Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP-and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.