Antimicrobial peptides from Mirabilis jalapa and Amaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

• Published in: Plant molecular biology Vol. 31; no. 5; p. 993
• Main Authors: De Bolle, M F, Osborn, R W, Goderis, I J, Noe, L, Acland, D, Hart, C A, Torrekens, S, Van Leuven, F, Broekaert, W F
• Format: Journal Article
• Language: English
• Published: Netherlands 01.08.1996
• Subjects: alternaria; Amino Acid Sequence; Antifungal Agents - isolation & purification; Antifungal Agents - metabolism; Antifungal Agents - pharmacology; antimicrobials; antimicrobianos; antimicrobien; Biological Transport; botrytis cinerea; Cell Compartmentation; code genetique; codigo genetico; disease resistance; Fluorescent Antibody Technique; genetic code; Immunoblotting; Microbial Sensitivity Tests; Mitosporic Fungi - drug effects; Molecular Sequence Data; nicotiana; Nicotiana - genetics; peptide; peptides; peptidos; Plant Diseases; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Proteins - pharmacology; plantas transgenicas; plante transgenique; Plants, Genetically Modified; Plants, Toxic; Protein Processing, Post-Translational; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; resistance aux maladies; resistencia a la enfermedad; Sequence Analysis; Tissue Distribution; Transformation, Genetic; transgenic plants
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/BF00040718

The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolle et al., Plant Mol Biol 28:713-721 (1995) and 22:1187-1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2 wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 (1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing either wild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. The in vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with either Botrytis cinerea or Alternaria longipes.