| Rice is the most important food crop in the developing countries. Crop losses due to insects, diseases, physiological and environmental factors can reach tens of billions of dollars each year. Rice blast caused by Magnaporthe grisea and sheath rot caused by Rhizoctonia oryzae are the two most important pathogens in rice. Rice blast itself can account for 10--15% annual loss in the absence of chemical solutions. Current efforts to confer disease resistance in rice through transgenic expression of antimicrobial genes from different sources have met with various successes. In this work, a novel approach was used to identify antimicrobial peptides with in vitro inhibitory activity against either M. grisea or R. oryzae. Fifth instar Manduca sexta were challenged with these pathogens to induce an immune response. Immune peptides showing antifungal activity against the pathogens were purified by HPLC column chromatography and their corresponding genes identified and cloned. Two genes, mag-1 showing in vitro antifungal activity against M. grisea, and rhizoc3, showing in vitro antifungal activity against R. oryzae were used to transform Nipponbare rice plants to try to enhance disease resistance to M. grisea and R. oryzae. T1 and T2 generations plants were screened for disease resistance, and those plants exhibiting higher resistance compared to control plants were characterized on the molecular and biochemical levels in order to correlate the exhibited resistance with the presence and expression of the gene. No significant correlation between disease resistance and gene expression was observed with the rhizoc3 transformed plants however a very limited correlation was found with the mag-1 transformed plants. This poor correlation might suggest that there may be problems expressing these proteins in an active form in plants.; Multiple expression systems (Pichia pastoris and Eschrichia coli) were tried to produce recombinant MAG-1 and Rhizoc3 for further characterization and antibody generation without success suggesting that these antimicrobial proteins may be toxic to Pichia and E.coli. |
