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Vegetable intelligence

Genetically modified sugarcane has insecticide property only when attacked by an insect

eduardo cesarTransgenic sugarcane tested in the Vegetation Houseeduardo cesar

One of the main pests that attack the sugarcane plantations is the sugarcane borer (Diatraea saccharalis), an insect that penetrates into the plant and digs out internal galleries, causing major damage to the producers. In order to control this enemy in an effective manner, researchers at the Luiz de Queiroz Upper School of Agriculture (Esalq) of the University of Sao Paulo (USP), in the town of Piracicaba, have managed, by way of genetic modification, to arrive at a sugarcane plant that releases proteins with insecticide activity only when attacked by the sugarcane borer.

The path towards producing a plant with these characteristics began with a detailed study and description of the sugarcane genes in order to know which were exclusively activated by the insects. Once this stage had been completed, it was then necessary to discover the DNA sequencing that activated these genes, the so called promoters, which permit the expression of the gene at the moment in which it is necessary. “A gene without a promoter is an inactive gene, a pseudo-gene” says professor Márcio de Castro Silva Filho, from the Molecular Biology Plants Laboratory, of Esalq’s Genetics Department, who headed up the research.

In order to express new genes in the sugarcane plant, with power over the borer, the researchers turned to the United States Department of Agriculture, which possesses various patented promoters. In 1998, the time at which the research began, there were no sugarcane promoters available in Brazil. The Brazilian researchers signed terms under which they promised to use the promoter sequencing only for research in the laboratory. “When we received the material, we began to make gene constructions, or that is to say, we placed the promoters behind the genes responsible for increasing the plant’s defense against the bore” advised professor Silva Filho. In this manner, the researchers managed to generate plants considered transgenic that had expressed the defense proteins. And with this they managed to prove that the plants associated to the promoters really possessed greater resistance against attack by the borer, the main pest, along with other sugarcane pests, responsible for damages of around US$ 500 million per year to Brazilian producers. This sugarcane is considered transgenic, although the promoters are of the species itself, because they were isolated from the plant’s genome and afterwards introduced into it.

The sugarcane borer cycle begins with butterflies, which lay small eggs on the lower part of the leaves. When these eggs hatch, minute larvae come out, of around 1 to 2 millimeters, which move off in the direction of the region close to the plant’s thatch (stalk), where they penetrate in and feed on the pulpy and sweet pith. Within the sugarcane, the larvae go through phase changes until they reach 3 to 4 centimeters when they come out of the plant, transform themselves into butterflies and begin the initial stage of a new life cycle of the insect. The galleries made by these gnawing insects practically fill the whole of the plant’s inside, bringing about a lowering of the vegetal mass and errors in germination, among other damage.

The holes opened up by the borers are also entrance doors for fungi that cause red decay, the disease responsible for the lowering of the sucrose production. When the raw material is destined for the production of alcohol, the problem is even more serious, since the invading microorganisms contaminate the liquor and compete with the yeast during fermentation.

In order to combat the sugarcane borer the major sugar/alcohol distilleries produce in their laboratories small wasps (Cotesia flavipes), which are released into the fields to live at the expense of the caterpillars. Small producers have no way of biologically controlling because there is not a sufficient production of these wasps on a commercial scale, without counting upon the fact that they have to be released into the plantations under ideal conditions of temperature and quantity in order to have an effect. It is starting from the moment in which the borer penetrates into the sugarcane that loses are inevitable, because in this phase there is no longer any chance of reverting to biological control nor of chemical, due to the high cost of insecticides and the low efficiency of the products, incapable of reaching the caterpillars in the inside of the plant.

Specific promoters
After having confirmed that the plants with the promoters had increased resistance against the pest, the researchers turned their attention to another challenge. They needed to discover new DNA sequences, as yet not patented, which would make the genes express themselves in defense against attacks by insects. And, as well as this, they wanted specific promoters, distinct from those discovered by the North Americans and ceded for research, known as constructive promoters, which express themselves throughout the life cycle of the sugarcane plant. “It’s this type of promoter that is being used by biotechnology companies in transgenic plants with resistance to insects” says Silva Filho.

Since the beginning, the research carried out at Esalq had the objective of identifying, in the sugarcane gene, promoters that were activated only when the plant would be attacked by caterpillars. After three years of study, the researchers managed to discover the promoter that controls the expression of gene defense, baptized as sugarin. Next a study on the cloning of the promoter was done and a patent deposited at the National Institute for Industrial Property (INPI), the patent request having been financed by FAPESP through the Intellectual Property Support Program (PAPI).

