Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1

Area of Science:

• Plant immunity
• Antimicrobial peptides
• Molecular cloning

Background:

• Plants utilize diverse molecules for defense against fungal pathogens.
• Defensins are key antimicrobial peptides in plant innate immunity.
• Previous work identified defensins in Scots pine seedlings.

Purpose of the Study:

• To express and purify recombinant Pinus sylvestris defensin 1 (PsDef1).
• To evaluate the antifungal activity of the recombinant defensin.
• To generate antibodies for studying PsDef1 expression levels.

Main Methods:

• Molecular cloning of PsDef1.
• Expression of mature PsDef1 fused to GST using the pET system.
• Proteolytic cleavage with Factor Xa to obtain active rPsDef1.
• Antifungal activity assays.
• Production of polyclonal antibodies.

Main Results:

• Recombinant PsDef1 (rPsDef1) was successfully expressed and purified.
• rPsDef1 exhibited strong antifungal activity against pathogenic fungi, comparable to endogenous defensin.
• rPsDef1 induced specific polyclonal antibodies.
• PsDef1 levels increased during germination and in response to Heterobasidion annosum infection.

Conclusions:

• Recombinant PsDef1 is biologically active and possesses significant antifungal properties.
• PsDef1 expression is upregulated during key developmental and defense-related processes in Scots pine.
• This study provides a tool (rPsDef1 and antibodies) for further investigation of plant defensins.