Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum

• Published in: Gene 2015-03, Vol.558 (1), p.143-151
• Main Authors: Rather, Irshad Ahmad, Awasthi, Praveen, Mahajan, Vidushi, Bedi, Yashbir S., Vishwakarma, Ram A., Gandhi, Sumit G.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antifungal; Antifungal Agents - chemistry; Antifungal Agents - isolation & purification; Antifungal Agents - pharmacology; Cloning, Molecular; Elicitor; MicroRNA; Modeling; Models, Molecular; Molecular Sequence Data; Ocimum basilicum; Ocimum basilicum - genetics; Ocimum basilicum - metabolism; Phylogeny; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - pharmacology; PR-5 protein; Sequence Alignment; Thaumatin; Yeasts - drug effects
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2014.12.055

Pathogenesis-related (PR) proteins are involved in biotic and abiotic stress responses of plants and are grouped into 17 families (PR-1 to PR-17). PR-5 family includes proteins related to thaumatin and osmotin, with several members possessing antimicrobial properties. In this study, a PR-5 gene showing a high degree of homology with osmotin-like protein was isolated from sweet basil (Ocimum basilicum L.). A complete open reading frame consisting of 675 nucleotides, coding for a precursor protein, was obtained by PCR amplification. Based on sequence comparisons with tobacco osmotin and other osmotin-like proteins (OLPs), this protein was named ObOLP. The predicted mature protein is 225 amino acids in length and contains 16 cysteine residues that may potentially form eight disulfide bonds, a signature common to most PR-5 proteins. Among the various abiotic stress treatments tested, including high salt, mechanical wounding and exogenous phytohormone/elicitor treatments; methyl jasmonate (MeJA) and mechanical wounding significantly induced the expression of ObOLP gene. The coding sequence of ObOLP was cloned and expressed in a bacterial host resulting in a 25kDa recombinant-HIS tagged protein, displaying antifungal activity. The ObOLP protein sequence appears to contain an N-terminal signal peptide with signatures of secretory pathway. Further, our experimental data shows that ObOLP expression is regulated transcriptionally and in silico analysis suggests that it may be post-transcriptionally and post-translationally regulated through microRNAs and post-translational protein modifications, respectively. This study appears to be the first report of isolation and characterization of osmotin-like protein gene from O. basilicum. [Display omitted] •ObOLP, an osmotin-like protein gene was isolated from Ocimum basilicum.•ObOLP exhibits significant induction on treatment with MeJA and wounding.•Recombinant ObOLP protein displays antifungal activity.•ObOLP may be expressed during herbivory and provide protection from fungal pathogens.