PRODUCTION OF A CHIMERIC PROTEIN ELICITOR MF2/MF3 AND EVALUATION OF ITS PROTECTIVE ACTIVITY AGAINST TOBACCO MOSAIC VIRUS

Abstract

or MF3 taken alone at a concentration twice exceeding the content of each protein in the tested mixture. These findings open the prospects of a combined use of both elicitors. However, preparation of such mixture requires production of both proteins via their heterologous expression in two different E. coli clones transformed with the corresponding encoding genes. Moreover, the corresponding procedures of isolation and purification would be required for each protein. In this work, the MF2/MF3 chimeric protein was first obtained and its protective activity in the tobacco plant ( Nicotiana tabacum L.)  tobacco mosaic virus (TMV, family Virgaviridae , genus To-bamovirus , species Tobacco mosaic virus) pathosystem was demonstrated. It was also established for the first time that the protective activity of MF2/MF3 is not inferior to the activity of a mixture of individual proteins taken in equivalent concentrations. The purpose of our study was obtaining of a chimeric MF2/MF3 protein via heterologous expression in Escherichia coli and a comparison of its protective activity with that of individual MF2 or MF3 and their mixture. The chimeric MF2/MF3 protein is a structure composed of two domains, in which MF2 protein was linked to the N-term i nal region of MF3 via the polyalanine linker (AAALIAA). The MF2/MF3 protein encoding sequences were constructed by the use of classic and overlap PCR. Recombinant MF2/MF3 protein was expressed by transformed E. coli Rosetta cells in the form of a 25-kDa water-soluble protein and purified to the homogeneity by immobilized metal affinity chromatography (IMAC). To evaluate the antiviral activity of the MF2/MF3 hybrid and to compare its protective effect with that of individual proteins and their mixtures, a biotest with pre-inoculation treatments of detached tobacco leaves of the necrosis-forming variety Xanthi NN, which were then infected with TMV, was used. Plants were grown in a climate