Protocol: an improved method to quantify activation of systemic acquired resistance (SAR)

Abstract

Background Plant responses triggered upon detection of an invading pathogen include the generation of a number of mobile signals that travel to distant tissues and determine an increased resistance in distal, uninfected tissues, a defense response known as systemic acquired resistance (SAR). The more direct means of measuring activation of SAR by a primary local infection is the quantification of pathogen multiplication in distal, systemic sites of secondary infection. However, while such assay provides a biologically relevant quantification of SAR, it is hampered by experimental variation, requiring many repetitions for reliable results. Results We propose a modification of the SAR assay based on the Arabidopsis – Pseudomonas syringae pathosystem exploiting the knowledge of source-sink relationships (orthostichies), known to centralize SAR-competency to upper leaves in the orthostichy of a lower primary infected leaf. Although many sources of variation such as genotypes of plant and pathogen, inoculation procedure, or environmental conditions are already taken into account to improve the performance of SAR assays, a strict leaf selection based on source-sink relationships is not usually implemented. We show how enacting this latter factor considerably improves data reliability, reducing the number of experimental repetitions for results. Conclusions Direct selection of leaves for both primary and secondary inoculation exclusively within the orthostichy of the primary infected leaf is a key element on reducing the number of experimental repetitions required for statistically relevant SAR activation results.

Multi-Omics Revealed Molecular Mechanisms Underlying Guard Cell Systemic Acquired Resistance

“…This study highlights the importance of identifying guard cell responses to bacterial pathogen invasion because stomatal pores are the entry ports that mount a first line of defense. Typically, SAR immune responses have been studied using whole leaves [ 38 , 39 , 40 , 41 ], but this can mask the response of low abundant cells like guard cells, and the changes of metabolites, lipids, and proteins in these highly specialized cells can be overlooked. Also of note is that by analyzing proteins, lipids, and metabolites together, we were able to have a correlated analysis with evidence at different levels.…”

“…A recent report on metabolite responses to biotic and abiotic stresses focused on sample collections from whole plants, apoplastic fluids, cell walls, phloem, leaves, stems, roots, and flowers [ 38 ], but did not sample guard cells, which are known to perceive and respond to bacterial pathogens and abiotic stresses. A recent protocol by Rufian et al [ 39 ] serves as an example of the two factors overlooked in SAR research. Firstly, they used infiltration to deliver the bacterial pathogen into apoplastic space, which completely bypasses stomatal immunity.…”

“…Pseudomonas syringae pv. tomato DC3000, the model pathogen for Arabidopsis SAR induction [ 22 , 39 ], was used for the experiments. Agar media plates were made using King’s B media protocol.…”

Pectobacterium Phage Jarilo Displays Broad Host Range and Represents a Novel Genus of Bacteriophages Within the Family Autographiviridae

“…To obtain the same number of cells in both cultures, DC3000 was grown to OD 600nm = 0.03, based on previous studies. 28 The bacterial suspensions were aliquoted in equal volumes. Phage Jarilo was added to the bacterial suspensions at a multiplicity of infection of 0.01.…”

An efficient, high‐throughput method for the simultaneous exposure of drought stress and bacterial infection in plants

“…Although several studies have successfully co‐imposed drought and bacterial stress, the utility of the methods used is limited by several factors (Ramegowda et al, 2013; Gupta et al, 2016; Sinha et al, 2016; Dixit et al, 2019). The well‐established Arabidopsis thaliana–Pseudomonas syringae pathosystem has been successfully used in combined‐stress studies, and infection protocols are available for P. syringae (Katagiri et al, 2002; Yao et al, 2013; Rufián et al, 2019). The most routinely used syringe infiltration method involves injecting bacteria directly into the apoplast, bypassing the natural infection pathway through the stomata (Tornero and Dangl, 2001; Katagiri et al, 2002; Liu et al, 2015).…”