Functional defence gene characterization: Heterologous gene expression in E. coli and yeast, functional assays
Approximately 100 target genes have been identified for functional biochemical characterization. Genes were identified based on transcriptome, proteome, and metabolite expression profiling of weevil-induced defence responses. They include 1-deoxy-D-xylulose 5-phosphate synthase (DXS), terpenoid synthases (TPS), prenyltransferases (PTs), cytochromes P450 (P450), dirigent proteins (DIR), chitinases (CHI), stilbene synthase (STS), chalcone synthases (CHS). The same genes are also targeted for SNP marker development in the Comparative Genomics research (see section on candidate gene identification).
Functional characterization involves the cloning of FLcDNAs, transformation into heterologous expression systems, production, and in some cases purification, of recombinant protein, and enzyme assays. We regularly use E. coli and yeast expression systems. In addition, for the P450s we have established an insect-cell expression system. Three DXS genes have been functionally characterized using E. coli complementation. This work was done in collaboration with the Max Planck Institute (Phillips et al. 2007). Characterization of new TPS genes is complete (13 mono-TPS, 4 sesqui-TPS, 1 di-TPS) and together with other characterized TPS (Keeling and Bohlmann 2006), we have established the TPS gene family as the largest functionally characterized conifer gene family.
We have identified several candidate Sitka spruce P450 genes for functional characterization and more than half have been cloned as FLcDNAs for recombinant expression. One Sitka spruce P450 has been functionally characterized. A new class of cytochrome P450s within the CYP85 clan was identified. Custom synthesis of substrates for P450s of the CYP720 family of diterpene resin acid biosynthesis is underway. 35 DIR genes have been cloned as FLcDNAs (see Ralph et al., 2006 and Ralph et al., 2007) and are currently being assessed for expression in insect cells and biochemical characterization at Washington State University.
For further information, please contact Joerg Bohlmann.
Transformation of Arabidopsis, Poplar and Spruce
This activity is in support of functional characterization of defence genes and genes associated with adaptation. Spruce and poplar transformation is being done in collaboration with the Canadian Forest Service and with the Umeå Plant Sciences Centre, respectively. Arabidopsis transformation is done in our own lab.
Target genes have been selected and a first set of transformation constructs have been made. Target genes identified for transformation include: 1-deoxy-D-xylulose 5-phosphate synthases (DXS), terpenoid synthases (TPS), prenyltransferases (PTs), cytochromes P450 (P450), dirigent proteins (DIR), chitinases (CHI), stilbene synthase (STS), chalcone synthase (CHS) as well as transcription factors and potential negative-regulators of defence signaling. These targets have been identified based on metabolite, proteome and gene expression profiling as well as on the basis of literature data.
The same genes are targeted for promoter discovery and testing of promoters in transgenic plants. Promoter cloning is underway. We have used Arabidopsis transformation for complementation tests of functions for P450s putatively involved in phytohormone biosynthesis but closely related within the CYP85 clan to P450 of diterpene resin acid defences. We will also started use Arabidopsis transformation to establish functions of ZIM (tify)-type regulators of conifer defense signaling. Other work under this activity established a heterologous insect-inducible promoter system (the potato PINII promoter) for controlled gene expression in transgenic spruce. We successfully used this promoter to drive wound-inducible TPS expression in spruce seedlings (Godard et al., 2007).