Protease inhibitor mediated resistance to insects

Protease inhibitors (PIs) are among the defensive molecules that plants produce in order to defend themselves against herbivores. A major aim of this thesis is to develop novel insect resistance traits usingheterologous, non-plant PIs. Prerequisite for the success of the thesis was to tackle number of different technological problems connectedto the gene expression and protein accumulation ofheterologousPIs which are normally not formed in organisms like yeast ( P.pastoris ) and potato ( S.tuberosum ).For yeast the thesis describes an improvement of gene expression of more than 20-fold through use of an optimized gene variant. For transgenic plants the isolation and use of novel gene regulatory elements (promoter and terminator) yielded very highheterologoustranscription levels for differenttransgenesin a nuclear environment. Levels of functional protein and insect resistance, however, were compromised by in-plantaproteolyticdegradation of a number of inhibitors. This degradation ofheterologousproteins in theendomembranesystem of plants was studied in detail and different approaches to improve the protein stability ofheterologousPIs in plants were carried out. Co-expression of different inhibitors in the form ofmultidomainproteins yielded the desired properties since they prevented their own degradation in-planta and at the same time targeted a wide range ofcysteineproteases of insect pests such as western flowerthripsFrankliniellaoccidentalis (Pergande). Adult femalethripswere found to sense the presence of specificcysteineprotease inhibitors in a matter of minutes after feeding on the plant and were strongly deterred by it. In a non-choice assay situation with purifiedmultidomaininhibitorsthripsfecundity was up to 80% suppressed, and on plants with high levels ofmultidomaincysteineprotease inhibitors thethripspopulation was similarly 80% smaller after two weeks. Thus, it seems that femalethripsselect host plants with low levels ofcysteineprotease inhibitors where the development of the future population will be optimal. The dual action ofcysteineprotease inhibitors both as deterrents and suppressors of fecundity may be implemented in resistance management strategies for protection against herbivorous insect pests.