Pectobacterium atrosepticum Transcriptional Responses

Pectobacterium atrosepticum strain SCRI1043 was obtained from the SCRI bacterial collection. The bacteria were grown in a variety of media at 15°C or 27°C as described in Table 3. Pectin, polygalacturonic acid and arabinogalactan were added to two of the growth media in order to trigger different responses in P. atrosepticum gene transcription. Two different types of pectin, citrus pectin (cip) and cabbage pectin (cap) were used, as this could in principle affect the transcriptional response of the bacterium. The temperature of 15°C was selected based on optimal expression of key enzymes involved in the breakdown of the plant cell walls (e.g. pectate and pectin lyase). The temperature of 27°C was selected as the optimum growth temperature for P. atrosepticum [53 (link)]. In addition, gene expression levels at different growth phases were tested, as these may vary during plant infection. Thus, bacteria were sampled from both exponential and stationary phase in LB medium (Table 3). For leaf infiltration, overnight LB-cultures of P. atrosepticum grown at 27°C were pelleted and resuspended in 10 mM MgSO₄. Leaflets from potato cv. Bintje were vacuum infiltrated with a suspension of 10⁷ bacterial cells/ml for ~15 minutes under low vacuum using a water pump. Negative control leaflets were infiltrated with 10 mM MgSO₄ without bacteria. After infiltration, leaflets were placed on moist filter paper in Petridishes (3–5 leaflets per dish), and incubated at 18°C. This temperature was selected to mimic conditions at which P. atrosepticum optimally causes blackleg and soft rotting symptoms [13 ]. Samples were harvested 21 hours after infiltration, at which point the leaves showed clear rotting symptoms, flash-frozen in liquid nitrogen and kept at -80°C until RNA extraction.