The resistance of insects to plant proteinase inhibitors

The research reported in this thesis describes the induction of proteinase inhibitor synthesis in solanaceous plants (tobacco and tomato), when lepidopteran larvae (Manduca sexta and Spodoptera exigua) are feeding on leaves. It is shown that the larvae circumvent the proteinase inhibitor defense of these plants by the induction of non-susceptible gut proteinases. A phage display method is presented, which may allow the isolation of PIs that are also active against the non-susceptible proteinases of insects. It is expected that the application of such PIs can complement the natural PI defense of plants, and result in the protection of transgenic plants against insects.Chapter one provides a general introduction to plant PIs It describes the different PI families identified in plants, their mode of action against serine proteinases and the available evidence for a defensive role in plants. The effects of dietary PIs in insects are reviewed, and the physiological mechanisms resulting in growth depression in vertebrates are discussed. Chapter two presents a simple, but powerful method to measure quantitatively activities of a wide range of serine proteinase inhibitors using a radial diffusion assay. The assay can detect as little as 2-20 pmol PI, the error is between 4-12%, and the detection range can vary by three orders of magnitude. In chapter three the induction of endogenous PIs in response to insect attack is compared to the response after mechanical wounding and virus infection. It is demonstrated that local induction of PIs after insect attack is very strong in mature tobacco and tomato plants, but that systemic induction is virtually absent. Instead of direct systemic PI induction, it is observed that wounding several leaves at once, creates locally a stronger wound response. This suggests the presence of a systemic factor, which regulates the strength of the local wound response by silent alarm. Chapter four describes the induction of proteinase activity insensitive to plant PIs in the gut of Spodoptera exigua larvae, when the insects are feeding on tobacco leaves containing either potato PI2 or endogenous tobacco PIs It is demonstrated that PIs decrease the proteinase activity in larval guts, but that this reduction is partially compensated for by the induction of PI-insensitive proteinase activity. The weight of larvae, fed with PI leaves, was not reduced, so that the induced PI-insensitive activity, apparently sufficiently, compensated proteinase activity lost by inhibition. Chapter five describes the analysis of gut proteinase activity of S. exigua larvae. Six major proteinase activities were identified and three were purified by anion exchange chromatography and further analyzed. One of the purified proteinases was a cysteine proteinase with optimal activity at pH 11 and is the first example of this class of proteinase to be isolated from a lepidopteran insect. Chapter six demonstrates that potato P12 can be displayed as a functional protein on M13-phages by fusion to a minor coat protein. It is shown that functional PI2-phages mixed with non-functional phages can be enriched 323,000-fold against trypsin after three selection rounds. Large engineered phage libraries of PI2-variants allow the selection of PI2 clones with high affinity for PI-insensitive proteinases; of insect pests. Finally, chapter seven discusses the results comprehensively to defend the thesis that insects acquire resistance against the PIs induced in their host plants. It is argued that the successful application of PIs for resistance breeding will require the selection of better PI genes and that phage display offers a suitable method for this purpose.