Extracellular Alkalinization Assay for the Detection of Early Defense Response

Natalia Moroz1, Alisa Huffaker2, Kiwamu Tanaka1

1 Department of Plant Pathology, Washington State University, Pullman, Washington, 2 Section of Cell & Developmental Biology, University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20057
Online Posting Date:  September, 2017
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Plant recognition of invading organisms occurs through identification of foreign molecules associated with attackers and of self‐derived, damage‐associated molecules. Perception of these molecules activates signaling processes including dynamic changes in ion balance, production of second messengers such as reactive oxygen species and nitric oxide, increased levels of plant hormones, and map kinase cascade activation. Together these signaling events stimulate transcriptional changes to initiate plant defense responses. Among the earliest detectable signaling events is a rapid increase in apoplastic pH, i.e., extracellular alkalinization. Here, an assay for quantification of this alkalinization response using suspension‐cultured cell lines for Arabidopsis, potato, and maize is described. This assay is an inexpensive, fast, simple, and reproducible method to quantify defense signaling output, providing a powerful tool for evaluating early plant responses to elicitors and pathogens. Results from the alkalinization assay are comparable to other more costly and time‐consuming methods for assessing defense signaling, such as measurement of the oxidative burst, calcium influx, and marker gene expression. This bioassay is a quantitative and robust method for evaluation of plant defense output. © 2017 by John Wiley & Sons, Inc.

Keywords: arabidopsis; culture; early defense response; extracellular alkalinization; maize; potato; suspension cell

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Table of Contents

  • Introduction
  • Basic Protocol 1: Suspension Cell Culture Preparation
  • Basic Protocol 2: Measurement of Extracellular Alkalinization
  • Basic Protocol 3: Calculation and Analysis of Extracellular Alkalinization
  • Support Protocol 1: Recovery of Suspension Culture from Callus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Suspension Cell Culture Preparation

  • For Arabidopsis suspension culture:
  • Arabidopsis suspension cells T87 (Arabidopsis Biological Resource Center, ABRC: https://abrc.osu.edu/cell‐culture)
  • Sterile NT‐1 medium, pH 5.8 (see recipe)
  • 22° to 26°C orbital incubator shaker (130 rpm)
  • For potato suspension culture:
  • Potato suspension cells derived from potato tubers (Solanum tuberosum L. cv. Russet Burbank)
  • Sterile Murashige and Skoog (MS) medium, pH 5.8 (see recipe)
  • 23°C orbital incubator shaker (130 rpm)
  • For maize suspension culture:
  • Maize suspension‐cultured cells derived from endosperm (Zea mays var. Black Mexican Sweet; Chourey & Zurawski, )
  • Sterile MS + 2,4‐D, pH 5.6 (see recipe)
  • 25°C orbital incubator shaker (130 rpm)
  • Laminar flow hood (for suspension cell transfer)
  • 250‐ml sterile glass conical flasks with plastic or metal lids
  • Aluminum foil

Basic Protocol 2: Measurement of Extracellular Alkalinization

  • Suspension cell culture (from protocol 1)
  • Elicitor(s) solutions and/or pathogen(s) suspensions (see reciperecipes)
  • MS or NT‐1 liquid medium (see recipe)
  • Autoclaved water
  • Automatic pipettors
  • Sterile 24‐well plates
  • Sterile, wide‐bore glass pipets or precut tips
  • Tabletop rotary shaker (180 rpm)
  • pH meter with an electrode

Basic Protocol 3: Calculation and Analysis of Extracellular Alkalinization

  • Computer with spreadsheet software (e.g., KaleidaGraph and Microsoft Excel)

Support Protocol 1: Recovery of Suspension Culture from Callus

  • Callus
  • MS or NT‐1 solid medium (see recipe)
  • MS or NT‐1 liquid medium (see recipe)
  • Plant growth chamber (Conviron)
  • Sterile Petri dishes (Fisherbrand, cat. no. FB0875712, 100 × 15–mm or Olympus Plastics, cat. no. 32‐107G, 90‐mm diameter)
  • Tweezers, sterile
  • Laminar flow hood
  • Alcohol lamp
  • 250‐ml flasks and lids, sterile
  • Aluminum foil
  • 23°C orbital shaker
  • Surgical tape
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Literature Cited

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