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

摘要

To study the interaction between plants and insects I performed the experimentsto find out the counter-defense mechanism of insects when insects were attacked by thedefense protein of plants. Jasmonate (JA) is one of the most important plant hormonesthat is involved in plant defense mechanism. I studied to find out the components of JAsignal transduction by T-DNA insertion mutant screening.In the first study, transcriptional regulation in cowpea bruchid guts duringadaptation to a plant defense protease inhibitor, cowpea bruchid, when fed on a dietcontaining the soybean cysteine protease inhibitor soyacystatin N (scN), activates anarray of counter-defense genes to adapt to the negative effects of the inhibitor and regainits normal rate of feeding and development. A collection of 1,920 cDNAs was obtainedby 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-freediet) cowpea bruchids. Subsequent expression profiling using DNA microarray and northern blot analyses identified 94 transcript species from this collection that areresponsive to dietary scN. The full-length cDNA of an scN-inducible cathepsin B-likecysteine protease was obtained. Its transcriptional response to scN during larvaldevelopment contrasts with the pattern of the cathepsin L family, the major digestiveenzymes. These results suggest cathepsin B-like cysteine proteases may play a crucialrole in cowpea bruchid adaptation to dietary scN.In the second study, screening of mutants that are altered in jasmonate-regulatedsignal transduction pathways using Arabidopsis thaliana was performed. Mutantscreening strategy using T-DNA insertion mutagenesis and AVP-LUC as a reporterenabled to find JA-signal transduction mutants of Arabidopsis thaliana, 9 underregulatedmutants and 6 over-regulated mutants. 20B15 showed reduced VSP1, THI2.1expression and increased PDF1.2 expression as compared to wild type when treated withJA. These data strongly suggested that 20B15 is a JA signaling mutant. 49R1, 49R2 and49R3 had same T-DNA insertion site (At1g53540) and showed about 10-fold higherAVP-LUC expression level than wild type when JA was treated. Genetic analysis showedthe mutation of these plants was recessive and tight linkage between mutant phenotypeand T-DNA insertion in At1g53540.