ANTIMICROBIAL PEPTIDES FROM MIRABILIS-JALAPA AND AMARANTHUS-CAUDATUS - EXPRESSION, PROCESSING, LOCALIZATION AND BIOLOGICAL-ACTIVITY IN TRANSGENIC TOBACCO

The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabil is jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are proce ssed from a precursor preprotein and preproprotein, respectively [De B olle ed al., Plant Mol Biol 28:713-721. (1995) and 22:1187-1190 (1993) , respectively]. In order to study the processing, sorting and biologi cal activity of these antimicrobial peptides in transgenic tobacco, fo ur different gene constructs were made: a Mj-AMP2 wild-type gene const ruct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacu olar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 ( 1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 m utant gene construct which was truncated in order to delete the sequen ce encoding the genuine carboxyl-terminal propeptide. Processing and l ocalization analysis indicated that an isoform of Ac-AMP2 with a cleav ed-off carboxyl-terminal arginine was localized in the intercellular f luid fraction of plants expressing either wild-type or mutant gene con structs. Mj-AMP2 was recovered extracellularly in plants transformed w ith Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly i n plants expressing the mutant precursor protein with the barley lecti n propeptide. The in vitro antifungal activity of the AMPs purified fr om transgenic tobacco expressing any of the four different precursor p roteins was similar to that of the authentic proteins. However, none o f the transgenic plants showed enhanced resistance against infection w ith either Botrytis cinerea or Alternaria longipes.