Ferry et al., 2005). These transgenic plants are now being grown on approximately 100 million hectares in 22 countries, and one of these being Australia with Bacillus thuringiensis cotton being one of the most common (Shewry et al., 2008). Genetically modified, or GM plants are now grown in over 200 countriesThe USA, Argentina, and Brazil represent the top three countries with the most areas of GM crops being planted (Table 1.) The four main GM crops include soybeans, maize, cotton, and canola oil. Examples of transgenic plants worldwide and their size of plantations can be seen in Table 1. This showing Australias dominance with Cotton, Rape and Carnations. Whilst it is easy to look toward economic gain that can be seen with the introduction with transgenic plants the health benefits from the decreased use of insecticide are also present. Crops can be tested for their Environmental Impact Quotient, EIQ, to tests for the chemical risks associated with the crops for consumers, the environment and the farm producing the crop. It was found that in the B. thuringiensis cotton in Australia when the proteins Cry1AC and Cry2Ab were tested for their EIQ value against pesticide, insecticide showed 135kg ai/ha, whilst the Bt variety with the two inserted genes showed a figure of 28ka ai/ha, a 64% decrease (Park et al., 2011).

Various protease and trypsin inhibitors have been used in creating transgenic plants. An example of how a crop is genetically modified is with respects to the tomato. So tomatoes can stay fresh while being shipped around the world, they are picked green and then artificially ripened via ethylene gas. Another strategy to create insect resistance is to integrate genes of protease inhibitors (PIs), which are small proteins that interfere with enzymes in the intestinal tracts of insects. These PIs have the potential to induce development disruption and increased larval mortality in a large range of insects, not differentiating between pests and beneficial insects. As a result, bees and other pollinators can be exposed to PIs through pollen and nectar. Researchers at the National Institute for Agricultural Research in France (INRA) investigated the effects on honey bees. They fed elevated levels of PIs together with a sugar solution to bees for three months and found that such bees died 15 days earlier than bees fed normal sugar. Another effect was that after only 15 days of ingesting PIs, bees had problems to distinguish between the smells of different flowers. The research concluded that negative effects of long-term exposure to PIs on the survival and behavior of honey bees cannot be excluded. (Tappeser, et al, 1997).

During the initial stages of insect resistance development, it was found that cowpea protease trypsin inhibitor (CpTI) gene showed resistance too many common insect pests such as the members of the families Lepidotera, Coleoptera and Orthoptera, which are all members of the Arthropod phylum, thus allowing a new avenue of genetically modified plants to be studied (Vasil, 2003). This was the first successful transformation of a particular gene into another plant species. CpTI is now used widely as an insect resistance gene due to its wide insect repellant catalogue and caused no negative effects if ingested by mammalian creatures. Other dominant examples include barley trypsin inhibitor (CMe) and squash trypsin inhibitor (CMTI). Both Bacillus thuringiensis and trypsin inhibitors focus their work around insect gut proteinases. Whilst Bacillus thuringiensis works via the binding of Bt toxin to particular Bt endotoxin-binding receptors in the insects mid-gut (Stewart, 2004), the trypsin inhibitors work from point of ingestion. When a protein enters the gut it is cleaved and eventuates into amino acid production. Trypsin inhibitors stop this cleavage reaction by binding to the active site on the proteins in the insect which results in death. Trypsin inhibitors fall under the class of serine proteinase inhibitors where two active sites allow for inhibition of trypsin and chymotrypsin (Jouanin et al., 1998). These inhibitors can be found in many higher plants.

Expression of the gene of these inhibitors of digestive enzyme have shown a similar level to that of Bt, but unfortunately in field has not shown the same levels of insect control. (Schuler et al., 1999)

However the use of trypsin inhibitors has hit a short fall with many geneticists showing evidence of insects becoming immune to the ingestion of proteinase inhibitors, unlike Bt which does not have evidence yet supporting any insect immunity (Jouanin et al., 1998). Because insects carry a wide variety of proteases what is being genetically inserted into the plant must cater for a wide variety of protease inhibition. Proteinase inhibitors have also been used in transgenic plants for the control of plant-parasitic nematode populations such as with the Globodera pallida. The CpTI was used in sequence with transgenic potato but only the less damaging males showed a decrease whilst there seemed to be no evidence toward female fertility loss (Fuller et al., 2008).

