Antimicrobial peptides in the seedling transcriptome of the tree legume Peltophorum dubium.

Antimicrobial peptides (AMPs) play an essential role in plant defense against invading pathogens. Due to their biological properties, these molecules have been considered useful for drug development, as novel agents in disease therapeutics, applicable to both agriculture and medicine. New technologies of massive sequencing open opportunities to discover novel AMP encoding genes in wild plant species. This work aimed to identify cysteine-rich AMPs from Peltophorum dubium, a legume tree from South America. We performed whole-transcriptome sequencing of P. dubium seedlings followed by de novo transcriptome assembly, uncovering 78 AMP transcripts classified into five families: hevein-like, lipid-transfer proteins (LTPs), alpha hairpinins, defensins, and snakin/GASA (Giberellic Acid Stimulated in Arabidopsis) peptides. No transcripts with similarity to cyclotide or thionin genes were identified. Genomic DNA analysis by PCR confirmed the presence of 18 genes encoding six putative defensins and 12 snakin/GASA peptides and allowed the characterization of their exon-intron structure. The present work demonstrates that AMP prediction from a wild species is possible using RNA sequencing and de novo transcriptome assembly, regarding a starting point for studies focused on AMP gene evolution and expression. Moreover, this study allowed the detection of strong AMP candidates for drug development and novel biotechnological products.

Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).

Snakins are antimicrobial peptides (AMPs) found, so far, exclusively in plants, and known to be important in the defense against a wide range of pathogens. Like other plant AMPs, they contain several positively charged amino acids, and an even number of cysteine residues forming disulfide bridges which are considered important for their usual function. Despite its importance, studies on snakin tertiary structure and mode of action are still scarce. In this study, a new snakin-like gene was isolated from the native plant Peltophorum dubium, and its expression was verified in seedlings and adult leaves. The deduced peptide (PdSN1) shows 84% sequence identity with potato snakin-1 mature peptide, with the 12 cysteines characteristic from this peptide family at the GASA domain. The mature PdSN1 coding sequence was successfully expressed in Escherichia coli. The purified recombinant peptide inhibits the growth of important plant and human pathogens, like the economically relevant potato pathogen Streptomyces scabies and the opportunistic fungi Candida albicans and Aspergillus niger. Finally, homology and ab initio modeling techniques coupled to extensive molecular dynamics simulations were used to gain insight on the 3D structure of PdSN1, which exhibited a helix-turn-helix motif conserved in both native and recombinant peptides. We found this motif to be strongly coded in the sequence of PdSN1, as it is stable under different patterns of disulfide bonds connectivity, and even when the 12 cysteines are considered in their reduced form, explaining the previous experimental evidences.