Pyramiding rice cystatin OCI and OCII genes in transgenic potato (Solanum tuberosum L.) for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say)

Abstract

Proteinase inhibitors oryzacystatins I and II (OCI and OCII) have shown potential in controlling pests that utilize cysteine proteinases for protein digestion. In order to achieve an additive effect against Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) digestive proteinases, we combined two individual OC genes in potato (Solanum tuberosum L.) cultivars Desiree, Dragaevka and Jelica. The OC genes were stacked into the potato genome by simultaneous co-transformation with both OCI and OCII genes or by sequential re-transformation of an OCI-expressing transgenic line with an OCII gene. Low constitutive and high wound-induced transcript levels of both OCI and OCII genes, directed by the inducible pin2 promoter, were detected in all doubly transformed lines from all three cultivars. Both recombinant OCs, detected by immunoblot, were in an active conformation as shown by their strong papain inhibitory activity. Although no significant differences in CPB larval mortality were observed when larvae fed on OCI/OCII transformed or control potato foliage, expression of both OCI and OCII in potato plants caused a reduction in CPB adult body weight and in weight of foliage consumed, indicating an additive effect. Overall, these results show that pyramiding of the OCI and OCII genes could be an effective strategy for breeding cultivars exhibiting elevated levels of resistance to CPB.