Tandem Tag Assay Optimized for Semi-automated in vivo Autophagic Activity Measurement in Arabidopsis thaliana roots.

Abstract

Autophagy is the main catabolic process in eukaryotes and plays a key role in cell homeostasis. In vivo measurement of autophagic activity (flux) is a powerful tool for investigating the role of the pathway in organism development and stress responses. Here we describe a significant optimization of the tandem tag assay for detection of autophagic flux in planta in epidermal root cells of Arabidopsis thaliana seedlings. The tandem tag consists of TagRFP and mWasabi fluorescent proteins fused to ATG8a, and is expressed in wildtype or autophagy-deficient backgrounds to obtain reporter and control lines, respectively. Upon autophagy activation, the TagRFP-mWasabi-ATG8a fusion protein is incorporated into autophagosomes and delivered to the lytic vacuole. Ratiometric quantification of the low pH-tolerant TagRFP and low pH-sensitive mWasabi fluorescence in the vacuoles of control and reporter lines allows for a reliable estimation of autophagic activity. We provide a step by step protocol for plant growth, imaging and semi-automated data analysis. The protocol presents a rapid and robust method that can be applied for any studies requiring in planta quantification of autophagic flux.

Plant phenotyping relevance

植物体内のオートファジー活性という生理状態を、蛍光イメージングと半自動解析で定量する手法を最適化し、プロトコルとして提示しているため。

Code and data availability

The paper's semi-automated autophagic flux analysis pipeline (ImageJ macros and R scripts) is publicly available in the authors' AuTToFlux GitHub repository, which also contains demo data for validating the analysis.