Production of an engineered killer peptide in Nicotiana benthamiana by using a potato virus X expression system

Abstract

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.

FIG. 1.

In vitro inhibitory activity of the KP synthetic decapeptide against bacterial and fungal pathogens. (A) The growth inhibition activity of synthetic KP (50-μg/ml concentration) against P. corrugata, P. syringae pv. tomato (NCPPB 1106, NCPPB 2563, and DAPP-PG 214), P. syringae pv. tabaci (Pt11528), and E. carotovora was measured by the reduction in the number of CFU compared to that with the irrelevant SP scramble peptide used as a control. Growth inhibition was calculated with the following equation: percentage of inhibition = (1 − number of CFU for KP treated/number of CFU for SP treated) × 100. (B) Effect of KP on spore germination of F. oxysporum and B. cinerea. Spore germination assay at a KP concentration of 50 μg/ml shows a 50% inhibition of B. cinerea and a 62% inhibition of F. oxysporum. Values are means of triplicate determinations ± standard errors of the means.

FIG. 2.

Schematic representation of PVX-derived vectors. The diagrams show the locations of the viral genes coding for the viral replicase (166K), the movement proteins (25K, 8K, and 12K), and the coat protein (CP) of the PVX-Sma variant. The PVX-Sma virus bears an N-terminal deletion of 22 amino acids compared to the wild-type PVX-201 (5) and was used for the antimicrobial peptide fusions. The viral genome is inserted between the constitutive promoter 35S, derived from cauliflower mosaic virus, and the transcription terminator of the nopaline synthase gene (NOS), which if important for the regulation of viral genome expression upon plant infection with plasmid DNA. (A) pPVX-KP is the plasmid carrying the virus genome engineered to express the KP sequence fused to the CP and was obtained by inserting a double-stranded oligonucleotide between the SmaI and NheI sites of the pPVX-Sma plasmid, derived from pPVX-201 (5). The SP sequence corresponding to the scramble peptide used as a negative control was also cloned between the SmaI and NheI sites of pPVX-Sma, yielding plasmid pPVX-SP (see Materials and Methods). (B) Phenotypes at 12 days postinoculation of leaves of Nicotiana benthamiana that were systemically infected with pPVX-KP and pPVX-SP. No difference was observed in the viral symptoms compared to those after infection with the virus without the insert (PVX-Sma) and the wild-type PVX-201. After the mechanical inoculation on a basal leaf, the virus moves from cell to cell in the infected tissue through the plasmodesmata and spreads systemically in the upper leaves through the phloem vein network. As shown, PVX produces typical mosaic symptoms on systemic leaves. Further experiments demonstrated that the modified CPs were highly stable and retained their ability to form CVPs after three cycles of reinfection.

FIG. 3.

Analysis of PVX-infected plants. Western blot analysis of total leaf protein from Nicotiana benthamiana leaves is shown. Gels were blotted to polyvinylidene difluoride membranes, and immunodetection was performed using anti-PVX CP mouse polyclonal antibodies (Adgen Limited, United Kingdom). An amount of 5 μg of total leaf protein was analyzed in each lane. Lane 1, proteins from upper leaves of pPVX-Sma-infected plants; lane 2, proteins from upper leaves of pPVX-KP-inoculated plants; lane 3, proteins from leaves of pPVX-SP-infected plants; lane 4, mock-inoculated plants used as negative controls. The results indicate the presence of single bands with the expected molecular weight (approximately 23,900) corresponding to KP-CP and SP-CP fusion proteins (lanes 2 and 3, respectively). The coat protein of the PVX-Sma virus bearing no peptide fusion is also shown as a control (lane 1), showing a lower band as expected (molecular weight, 23,000). The arrow indicates the position of the 25-kDa band of the molecular mass marker (Rainbow Markers; Amersham).

FIG. 4.

In vitro inhibitory activity of the PVX-KP chimeric virus particles against bacterial and fungal pathogens. (A) Growth inhibition activity of PVX-KP (50 μg/ml) against P. corrugata, P. syringae (pv. tomato NCPPB 1106, NCPPB 2563, and DAPP-PG 214), and E. carotovora was measured by the reduction in the number of CFU compared to the controls treated with the irrelevant PVX-SP. Growth inhibition was calculated by the following equation: percentage of inhibition = (1 − number of CFU for PVX-KP treated/number of CFU for PVX-SP treated) × 100. The in vitro activity against the human pathogens Candida albicans and Staphylococcus aureus at a concentration of 100 μg/ml is also reported (shaded bars). (B) Effect of PVX-KP on spore germination of Fusarium oxysporum and Botrytis cinerea. The reduction in the number of germinated spores was compared to that for controls treated with the irrelevant PVX-SP. Spore germination assay at 50-μg/ml PVX-KP shows a 90% inhibition of B. cinerea and a 95% inhibition of F. oxysporum. Values are means of triplicate determinations ± standard errors of the means.

FIG. 5.

In vitro activity of the recombinant PVX-KP virus against P. syringae pv. tomato DAPP-PG 214 compared to the chemically synthesized KP decapeptide. Growth inhibition was monitored counting the number of surviving bacteria (CFU) at different incubation times. After incubation for 1 h with 50 μg/ml of PVX-KP, the rate of bacterial killing was 99%, whereas with KP decapeptide (50 μg/ml), the killing rate was 21% at 1 h and reached 92% after 4 h. It should be noted that the concentration of 50 μg/ml of PVX-KP corresponds to a theoretical concentration of fused peptide of 2 μg/ml. As negative controls, bacteria were incubated either with PBS, with the chemically synthesized SP scramble peptide, or with the PVX-SP virus displaying the scramble peptide. Values for each time point are means of triplicate determinations ± standard errors of the means.

FIG. 6.

In planta assays. N. benthamiana plants were inoculated with the viral constructs pPVX-KP, bearing the killer peptide, and pPVX-SP, bearing the irrelevant scramble peptide as a control. After 5 days, the upper leaves of these plants were challenge infiltrated with diluted cultures of P. syringae pv. tabaci (Pst) (10⁵ CFU/ml LB medium) by infiltrating bacteria into the lamina with a needleless 5-ml syringe. (A) Representative pictures of the chlorotic and necrotic symptoms observed after 8 days from the infiltration experiments conducted with P. syringae pv. tabaci (top). The symptoms appear weaker with pPVX-KP-inoculated plants than with pPVX-SP-inoculated plants, used as negative controls. Mock-infiltrated plants (H₂O), used as negative controls (bottom), show no necrotic area, as expected. (B) The number of surviving bacteria following the leaf infiltration with P. syringae was determined. Leaf disks (1-cm diameter) were collected within a single infiltrated area from each treated plant and were quickly ground in 500 μl LB. Homogenates were plated on LB agar at three dilutions (10⁻¹, 10⁻², and 10⁻³). P. syringae colonies were counted after incubation at 28°C for 24 to 48 h. A reduced number of surviving bacteria is observed in the PVX-KP-infected plants compared to plants infected with PVX-SP and PVX-Sma, lacking the peptide, used as controls. A plant not infected with virus (N.I.) but infiltrated with P. syringae was also used as a control. Data for plants infiltrated with distilled water are also reported (H₂O). Values are means ± standard errors of the means from three independent experiments.