Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

Anna Nadal,Esther Badosa,Maria Montero,Emilio Montesinos,Laura Montesinos,Joaquima Messeguer,Nuri Company,Maria Pla

doi:10.1186/1471-2229-12-159

Abstract

BackgroundThe Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept.ResultsOur design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines.ConclusionsConstitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

Highlights

The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity
The length of the BP100 undecapeptide, a short molecule, needs to be increased above a minimum threshold to achieve expression in plant systems. Accumulation of this bactericidal peptide in plants could potentially result in BP100 degradation by plant proteolytic activities and/or produce plant cell damage even though cytotoxicity and protease susceptibility were minimized during the BP100 selection procedure
We found that transformation of rice with genes encoding α-helical cationic antimicrobial peptide (AMP) such as those derived from the synthetic 11 amino acids-long BP100 had, in many cases, a highly negative effect on the efficiency of transformation

Summary

Introduction

The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. Antimicrobial peptides (AMPs) are short sequence peptides, normally less than 50 amino acid residues, reported in living systems They are components of the defense system against pathogens in plants and animals or are produced by microorganisms in antibiosis processes (see reviews in [1,2,3] bacteria; [4,5] fungi; [6,7] insects; [8,9,10] amphibian and mammals, and [11] plants). AMPs offer major perspectives as a novel class of therapeutic agents, especially against fungal infections and antibiotic-resistant bacterial pathogens in humans and animals [7,9] This great potential extends to plant disease-protection products [13,14,15], as substitutes of antibiotics in animal feed, biopreservatives in food, cosmetics and biomaterials, and as antifouling agents [16,17]. AMPs have proved successful as biopesticides, with commercial development of several microorganisms secreting these compounds [14]

Objectives

Methods

Results

Discussion

Conclusion