To generate specific nematode resistance effective control strategies are needed such as genes that encode an protease inhibitor, peptide or interfering RNA or promoters that direct specific patterns of expression for that effector (Atkinson et al., 2003). Most of the characterized genes (R-genes with a transgene) confer resistance to the three most spread and economically important nematode groups Meloidogyne, Globodera and Heterodera species (Molinari, 2010). The use of Bacillus thuringiensis as an inhibitor of the nematode has proved successful also. Both the β-exotoxin and the δ-endotoxin manage to bind to the specific receptor in the midgut of the nematode and in some species cause death after a couple of days (Atkinson et al., 2003). New research has shown success in the form of RNAi (plant-delivered RNA interference), where silencing of the nematode genes is a possibility (Atkinson et al., 2003). Whilst the nematode maintains to be a pest to the crop industry, research is being conducted to decrease the populations that are inhibiting much plant growth. Without the use of transgenics, continued use of the toxic fumigant nematicide, methyl bromide, will be the strongest option in the control of these pest (Molinari, 2011). With its environmental and human health risks weighing high and its costs being quite substantial, transgenic plants hold the key to the control of these species.

Nematodes are plant-parasitic pests that occur in temperate and tropical crop regions and account for 15% of the total nematode populations in the world (Fuller et al., 2008). They cost $125 billion worth of crop losses globally and among them the most damaging are root-knot nematodes (Meloidogyne species) and cyst nematodes (Heterodera and Globodera species) (Fuller et al., 2008). Endoparasitic nematodes are believed to be the most advanced form of nematodes present in the world.

There are many different techniques used for the generation of transgenic plants. Foreign genes may be introduced via direct gene transfer of naked DNA or by using a carrier organism in a method known as vector mediated transfer. Other factors must also be considered such as isolation of transformed plants and the ability to monitor gene expression. In this respect the use of both selectable marker genes and reporter genes, when transferring foreign DNA, must also be considered.

Vector mediated transfer is a method that requires an organism to deliver the gene of interest, such as a gene encoding resistance to insect pathogens, into the host plant. In this instance the natural plant pathogen known as Agrobacterium tumefaciens is often used. This gram negative bacterium lives in the soil and causes 'crown gall' disease in wounded plants. It carries a large 200kb tumour inducing (Ti) plasmid that has a region of transfer (T) DNA, (Narasimhulu et al, 1996).

This T-DNA is transferred to the host plant as part of the normal infection process and become integrated into its genome and expressed as shown in table 1. The T-DNA contains certain oncogenes (ONC) which induce the expression of plant growth regulators (auxins and cytokines) (Zupan et al., 1995). These regulators stimulate cell divisions and the formation of tumours or 'crown galls'.

Figure : Showing transfer DNA from Agrobacterium tumefaciens Ti plasmid being integarated into the plants chromosome region

The T-DNA also encodes genes for 'opine' synthesis that when produced can only be metabolised by

the Agrobacterium and thereby act as an exclusive food source for the bacteria. Certain virulence genes (vir) aid transmission of the T-DNA firstly by encoding nucleases which cleave at the left and right border repeats of the Ti plasmid to release the T-DNA, and then by the production of proteins which bind to the naked T-DNA preventing its degradation in transit, (Zupan et al., 1995). Other functions of the virulence genes are the production of proteins which enable pore formation between the bacterium and the plant, and are involved with signal detection from wounded plants, (Zupan et al., 1995).

Figure 2: The Ti plasmid is too large to be used directly as a vector, so smaller vectors have been constructed that are suitable for manipulation in vitro.

In cases where Agrobacterium is used for vector - mediated gene transfer, the Ti plasmid is isolated and modified. ONC genes are removed from between the left and right border repeats and the gene of interest (such as a gene encoding resistance to insect pathogens), along with a selectable marker gene, are inserted into the T-DNA. The modified Ti plasmid is then put back into the Agrobacterium and used to infect target plants. Infection is achieved by co-culturing leaf discs with the bacterium which

can later be killed with antibiotics. Not all plants are able to be transformed in this manner and although success has been reported in barley and rice, cereals such as wheat and maize have met difficulties (Jones and Burgess, 2011). For this reason various alternatives to vector - mediated gene transfer have been generated.

