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 gene's 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. The assembly is based on specific recognition sites for the individual donor fragments, derived i.e., from a promoter, coding sequence, resistance gene or a protein tag. 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 this pathway is locally activated in response to colonization by the fungus.

Spatially distinct phytohormone responses of individualArabidopsis thalianaroot cells to infection and colonization byFusarium oxysporum

“…To monitor the infection and colonization of A. thaliana roots by Fo 5176, we cultivated 10-day old seedlings in square petri dishes, added fungal spores and then imaged hyphal growth, infection and colonization over the next 14 days. We used a transgenic Fo 5176 line expressing a cytoplasmic tdTomato fluorophore to visualize the fungus (Calabria et al ., 2022). One of the first things noticeable when imaging infected plants was that the tissue surrounding the colonization site appeared to undergo immediate cell death.…”

“…(2018) demonstrated the requirement of the JA-receptor COI1 for PEP1-induced growth inhibition (as an indirect readout for activated immune signaling) and apoplastic ROS-burst via a member of the RBOH family of NADPH oxidases. In our previous work we showed that all of these components, PEP1 , PEPR1 and 2 , BIK1 , and RBOHD , are also locally induced in the vascular cells neighboring the Fo 5176-colonized zone (Calabria et al ., 2022). Of these components, PEP1 and PEPR2 show the strongest induction, with both not just induced in cells of the defense response zone, but also in a long stretch covering the entire region from the HR zone and the defense response zone (Calabria et al ., 2022).…”

“…In our previous work we showed that all of these components, PEP1 , PEPR1 and 2 , BIK1 , and RBOHD , are also locally induced in the vascular cells neighboring the Fo 5176-colonized zone (Calabria et al ., 2022). Of these components, PEP1 and PEPR2 show the strongest induction, with both not just induced in cells of the defense response zone, but also in a long stretch covering the entire region from the HR zone and the defense response zone (Calabria et al ., 2022). It is therefore a possibility that this PEP pathway activates ET biosynthesis and signaling.…”

“…In this case, the PEP pathway would act as an amplifier of both, JA- and ET-signaling (Liu et al ., 2013; Holmes et al ., 2018; Calabria et al ., 2022). RBOHD activation appeared to be strongest in the HR and boundary zones, and this is also where we observed ‘root browning’ (Calabria et al ., 2022). Thus, RBOHD may play a dual role in promoting cell death in the HR zone and establishing the boundary.…”

“…For our work, we have set up a microscopy-based live-imaging approach monitoring transcriptional activity of different pathways in planta , over time and on an individual cell level. We have previously published our pGG-PIP GreenGate entry vector collection with regulatory sequences for plant immunity genes, including reporters for the different phytohormone pathways (Calabria et al ., 2022). Using this approach, we detected local responses of genes involved in SA and JA biosynthesis, signaling and regulation, targeted to specific tissues and to a very limited number of root cells upon infection with Fo 5176.…”