Indian Journal of Biotechnology Vol 16, April 2017, pp 195-198 Relative efficiencies of transient transformation of Arabidopsis thaliana and Nicotiana tabacum upon heterologous expression of membrane proteins Panchsheela Nogia, Vandana Tomar, Gurpreet Kaur Sidhu, Rajesh Mehrotra and Sandhya Mehrotra* Department of Biological Sciences, Birla Institute of Technology and Science, Pilani 333031, India A cyanobacterial gene encoding a protein with multiple transmembrane helices, when transformed into Arabidopsis thaliana and Nicotiana tabacum, demonstrated several differences in the relative responses of both the host species, particularly in efficiencies of regeneration of transformed tissue from leaf explants. The gene of interest (GOI) encoded an integral membrane protein containing 12 transmembrane helices and functioned as a sodium dependent inorganic ion transporter in a native cyanobacterium. Agrobacterium tumefaciens-mediated transformation was carried out by coculture method. The leaves of N. tabacum successfully regenerated into transformed callus tissue and gave positive results upon GUS assay; while the A. thaliana leaves did not give any transformed callus and, a few days post agro-infection, all the explants displayed tissue necrosis. The results obtained point towards A. thaliana's recalcitrance to Agrobacterium-mediated transformation by coculture method and the difficulty of expressing hydrophobic membrane proteins in heterologous systems. Keywords: Agrobacterium tumefaciens, Arabidopsis thaliana, hydrophobic membrane protein, Nicotiana tabacum, transformation efficiency Introduction Transient expression studies provide a base for analyzing success rate of transgene expression in a heterologous host system. Arabidopsis thaliana and a few Nicotiana species (N. tabacum, N. benthamiana & N. attenuata) are widely used by researchers as model host plants to study protein expression following various transformation methods. A. thaliana, in terms of its competency with different transformation methods, has been well studied using a variety of explants and its performance has been investigated in various transient transformation experiments, such as, young seedlings transformed by vacuum infiltration 1 , leaves transformed by agroinfiltration 2 and coculture 3 method. However, nowadays, these methods are rarely used due to the poor demonstration of A. thaliana plants in transient expression studies. Instead, A. thaliana is now most widely used to develop stable plants by floral dip method 4 , which has proved to be a highly successful and efficient transformation method. Several Nicotiana species, on the other hand, are relatively easy to transform by Agrobacterium-mediated coculture, agroinfiltration and particle bombardment methods, and are found to be effective and resilient for both transient as well as stable protein expression experiments 5 . The biochemical nature of proteins also influences their expression in heterologous systems. Of various categories, the membrane proteins being of hydrophobic nature offer the greatest challenge. The membrane proteins play various important roles in the living cells and function as transporters, receptors, enzymes etc. In most organisms, they constitute about 20-30% to the total protein. High-level expression of membrane proteins remains a challenge to this day because of their low natural abundance and tendency to form aggregates 6 . These features also make them difficult targets for structural characterization. For optimized expression of hydrophobic proteins, various host systems are tested but it is still a major challenge. Possible reasons behind the lack of success can be that these proteins may have some lethal effects on the host or have undergone alterations at the molecular level upon expression. In addition, the host machinery may or may not allow proper folding and activation of the protein. Researchers have been using genes from different organisms and have tried their expression in various bacterial, plant and other eukaryotic heterologous host systems but the success rate is always variable with respect to both the type and complexity of host and protein 7 . Specifically, in case of heterologous systems, we need to optimize all the parameters related to host growth and transformation according to the nature of the protein. Apart from the discussed aspects so far, —————— *Author for correspondence sandhyamehrotrabits@gmail.com