Naclo

Susceptibility of the L. monocytogenes isolates was tested to six biocides commonly used to sanitize food contact surfaces, namely, the quaternary ammonium compound BAC (≥95%; Sigma-Aldrich, Steinheim, Germany), GDA (50%; Carl Roth, Karlsruhe, Germany), IPA (≥99.9%; Carl Roth), the chlorine-releasing compound NaClO (12% Cl, techn.; Carl Roth), the oxidizing agent PAA (36 to 40% [wt/vol]; Sigma-Aldrich), and a biocidal product (Budenat Intense D443; Buzil-Werk Wagner, Memmingen, Germany) containing APD (7.5% [wt/wt]) as an active ingredient. The biocides were serially diluted in 2-fold steps just before the experiment using standardized hard water as defined in EN 1276, as follows: 10 to 0.08 mg/liter BAC, 5,650 to 44 mg/liter GDA, 249,600 to 3,900 mg/liter IPA, 8,000 to 62.5 mg/liter free chlorine (NaClO), 2,875 to 22 mg/liter PAA, and 48 to 0.7 mg/liter APD in Budenat.

Arabidopsis of the accession Columbia (Col-0) was used as wild-type in this work. All transgenic plants were established in the Arabidopsis ecotype Col-0 (Supplementary Fig. S1 at JXB online). The T-DNA double knock-out mutant lines KO6/62 and KO6/76 were made by crossing single knock-out lines (Stiti et al., 2011 (link)). Transgenic plants were selected on MS agar plates containing 50 mg l⁻¹ kanamycin. All plants were grown under approximately 120–150 μE m⁻² s⁻¹ light at 22 °C with a day/night cycle of 8/16 h (short day) if not otherwise stated. To induce flowering, 4-week-old plants were moved to a growth chamber with a 16/8 h photoperiod (long day). For plant growth under sterile conditions, seeds were surface sterilized in 70% (v/v) ethanol for 2 min, then in 7% (v/v) NaClO (Carl Roth; Karlsruhe, Germany) containing 0.1% (w/v) SDS for 10 min and finally seeds were rinsed four times with sterile distilled water and sown on MS-agar plates.

To investigate the response to colonization by S. Typhimurium 14028s, lettuce plants were exposed to Salmonella under sterile conditions. To this end, lettuce seeds were surface-sterilized by washing 5 times for 1 min with sterile tab water, followed by a sterilization step with 3% NaClO (Carl Roth GmbH + Co. KG) for 4 min and subsequently 4 washing steps with sterile tab water for 1 min. The sterilized seeds were placed on ¼ Murashige and Skoog agar medium (MS, Duchefa Biochemie, Haarlem, The Netherlands) at pH 5.6, including vitamins and 5 g/L sucrose. The seeds were incubated overnight in the dark and subsequently for additional 4 days in a climate chamber at 21°C, 16 h light. At the one-leaf stage, plants were transferred to 5 mL ¼ MS medium in 6-well plates (3 plants per well, in 3 replicates) and further incubated for 24 h. After this pre-adaption, MS medium was inoculated with S. Typhimurium 14028s resuspended in 10 mM MgCl₂ to obtain a final cell count of 10⁸ CFU/mL, 10 mM MgCl₂ solution was used as negative control. After incubation for 24 h, plants were harvested, immediately frozen in liquid nitrogen, ground with sterile mortar and pestle and stored at -80°C until the RNA extraction.