Survival of <i>Galleria melonella</i> larvae after injection with <i>P</i>. <i>aeruginosa</i> PAO1 or A19.

<p>Fifth-instar <i>G</i>. <i>mellonella</i> larvae were injected in the left hind proleg with different amounts of <i>P</i>. <i>aeruginosa</i> PAO1 and A19 bacteria. The image was captured 18 hours post infection. Healthy larvae are cream colored, and darker pigmentation indicates infection. Dead larvae can be recognized from their dark brown-black color.</p

Sensitivity of clinical isolates to three plant-expressed pyocins.

<p>Venn diagram describing pyocin susceptibilities of clinical <i>Pseudomonas aeruginosa</i> isolates. Left–susceptibility of all 100 tested isolates, right–susceptibility of 21 antibiotic-resistant isolates.</p

Pyocin expression in plants.

<p><b>A–schematic presentation of T-DNA region with pyocin expression cassette.</b> RB–right T-DNA border, AtAct23Prom–<i>A</i>. <i>thaliana</i> actin promoter, RdRp–RNA-dependent RNA polymerase, MP–truncated TMV movement protein, LB–left T-DNA border. <b>B–expression of pyocins in <i>N</i>. <i>benthamiana</i> leaves.</b> Plant material (50 mg) was harvested at 5 or 7 days post spraying (dps) (pooled samples of three leaves—pyocins S5, PaeM, PaeM4, L1, L2 at 5 dps, L3 at 7 dps), ground in liquid nitrogen, extracted with 50 mM Tris-HCl, 150 mM NaCl (pH 7.0) (S5, PaeM and PaeM4) or 50 mM HEPES, 10 mM CH₃COOK, 5 mM Mg(CH₃COO)₂, 2 mM DTT, 1 mM EDTA, pH 5.0 (L1, L2, L3) and denatured at 98°C for 10 min. Solutions containing 5 μg of protein were resolved in 12% polyacrylamide gel for Coomassie staining. Mw–PageRuler Prestained protein ladder (ThermoFisher Scientific Baltics), Wt–crude extract of non—sprayed <i>N</i>. <i>benthamiana</i> leaves, S5, M, M4, L1, L2, L3 –extracts of <i>N</i>. <i>benthamiana</i> leaves, sprayed with pyocin expression constructs (pyocin S5, PaeM, PaeM4 and lectin–like pyocins L1, L2, L3). Bands corresponding to recombinant pyocins are marked by arrows. <b>C–purified pyocins</b>. Pyocins were purified by two-step chromatography as described in Purification section of Methods, and resolved in 12% polyacrylamide gel for Coomassie staining.</p

Sensitivity of PAO1 and PAO1 (FiuA) to pyocins.

<p>Plant-produced pyocins S5, L1, PaeM4, PaeM_JJ692 and PaeM _NCTC10332 were applied (5 μg each) on <i>P</i>. <i>aeruginosa</i> PAO1, PW1861 (PAO1 (FiuA)) and PW1862 (PAO1 (FiuA)) lawn and the plates were incubated overnight at 37°C.</p

Agar drop plate assay on different <i>P</i>. <i>aeruginosa</i> isolates with plant-produced pyocins.

<p>Aliquots of 10 μl (10 μg) of purified pyocins were spotted on <i>P</i>. <i>aeruginosa</i> agar lawn and incubated overnight. <b>A–</b><i>P</i>. <i>aeruginosa</i> agar lawn grown on CAA medium, <b>B–</b><i>P</i>. <i>aeruginosa</i> agar lawn grown on LB medium.</p

Survival of larvae pre-infected with <i>P</i>. <i>aeruginosa</i> PAO1 and A19 and treated therapeutically with pyocins.

<p><i>G</i>. <i>melonella</i> larvae were infected with 500 CFU of <i>P</i>. <i>aeruginosa</i> A19 or PAO1 and treated with pyocins 3 hours after infection. <b>A–</b>larvae infected with A19 strain and treated with 10 μg of the indicated pyocin. <b>B–</b>larvae infected with PAO1 strain and treated with 10 μg of the indicated pyocin. <b>C–</b>larvae infected with A19 strain and treated with 1 μg of the indicated pyocin. Killing curves were plotted and estimation of differences in survival analyzed by the Kaplan-Meier method using XLSTAT software.</p

Pyocin activity against biofilms.

<p>One day-old <i>P</i>. <i>aeruginosa</i> biofilms grown in CAA medium were treated with pyocins. <b>A–</b>A19 strain treated with 10 μg mL^-1 of pyocins, when each pyocin is used separately, and 2.5 μg of each pyocin used as a mixture. <b>B</b>–PW2389 (PAO1(mucB)) treated with 100 μg mL^-1 of pyocins. <b>C</b>–PAO1 and PW2389 treated with 10 μg mL^-1 of pyocins. Data are the mean ± SD of at least three separate independent experiments. * Denotes statistical significance (p≤0.001) for comparison of treatment with antimicrobials versus control by a one-way ANOVA test with Bonferroni correction applied. ** no statistical significance (p>0.001).</p

Clustal W alignment of <i>E</i>. <i>coli</i> ColM, <i>P</i>. <i>aeruginosa</i> PaeM, syringacin M and PaeM4 amino acid sequences.

<p>Residues essential for activity of ColM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185782#pone.0185782.ref019" target="_blank">19</a>] are marked by asterisks; residues essential for activity of PaeM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185782#pone.0185782.ref020" target="_blank">20</a>] are marked by black asterisks.</p

Pyocin antibacterial assays in liquid culture.

<p><i>P</i>. <i>aeruginosa</i> strains were cultivated in CAA medium, treated with 5 μg mL^-1 or 50 μg mL^-1 of one or several pyocins and incubated with shaking for 6.5 hours. The antimicrobial activity of pyocins was evaluated by determining cell numbers of bacterial test culture. Serial dilutions of 10, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵ were made, plated on LB agar plates, incubated overnight at 37°C and the CFU calculated. Data are the mean ± SD of three independent experiments. * Denotes statistical significance (p≤0.001) for comparison of treatment with antimicrobials versus control by a one-way ANOVA test with Bonferroni correction applied.</p

Expression and Anthocyanin Biosynthesis-Modulating Potential of Sweet Cherry (<i>Prunus avium</i> L.) MYB10 and bHLH Genes

<div><p>Anthocyanins are essential contributors to fruit coloration, an important quality feature and a breed determining trait of a sweet cherry fruit. It is well established that the biosynthesis of anthocyanins is regulated by an interplay of specific transcription factors belonging to MYB and bHLH families accompanied by a WD40 protein. In this study, we isolated and analyzed <i>PaWD40</i>, <i>PabHLH3</i>, <i>PabHLH33</i>, and several closely related MYB10 gene variants from different cultivars of sweet cherry, analyzed their expression in fruits with different anthocyanin levels at several developmental stages, and determined their capabilities to modulate anthocyanin synthesis in leaves of two <i>Nicotiana</i> species. Our results indicate that transcription level of variant <i>PaMYB10</i>.<i>1-1</i> correlates with fruit coloration, but anthocyanin synthesis in <i>Nicotiana</i> was induced by another variant, <i>PaMYB10</i>.<i>1-3</i>, which is moderately expressed in fruits. The analysis of two fruit-expressed bHLH genes revealed that <i>PabHLH3</i> enhances MYB-induced anthocyanin synthesis, whereas <i>PabHLH33</i> has strong inhibitory properties.</p></div