Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria

Abstract

Antimicrobial peptides (AMPs) are small peptides with less than 50 amino acids and are part of the innate immune response in almost all organisms, including bacteria, vertebrates, invertebrates and plants. AMPs are active against a broad-spectrum of pathogens. The inducible expression of AMPs in plants is a promising approach to combat plant pathogens with minimal negative side effects, such as phytotoxicity or infertility. In this study, inducible expression of the de-novo designed AMP SP1-1 in Micro Tom tomato protected tomato fruits against bacterial spot disease caused by Xanthomonas campestris pv. vesicatoria. The peptide SP1-1 was targeted to the apoplast which is the primary infection site for plant pathogens, by fusing SP1-1 peptide to the signal peptide RsAFP1 of radish (Raphanus sativus). The pathogen inducibility of the expression was enabled by using an optimized inducible 4XW2/4XS promoter. As a result, the tomato fruits of independently generated SP1-1 transgenic lines were significantly more resistant to X. campestris pv. vesicatoria than WT tomato fruits. In transgenic lines, bacterial infection was reduced up to 65% in comparison to the infection of WT plants. Our study demonstrates that the combination of the 4XW2/4XS cis-element from parsley with the synthetic antimicrobial peptide SP1-1 is a good alternative to protect tomato fruits against infections with X. campestris pv. vesicatoria.

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

X. campestris pv vesicatoria (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.

Fig 2. Induction of pathogen-inducible promoter 4XW2 in response to Pep25 and X. campestris pv. vesicatoria.

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 X. campestris pv. vesicatoria 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 (http://imagej.nih.gov/ij/). Scale bar: 20 μm. Asterisks indicate significant different in comparison to the corresponding control treatment, *P<0.05, **P<0.01, ***P<001.

Fig 3. 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.

Fig 4. X. campestris pv. vesicatoria induces expression of SP1-1 in tomato fruits.

Expression of SP1-1 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 SP1-1 after Mock treatment (MgCl₂) was set to 1. Fold change of expression of SP1-1 in X. campestris pv. vesicatoria 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

Fig 5. Resistance of tomato fruits of T0 transgenic plants against X. campestris pv. vesicatoria infection.

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