Metabolic Engineering of Arabidopsis thaliana and Carica papaya Using Native Papaya Promoters to Control Stilbene Synthase Gene Expression

Abstract

Papaya is an important fruit crop in the tropics, but it has inadequate resistance to the devastating pathogen, Phytophthora palmivora that causes root and stem rot. To develop disease resistance, papaya was engineered with the gene encoding grapevine stilbene synthase (Vst1), the key enzyme synthesizing the antimicrobial and antifungal phytoalexin, resveratrol. However, Vst1 expression under its own promoter is suboptimal in papaya. Thus, to improve expression, four pathogen-induced native papaya promoters highly expressed in the roots and stems were used to control Vst1 expression for resveratrol glucoside (piceid) synthesis. The promoters were isolated from genes encoding peroxidase (Cp9), beta-1,3-glucanase (Cp29) ferulate-5-hydroxylase (Cp35), and hypersensitive-induced response protein (Cp45). These promoters and the constitutive promoter, CaMV35S, were fused to the eGFP gene and transformed into the model plant, Arabidopsis thaliana. This allowed an efficient and rapid evaluation of promoter functionality, strength, and tissue expression. In silico analysis predicted the presence of several cis-regulatory promoter elements associated with stress and defense responses. The Arabidopsis transcriptional machinery readily recognized the promoters at the precise transcription initiation sites as used in papaya. Qualitative and quantitative eGFP measurements (fluorescence, mRNA and protein levels) indicated variations in tissue expression and promoter strength. Comparison of eGFP mRNA and protein levels indicated post-transcriptional regulation. Predicted mRNA 5′-UTR secondary structures potentially affected the translational efficiency of the mRNAs during plant development, most notably for Cp9 and Cp35. Two differentially expressed promoters (Cp9 and Cp29) were fused to Vst1 and genetically transformed into Arabidopsis. They synthesized variable piceid concentrations as detected through RP-HPLC analysis and verified through tandem mass spectrometry (MS/MS). Competition for precursor substrates (pcoumaroyl CoA, malonyl CoA) between chalcone synthase and stilbene synthase resulted in reduction of anthocyanin and seed tannin levels. A new transformation protocol was developed using suspension derived-cultures facilitated Agrobacterium-mediated transformation that generated several transgenic papaya lines. PCR, Southern Blot, RTPCR, qRT-PCR, and RP-HPLC analyses confirmed independent transgene integration and copy number, Vst1 mRNAs driven by the native promoters, and promising basal piceid levels. Transgenic papaya plants are being regenerated from the calli that have potential disease resistance against Phytophthora palmivora and other papaya diseases.