High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Summary

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Abstract

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.

Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

Introduction

Pseudomonas syringae is a Gram-negative pathogenic bacterium that infects a wide range of plant hosts. Bacteria enter the host plant through the stomata or physical wounds and proliferate in the apoplast¹. Plants have evolved a two-tiered immune response to protect against infection by bacterial pathogens. The first level occurs at the plant cell surface, where pattern recognition receptors on the plant cell membrane perceive highly conserved pathogen-associated molecular patterns (PAMPs) in a process called PAMP-triggered immunity (PTI)². During this process, the host plant upregulates defense response pathways....

Protocol

1. Preparation and use of biosafety cabinet

1. Wipe down biosafety cabinet with 70% ethanol.
2. Close the sash and turn on the ultraviolet light in the biosafety cabinet for 15 min.
3. After 15 min, turn off the ultraviolet light in the biosafety cabinet. Lift the sash and turn on the blower for 15 min.
4. Wipe all items to be used in the biosafety cabinet with 70% ethanol prior to putting the items into the sterilized cabinet.
5. Clean gloves or bare hands with 70% ethanol before wor.......

Discussion

A protocol for flood inoculation with PstDC3000 or Pst19 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined²². For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf.......

Materials

Name	Company	Catalog Number	Comments
3M Tape Micropore 1/2" x 10 YD CS 240 (1.25 cm x 9.1 m)	VWR International	56222-182
3mm borosilicate glass beads	Friedrich & Dimmock	GB3000B
Bacto peptone	BD	211677
Bacto agar	BD	214010
Biophotometer Plus	Eppendorf	E952000006
Biosafety cabinet, class II type A2
BRAND Disposable Plastic Cuvettes, Polystyrene	VWR International	47744-642
Chenille Kraft Flat Wood Toothpicks	VWR International	500029-808
cycloheximide	Research Products International	C81040-5.0
Dibasic potassium phosphate anhydrous, ACS grade	Fisher Scientific	P288-500
Dimethylformamide
Dissecting microscope (Magnification of at least 10x)
Ethanol - 190 Proof
Falcon polystyrene 96 well microplates, flat-bottom	Fisher Scientific	08-772-3
Glass Alcohol Burner Wick	Fisher Scientific	S41898A / No. W-125
Glass Alcohol Burners	Fisher Scientific	S41898 / No. BO125
Glycerol ACS reagent	VWR International	EMGX0185-5
Kimberly-Clark™ Kimtech Science™ Kimwipes™ Delicate Task Wipers	Fisher Scientific	06-666-A
Magnesium chloride, ACS grade	VWR International	97061-356
Magnesium sulfate heptahydrate, ACS grade	VWR International	97062-130
Microcentrifuge tubes, 1.5 mL
Microcentrifuge tubes, 2.2 mL
Mini Beadbeater-96, 115 volt	Bio Spec Products Inc.	1001
Murashige & Skoog, Basal Salts	Caisson Laboratories, Inc.	MSP01-50LT
Pipet-Lite XLS LTS 8-CH Pipet 20-200uL	Rainin	L8-200XLS
Pipet-Lite XLS LTS 8-CH Pipet 2-20uL	Rainin	L8-20XLS
Polystyrene 100mm x 25mm sterile petri dish	VWR International	89107-632
Polystyrene 150mm x 15mm sterile petri dish	Fisher Scientific	FB08-757-14
Polystyrene 150x15mm sterile petri dish	Fisher Scientific	08-757-148
Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach)	Staples	1013131
Rifampicin	Gold Biotechnology	R-120-25
Silwet L-77 (non-ionic organosilicone surfactant co-polymer C13H34O4Si3 surfactant)	Fisher Scientific	NCO138454
Tips LTS 20 μL 960/10 GPS-L10	Rainin	17005091
Tips LTS 250 μL 960/10 GPS-L250	Rainin	17005093
VWR dissecting forceps fine tip, 4.5"	VWR International	82027-386