Resum

This work is included in a largest project focused on the improvement of transgenic rice plants resistance against pests and phytopathogens. Our goal is the obtention of an improvement of the rice resistance against the lepidopterean Chilo suppressalis, the most important rice pest. The chosen strategy was the expression of the mpi gene (Maize Proteinase Inhibitor, coding for a proteinase inhibitor) on rice, by both biolistic and Agrobacterium transformation methods. The proteinase inhibitors are part of the natural defence system of plants against insect predation. Expression of this gene, and accumulation of the MPI protein, is stable through successive generations of rice transgenic lines. We have demonstrated that constitutive expression of the mpi gene confers resistance towards Chilo suppressalis larvae infestation in transgenic rice. Bioassays performed with transgenic rice plants expressing the mpi gene constitutively that were infested with Chilo larvae, showed that ingestion of the MPI lead to a significant reduction of the larvae weight with respect to the control larvae group (as far as 60 %). It is also important to point out that larvae fed on transgenic MPI plants showed a significant delay in their development, preventing the achievement of the L4 larval stage in most of them.

The mpi promoter is functional in rice and confers wound-inducibility of gus reporter gene. The 2K fragment of mpi promoter confers a more rapid and intense induction than shortest C1 fragment. Moreover, 2K fragment of mpi promoter doesn't confer expression of gus reporter gene in the rice seed endosperm.

On the other hand, the maize mpi gene is correctly expressed in rice plants under control of his own regulatory regions. The observed expression levels allow obtaining similar effects on Chilo larvae than those obtained with constitutive mpi expression.

Besides, we fixed the optimal pH of digestive proteinase activities of Chilo suppressalis and Cacyreus marshalli (Geranium pest) as alkaline (pH 10.5 and 10 respectively). In Chilo digestive system participate serine proteinases activities of trypsin and chimotryspsin type, and also carboxy and aspartic proteinases. We also observed an adaptation effect on Chilo digestive system of larvae feed on transgenic rice plants, with an increase of 40% of total proteolytic activities in comparison to the control larvae activities. Nevertheless, this increase is not effective to overcome the MPI inhibitor effect.

The analysis of AFP (Antifungal Protein) allows to conclude that this protein shows an elevated antifungal activity against F. verticillioides and M. grisea at low nM concentrations. Besides, AFP shows activity against the oomycete Phytophthora infestans at low mM concentrations. The presence of AFP protein leads to important morphological abnormalities of these phytopathogens. We have not detect toxic effects of AFP protein towards rice protoplasts at high AFP concentrations. The topic application of AFP protein on rice plants confers protection to Magnaporthe grisea infection.

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