EXPRESSION OF BIOLOGICALLY-ACTIVE HORDOTHIONINS IN TOBACCO - EFFECTS OF PRE-SEQUENCES 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 barl ey endosperm which are processed from larger precursor proteins, consi sting of an amino-terminal signal peptide (SP), the mature highly basi c HTH and a carboxy-terminal acidic peptide (AP). Different HTH precur sor proteins were expressed in tobacco to study the effects of the pre -sequences (SP) and pro-sequences (AP) on expression, processing, sort ing and biological activity and hence the feasibility of engineering b acterial disease resistance into crops which lack these proteins. Maxi mum HTH expression levels of approximately 0.7% (11 mu mol/kg) of tota l soluble protein in young tobacco leaves were obtained using a semi-s ynthetic gene construct encoding a complete chimaeric HTH precursor pr otein. Tenfold lower HTH expression levels (maximum 1.3 mu mol/kg) wer e obtained using synthetic gene constructs without the AP-coding seque nce and no expression was found in plants containing synthetic HTH gen e constructs without SP-and AP-coding sequences. In both cases where e xpression was found, the precursors were apparently correctly processe d, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant pheno type 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 intercellu lar spaces of leaves or culture medium by protoplasts. Our results ind icated that the AP is not involved in sorting and suggested that it mi ght facilitate transport through membranes. The in vitro toxicity of H TH isolated from transgenic tobacco plants expressing the two differen t precursor proteins for the bacterial plant pathogen Clavibacter mich iganensis subsp. michiganensis appeared similar to that of the HTH pur ified from barley endosperm.