Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants: evidence for a Kex2p pathway in plants.

Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. We show here that one of these toxins, the KP6 killer toxin, is synthesized by transgenic tobacco plants containing the viral toxin cDNA under the control of a cauliflower mosaic virus promoter. The two components of the KP6 toxin, designated alpha and beta, with activity and specificity identical to those found in toxin secreted by U. maydis cells, were isolated from the intercellular fluid of the transgenic tobacco plants. The beta polypeptide from tobacco was identical in size and N-terminal sequence to the U. maydis KP6 beta polypeptide. Processing of the KP6 preprotoxin in U. maydis requires a subtilisin-like processing protease, Kex2p, which is present in both animal and fungal cells and is required for processing of (among other things) small secreted polypeptide hormones and secreted toxins. Our findings present evidence for Kex2p-like processing activity in plants. The systemic production of this viral killer toxin in crop plants may provide a new method of engineering biological control of fungal pathogens in crop plants.     

In vivo analysis of intron processing using splicing-dependent reporter gene assays

The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cultured cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.