Structure, dynamics, and function of PsDef2 defensin from Pinus sylvestris.

Plant defensins demonstrate high structural stability at extreme temperatures and pH values and, in general, are non-toxic to mammalian cells. These properties make them attractive candidates for use in biotechnology and biomedicine. Knowing the structure-function relationship is desirable to guide the design of plant defensin-based applications. Thus far, the broad range of biological activities was described only for one defensin from gymnosperms, the defensin PsDef1 from Scots pine. Here, we report that closely related defensin from the same taxonomy group, PsDef2, differing from PsDef1 by six amino acids, also possesses antimicrobial, antibacterial, and insect α-amylase inhibitory activities. We also report the solution structure and dynamics properties of PsDef2 assessed using a combination of experimental nuclear magnetic resonance (NMR) techniques. Lastly, we perform a comparative analysis of PsDef2 and PsDef1 gaining a molecular-level insight into their structure-dynamics-function relationship.

Seed-derived defensins from Scots pine: structural and functional features.

MAIN CONCLUSION: The recombinant PsDef5.1 defensin inhibits the growth of phytopathogenic fungi, Gram-positive and Gram-negative bacteria, and human pathogen Candida albicans. Expression of seed-derived Scots pine defensins is tissue-specific and developmentally regulated. Plant defensins are ubiquitous antimicrobial peptides that possess a broad spectrum of activities and multi-functionality. The genes for these antimicrobial proteins form a multigenic family in the plant genome and are expressed in every organ. Most of the known defensins have been isolated from seeds of various monocot and dicot species, but seed-derived defensins have not yet been characterized in gymnosperms. This study presents the isolation of two new 249 bp cDNA sequences from Scots pine seeds with 97.9% nucleotide homology named PsDef5.1 and PsDef5.2. Their deduced amino acid sequences have typical plant defensin features, including an endoplasmic reticulum signal sequence of 31 amino acids (aa), followed by a characteristic defensin domain of 51 aa. To elucidate the functional activity of new defensins, we expressed the mature form of PsDef5.1 in a prokaryotic system. The purified recombinant peptide exhibited activity against the phytopathogenic fungi and Gram-negative and Gram-positive bacteria with the IC50 of 5-18 µM. Moreover, it inhibited the growth of the human pathogen Candida albicans with the IC50 of 6.0 µM. Expression analysis showed that transcripts of PsDef5.1-2 genes were present in immature and mature pine seeds and different parts of seedlings at the early stage of germination. In addition, unlike the PsDef5.2, the PsDef5.1 gene was expressed in the reproductive organs. Our findings indicate that novel defensins are promising candidates for transgenic application and the development of new antimicrobial drugs.