A Proteomic Approach to Evaluate the Effects of Endogenous Expression of Cryptogein Gene in Crypt-Transgenic Plants of Bacopa monnieri

Abstract

Cryptogein, a 10 kDa sterol scavenging protein secreted by the oomycete Phytophthora cryptogea [1], belongs to 'elicitin' class of elicitors [2]. Cryptogein has been used to study the underlying molecular mechanisms of disease resistance in tobacco. Application of purified cryptogein protein, even in nanomolar quantity causes hypersensitive response (HR)-like necrosis in tobacco plants [3] and also induces systemic acquired resistance (SAR) to P. parasitica var nicotianae [4].There are several reports on transformation with cryptogein (crypt) gene aimed to achieve the resistance against plant pathogens. Over expression of β-cryptogein induces HR in tobacco and triggers resistance against P. parasitica var nicotianae [5,6] and other pathogens. Effective increase in biomass and/ or secondary metabolites accumulation in crypt-transgenic cultures in a number of plant species have also been observed [7][8][9], where the crypt gene was either under the constitutive promoter [7] or under the inducible promoter [8]. AbstractCryptogein, a proteinaceous elicitor has been reported to affect growth and secondary metabolism in several plant species. We established two transgenic plant lines of Bacopa monnieri L., an indigenous medicinal species, following Agrobacterium tumefaciens mediated transformation. The transgenic plant lines obtained following transformation with strains LBA4404-nptII-crypt (plant line BmAt-ncrypt) and LBA4404-nptII (plant line BmAt-n), were maintained on selection media i.e. MS medium containing 100 mg/l Kanamycin. To evaluate the effects of endogenous expression of cryptogein gene in crypt-transgenic plants of B. monnieri, in the present study, a proteomic approach was employed. Using 2D-PAGE followed by MALDI TOF MS/MS, we found 54 spots with reproducible differences in abundance (≥ +2 or ≤ -0.5-fold) between the plant lines. Bioinformatics analyses helped their identification, functional annotation and categorization.Significantly upregulated gene expression (p≤ 0.5) of most of the key enzymes of triterpenoid saponin biosynthetic pathway (mevalonate kinase, mvk; mevalonate diphosphate decarboxylase, mdc; squalene synthase, sqs; β-amyrin synthase, bas and UDP-glycosyl transferase 2, ugt 2) in crypt-transgenic plant lines (BmAt-ncrypt) of B. Monnieri was observed as compared to non-transformed (Bm-NT) and empty vector control (BmAt-n) plant lines. Cryptogein-induced ROS production, a typical stress response of plant cells towards cryptogein, was significantly increased (p≤ 0.5) in specific activities and higher abundance of several anti-oxidant enzymes, such as SOD (3.0-5.0-fold), APX (2.3-3.0-fold) and CAT (2.6-fold) in BmAt-ncrypt. The upregulation of CaM (1.6-fold) and calcium-transporting ATPase-2 (2.0-fold) in BmAt-ncrypt indicate possible calcium-dependant cryptogein elicitation. A 3.0-fold increase in EIN3 levels with down-regulation of F-box protein, its regulator also indicate induction of JA-ET signalling pathway in crypt-transgenic plants. This study not only demonstrates involvement of ...