title: Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness.
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.
authors.affiliation: Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Nadal
authors.forename: Maria
authors.initials: M
authors.lastname: Montero
authors.forename: Nuri
authors.initials: N
authors.lastname: Company
authors.forename: Esther
authors.initials: E
authors.lastname: Badosa
authors.forename: Joaquima
authors.initials: J
authors.lastname: Messeguer
authors.forename: Laura
authors.initials: L
authors.lastname: Montesinos
authors.forename: Emilio
authors.initials: E
authors.lastname: Montesinos
authors.forename: Maria
authors.initials: M
authors.lastname: Pla
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D060467:Disease Resistance / Q000276:immunology; D018628:Gene Dosage / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006720:Homozygote; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D012275:Oryza / Q000235:genetics* / Q000382:microbiology / Q000502:physiology* / Q000648:ultrastructure; D018384:Oxidative Stress / Q000187:drug effects; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic / Q000187:drug effects; D019076:Transgenes / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D009842:Oligopeptides; D012333:RNA, Messenger; C540525:lysyl-lysyl-leucyl-phenylalanyl-lysyl-lysyl-isoleucyl-leucyl-lysyl-tyrosyl-leucinamide; D006861:Hydrogen Peroxide