Induction of pathogen-inducible promoter 4XW2 in response to Pep25 and <i>X</i>. <i>campestris</i> pv. <i>vesicatoria</i>.

<p>The 4XW2-GFP-GUS was transiently transformed into TH3 tomato protoplasts using a modified PEG method. GFP fluorescence was monitored 12–16 h after water (A and B), pep25 (C and D) and <i>X</i>. <i>campestris</i> pv. <i>vesicatoria</i> treatment (E and F). (G) Number of protoplasts showing GFP fluorescence is given in percent. (H) Quantification of GFP fluorescence intensity was done using the Image J software (<a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>). Scale bar: 20 μm. Asterisks indicate significant different in comparison to the corresponding control treatment, *P<0.05, **P<0.01, ***P<001.</p

<i>X</i>. <i>campestris</i> pv. <i>vesicatoria</i> induces expression of <i>SP1-1</i> in tomato fruits.

<p>Expression of <i>SP1-1</i> was analyzed by quantitative RT-PCR in tomato fruits of T583-4, T583-5 and T583-6 36 h after inoculation and normalized to two internal reference genes (ubiquitin and actin). Expression of <i>SP1-1</i> after Mock treatment (MgCl₂) was set to 1. Fold change of expression of <i>SP1-1</i> in <i>X</i>. <i>campestris</i> pv. <i>vesicatoria</i> treated samples is given relative to the expression in Mock treated samples. Data are the mean ±SD of two to three biological replicates. Significant differences from the control are indicated: ***, P<0.001 **, P<0.01 and *, P<0.05</p

Resistance of tomato fruits of T0 transgenic plants against <i>X</i>. <i>campestris pv</i>. <i>vesicatoria</i> infection.

<p>(A) Infection symptoms of WT and transgenic T0 tomato fruits overexpressing <i>SP1-1</i> (T583-4, T583-5 and T583-6) after Mock treatment (MgCl₂) or inoculation with <i>X</i>. <i>campestris</i> pv. <i>vesicatoria</i> (10⁶ CFU/ml). Pictures are taken two days after treatment. (B) Incidence of infection symptoms two days after inoculation with <i>X</i>. <i>campestris</i> pv. <i>vesicatoria</i> is given in percentage. The values represent the mean of three independent experiments +/- SE.</p

Transgenic tomato Micro Tom lines expressing the AMP SP1-1.

<p>(A) Schematic illustration of PMIGW-4XW2/4XS::SP1-1 vector construct. The vector contains a CaMV35S promoter-driven phosphomannose isomerase (PMI) gene for mannose selection. B1, B2, B3 and B4 represent attB Gateway recombination sites. SP, signal peptide RsAFP1 from radish; SP1-1, synthetic antimicrobial peptide; TNos, nopaline synthase gene terminator; RB, right border; LB, left border; W2 <i>cis</i>-acting elements from the parsley PR1 gene; S, <i>cis</i>-elements from the parsley EL17 gene; CaMV35S, minimal promoter containing the sequence -46 to +8 from the cauliflower mosaic virus 35S promoter; T35S, terminator from the cauliflower mosaic virus 35S. (B) Molecular characterization of transformed plants. PCR was done using DNA from young leaves as template and RsAFP1-TNos-specific primers. PCR fragments were obtained for T583-4, T583-5 and T583-6. PMIGW-4XW2/4XS::SP1-1 transformation vectors and WT Micro Tom tomato plants were used as positive and negative controls respectively. (C and D) Morphological phenotypes of six weeks old WT and transgenic T1 Micro Tom tomato plants. Transgenic and WT plants were grown in climate chambers for 6 weeks. (E) Detached leaves of line T583 were infiltrated with water (H) or pep25 peptide (P). Total RNA was extracted before induction (BI) and 0, 3, and 22 h after induction. RT-PCR was carried out with gene-specific primers for RsAFP1-SP1-1 and actin and fragments of 163 bp and 586 bp were expected, respectively. Genomic DNA of the transgenic line T583 was used as control (+). Total RNA from WT plants and water (-) was used as negative control. The SP1-1 band is marked with an arrow. M, 100 bp DNA ladder.</p

Antibacterial activity of synthetic peptide SP1-1 in the presence of tomato apoplastic fluid.

<p><i>X</i>. <i>campestris</i> pv <i>vesicatoria</i> (10⁵ cfu/ml) was incubated with 0 or 10 μg/ml of peptide SP1-1 in the presence or absence of 10 μg/ml of tomato apoplastic fluid. Bacterial growth was determined by measuring OD_600nm (OD 0.2 = 10⁸ cfu/ml) 15 hours after APO; tomato apoplastic fluid. Values represent the mean of at least three biological replicates ± standard error of the mean. Asterisks indicate significant different in comparison to the corresponding control treatment, *P<0.05, **P<0.01.</p

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

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

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

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