Pseudomonas syringae

To determine if exposure of Arabidopsis seedlings to bacterial volatiles elicited ISR against Pseudomonas syringae pv. maculicola ES4326, we developed a 24-well microtitre-based disease assay system (Fig. 1A). The 24-multimicrotitre plate of bacterial suspensions of PGPR strains B. subtilis GB03 and P. polymyxa E681 at OD₆₀₀ = 1 (10^8–9 cfu/ml) was inoculated in an empty well (without plant). For assessing ISR by bacterial volatile and its derivatives, the I-plate system was employed. After application of 30 µl of 10 mM and 100 µM decane, undecane, and dodecane, the I-plate was tightly sealed with parafilm. Seven days after inoculation with PGPR or VOCs, 2.5 ml of a bacterial suspension (at OD₆₀₀ = 1) of P. syringae pv. maculicola ES4326 grown on King's B medium was added to each well. A whole seedling in each well was then soaked in the bacterial suspension for 5 min, and the suspension was removed. The plant was rinsed with sterile distilled water three times. The 24-multimicroliter plate with challenged Arabidopsis seedlings was then placed in a growth chamber that was maintained at 21°C in 12/12 day and night condition. Disease severity was measured four to seven days after pathogen challenge.

Draft genome sequences for each strain were searched by tBLASTn (at first with an with e-value cutoff of 10⁻⁵, but later with no cutoff) for the presence of known TTEs. This list was constructed by combining protein sequences for known P. syringae TTEs (http://pseudomonas-syringae.org/) with our list of novel TTEs identified from a subset of these genomes (see previous section). The potential TTE sequence was then pulled out of the draft genome sequence to the next possible stop codon and, if there was no identifiable start codon based on similarity to known TTEs, up to the earliest possible start codon after an upstream stop codon. The position of the start codon was further refined by relationship to an identifiable hrp-box or by comparison to other known sequences. When possible, if there was a frameshift or early stop codon that led to early protein termination (such that the locus was split into two halves that were each orthologous to a given TTE) or if there was a scaffold break disrupting a potential TTE, genomic sequences were bridged or verified by PCR-based sequencing. In some cases, the presence of TTEs could not be verified because PCR-based sequencing failed on 3 separate attempts, or because only a partial sequence of the TTE was present on a contig with no ability to bridge a gap. In some cases, there were loci in the genomes that matched by BLAST, but were significantly diverged from previously identified TTEs or were novel chimeras. In these cases, at least one subfamily member from these TTE families were cloned and tested for translocation (Dataset S9). Sequences identified as HrpL-regulated by screen or as potential TTE by similiarity, but which were not tested for translocation, are also listed in Dataset S9.
Draft genome sequences were also searched for pathways involved in construction of six well known phytotoxins associated with P. syringae (coronatine, phaseolotoxin, tabtoxin, syringomycin, syringopeptin, syringolin) as well as genes for ethylene production (efe), auxin production and modification (iaaM, iaaH, and iaaL) and an enzyme whose activity leads to secretion of an HR inducing factor, syringolide (avrD). Protein sequences for loci involved in toxin metabolic pathways in various strains were obtained from NCBI and used as a tBLASTn query on each draft genome sequence. A strain was considered to possess a toxin or gene if a majority of the protein sequences for each pathway had significant BLAST hits (<1e⁻⁵) with an average similarity of 80% or greater. If some, but not all, of the pathway for a particular toxin was present, the strain was considered to potentially possess the toxin.

Phi6 bacteriophage and its host bacterium, Pseudomonas syringae serovar phaseolicola HB10Y were obtained from Ann Vidaver, University of Nebraska during earlier research of Prof. Bamford [28 (link)]. HB10Y was aerobically grown in Luria-Berthani (LB) broth at 22 °C, 23 °C, or 28 °C depending on the experiment (see further).

The antibacterial activities were evaluated by the conventional broth dilution assay [45 (link)]. Five phytopathogenic bacteria (Xanthomonas citri pv. malvacearum, X. axonopodis, Comamonas terrigena, Pseudomonas syringae, and Dickeya chrysanthemi), four animal pathogenic bacteria (Escherichia coli, P. aeruginosa, Staphylococcus aureus, and Bacillus subtilis), and eight marine fouling bacteria (Aeromonas hydrophila, A. salmonicida, Enterobacter cloacae, P. fulva, Vibrio anguillarum, V. harveyi, Photobacterium halotolerans, and P. angustum) were used, and cipofloxacin (CPFX) and DMSO were used as the positive and negative control, respectively. The antibacterial activity assay was carried out by using previously described methods [22 (link),23 (link)]. The tested concentrations of isolated compounds and CPFX were 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.13 µM, 1.56 µM, 0.78 µM, 0.39 µM, and 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.13 µM, 1.56 µM, 0.78 µM, 0.39 µM, 0.20 µM, 0.10 µM, 0.049 µM, and 0.024 µM, respectively.