Molecular Identification and Antifungal Activity of a Defensin (PaDef) from Spruce

Abstract

Defensins, as part of the PR protein family, not only have antimicrobial activity in vitro, but also play an important role in plant disease resistance. To explore the molecular function of defensin in spruce, a defensin gene PaDef was isolated and characterized from Picea asperata. The full-length cDNA sequence of the PaDef was 264 bp and contained an ORF of 252 bp, encoding a total of 83 amino acids. The deduced PaDef protein belongs to the gamma-thionin family and contains the Knot1 domain and eight conserved cysteine residues. Blast and phylogenetic analysis showed that the PaDef protein shared high homology with other plant defensin proteins. The tertiary structure of PaDef was obtained by homologous modelling using the structure of Pinus sylvestris defensin as a template and exhibited a βαββ structure. SDS-PAGE analysis showed that the molecular mass of recombinant PaDef expressed in Escherichia coli was 23.8 kDa. Plate assay showed that purified PaDef exhibited strong antifungal activity inhibiting the growth of Pestalotiopsis neolitseae, but not Colletotrichum gloeosporioides and Botrytis cinerea. Microscopic observation revealed that purified PaDef had a negative effect on the hyphal morphology of all three phytopathogens. qRT-PCR analysis indicated that the PaDef gene had the highest expression level in needles of P. asperata compared with roots, phloem, twigs, and could be significantly upregulated by the pathogenic fungus Lophodermium piceae, which causes needle cast disease. These results provide a molecular basis for determining the molecular function of defensin PaDef in P. asperata and its potential as an antifungal agent.