“The sugarin promoter has major biotechnological potential, because we believe that it functions in a similar manner to other plants that are relatives of the sugarcane, such as maize and rice” says Silva Filho. If the plant is not attacked by the insect, the sugarcane is the same as a conventional plant, which does not go through any genetic modification, in a different manner to the varieties of transgenic maize and cotton resistant to insects, released for commercial purposes in Argentina, China and the United States, which basically use genes isolated from a soil bacterium called Bacillus thuringiensis (BT). These plants produce a toxin, derived from a bacterial gene during all of the plant’s cycle, even if it were not being attacked.

The difference of the Esalq sugarcane with other transgenic plants was proven in various experiments that evaluated situations in which the defense gene had expressed itself. One of them consisted in a wound on the plant, such as a tear on a leaf, for example. “Normally, the majority of defense genes that are activated by insects also enter into action when a wound occurs” says Silva Filho. In the case of the sugarcane modified with the sugarin promoter, the plant responds only to the insect. The researchers still do not know for certain how the plant manages to know that the lesion is caused by an insect and not by a wound. They are still attempting to describe the molecules that are involved in this specific response. One of the hypotheses is that the substances present in the insect’s saliva can activate the gene expression.

In 1998, in order to understand this close relationship between plant and herbivore insect, the researchers began extensive research that culminated in 2002 and was also financed through FAPESP within the Thematic Project program. “We began to carry out coverage of both sides” says Silva Filho. On the one hand, the research sought to understand the defense mechanisms that the plant uses against the insect to avoid it being used as food or a host. And there are many mechanisms, since the plant cannot leave its position to defend itself. On the other hand, the insects also have their strategies to fool the plant’s defense. The strategies used by each one of the opponents are fundamental for advancing our methods of obtaining greater productivity in the field. In the end, both insects and plants are in a process of joint evolution that dates back to hundreds of thousands of years.

Late response
Starting from this Thematic Project, a new line of research was initiated in the Esalq laboratory involving interaction between the plant and the insect. This resulted in the isolation and description of the sugarin promoter, a study that was the doctorate thesis of Patrícia Pompermayer, under the guidance of Silva Filho and one of the patent’s co-authors. Having completed this stage, the research is now in the phase of detailing out the mechanism in which the sugarcane promoter functions, a study that is being conducted by Anne Hackbart de Medeiros, also being supervised by professor Silva Filho and another of the patent’s co-authors. “We can see in this phase that the peak of activation of the gene is around 24 hours after the attack by the borer” says Silva Filho. Some plants respond immediately. Others take a little longer, as in the case of sugarin. This apparent slowness in activating the defense mechanism is being studied by the group.

At the same time that the researchers are finalizing their studied on sugarin, they are preparing to request the National Technical Biosafety Commission (CTNBio), the government organ that controls the planting of transgenic crops in the country, for authorization to carry out experiments with seedlings  that are already prepared in the case of this field vegetable, in an area within Esalq. Under these conditions it will be possible to know if the promoter activates the expression of the plant’s defense also when an attack begins by the sugarcane spittlebug (Mahanarva fimbriolata), a sucking insect that has had its infestation index increased because of the mechanical cutting of the sugarcane crop – at the end of the process the machine leaves a covering of straw, ideal for the proliferation of this pest.

The Projects
1. Biochemical, entomological and molecular description of the interaction between inhibitors of digestive proteinases and insects of the Lepidoptera order (nº 97/04934-3); Modality  Thematic Project; Coordinator Marcio de Castro Silva Filho – USP; Investment R$ 198,265.10 and US$ 139,201.90 (FAPESP)
2. Plant-insect interaction:  a co-evolution process involving distinct adaptive schemes (nº 02/11462-0); Modality Regular Line of Research Assistance; Coordinator Marcio de Castro Silva Filho – USP; Investment R$ 108,250.00 and US$ 6,000.00 (FAPESP)
3. Patenting of a sugarcane promoter induced by herbivore insects (nº 04/09979-0); Modality Intellectual Property Support Program (PAPI); Coordinator Marcio de Castro Silva Filho – USP; Investment R$ 6,000.00 (FAPESP)