pGG-PIP: A GreenGate (GG) entry vector collection with Plant Immune system Promoters (PIP)

Abstract

The regulatory sequences controlling the expression of a gene (i.e., the promoter) are essential to properly understand a genes function. From their use in mutant complementation assays, to studying their responsiveness to different stimuli via transcriptional reporter lines or using them as proxy for the activation of certain pathways, assays using promoter sequences are valuable tools for insight into the genetic architecture underlying plant life. The GreenGate (GG) system is a plant-specific variant of the Golden Gate assembly method, a modular cloning system that allows the hierarchical assembly of individual donor DNA fragments into one expression clone via a single reaction step. Here, we present a collection of 75 GG entry vectors carrying putative regulatory sequences for Arabidopsis thaliana genes involved in many different pathways of the plant immune system, designated Plant Immune system Promoters (PIP). This pGG-PIP entry vector set enables the rapid assembly of expression vectors to be used for transcriptional reporters of plant immune system components, mutant complementation assays when coupled with coding sequences, mis-expression experiments for genes of interest, or the targeted use of CRISPR/Cas9 genome editing. We used pGG-PIP vectors to create fluorescent transcriptional reporters in A. thaliana and demonstrated the potential of these reporters to image the responsiveness of specific plant immunity genes to infection and colonization by the fungal pathogen Fusarium oxysporum. Using the PLANT ELICITOR PEPTIDE (PEP) pathway as an example, we show that several components of this pathway are locally activated in response to colonization by the fungus.