Isolation and immunolocalization of a Pinus nigra lectin (PNL) during interaction with the necrotrophs—Heterobasidion annosum and Fusarium avenaceum

Abstract

Pinus nigra ARN. lectin (PNL) was isolated from protein bodies of European black pine seeds by affinity chromatography of protein bodies extract on GlcNAc- C -glycosylated Spheron 300 beads. In vitro tests confirmed that PNL has the ability to agglutinate rat erythrocytes, GlcNAc- C -glycosylated HEMA BIO 1000 microbeads and Trichoderma viride spores. Western blot immuno-assays using antibodies against PNL antigen demonstrated specificity of the antisera. Immuno dot assays with polyclonal antibody against the purified PNL exhibited strong cross reaction to both Scots pine and Norway spruce seed extracts but not to fungal cell wall extracts from either Heterobasidion annosum orFusarium avenaceum or Phlebiopsis gigantea. The indirect immunofluorescence assay showed a strong interaction of PNL with hyphal materials of the necrotrophs (H. annosum and F. avenaceum). Results from immunolocalization experiments suggest that PNL is not degraded during seed germination and development since it was localized in all parts of juvenile pine seedlings. The protein was mainly observed on the cytoplasmic membranes and on the primary cell walls. In infected seedlings, a strong labelling of hyphal materials with PNL antisera was recorded only at the early stages of infection but not at the later stages of hyphal invasion. At advanced stages of the infection, most of the fungal hyphae were covered by host phenolic-like substances which probably masked the interaction of the antiserum with pathogen cell wall materials; hence the low labelling. The potential function of PNL as a recognition molecule during interaction of conifer trees with necrotrophic parasites is discussed.