Ultrastructural detection of β-1,3-glucans in tobacco root tissues infected by Phytophthora parasitica var. nicotianae using a gold-complexed tobacco β-1,3-glucanase

Abstract

A β-1,3-glucanase, purified to homogeneity from tobacco mosaic virus-infected tobacco plants, was successfully conjugated to colloidal gold at pH 5·5 and used for localizing β-1 ,3-glucans in tobacco root tissues infected by Phytophthora parasitica var. nicotianae. Specificity of the enzyme for β-1,3-glucans was demonstrated by competition in binding using several polysaccharides as potential substrates. Of the various sugars tested only laminarin, a polymer of β-1,3-glucans was found to inhibit the enzyme-binding activity efficiently. In addition, the abolition of labelling over isolated fungal cell walls previously treated to remove β-1,3-glucan further confirmed the strict affinity of the tobacco β-1,3-glucanase for β-1,3-glucan molecules. In healthy tobacco root tissues, β-1,3-glucans occurred as small deposits in sieve elements and were seldom detected in other tissues. However, in fungus-infected tobacco root tissues, large amounts of β-1,3-glucans (callose) were found at sites of attempted pathogen penetration such as sieve tube members, plasmodesmata, and intercellular spaces. Papillae, thought to be physical barriers formed in response to infection, contained β-1,3-glucans distributed mainly over an amorphous matrix. The occurrence of β-1,3-glucan molecules in host cell walls adjacent to fungal cells raises the question of their origin. The possibility that they are released from the fungal cell wall through the action of plant β-1,3-glucanases is discussed. The present study demonstrates the potential value of the tobacco β-1,3-glucanase (PR-N) for ultrastructural studies on β-1,3-glucans in plant biology and pathology.