TITLE: Pseudomonas syringae seed infections
AUTHORS: William Rooney, Janet Laird (University of Glasgow), Mechna Chowdhury (University of Glasgow), Catriona MacIntosh (University of Glasgow), Xiaoyi Deng (University of Glasgow), Phoebe McBride (University of Glasgow), Joel Milner (University of Glasgow)

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Seed contamination represents a critical route for the transmission of bacterial plant diseases. Here we have created a step-by-step protocol for </span><span style = "font-style:italic;">in vitro</span><span> seed infections of </span><span style = "font-style:italic;">Pseudomonas syringae. </span><span>This protocol provides a robust method for </span><span style = "font-style:italic;">P. syringae</span><span> infection in cucumber. This synthetic seed infection assay is generally applicable for </span><span style = "font-style:italic;">P. syringae</span><span> as we have successfully adapted it for both tomato and soybean.</span></div></div>

[STEPS]
?. [Sterlising seeds]
Transfer seeds to a sterile tube. For cucumber seeds used in this protocol, a 50 mL conical centrifuge tube will suffice. This protocol can be adapted for bigger and smaller seeds.
?. [Sterlising seeds]
Add the sterilisation solution (50% bleach (v/v water), 0.002% tween 20) and mix by inversion for 8-10 minutes. Decant the sterilisation solution and wash seeds in water to remove residual bleach.
?. [Sterlising seeds]
Once the bleach is no longer present transfer seeds into a sterile conical flask
?. [Overnight culture ]
Prepare a 5 mL Kings Broth liquid culture (20 gL-1 peptone 10 gL-1 glycerol, 1.5 gL-1 K2HPO4, 1.23 gL-1 MgSO4  pH 7.2)
?. [Overnight culture ]
Inoculate a single colony of Pseudomonas syringae into a 5 mL liquid culture and incubate overnight in a shaking incubator for 16-18 hours, 28°C at 180-200 rpm.
?. [Seed infection]
Centrifuge the overnight culture using a bench top centrifuge 4,000 g for 15 mins
?. [Seed infection]
Re-suspend the pellet in sterile 10 mM MgCl2 buffer and adjust the OD600 to 0.2. An OD600 is approximately ∼ 1 x 108 colony-forming units per mL (cfu mL-1)
?. [Seed infection]
Dilute the bacterial inoculate by 1/100 (∼1 x 106 cfu mL-1) and pour into the conical flask containing the seeds. Ensure the seeds are immersed in the inoculate.
?. [Seed infection]
Place the flask into a bell jar attached to a vacuum pump. Turn on the vacuum pump and allow the seeds to experience -1 atmospheres of pressure for 30 mins.
?. [Sterlising seeds]
?. [Seed infection]
?. [Seed infection]
?. [Seed infection]
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Switch off the pump, release pressure immediately and leave seeds for 5 minutes to settle.
?. [Seed infection]
Remove the flask and decant the inoculate, transfer seeds onto the sterile blue roll and remove excess moisture.
?. [Seed infection]
Wet some blue roll in a petri dish with sterile water and place the seeds on top of the blue roll.
?. [Seed infection]
?. [Seed infection]
At this point, you can take a sample for a 0-hour time point or incubate seeds in a growth chamber. For our purposes, the growth chamber conditions we used were: 16 h light/8 h dark (at 26 and 22 °C, respectively) and at a light level of 80 μmol/m2/s.
?. [Measuring bacterial load in seeds]
Take note of the sample weight grind using a sterile mortar and pestle.
?. [Measuring bacterial load in seeds]
Dilute sample in 1000 μL of sterile 10 mM MgCl2 and continue grinding.
?. [Measuring bacterial load in seeds]
Next transfer the "grindate" to a microcentrifuge tube. Top tip: cutting the tip of the pipette tip will make it easier to transfer
?. [Measuring bacterial load in seeds]
Let the insoluble material float to the bottom and use the supernatant to measure bacterial load by spotting 10 μL onto Kings Broth agar plates.Top tip: If you are recovering too few bacteria you can increase the volume of supernatant used on the plate. Conversely, if you have too much serially diluted the bacterial (1:10).
?. [Measuring bacterial load in seeds]
To calculate bacterial load:bacterial load = [colony-forming units on plate] x [dilution factor] / [seed weight]Example:5 [colony forming units] x 1,000 [dilution factor] / 250 mg = 20 colony forming units/mg of tissue.
?. [Sterlising seeds]
Wash seeds in a 75% ethanol solution for 2 minutes and decant the ethanol.