Direct gene transfer (DGT) methods provide an alternative to the use of organisms as vectors for the generation of transgenic plants. Most DGT methods aim to target protoplasts rather than whole plant cells. Protoplasts are living units of plant cells containing the nucleus and cytoplasm surrounded by a cell membrane, (Jones and Burgess, 2011). They can be isolated through the enzymatic degradation of plant cell walls using various pectinases, hemicellulases and cellulases, (Jones and Burgess, 2011). Once protoplasts are isolated the plasma-membrane is exposed and foreign DNA can be introduced. This can be achieved via the application of a pulse or electrical discharge (electroporation) causing pores to open in the membrane. Alternatively the membrane can be chemically disrupted such as with the use of polyethylene glycol (PEG) (Christou, 1996). Both methods induce uptake of foreign DNA by the protoplasts, however only in a small number will the DNA become stably integrated into the genome and expressed. Transformed protoplasts must therefore be selected for by using a selectable marker gene such as one which encodes resistance to a certain antibiotic or herbicide. Protoplasts must then be plated on media where only transformants can survive. From these protoplasts callus formation can then be induced and transformed plants can be regenerated, (Jones and Burgess, 2011).

Another DGT method used for the integration of foreign DNA is particle bombardment. This physical approach requires the use of metal particles, usually tungsten or gold of approximately 1 micron in size, (Christou, 1996). These particles are sterilised then coated in either circular or linear DNA. Particles are then accelerated through plant tissue in a sterile vacuum. DNA can be shot through about 6 layers of tissue and the process adjusted to result in optimum expression without causing too much damage. Recombinant wheat plants have been successfully generated using this method, (Vasil et al, 1992).

Other methods of DGT include microinjection. The process involves the injection of DNA into a protoplast or cell nucleus. Plant cells are held in place using a holding pipette and a specialised injection pipette is used to insert the DNA, (Neuhaus and Spangenberg, 1990). Injected cells must then be collected and grown in isolation. Reported success has been seen in rapeseed where microinjection of DNA into early stage embryoids of rapeseed microspores, lead to the development of haploid transgenic plants following embryogenesis (Neuhaus et al., 1987).

Both DGT and vector-mediated gene transfer may be considered as effective strategies to create new plant varieties as opposed to traditional plant breeding techniques. This is because to generate a new plant containing a certain gene from another plant requires extensive breeding programs and backcrossing techniques which can be time consuming and limited in their effectiveness. Use of a vector or DGT to introduce an extra gene of interest may be seen as causing less genetic disturbance to what may already be described as an 'ideal' plant compared to the large scale genetic rearrangements caused by out-breeding to acquire a desired trait.

Although it is obvious that there are many beneficial byproducts of making plants resistant to insecticides, there are also barriers as well to this methodology. Barriers to genetically modifying plants include their ecological effects and public acceptance of the transgenic plant itself. Current environmental and ecological risks include adverse affects on non target organisms like beneficial insects, the soil, and endangered species. This is analysed via biological fate analysis and is still being researched. Current insect resistance management includes having the highest dose of protein possible expressed in a plant that is also consistent with good agronomic qualities and combining use of the product with a refugia (alternative hosts and non Bt plants). Dr. George L. Teetes states, "Man has through plant domestication and cultivation practices interfered in many ways with species diversity and natural defense mechanisms of plants" (Teetes, 2009). Because pest insect populations are usually large in size and they breed quickly, there is always a risk that insecticide resistance may evolve, especially when insecticides are misused or over-used.

In the future, scientists suspect use of multiple insect control proteins (called pyramiding,) increased education on sound Integrated Pest Management (IPM) practices, and will increase the monitoring for resistance. Monitoring tests the effectiveness of resistance management tactics, provides an early warning of impending resistance problems, and evaluates changes in distribution and the severity of the resistance problem.