Bloodworm Resistant Rice

Abstract

The aim this project was to determine if it was feasible to control bloodworm infestations in rice using transgenic plants expressing an insecticidal toxin. Without control bloodworm can cause massive damage to the plant stand, up to 85% plant loss in years of high infestation. Currently bloodworm are controlled through the use of chemicals applied at the time the paddy is planted. The advantage of a transgenic plant approach is that it will eliminate, or at least severely reduce, the need for pesticide application and the consequent impact on beneficial insects and animals. Avoidance of pesticide application will improve water quality. We have shown that the insecticidal toxins from Bacillus thuringiensis israelensis (Bti) are toxic to bloodworm. Whole Bti was highly toxic to bloodworm with an LC50 of 45ng of toxin per ml of medium, which is a far greater toxicity than that of other Bt strains for their target insects. I have cloned the individual toxin genes from Bti and expressed five in Bt (cry4A, cry4B,cry10A, cry11A and cyt1A) and the relative toxicities of the individual toxins and their mixtures were determined. The most toxic protein was Cry11A, closely followed by Cry4B, with LC50s of 550 and 980 ng toxin per ml, respectively. After the identification of cry11A as a candidate gene, a construct was made to transform rice and test the expression of the native coding sequence. One callus line (of twenty) was found to contain detectable quantities of Cry11A protein. However, once regenerated into a transgenic plant this line does not express the toxin well. This was expected because native Bt cry genes are not well expressed in plant tissue because of the differing codon usages by Bt and rice and potential negative acting elements of the gene itself when expressed in another organism. A synthetic rice optimised gene was designed to increase the amount of protein produced within rice plants and so obtain plants with enough toxin production to be insecticidal. The synthetic gene was coupled to a strong constitutive promoter and transformed into rice to ascertain if sufficient expression could be obtained to kill bloodworm. Rice transformation was performed on callus, which is an undifferentiated lump of rice cells not capable of survival outside tissue culture. Ten transgenic callus lines were bioassayed at Yanco. All of the calli had at least some activity against bloodworm and two look to have very good levels of activity against bloodworm. This is significant for several reasons: The expression levels achieved are able to kill bloodworm, meaning that it is possible to express enough of the toxin to be lethal to insects. The resynthesised gene retains the toxicity of the parental gene. The large number of lines that are demonstrating toxicity indicates that even moderate expression in planta enables control of bloodworm at least in the laboratory. This gives a greater range of options for deciding on the level of expression acceptable in the field. The speed at which the insects die implies that they stop feeding quickly. In some insecticidal plants it takes days to stop feeding and die, in this time they are able to do considerable damage to the plant. With a rapid acting toxin, it is more likely that the damage caused between feeding and death will be minimal. 2 The demonstration of bloodworm activity is a major step forward and we are now in a strong position to continue the project, Plants expressing Cry11A have been generated and demonstrated to have high level bloodworm activity. All insects exposed to the Cry11Asyn transgenics were killed. A root specific promoter has been cloned and used to drive the expression of Cry11Asyn. Root specific expression is seen as a desireable characteristic because it limits the expression of the transgene in seed and therefore peoples exposure to Cry11A protein. Genes with potentially interesting expression profiles were identified through EST expression data in public databases, the expression pattern and level were experimentally determined using real time PCR and subsequently cloned. Expression analysis will be performed once plants are generated.