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

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.