Final Report Summary - VIPHASE (Mode of action of Bacillus thuringiensis VIP proteins in Helicoverpa armigera and Spodoptera exigua)
The object of VipHaSe was to study the mechanism of action of different Vip3A toxins in Helicoverpa armigera and Spodoptera exigua larvae and in particular to understand which key steps in the mode of action account for the differences in susceptibility. As a first step the toxicity was studied in terms of mortality of the proteins (lysate of E. coli expressing Vip3Aa, Vip3Af, Vip3Ae, Vip3Ad or ISP3F) by means of surface contamination bioassays with neonate larvae. The results obtained showed that the mortality induced by the most active Vip3A proteins (Vip3Aa, Vip3Af, Vip3Ae) was much lower compared to the toxicities displayed by the Cry toxins in both species, but the toxicity in terms of growth inhibition was very high. These results led us to hypothesize that there is a different mechanism of action between the two classes of proteins, Cry and Vip. On the other hand, in Spodoptera frugiperda, the bioassays revealed a high toxicity in terms in both mortality and growth inhibition of Vip3Aa, Vip3Af and Vip3Ae proteins. To perform biochemical analysis of the mechanism of action, highly purified proteins were necessary. vip3Aa, vip3Af and vip3Ae genes have been modified to contain a His tag sequence at the N terminus of the proteins and have been cloned in pMa5-8 and expressed in Escherichia coli WK6. The His tag facilitated the purification because the protoxin could be separated by affinity chromatography from the mixture of proteins contained in the lysate of E. coli expressing Vip3A proteins. Although a protocol to obtain high yields of highly pure proteins was successfully developed, toxicity bioassays in S. frugiperda and S. exigua neonate larvae showed a partial loss of activity (about ten-fold) in S. frugiperda and a complete loss of activity in S. exigua. Thus, a different protocol of purification by anion exchange chromatography was developed, unfortunately with results similar to the previous purification. It had to be taken into account that handling these proteins during purification processes led to a decrease in activity.
A polyclonal antibody raised in rabbit against Vip3Aa protoxin was produced. The quality of the antibody has been tested by Western blot analysis, and it was suitable for the detection of Vip3Aa and also other Vip3A proteins. It was very useful to confirm the presence of the Vip3A proteins in the different fractions obtained during the purification processes. It was also useful for preliminary studies of the Vip3A receptors and Vip3A mechanism of action at cellular level in progress in the host lab. Since in Spodoptera frugiperda the bioassays revealed a high toxicity in terms of mortality of Vip3Aa, Vip3Af and Vip3Ae proteins, we decided to investigate which step in the mode of action was responsible for the differences in mortality of these two closely related Spodoptera species. The activation with midgut juice crude extracts did not show differences in the activation profiles. The separation of serine-proteases was carried out by chromatographic techniques from the midgut juice of both species. The detailed study of the activation process by the different fractions with serine-protease activity in the two species is in progress.
A polyclonal antibody raised in rabbit against Vip3Aa protoxin was produced. The quality of the antibody has been tested by Western blot analysis, and it was suitable for the detection of Vip3Aa and also other Vip3A proteins. It was very useful to confirm the presence of the Vip3A proteins in the different fractions obtained during the purification processes. It was also useful for preliminary studies of the Vip3A receptors and Vip3A mechanism of action at cellular level in progress in the host lab. Since in Spodoptera frugiperda the bioassays revealed a high toxicity in terms of mortality of Vip3Aa, Vip3Af and Vip3Ae proteins, we decided to investigate which step in the mode of action was responsible for the differences in mortality of these two closely related Spodoptera species. The activation with midgut juice crude extracts did not show differences in the activation profiles. The separation of serine-proteases was carried out by chromatographic techniques from the midgut juice of both species. The detailed study of the activation process by the different fractions with serine-protease activity in the two species is in progress.