Journal: PLoS ONE
Article Title: Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria
Figure Lengend Snippet: Transgenic tomato Micro Tom lines expressing the AMP SP1-1. (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 cis -acting elements from the parsley PR1 gene; S, cis -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.
Article Snippet: PCR amplifications from genomic DNA were done using the PCR super mix (Invitrogen) following the manufacturer´s instructions.
Techniques: Transgenic Assay, Expressing, Plasmid Preparation, Construct, Selection, Sequencing, Transformation Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control