Endogenous proteinase inhibitors induced in tobacco in response to herbivory: effects on the interpretation of insect resistance of transgenic tobacco plants

Abstract

Relatively little is known about the endogenous proteinase inhibitors induced in tobacco in response to insect herbivory, despite several reports on insect resistance based on transgenic tobacco, constitutively expressing trypsin inhibitors (Hilder et al., 1987). No reference has been made to potential changes in the inhibitor level during the 5–7 days of infestation with insects. A report by Walker-Simmons & Ryan (1977) surveyed wound-induced accumulation of trypsin inhibitor activities in several plant genera. They described that tobacco (Nicotiana tabacum L.) induces inhibitors to higher levels than tomato (Lycopersicon esculentum Miller) in response to crude elicitors from plants. In our experiments we found that after an insect attack of seven days very high levels of endogenous trypsin, chymotrypsin and subtilisin inhibitors were induced in tobacco (N. tabacum cv. Petit Havana SR1). These endogenous levels exceeded the reported transgenic inhibitor levels. This apparent major endogenous inhibitor contribution should be taken into account when considering the total inhibitor levels required in planta to reduce insect growth. We decided to investigate how these inhibitors are regulated in response to a simulated insect attack in tobacco (N. tabacum cvs Samsun NN and Petit Havana SR1) and tomato (L. esculentum cv. Moneymaker). For studies of insect resistance in plants it is necessary to know the differential as well as the overall inhibitor level at which insect larval growth is affected. As far as we know no one has followed overall inhibitor activity levels for any length of time. We developed a quantitative assay to monitor overall inhibitor activity levels against trypsin, chymotrypsin and subtilisin, which allows rapid estimation of inhibitor levels in large numbers of samples.