revision (for Nancy)

Quantification of bacterial growth (P.syringae) in Arabidopsis

(Modified from Tornero and Dangl, (2001) Plant J. 28(4), 475-481) and The Arabidopsis Thaliana-Pseudomonas Syringae Interaction, Fumiaki Katagiria, Roger Thilmonyb, and Sheng Yang

Heb, The Arabidopsis book, Online) Plant growth

Grow plants under short-day conditions for 3-5 weeks (before bolting).

Bacterial strains and media Pseudomonas syringae pv. tomato strains, as frozen stocks (-80C freezer)

DC3000 Rif 50 Disease for Columbia ecotype DC3000 (pDSK600-empty) Rif50 Spect100 Strep50 Disease for Columbia ecotype DC3000 (pDSK600-avrRpt2) Rif50 Spect100 Strep50 Resistance for Columbia ecotype Spect: Spectinomycin; Strep: Streptomycin; Rif: Rifampicin.

King’s B medium (KB) Medium for plates (per liter): 10g Bacto-tryptone 10g Bacto-peptone 1.5g K2HPO4 3.08 g MgSO4.7H2O 15g agar pH to 7.2 (~4ml KOH 1M)

Medium for liquid (per liter): 20g Bacto-Proteose peptone #3 10g or 10 mL Glycerol (specific for microbes*) 1.5g K2HPO4 (2g if 3H20) 1.5g g MgSO4.7H2O pH to 7.2

10mM MgCl2 = 2.032g/l

Bacterial growth

In the evening of day –3 (three days prior the infection) or morning of day -2, seed a fresh plate from the stocks and incubate for ~2 days at 28°C.

In the morning of day –1, use a spreader to distribute a generous sample of the growing bacteria over a fresh plate to obtain a lawn the next day (incubate 24h at 28°C).

On day 0 (infection day) add 10ml of 10mM MgCl2 to the plate, wait 10min and pipette the suspension out of the plate. Measure OD600, adjust OD600 to 0.05 (~1-2.5x107). Blank using 10mM MgCl2. Infiltration

Inoculate the plants in the morning when stomatas are open. Label the petioles of leaves to be inoculated with a permanent marker. Include a Mock

treated plant as a control (10mM Mg MgCl2). Inoculate using a 1ml syringe (no needle) and infiltrate 10-50ul per leaf. Don’t worry about

the volume too much, all the leaves fill up with similar quantities and we take Day0 samples to ensure all leaves have similar amounts of bacteria). Inoculate the abaxial (bottom) leaf surface while pressing with a finger against the syringe tip on the adaxial side. Use the space between vessels, so one side of the leaf. Inoculate 4 leaves / plant.

After inoculation the plants are moved to the chamber and covered with a transparent lid (stomatal opening depends on high humidity). Then, samples for day 0 are taken 1hr after inoculation. Don’t go over 3 days as some of the leaves in the compatible interactions are

desiccated but carry viable bacteria. It’ll be a source of error as we’ll express growth per fresh weight.

Extraction and counting

Use 3 replicates/timepoint and 4 leaves/replicate. For each replicate, transfer 4 leaves to an eppendorf tube, determine the leaf weight, add

1ml 10mM MgCl2 and grind by hand using a plastic pestle (I prefer to add 500ul to grind, then add the remaining 500ul and mix briefly).

Prepare 10-fold serial dilutions (180µl MgCl2 + 20µl sample) and plate them (20µl of each) onto KB plates with the appropriate antibiotics. Choose dilutions containing 1-20 colonies after 40h of incubation at 28°C. Express total number of bacteria as colony forming units/mg fresh weight (cfu/mg FW) = k (N x 10d-1)/weight of tissue.

k* = 500 (constant) N= number of colonies d = dilution number

*One colony in the first dilution would indicate that the concentration in the first well was 1cfu/20µl. As this is from a 10-fold dilution, it is equivalent to 10cfu/20µl from the original tube. Assuming that the volume of the plant is negligible in comparison to the 1000µl of buffer, this equals a total of 500 cfu in the tube.

Taken from the Arabidopsis Book.

Syringe infiltration of Arabidopsis leaves. (A) The abaxial (under) side of the Arabidopsis leaf to be syringe-infiltrated. (B) Placement of the syringe on the right side of the leaf, avoiding the midvein. (C) Gentle infiltration of a portion of the leaf’s intercellular space. (D) The syringe-infiltrated leaf. Note that the infiltrated area appears water-soaked