Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two-spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato --> herbivore --> predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a approximately 30-kDa target proteinase in the prey's midgut, forming a sodium dodecyl sulphate (SDS)-stable complex detected on immunoblots with an anti-OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetle's midgut, digestive protease activity in the predator, known to include OCI-sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine-type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predator's OCI-sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30-kDa-sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI-expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.     

Growth compensation and faster development of Colorado potato beetle (Coleoptera: Chrysomelidae) feeding on potato foliage expressing oryzacystatin I

Feeding, growth, development, and food conversion efficiency of Colorado potato beetle larvae reared on foliage from a “Kennebec” potato line expressing oryzacystatin I (OCI) at about 1% of its total soluble proteins were compared to those of larvae feeding on untransformed foliage from the same line. During stages L1 to L3, larvae feeding on OCI consumed leaf material 14% faster, gained weight 28% faster, and weighed 20% more at the end of the L3 stage, compared to controls. Continued exceptional performance on OCI during the final L4 stage was expressed as faster development than controls, an effect that persisted during pupal development and resulted in emergence of similar weight adults 1 day earlier than controls. Larvae initially maintained on control foliage and switched to OCI foliage during L4 did not overcompensate as those on OCI foliage throughout development, but performed similarly to larvae on control foliage throughout. Total azocaseinase activity in midgut extracts from these 4th instars 1 d after switching to OCI foliage was sensitive to inhibition by a recombinant form of OCI expressed in Escherichia coli, but was no longer sensitive 4 d after switching, indicating a gradual adaptation of the insect digestive protease system, based on the production of OCI insensitive proteases. Despite OCI potato foliage being consumed faster by small larvae using it for food, there was no indication that it was less efficient than untransformed foliage as food protein. Arch. Insect Biochem. Physiol. 40:69–79, 1999. © 1999 Wiley‐Liss, Inc.     

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect‐resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad‐spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI‐expressing and control (comparator) lines, followed by a two‐dimensional gel electrophoresis (2‐DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI‐expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2‐DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2‐DE showed unaltered deposition patterns in SlCDI‐expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI‐expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel‐generation transgenic plants.