Expression and Functional Analysis of Two Osmotin (PR5) Isoforms with Differential Antifungal Activity from Piper colubrinum: Prediction of Structure–Function Relationship by Bioinformatics Approach

Abstract

Osmotin, a pathogenesis-related antifungal protein, is relevant in induced plant immunity and belongs to the thaumatin-like group of proteins (TLPs). This article describes comparative structural and functional analysis of the two osmotin isoforms cloned from Phytophthora-resistant wild Piper colubrinum. The two isoforms differ mainly by an internal deletion of 50 amino acid residues which separates them into two size categories (16.4 kDa-PcOSM1 and 21.5 kDa-PcOSM2) with pI values 5.6 and 8.3, respectively. Recombinant proteins were expressed in E. coli and antifungal activity assays of the purified proteins demonstrated significant inhibitory activity of the larger osmotin isoform (PcOSM2) on Phytophthora capsici and Fusarium oxysporum, and a markedly reduced antifungal potential of the smaller isoform (PcOSM1). Homology modelling of the proteins indicated structural alterations in their three-dimensional architecture. Tertiary structure of PcOSM2 conformed to the known structure of osmotin, with domain I comprising of 12 β-sheets, an α-helical domain II and a domain III composed of 2 β-sheets. PcOSM1 (smaller isoform) exhibited a distorted, indistinguishable domain III and loss of 4 β-sheets in domain I. Interestingly, an interdomain acidic cleft between domains I and II, containing an optimally placed endoglucanase catalytic pair composed of Glu-Asp residues, which is characteristic of antifungal PR5 proteins, was present in both isoforms. It is well accepted that the presence of an acidic cleft correlates with antifungal activity due to the presence of endoglucanase catalytic property, and hence the present observation of significantly reduced antifungal capacity of PcOSM1 despite the presence of a strong acidic cleft, is suggestive of the possible roles played by other structural features like domain I or/and III, in deciding the antifungal potential of osmotin.

Cloning and characterization of Osmotin (OsmSt) alleles from potato cultivar ‘Kufri Chipsona 1’

“…Osmotin and its various isoforms have been cloned from many plants (Capelli et al 1997, Pla et al 1998, Jami et al 2007, Singh et al 2017, Mani et al 2012, Li et al 2015. The presence of osmotin isoforms in same plant have been previously reported in Piper colubrinum (Mani et al 2012). The two isoforms were reportedly distinct from each other in terms of size, pI value and molecular structure.…”

“…Generally, the presence of 16 cysteine residues have been reported in osmotin (Kumar et al 2015), but in the present study, only 15 cysteine residues were present which lead to formation of 7 disulphide bridge only (Figure 3). The presence of disulphide bridges is known to provide high stability to the protein (Mani et al 2012). The instability index (II) calculated for OsmSt1 and OsmSt2 using ProtPram tool of Expasy server were found to be about 38.90 which indicates that the proteins are highly stable (https://web.expasy.org/protparam/).…”

“…On the basis of their biochemical properties and mechanism of action, PR proteins are divided into 17 families including β-1,3 glucanases (PR-2), chitinases 4,8 and 11), thaumatin-like proteins (PR-5), ribosome inactivating proteins (PR-10), defensins (PR-12), thionins (PR-13) and lipid transfer proteins (PR-14) (Kaur et al 2017a). Among these, PR-5 protein family is known for its antimicrobial activity, osmoregulation and role in plant development (Mani et al 2012). PR-5 family is named as thaumatin-like protein (TLPs) due to its close similarity with sweet tasting protein thaumatin of plant Thaumatococcus danielli .…”

“…The protein have been cloned from various plants such as Nicotiana tabaccum (Lui et al 1994, Evers et al 1999, Zuker et al 2001, Ouyang et al 2005, Qin et al 2006, Noori and Sokhansanj 2008, Parkhi et al 2009, Goel et al 2010, Husaini et al 2012, Subramanyam et al 2011, Solanum nigrum (Vasavirama and Kirti 2012), Capsicum annuum (Choi et al 2013), Solanum tuberosum (Rivero et al 2012), etc. The over-expression of osmotin have been reported to exhibit anti-fungal activity against diverse range of pathogens such as Puccinia triticina (Li et al 2015), Fusarium graminearum , Mackintosh et al 2007, Phytophthora capsici, Fusarium oxysporum (Zuker et al 2001, Mani et al 2012, Phytophthora infestans (Liu et al 1994, 1996, Li et al 1999, Rhizoctonia solani (Rivero et al 2012), etc. The anti-fungal property of the protein attributes to its capability to bind to domains present on the fungal plasma membrane (Thevissen et al 2005) which leads to dissipation of membrane integrity and can also inhibit spore germination through spore lysis (Koiwa et al 1997).…”

Eleven <i>StOSMs</i> Genes in the Potato Genome Response to Water Deficiency

“…Subsequently, it was showed that abscisic acid (ABA), low temperature and NaCl can induce the accumulation of three StOSM mRNAs (pA13, pA35, and pA81) in S. commersonii grown in vitro (Zhu et al 1995). Later on StOSM was shown to exhibit antifungal activity against a range of fungal pathogens (Rivero et al 2012, Mani et al 2012and Patade et al 2013. Recently, StOSM overexpression in transgenic Arabidopsis and pepper results in H 2 O 2 accumulation and the response of hypersensitive cell death in the leaf and reduces susceptibility to pathogen infection (Choi et al 2013).…”