Antibacterial activity of synthetic peptides in presence of apoplast fluid and in tomato fruits.

<p>(A) Approximately 10⁵ cfu/ml bacteria (<i>P. syringae</i> pv <i>tomato</i>) were incubated with 0 or 10 µg/ml peptide in the presence or absence of different concentrations (10 µg/ml or 30 µg/ml) of tomato apoplastic fluid. After 14–16 h the bacterial growth was determined by measuring OD_{600 nm}. APO, tomato apoplastic fluid. Values represent the mean of at least three biological replicates ± standard error of the mean. *indicates significantly different in comparison to the corresponding control treatment, <i>P</i><0.05. **indicates significantly different in comparison to the corresponding control treatment, <i>P</i><0.01. (B) <i>X. vesicatoria</i> (0.5×10⁵ cfu/ml) were treated with different concentrations of peptide SP10-5 and immediately injected into tomato fruits. After incubation for 5 d at room temperature infection symptoms were monitored. Above the values of incidence of infection symptoms is given in percentage. The total number of inoculation sides of three biological replicates were 22.</p

Sequences and structural-chemical properties of peptides of the 2^nd generation.

a<p>Estimated using the program Vector NTI 9.1 (Invitrogen).</p>b<p>Calculated using ProtParam tool (<a href="http://www.expasy.org/tools/protparam.html" target="_blank">http://www.expasy.org/tools/protparam.html</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Gasteiger1" target="_blank">[30]</a>), H [peptide], grand average hydrophobicity of full peptide.</p>c<p>H [cluster], hydrophobicity of the hydrophobic cluster of the peptides with the calculation based on the hydrophobicity scales for amino acids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Eisenberg1" target="_blank">[29]</a>. pI, isoelectric point. Special features: Important alterations in comparison to the leading structure are highlighted.>increased,</p

Sequences and structural-chemical properties of peptides of the 1^st generation.

a<p>Estimated using the program Vector NTI 9.1 (Invitrogen).</p>b<p>Calculated using ProtParam tool (<a href="http://www.expasy.org/tools/protparam.html" target="_blank">http://www.expasy.org/tools/protparam.html</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Gasteiger2" target="_blank">[84]</a>), H [peptide], grand average hydrophobicity of full peptide.</p>c<p>H [cluster], hydrophobicity of the hydrophobic cluster of the peptides with the calculation based on the hydrophobicity scales for amino acids (Eisenberg, 1984).</p>d<p>Secondary structure prediction according to NNPREDICT; H, helix; E, strand; -, no prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071687#pone.0071687-Kneller1" target="_blank">[31]</a>. pI, isoelectric point.</p

Growth inhibition of phytopathogens on tomato leaves.

<p>Tomato leaves were inoculated with (A) virulent <i>P. syringae pv. tomato</i> DC3000 (10⁷ CFU/ml) or (B) spores of <i>A. alternata</i> or <i>C. herbarum</i> (10⁴ spores/ml). Afterwards different concentrations of antimicrobial peptides were sprayed onto the leaves. Bacterial growth was monitored 30 min after peptide treatment by determining colony-forming units per defined leaf area. Fungal growth was analysed 48 h after peptide treatment by quantification of fungal DNA content in the leave tissue. Fungal growth on leaves treated with peptide dilution buffer was set to 100%. Values represent the mean of at least three biological replicates ± standard error of the mean. *indicates significantly lower than the control treatment, <i>P</i><0.05.</p

Structural analyses of selected peptides in presence of lipophilic compounds.

<p>Circular dichroism (CD) spectra of SP1-1, SP8, SP10-10 and SP13. (A) CD spectra of peptides in ddH₂O reveal that they are random coil. (B) In the presence of 1,2-dimyristoyl-sn-glycero-3-phospho-sn-glycerol (DMPG) micelles the peptides displayed an α-helical structure. Peptide and DMPG concentrations used for the measurements were 130 µM and 1 mM, respectively.</p

Effect of designed antimicrobial peptides on the viability of Arabidopsis mesophyll protoplasts <i>in-vitro</i>.

<p>Protoplasts were incubated for 1 h with different concentrations of SP1-1, SP10-2 and SP10-5 and analysed with a microscope (x 200). Cells with spherical shape without any sign of cytoplasmic degradation were defined as viable. A change of the cell shape, chloroplast release and/or agglomeration of protoplasts indicates a toxic effect of the peptides on plant cells (arrows).</p

Antimicrobial activities (MIC) and hemolytic activities of peptides of the 2^nd generation.

<p>Values reflect the MIC (µg/ml) after different incubation periods as indicated in brackets after the organism.</p>a<p>Shown are the peptide concentrations leading to 25% hemoglobin release from human blood cells.</p><p>>200 describes a slight hemolytic activity at 200 µg/ml but still below the above mentioned threshold, no value indicates none detectable hemolytic activity up to the highest concentration tested.</p

Bacterial membrane depolarization 60 minutes after AMP treatment.

<p>Depolarisation of bacterial membranes was determined by loading 5×10⁷ cfu/ml gram-positive <i>C. michiganensis</i> or gram-negative <i>P. syringae pv. syringae</i> with DiSC3(5) and measuring fluorescence intensity (FI) 60 min after addition of 0.5, 1, 5 or 10 µg/ml peptides (λ_ex: 622 nm λ_em: 670 nm). 1% Sodium dodecyl sulfate (SDS) and SP8 were used as positive and negative control, respectively. Shown are mean values ± standard error of the mean of three independent measurements normalized against FI after buffer treatment.</p

Antimicrobial activities (MIC) of designed first generation peptides against plant pathogens and hemolytic activities.

a<p>Shown are the peptide concentrations (µg/ml) leading to 25% hemoglobin release from human blood cells.</p><p>>200 describes a slight hemolytic activity at 200 µg/ml but still below the above mentioned threshold.</p