Isolation and Characterization of Insecticidal Cyclotides from Viola communis

Cyclotides are cysteine-rich plant-derived peptides composed of 28-37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides. The presence of cyclotides in Viola communis was inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communis and other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.