Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 3.25

FEEDING OF H. zeae LARVAE ON DIETS CONTAINING PROTEINASE INHIBITORS INDUCES THE EXPRESSION OF DIFFERENT PROTEINASES

Volpicella M.*, Ceci L.R.**, Beekwilder J.***, Jongsma M.A.***, Gallerani R.*

*) Dept. Biochem. and Mol. Biol., University of Bari

**) Unit for the study of mitochondria and energetic metabolism-CNR Trani (BA)

***) Business Unit Cell Cybernetics, Plant Research International Wageningen (NL)

insect adaptation, plant defence, trypsin, proteinase inhibitor, mass-spectrometry

Proteinase inhibitors (PIs) are small proteins that, acting as substrate, form stable complexes with proteinases, strongly reducing their activity. In plants they act in many cases as defensive molecules against chewing insects. Indeed, insect digestive proteinases are the targets of specific plant PIs (1). PIs attenuate nutrient assimilation in insect guts by inhibiting the activity of digestive proteinases causing severe retardation in growth and diffusion of insect larvae. Transgenic plants expressing heterologous PIs, have been demonstrated effective in reducing growth and diffusion of insect larvae (2). However it has became evident that insects can adapt their gut enzyme content upon ingestion of PIs, by producing new and “insensitive” proteinases (3). Also the PI content of some plants consists of several inhibitors coded by multigenic families, but with different specificities (4).

A rationale behind the adaptive capacity of insects has not been yet clarified at the moment, hampering any attempts to design new PIs effective against insensitive proteases. The availability of inhibitors highly active against specific proteinases would be of valuable interest for both fundamental and applicative studies in agriculture and medicine. The isolation and identification of insect insensitive proteinases will be of great help in understanding the molecular mechanism of insect adaptation and its influence in the stability of proteinase-inhibitor complexes.

In this communication, analyses on the proteolytic content of Helicoverpa zeae guts after feeding on control and PI-containing diets are reported. Isolation of H. zeae proteinases was carried out by affinity chromatography on a MTI2-CNBr-Sepharose. They were identified as trypsin or chymotrypsin like proteinases by digestive analysis of the specific Ser-Arg-Arg-Pro-Leu-p-nitroanilide and Arg-Arg-p-nitroanilide substrates. The isolated proteinases were also characterised by using two and one dimensional gel electrophoresis and some of them were sequenced by Qtof mass spectrometry. Sequencing results, matched with available cDNA sequences, allowed the assignment of biochemical function to specific trypsin genes of the Lepidopteron.

This study, beside identification of the H. zeae "insensitive" proteinases, will be useful for the isolation of specific proteinases to be used in the screening of a phage display library of the Mustard Trypsin Inhibitor 2 (MTI-2) already established (5). With the phage display selection, we intend to isolate variants of MTI-2 with new and higher activities against specific insensitive serine proteinases.

1 Ryan, C.A. Annu. Rev. Phytopathol. 1990, 28, 425-449.

2 Jouanin, L., Bonadé-Bottino, M., Girard, C., Morrot, G., Giband, M. Plant Science 1998, 131, 1- 11.

3 Jongsma, M.A., Bolter, C. J. Insect Physiol. 1997, 43, 885-895.

4 De Leo, F., Volpicella, M., Licciulli, F., Liuni, S., Gallerani, R. and Ceci, L.R. Nucl. Acids Res., 2002, 30: 347-348.

5 Ceci, L.R., Volpicella, M., Rahbe, Y., Gallerani, R., Jongsma, M.A., and Beekwilder, J. The Plant Journal 2002, Submitted.

ACKNOWLEDGEMENTS: Research funded by EU-FAIR contract n° FAIR6-CT98-4239.