Molecular characterization and heterologous expression of a pathogen induced PR5 gene from garlic (Allium sativum L.) conferring enhanced resistance to necrotrophic fungi

摘要

Pathogenesis-related protein-5 (PR5) is encoded by a complex group of gene family that are involved in host defense against biotic and abiotic stresses as well as regulation of physiological processes in a wide range of plants and animals. In the present study, we isolated and characterized a PR5 gene, designated as AsPR5, induced in response to Fusarium oxysporum f. sp. cepae (FOC) infection in garlic (Allium sativum). AsPR5 cDNA encoded a protein of 223 amino acids including a 22 amino acid signal peptide at the N-terminus suggesting that it is an apoplast secreted acidic protein. AsPR5 protein contains 16 conserved cysteine residues and five additional conserved amino acids- an arginine, a glutamic acid and three aspartic acids related with antifungal activity of most plant thaumatin like proteins. Semi-quantitative RT-PCR showed that the transcript levels of AsPR5 was higher in stem tissues, the primary site of FOC infection, followed by leaves, roots and flowers. Temporal expression analysis using qPCR revealed high expression of AsPR5 upon infection with FOC as well as various phytohormones (JA, ET and ABA) and stress stimuli (wounding, high temperature and salinity) indicating its involvement in both biotic and abiotic stresses. Analyses of purified recombinant protein against different fungal pathogens showed improved antifungal activity compared to other reported PR5 proteins. Furthermore, the Arabidopsis plants ectopically expressing AsPR5 showed enhanced resistance to fungal pathogen, Botrytis cinerea and constitutively higher expression of defense responsive genes such as Lox3, PDF1.2, PAD3, AOS, and AOC. These results suggest that, besides antifungal activities against the necrotrophs, AsPR5 also play significant role in activating multiple defense pathways for enhancing stress resistance in crop plants.