Endochitinase activity in the apoplastic fluid of Phellinus weirii‐infected Douglas‐fir and its association with over wintering and antifreeze activity

Extracellular proteins were extracted from Phellinus weirii infected Douglas‐fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase‐like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two‐dimensional gel electrophoresis (2‐D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27–30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii‐infected winter Douglas‐fir needles showed anti‐freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response.