Bean alpha-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions

Abstract

Two alpha-amylase inhibitors, called alphaAI-1 and alphaAI-2, that share 78% amino acid sequence identity and have a differential specificity toward mammalian and insect alpha-amylases are present in different accessions of the common bean (Phaseolus vulgaris). Using greenhouse-grown transgenic peas (Pisum sativum), we have shown previously that expression of alphaAI-1 in pea seeds can provide complete protection against the pea weevil (Bruchus pisorum). Here, we report that alphaAI-1 also protects peas from the weevil under field conditions. The high degree of protection is explained by our finding that alphaAI-1 inhibits pea bruchid alpha-amylase by 80% over a broad pH range (pH 4.5-6.5). alphaAI-2, on the other hand, is a much less effective inhibitor of pea bruchid alpha-amylase, inhibiting the enzyme by only 40%, and only in the pH 4.0-4.5 range. Nevertheless, this inhibitor was still partially effective in protecting field-grown transgenic peas against pea weevils. The primary effect of alphaAI-2 appeared to be a delay in the maturation of the larvae. This contrasts with the effect of alphaAI-1, which results in larval mortality at the first or second instar. These results are discussed in relationship to the use of amylase inhibitors with different specificities to bring about protection of crops from their insect pests or to decrease insect pest populations below the economic injury level.

Figure 1

Pea weevil emergence in nontransgenic and transgenic peas (cv Greenfeast) at a field trial in Wagga Wagga in 1996. The extent of infestation, as determined by larval entry, was 80% of the seed. The percent adult emergence reflects the number of these larvae that matured into adults and was measured at 75 DPH. Error bars are 95% binomial confidence intervals (23). Results are shown for three replicate plots of nontransgenic and transgenic pea containing αAI-1.

Figure 2

Pea weevil emergence in nontransgenic and transgenic peas (cv. Laura) at three sites across Australia in 1997. Comparison of percent adult emergence in three replicates each of two different lines of transgenic pea containing αAI-1 and one line of nontransgenic pea. Error bars are 95% binomial confidence intervals (23).

Figure 3

Emergence of adult pea weevils in nontransgenic and αAI-1 and αAI-2 transgenic peas of cv. Laura. The percent adult emergence in the seed samples was calculated at various times after harvest. Error bars are 95% binomial confidence intervals (23).

Figure 4

Quantification of αAI-1 and αAI-2 in transgenic peas by immunoblot assay. Known amounts of purified bean αAI-1 and αAI-2 and 100 μg of protein extracted from representative seeds of nontransgenic Laura (untr), αAI-1 line, and the αAI-2 line were separated an SDS/20% polyacrylamide gel and transferred to nitrocellulose. The α-amylase inhibitors were detected by using an antibody to αAI-1 prepared in rabbit and detected by using chemiluminescence. The multiple bands result from the antibody reacting with the 25-kDa pre-pro-αAI-1 and with the differentially glycosylated isoforms of mature αAI-1 (25).

Figure 5

The influence of pH on the activities of B. pisorum α-amylase and the α-amylase inhibitors in vitro. (A) pH dependence of B. pisorum α-amylase activity. (B) pH dependence of inhibitor activity from αAI-1 and αAI-2 transgenic pea against B. pisorum amylase. Twenty-five micrograms of protein extract was used to determine the percent inhibition of B. pisorum amylase at different pH values as described in Materials and Methods.