High-Throughput Protoplast Trans-Activation (PTA) Screening to Define Transcription Factors in Auxin-Mediated Gene Regulation

Abstract

Genome sequencing and annotation studies clearly highlight the impact of transcriptional regulation in plants. However, functional characterization of the majority of transcriptional regulators remains elusive. Hence, high-throughput techniques are required to facilitate their molecular analysis. Here, we provide a detailed protocol to conduct a high-throughput protoplast trans-activation (PTA) screening, which enables simultaneous analysis of up to 95 individual transcription factor activities on a customizable promoter:LUCIFERASE reporter. This system is well suited to decipher complex transcriptional networks such as that triggered by the phytohormone auxin.

A Protoplast Transient Expression System to Enable Molecular, Cellular, and Functional Studies in Phalaenopsis orchids

“…Hence, this protocol provides a useful platform for in-depth studies of gene functions and molecular networks that were previously difficult in Phalaenopsis orchids. Recently, protoplast transient expression systems have been extended to studies on dissection of miRNA pathways ( Martinho et al, 2015 ), protein-DNA binding ( Lee et al, 2017 ), microbe-associated molecular patterns-triggered immunity ( Fraiture et al, 2014 ; Kanofsky et al, 2016 ), ribonuclease-mediated mRNA decay ( Hayashi et al, 2016 ), and auxin-mediated transcriptional regulatory networks ( Wehner et al, 2017 ). It is therefore conceivable that our established protocol may be applied to diverse aspects of orchid biology studies.…”

“…Protoplast-based high-throughput systems have been established for dissection of signaling pathways, analysis of transcription factor functions, and identification of kinase-associated phosphoisoforms ( Wehner et al, 2011 ; Zhang et al, 2015 ; Dory et al, 2016 ; Wehner et al, 2017 ). Therefore, the orchid protoplast transient expression system provides a molecular tool to characterize functions of proteins identified from open reading frames from recently released transcriptome datasets ( Cai et al, 2015 ; Fang et al, 2016 ; Chao et al, 2017 ) and potentially allows discovery and/or validation of hierarchical gene regulatory networks.…”