Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017;1578:207-221.
doi: 10.1007/978-1-4939-6859-6_17.

Quantitative Evaluation of Plant Actin Cytoskeletal Organization During Immune Signaling

Affiliations
Free PMC article

Quantitative Evaluation of Plant Actin Cytoskeletal Organization During Immune Signaling

Yi-Ju Lu et al. Methods Mol Biol. .
Free PMC article

Abstract

High spatial and temporal resolution microscopy-based methods are valuable tools for the precise real-time imaging of changes in cellular organization in response to stimulus perception. Here, we describe a quantitative method for the evaluation of the plant actin cytoskeleton during immune stimulus perception and the activation of defense signaling. As a measure of the biotic stress-induced changes in actin filament organization, we present methods for analyzing changes in actin filament organization following elicitation of pattern-triggered immunity and effector-triggered immunity. Using these methods, it is possible to not only quantitatively evaluate changes in actin cytoskeletal organization following biotic stress perception, but to also use these protocols to assess changes in actin filament organization following perception of a wide range of stimuli, including abiotic and developmental cues. As described herein, we present an example application of this method, designed to evaluate changes in actin cytoskeletal organization following pathogen perception and immune signaling.

Keywords: Actin cytoskeleton; Confocal microscopy; ETI; Immune signaling; PAMP; PTI; Pseudomonas syringae; Quantitative evaluation.

Figures

Fig. 1
Fig. 1
Treatment of Arabidopsis seedlings with pathogen and pathogen elicitors for induction of immunity and elicitation of changes in host actin cytoskeletal organization. (a) Fiberglass insect screen. (b) 10–12-day-old seedling in pots covered with fiberglass insect screen. (c, d) 10–12-day-old Arabidopsis are dip-inoculated in a suspension of Pseudomonas syringae. (e) Pathogen elicitor treatment of detached Arabidopsis cotyledons
Fig. 2
Fig. 2
Twenty-five Z-series image stacks collected using confocal laser scanning microscopy. The image stacks are collected in 0.5 μm intervals. The projection, which is the sum of the merged image stacks, is shown at the bottom left
Fig. 3
Fig. 3
A sample of four images stacks analyzed for quantitative evaluation of the Density metric. (a, b) are confocal and bright field images, respectively, generated by the confocal laser scanning microscopy. The bright field image, which is also created during the Density analysis (Subheading 3.5.2) is shown, yet is not used for quantitative evaluation (Subheading 3.5.2, step 3). (c) The original, unprocessed, confocal image. (d) The same image as (c) following processing using the Gaussian Blur filter. (e) The same image as in (d) following processing with the high bandpass (HBP) filter. (f) The HBP filter processed stacks for each of the 25 collected images, merged, to yield a processed 3D image. This processed image is used as the input file for quantitative Density analysis
Fig. 4
Fig. 4
Quantitative analysis of skewness and density metrics following pathogen elicitation of 12–14-day-old Arabidopsis seedlings. (a) Representative picture of finalized image. (b) Actin skewness and density are analyzed quantitatively. The data presented in the left graft (Bundling, skewness) represents the raw output values generated in Subheading 3.5.1. For presentation of the data shown in the graph on the right (% occupancy, density), the final output values (Subheading 3.5.2) are calculated whereby “% occupancy” is represented as: [100 × (Area ÷ 640,000)]. This calculation was originally described by Higaki and colleagues [8]

Similar articles

See all similar articles

Cited by 1 article

Publication types

MeSH terms

LinkOut - more resources

Feedback