Heterologous expression of Triticum aestivum WRKY transcription factors in Pichia pastoris : interaction with promoters of defence genes

Abstract

WRKY transcription factors are the largest family of plant transcription factors. They are responsible for the regulation of development, growth, senescence, signal transduction, and stress resistance (biotic and abiotic). Three family groups have been characterised based on the number and location of the WRKY conserved domain. Three wheat WRKY transcription factors candidates, and a mutant variant, were selected for this project (TaWRKY53b, TaWRKY19, TaWRKY3, and TaWRKY3m). Previous work from the Molecular Biology and Biotechnology group has shown the differential expression of these TaWRKY genes following periods of biotic and abiotic stress. The study of these proteins would be greatly accelerated if they could be isolated in large quantities. Isolation from source material is not feasible nor are these transcription factors amenable to heterologous expression in prokaryotic expression systems. Here we show that wheat WRKY proteins can be expressed and purified from the methyltrophic eukaryotic yeast Pichia pastoris, to provide functional protein with the capacity to bind DNA. Candidate TaWRKYs were first studied in silico. TaWRKY53b and TaWRKY19 were identified as Group I carrying two WRKY DNA binding domains [DBD] (trans and Cis domains) whereas TaWRKY3 a group IIc contained only one WRKY DBD. All were determined to be localized in the nucleus. Expression of the TaWRKY genes was regulated by bZIP, ethylene-responsive and other transcription factors (Chapter 2). Protein to protein interactions using STRING analyses were less informative due to gaps in current protein databases. In Chapter 3, wheat WRKY transcription factor (TaWRKY53b, TaWRKY19, TaWRKY3, and TaWRKY3m) protein coding sequences were cloned into the pGAPZaA expression vector. The resulting vector was transformed into Pichia pastoris for constitutive protein expression. Each WRKY protein carries a 6x His tag for protein identification and purification. Small-scale expression showed the production of all WRKY proteins. TaWRKY19 and TaWRKY53b were expressed in 5 litre bench-top fermenter in basal salt media (BSM) and proteins were purified using a pipeline of hydrophobic interaction chromatography (HIC) and immobilized metal affinity chromatography (IMAC) column. TaWRKY3 and TaWRKY3m were not compatible with HIC purification and phosphates in the BSM precipitate during IMAC. Replacing BSM with M3 media allow one-step IMAC purification of both TaWRKY3 and TaWRKY3m directly from low pH fermentate (Chapter 4). Purified recombinant WRKYs were used to demonstrate binding to cis-regulatory elements from plant promoters. Electrophoretic mobility shift assays (EMSA) were performed against synthetic W-box and mutated W-box repeats and against promoter fragments of TaPR1-23 and PcPR1 each containing W-box elements. TaWRKY3 dimerized during EMSA thus reducing the conclusively of the interaction. The mutation in the WRKY DNA binding domain of TaWRKY3m resulted in no W-box binding. TaWRKY19 and TaWRKY53b preferentially bound the promoter fragments (TaPR1-23 and PcPR1) suggesting that addition sequences outside of the core W-box element are required for optimal binding. Recombinant WRKYs did not bind the mutated W-box repeats (Chapter 5) Overall, this project had demonstrated the use of P. pastoris to express soluble, correctly folded recombinant TaWRKY transcription factor proteins. It also showed that these recombinant transcription factors interact specifically with target gene promoter elements. Furthermore, the project highlights the regulatory network controlling the expression of WRKYs in wheat. This approach can now be used to generate functional and highly purified recombinant WRKY transcription factors that can be used to identify the genes they regulate and thus elucidate the specific stress responses and provide targets for wheat breeding strategies.