Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defense protease inhibitor and screening of mutants that are altered in jasmonate-regulated signal transduction pathways using Arabidopsis thaliana

Abstract

To study the interaction between plants and insects I performed the experiments to find out the counter-defense mechanism of insects when insects were attacked by the defense protein of plants. Jasmonate (JA) is one of the most important plant hormones that is involved in plant defense mechanism. I studied to find out the components of JA signal transduction by T-DNA insertion mutant screening. In the first study, transcriptional regulation in cowpea bruchid guts during adaptation to a plant defense protease inhibitor, cowpea bruchid, when fed on a diet containing the soybean cysteine protease inhibitor soyacystatin N (scN), activates an array of counter-defense genes to adapt to the negative effects of the inhibitor and regain its normal rate of feeding and development. A collection of 1,920 cDNAs was obtained by differential subtraction with cDNAs prepared from guts of the 4th instar larvae of scNadapted (reared on scN-containing diet) and scN-unadapted (reared on regular scN-free diet) cowpea bruchids. Subsequent expression profiling using DNA microarray and northern blot analyses identified 94 transcript species from this collection that are responsive to dietary scN. The full-length cDNA of an scN-inducible cathepsin B-like cysteine protease was obtained. Its transcriptional response to scN during larval development contrasts with the pattern of the cathepsin L family, the major digestive enzymes. These results suggest cathepsin B-like cysteine proteases may play a crucial role in cowpea bruchid adaptation to dietary scN. In the second study, screening of mutants that are altered in jasmonate-regulated signal transduction pathways using Arabidopsis thaliana was performed. Mutant screening strategy using T-DNA insertion mutagenesis and AVP-LUC as a reporter enabled to find JA-signal transduction mutants of Arabidopsis thaliana, 9 underregulated mutants and 6 over-regulated mutants. 20B15 showed reduced VSP1, THI2.1 expression and increased PDF1.2 expression as compared to wild type when treated with JA. These data strongly suggested that 20B15 is a JA signaling mutant. 49R1, 49R2 and 49R3 had same T-DNA insertion site (At1g53540) and showed about 10-fold higher AVP-LUC expression level than wild type when JA was treated. Genetic analysis showed the mutation of these plants was recessive and tight linkage between mutant phenotype and T-DNA insertion in At1g53540.