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

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness Nadal et al. Nadal et al. BMC Plant Biology 2012, 12:159 http://www.biomedcentral.com/1471-2229/12/1/159 Nadal et al. BMC Plant Biology 2012, 12:159 http://www.biomedcentral.com/1471-2229/12/1/159 RESEARCH ARTICLE Open Access Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness Anna Nadal1†, Maria Montero1†, Nuri Company1, Esther Badosa1, Joaquima Messeguer2, Laura Montesinos1, Emilio Montesinos1 and Maria Pla1* Abstract Background: 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. 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. Results: Our 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. Conclusions: Constitutive 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. Keywords: Antimicrobial peptide AMP, BP100, Transgenic rice, Oryza sativa, Hostplant fitness, Pathogen-resistant rice * Correspondence: † Equal contributors 1 Institute of Food and Agricultural Technology (INTEA), University of Girona, Campus Montilivi, EPS-1 17071, Girona, Spain Full list of author information is available at the end of the article © 2012 Nadal et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nadal et al. BMC Plant Biology 2012, 12:159 http://www.biomedcentral.com/1471-2229/12/1/159 Background 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 [1-3] bacteria; [4,5] fungi; [6,7] insects; [8-10] amphibian and mammals, and [11] plants). Around 1,000 AMPs have been reported [12]. They can structurally be linear peptides (often adopting α-helical structures); cysteine-rich open-ended peptides with disulfide bridges; cyclopeptides forming a peptide ring, or pseudopeptides. 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 [13-15], 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]. In recent years, novel peptides have been designed, based on natural AMPs, with the aim of optimizing the activity against selected target pathogens (including microorganisms against which no AMP or antibiotic are known) while decreasing toxicity to non-target organisms and increasing stability. Short truncated compounds (minimal domain), chimerical constructions and improved sequence analogs have been reported. Examples are mellitin derivatives blocking plant viruses [18], the anti-fungal and anti-bacterial lactoferricin B derivatives [13], antifungal cecropin A and cecropin A-mellitin derived peptides [19-21], and the de novo designed antifungal hexapeptide PAF26 [22,23] and bactericide cyclic decapeptide BPC194 series [19,20]. Genetically modified (GM) plants with different degrees of resistance to pathogens have been obtained by expression of native or synthetic analogues of AMPs, either constitutively or in response to pathogen attack (reviewed in [13,14]). These include AMPs naturally produced by insects [24-29] and amphibians [30]; fungal [15,31] and plant defensins [32-36]; and modified AMP analogues such as the magainine derived Myp30 [37] and MSI-99 [38-40], MsrA3, derived from temporin A [41] and MsrA2, derived from dermaseptin B1 [42], the chimeric peptides MsrA1 and CEMA derived from cecropin A and mellitin [43,44] and the synthetic D4E1 peptide [45,46]. The expression of these AMPs in plants including tobacco, rice, potato, tomato, grapevine and cotton, have been found to give moderate resistance to relevant plant pathogenic bacteria or fungi. Combinatorial chemistry approaches have been used to assist the design of new AMPs with superior properties. Page 2 of 21 The CECMEL11 peptide library, a 125-member linear undecapeptide library contains groups of sequences with high activity against a number of reporter bacterial and fungal phytopathogenic species, several also exhibiting lowsensitivity to protease degradation and hemolytic activity [47,48]. These peptides were cecropin A-mellitin hybrids and had the structure of an amp (...truncated)