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In recent years, there has been substantial progress in the development of methods to analyze inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). However, many of these techniques are labor- and cost-intensive and can usually only be carried out by laboratories specialized in InsPs/PP-InsPs analysis. In this chapter, we present a simple method that exploits the fact that phosphorylation and/or dephosphorylation of certain InsP/PP-InsP species induces the activation of promoters driving the expression of genes involved in phosphate starvation response (PSR). By linking PSR-inducible promoters to the RUBY reporter, which allows continuous and noninvasive visualization of promoter activation, we provide a new analytical tool to monitor enzymatic activities that alter PP-InsP levels, eliminating the need for complex biochemical analysis. We present a step-by-step protocol showing how simple coinfiltration of promoter-RUBY T-DNA constructs together with PP-InsP pyrophosphatase-encoding T-DNAs into Nicotiana benthamiana leaves enables evaluation of the enzymatic activity of expressed pyrophosphatases, and mention possible downstream analyses by methods described in other chapters of this special issue.

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A Simple RUBY Reporter System in Nicotiana benthamiana for Studying Inositol Pyrophosphate Dynamics

  • 实验方案

  • 发表日期：

    2025年

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  • Marília Kamleitner,
  • Annett Strauß,
  • Natalie Faiß,
  • Klea Lami,
  • Thomas Lahaye,
  • Gabriel Schaaf,
  • Verena Gaugler

摘要

In recent years, there has been substantial progress in the development of methods to analyze inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). However, many of these techniques are labor- and cost-intensive and can usually only be carried out by laboratories specialized in InsPs/PP-InsPs analysis. In this chapter, we present a simple method that exploits the fact that phosphorylation and/or dephosphorylation of certain InsP/PP-InsP species induces the activation of promoters driving the expression of genes involved in phosphate starvation response (PSR). By linking PSR-inducible promoters to the RUBY reporter, which allows continuous and noninvasive visualization of promoter activation, we provide a new analytical tool to monitor enzymatic activities that alter PP-InsP levels, eliminating the need for complex biochemical analysis. We present a step-by-step protocol showing how simple coinfiltration of promoter-RUBY T-DNA constructs together with PP-InsP pyrophosphatase-encoding T-DNAs into Nicotiana benthamiana leaves enables evaluation of the enzymatic activity of expressed pyrophosphatases, and mention possible downstream analyses by methods described in other chapters of this special issue.