Production of BP178, a derivative of the synthetic antibacterial peptide BP100, in the rice seed endosperm

Abstract

Background: BP178 peptide is a synthetic BP100-magainin derivative possessing strong inhibitory activity against plant pathogenic bacteria, offering a great potential for future applications in plant protection and other fields. Here we report the production and recovery of a bioactive BP178 peptide using rice seeds as biofactories.

Results: A synthetic gene encoding the BP178 peptide was prepared and introduced in rice plants. The gene was efficiently expressed in transgenic rice under the control of an endosperm-specific promoter. Among the three endosperm-specific rice promoters (Glutelin B1, Glutelin B4 or Globulin 1), best results were obtained when using the Globulin 1 promoter. The BP178 peptide accumulated in the seed endosperm and was easily recovered from rice seeds using a simple procedure with a yield of 21 μg/g. The transgene was stably inherited for at least three generations, and peptide accumulation remained stable during long term storage of transgenic seeds. The purified peptide showed in vitro activity against the bacterial plant pathogen Dickeya sp., the causal agent of the dark brown sheath rot of rice. Seedlings of transgenic events showed enhanced resistance to the fungal pathogen Fusarium verticillioides, supporting that the in planta produced peptide was biologically active.

Conclusions: The strategy developed in this work for the sustainable production of BP178 peptide using rice seeds as biofactories represents a promising system for future production of peptides for plant protection and possibly in other fields.

Keywords: Antimicrobial peptide; Oryza sativa; Pathogen resistance; Peptide recovery; Protein bodies; Rice biofactory.

Fig. 1

Schematic representation of the plant expression vectors for the expression of the BP178 gene in the rice endosperm. a Three different constructs in pCAMBIA vector (pC) in which the synthetic gene was cloned between the endosperm-specific promoter including the signal peptide coding sequence of the corresponding seed storage protein (SP) and the nopaline synthase terminator (nos). Relevant restriction enzyme sites for cloning purposes are indicated. The hptII gene encoding resistance to hygromycin under the control of the CaMV35S promoter and terminator was contained into the T-DNA region of pC. LB, left border; RB, right border of T-DNA. Arrows indicate the orientation of the sequence. b The chimeric BP178 peptide corresponding to the fusion of BP134 (KKLFKKILKYL-OH, a cecropin A (1–7)-melittin (2–9) hybrid, in green) linked through the hinge sequence (AGPA, in orange) to a magainin fragment peptide (GIGKFLHSAKKFGKAFVGEIMNS –OH, in blue); and extended with ER retention signal (KDEL, in pink). For details, see Additional file 1: Figure S1

Fig. 2

BP178 peptide accumulates in the endosperm of Glb1:BP178 transgenic rice seeds without altering accumulation levels of native storage proteins. a Coomassie Blue staining of PB extracts from the indicated seeds. EV indicates seeds from plants transformed with the pCAMBIA 1300 empty vector. b Ponceau staining of protein samples (50 μg) c Western-blot analysis and quantification of the BP178 peptide in the protein body-enriched fractions (50 μg) from mature seed endosperms of the indicated pGlb1:BP178, pGluB4:BP178, pGluB1:BP178 and empty vector (EV) lines. The accumulation levels of BP178 peptide were quantified with the Quantity tools of the Chemidoc program using the 0.3, 0.15 and 0.05 μg amounts of synthetic BP178 peptide as a reference (taking into account the sum of the three bands observed). Numbers below the western blot lanes correspond to μg of BP178 peptide. d Western-blot analysis of protein body-enriched fractions (50 μg) from embryos, leaves or roots of the indicated pGlb1:BP178, and empty vector (EV) lines. For comparative purposes, 0.30, 0.15 and 0.05 μg of synthetic BP178 peptide were run simultaneously in the Tricine-SDS gels. Immunodetection was performed using specific polyclonal anti-BP178KDEL antibodies and coloured phosphatase alkaline reaction

Fig. 3

Stability of the BP178 peptide accumulated in 3 years-old seeds. The endosperm tissue was manually dissected from mature seeds (0-days) and germinating rice seeds (3 and 7 days of germination). Protein extracts (35 μg) were separated by Tricine-SDS gels, transferred onto PVDF membrane, and incubated with the anti-BP178 antibody followed by anti-rabbit IgG (Fc) alkaline phosphatase conjugated secondary antibody. EV, rice plants with the empty pCAMBIA 1300 vector were used as a negative control, and 3.2 μg of synthetic BP178 were used as reference control. Arrows, indicate BP178 A-related bands in rice extracts. Numbers in the left side indicate the size of the molecular markers (kDa). Lower panels show Ponceau staining of protein samples

Fig. 4

Purification, quantification and antibacterial activity of the BP178 peptide recovered from the pGlb1:BP178 rice seeds. a Schematic procedure for purification. BP178 peptide was recovered at high purity in the acetone supernatant (2) b Tris-Tricine SDS-PAGE and western-blot analysis using anti-BP178 antibodies of fractions recovered during the purification process described in (a) starting from protein body fractions (50 μg) from seeds from pGlb1:BP178 line 7.2. Numbers in the top of panel (b) correspond to: 1, Supernatant; 2, Pelleted protein bodies; 3, Pellet. c Peptide yield from seeds of the indicated pGlb1:BP178 events. The amount of BP178 was calculated by comparing the signal intensity of known amounts of standard (synthetic peptide). Values correspond to the mean of at least 3 independent assays and the standard error is indicated. d Antimicrobial activity against Dickeya sp. of two batches of purified BP178-fractions from pGlb1:BP178 lines. Empty vector (EV) and reference synthetic BP178 peptide were used as control. Mean values of two replicates and the standard error are indicated

Fig. 5

Resistance of BP178 rice seeds to Dickeya sp. infection. a Phenotypical appearance of wild-type (Senia and Ariete cultivars), empty vector (EV) and the transgenic seedlings carrying the indicated transgenes after 7 days in contact with Dickeya sp. 1552.10.1 bacterial suspensions (10⁷ cfu/mL). Pictures are representative of at least 2 independent assays in which 12 seeds per line were analysed. b Germination capability of Dickeya-infected T3 rice seeds expressing the BP178 gene. Mean germination index (12 seeds) in inoculated (dark grey) or non-inoculated (pale grey) seeds at 7 days after inoculation. Values correspond to the mean germination index of two independent assays. The standard error is also indicated. The asterisks indicate statistically significant differences compared to the WT and empty vector mean (Tukey’s test, *p < 0.001)

Fig. 6

Resistance of BP178-rice seeds to Fusarium verticillioides infection. a Phenotypical appearance of wild-type (WT) and transgenic seedlings carrying the indicated transgenes or the empty vector (EV) 7 days after germination in contact with F. verticillioides spore suspensions (10⁵ spores/mL). Pictures are representative of 3 independent assays b Germination capability of the fungal inoculated transgenic seeds referred to wild-type seeds. Values correspond to the mean germination rate and standard errors of three independent assays in which at least 3 independent lines per transgene were analysed. Asterisks denote statistically significant differences with wild-type and empty vector plants (Tukey’s test, *p < 0.001)