Objective
PROTEASE INHIBITORS provide plants with a natural DEFENSE AGAINST INSECT PREDATORS. They complex with insect gut proteases, when they are ingested and thus prevent the breakdown of plant protein. Insects can not survive when their proteases are effectively inhibited, but it was shown recently that they avoid the adverse effects of protease inhibitors with the expression of protease INHIBITOR-INSENSITIVE PROTEASES.
Plant protease inhibitor defense can be made more effective by the transformation of plants with genes encoding protease inhibitors that are specific for the insect enzymes that can not be inhibited by the host plant repertoire. A highly effective cysteine protease inhibitor is the multi-domain inhibitor MULTICYSTATIN FROM POTATO. It has the unique property of spontaneously forming natural protein crystals in the plant cell that are visible by microscope, but its structural properties remain unknown. Its EIGHT NEARLY IDENTICAL REPEATED DOMAINS give it the potential to form large complexes with the insect proteases and increased avidity for the insect cysteine proteases to which it binds. Moreover, its multi-domain structure affords it with better stability against proteolytic breakdown by gut carboxypeptidases. The specificity of this inhibitor in its present form is not suitable, however, to inactivate all cysteine proteases of the Colorado potato beetle - the major insect pest of this crop in Europe - and it is desirable to replace the phytocystatin domains with domains from other sources or to IMPROVE their SPECIFICITY.
Our aim is to elucidate the STRUCTURE of the natural 8-domain multicystatin cysteine protease inhibitor from potato in order to understand how it can inhibit 8 molecules of papain simultaneously and to construct similar multi-domain molecules with domains, which are known to be active against phytocystatin-insensitive Colorado potato beetle proteases. Prior work has demonstrated that specific non-plant type I and III cystatins are capable of effectively blocking the phytocystatin insensitive proteases. PHAGE DISPLAY will be used to generate more specific phytocystatin domains effective against the beetle proteases and also to optimize the links between the non-plant cystatins when multi-domain constructs are made. A domain of a small carboxypeptidase inhibitor -preferably minimized- will be added to the multi-domain constructs in order to control these peptidases as well, which are also essential for the insect and known to present a problem for the stability of cystatins in insect guts. Constructs containing multiple domains of type I and III cystatins, selected specific phytocystatins and carboxypeptidase inhibitors will be expressed in TRANSGENIC POTATO PLANTS during the second final year.
The obtention of a fully functional multi-domain structure, and its efficient expression in plants, is a challenge from the protein and genetic engineering points of view. The practical output of the project will be transgenic potato plants with enhanced resistance against Colorado potato beetle. The involvement of a potato breeder ensures that the economic potential will be exploited in a later phase.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences microbiology virology
- medical and health sciences medical biotechnology genetic engineering
- natural sciences physical sciences optics microscopy
- natural sciences biological sciences zoology entomology
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
6700 AA Wageningen
Netherlands
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