Characterization of the maize sesquiterpene cyclase genes involved in the defense response to insect damage

Rutgers University New Brunswick, New Brunswick NJ

Investigators

Abstract

Plants possess a variety of mechanisms to protect themselves from attack by herbivores. One system that is induced upon attack by herbivorous insects is the production of terpenoids that can serve in either direct or indirect defense against the herbivore. It has previously been shown that the maize sesquiterpene cyclase gene stc1, located in 9S, is induced in corn seedlings being foraged by beet armyworm (BAW) larvae. The product of this gene is a volatile terpenoid that most likely serves to attract wasps that parasitize the BAW larvae. Preliminary work leading to this proposal has now shown that stc2, the ortholog of stc1 in 6L, is induced by a different insect pest, the southwestern corn borer (SWCB). The P.I.s propose a genetic, molecular, and biochemical dissection of the two orthologous maize genes involved in this novel type of inducible plant defense system, as follows: 1. Characterization of the induction of the stc2 gene by the SWCB and other related corn borers. These experiments will establish the pattern of stc2 induction in response to regurgitant from the SWCB and whether the plant response is specific to that insect species or not. 2. Determination of the complete structure of the stc2 gene by isolating a full-length stc2 cDNA from seedling sheaths that have been foraged by SWCB. This will enable identification of the N-terminus of the protein, confirmation of the predicted intron-exon structure of the transcript, and expression of the STC2 enzyme in a heterologous system for biochemical characterization of its activity. 3. Assessment of the possible role of stc2 as an insect resistance gene by isolating and sequencing the stc2 gene from the non-inducible, SWCB-susceptible Ki3 inbred line. This would identify the basis of the noninducibility of stc2 in Ki3 and provide further evidence that stc2 corresponds to a quantitative trait locus (QTL) for SWCB resistance that maps very close to the location of stc2 in 6L. 4. Determination of the nature of the terpenoid product catalyzed by the heterologously expressed STC1 and STC2 enzymes. These experiments will identify the nature of the terpenoids made by these enzymes and confirm whether the product of STC1 is a sesquiterpenoid, as suggested by the earlier biochemical genetics data. 5. Identification of the most likely subcellular localization of the STC proteins with constructs that fuse their putative chloroplast transit peptides to GFP. This will confirm whether the predicted N-terminal targeting sequence in each of the three sequenced stc1 alleles behaves as a chloroplast transit peptide in vivo. The proposed activity will have clear broader impacts. The work combines genetics, molecular biology, and biochemistry to analyze the response of maize plants to insect pests. It will serve as excellent training ground for student and postgraduate researchers interested in applying biochemistry and molecular genetics to address practical problems in plant biology. Both the P.I and co-P.I. are members of minorities and their labs are heavily populated by under-represented groups. The project is relevant to long-term improvement in U.S. agriculture in that it addresses mechanisms by which maize defends itself from insect attack. The identification of genes involved in insect resistance will aid breeders in developing naturally resistant crop species. Understanding natural defense mechanisms in major crops will allow alternative insect control strategies and reduce our dependence on potentially toxic pesticides. Thus, the problem to be investigated is of scientific, economic and environmental significance.