Studies of pathogenesis-related proteins in the strawberry plant: partial purification of a chitinase-containing protein complex and analysis of an osmotin-like protein gene

Abstract

Plant chitinases and osmotin-like proteins (OLPs) are both pathogenesis-related (PR) proteins, which are implicated in plant responses to pathogen attacks and environmental stresses. In this dissertation, a chitinase-containing protein complex was purified to near homogeneity from strawberry leaf extracts. This protein complex contained at least five different chitinase molecules as revealed by activity gel assays. A previous study showed that winter rye leaves contain seven protein complexes, which consist of various combinations of a chitinase, two glucanase-like proteins (GLPs) and a thaumatin-like protein (TLP). Western blot analysis of the strawberry chitinase complex, however, did not detect the presence of any GLP or TLP in the complex. The second part of this dissertation research dealt with studies of strawberry OLP genes. A genomic clone containing an OLP gene, designated FaOLP2, was isolated and completely sequenced. FaOLP2 contains no intron, and has a potential to encode a precursor protein of 229 amino acid residues with a 27-amino acid signal peptide at the N-terminus. Southern blot analysis showed that FaOLP2 represents a small multi-gene family. The expression of FaOLP2 in different strawberry organs was analyzed using real-time PCR. The result showed that FaOLP2 expressed at different levels in leaves, crowns, roots, green fruits and ripe red fruits. Furthermore, the expression of FaOLP2 under different abiotic stresses was analyzed at different time points. All of the three tested abiotic stimuli, abscisic acid, salicylic acid and mechanical wounding, triggered significant induction of FaOLP2 within 2-6 h post-treatment. Comparing the three stimuli, FaOLP2 was more prominently induced by salicylic acid than by abscisic acid or mechanical wounding. The positive responses of FaOLP2 to these stress factors suggested that FaOLP2 may be involved in the protection of strawberry against pathogen attacks and against osmotic-related stresses. In addition to FaOLP2, the expression of a previously cloned OLP gene (FaOLP1) upon fungal infection was examined at different time points post-infection. Each of the two tested fungal species, Colletotrichum fragariae and Colletotrichum acutatum, triggered a substantial induction of FaOLP1 at 24-48 h post-inoculation, indicating that FaOLP1 could be involved in strawberry defense against fungal infection.