Analysis Of Rice Genes Induced By Striped Stemborer (Chilo Suppressalis) Attack Identified A Promoter Fragment Highly Specifically Responsive To Insect Feeding

Rice striped stemborer,Chilo suppressalis(Walker),is one of the most damaging Lepidoptera pests of rice worldwide.There are few documents on molecular defensive mechanism of rice against C.suppressalis,and no reports about identification of rice promoter specifically responsive to C.suppressalis attack.The objective of this study was to identify rice genes responding to C.suppressalis and rice gene promoter that is specifically induced by feeding of C.suppressalis.mRNA from rice sheath with and without chewing by C.suppressalis was used as tester and driver respectively to construct two subtractive cDNA libraries.Libraryâ… was constructed using mRNA isolated from the feeding treatment as the tester with the undamaged control as the driver,and the reverse was done for libraryâ…¡.Thus,libraryâ… andâ…¡were expected to yield up-and down-regulated genes,respectively.A total of 864 clones randomly selected from the two libraries were sequenced,and 271 unique sequences longer than 100 bp were obtained.Functions of these ESTs were analyzed with BLASTN and BLASTX.These genes played roles in photosynthesis,primary metabolism, secondary metabolism,signal transduction,protein degration,electron transport and so on. This result indicated that the rice response triggered by C.suppressalis is likely to be involved comprehensive transcriptional reorganization.These 271 cDNA clones together with other 113 ESTs from the normalized cDNA library and internal controls were arrayed on a nylon membrane.Total RNA of rice sheath harvested at 6 hours after C.suppressalis attack and control was reversely-transcribed and labeled with ³²P as the probes,and hybridized with the membranes.The hybridization was repeated twice,which identified 12 up-regulated and 5 down-regulated clones.The differentially expressed genes verified from the macroarray were much less than those from subtractive libraries,but only 2 differentially expressed genes were the exception which presented in the opposite library they should be.Four of these up-regulated clones were randomely selected to be analyzed by Northern blot.Three of the selected clones were induced both by C.suppressalis infestation and wounding.One cDNA(B1-A04) encoding a putative subtilisin/chymotrypsin inhibitor was found to be rapidly and highly induced by C. suppressalis innfestation,compared with mechanical wounding.The putative promoter region of B1-A04,spanning from-1569 to +446 relative to the transcriptional initiation site was isolated,fused to the GUS gene and introduced by Agrobacterium-mediated transformation to rice.In non-infested plants,the GUS activity driven by this promoter fragment was detected in culms and panicles,but not in leaves and sheaths.At 6 h after insect feeding,GUS activity was significantly induced in sheaths and culms,but not in leaves.GUS activity and native B1-A04 gene were not induced by JA and ABA treatment.These results showed that this promoter fragment had an organ-specific(i.e.culm and panicle) as well as developmental stage-specific(booting stage) expression pattern.A serial deletion analysis revealed two regions(-1569 to-1166 and-1166 to-582) that negatively regulate the gene expression in sheaths of non-infested plants but not in insect-infested plants.An electrophoretic mobility shift assay(EMSA) identified 7 DNA fragments with various binding activities with nuclear proteins from mechanically wounded,insect-infested and untreated plants,and their possible roles in gene regulation were speculated.This promoter fragment should have utility in development of insect resistant transgenic crops.