Cloning and cell-free translation of Phytoplasma solani Bax Inhibitor 1

Introduction Phytoplasmas are pleomorphic wall-less bacteria, members of the class Mollicutes. They are associated with more than 700 diseases, affecting hundreds of plants genera. Plants have developed different defense mechanisms against pathogens. Programmed Cell Death (PCD) is one of these. This mechanism allows plants to remove the infected cells, while maintaining the integrity of the remaining organism. The membrane protein Bax Inhibitor-1 (BI-1) is an important PCD regulator able to act in animals, plants, phytoplasmas and yeasts. Little is still known about the mechanism of action of the phytoplasmatic BI-1 protein within the host plant and its interaction with the cellular components. Being Phytoplasmas non-cultivable organisms and because its low expression levels, BI-I has never been purified. The purpose of this research was to clone and express the ‘Candidatus Phytoplasma solani’ Bax Inhibitor-1 in a cell-free (CF) system to produce the protein in vitro for future structural and functional studies. Methods cDNA was synthesized from the total RNA extracted from infected plants. BI-1 gene was amplified by PCR using specific primers introducing SmaI and NcoI restriction sites, and ligated in pIVEX2.4d vector using T4 DNA ligase. Sequencing confirmed the correct ORF maintenance. Cell free translation was performed in one mL of translationally active E. coli extracts. Results The synthesized BI-1 protein was purified by affinity chromatography, using Ni-NTA resin. The correct size of the purified protein was demonstrated by SDS-PAGE. Only one band with the expected Mr was visible the gel confirmed the BI-1 synthesis. The reaction yield was assessed, too. Conclusion Results indicate that BI-1 can be expressed in vitro using a cell-free system. Further work is necessary to test the optimal expression conditions to stabilize the structure of this membrane proteins, thus allowing the assessment of its biological activity.