No abstract

URF13, the product of the mitochondrial T-urfl3 gene, confers on Texas cytoplasmic male-sterile maize (Zea mays L.) a unique susceptibility to a fungal pathogen (Bipolais maydis race T) and sensitivity to its pathotoxin. Expression of URF13 in Escherichia coil imparts pathotoxin sensitivity to the bacterium. We show by ion uptake studies in E. coli that a pathotoxin-URF13 interaction causes membrane permeability. Similarly, mitochondrial dysfunction caused by membrane permeabilization probably accounts for increased colonization of maize carrying the Texas cytoplasm by toxinproducing pathogens. Site-directed mutagenesis studies show that approximately one-quarter of the amino acids at the carboxyl end of URF13 can be eliminated without affecting toxin sensitivity. We have identified two dicyclohexylcarbodiimide (DCCD) binding sites in the URF13 protein and show that one of the sites is involved in conferring DCCD protection against the pathotoxin. Substitutional mutations at this DCCD binding site also eliminate toxin sensitivity.

The first open reading frame (ORF 1) of potato virus X (PVX) encodes a putative replicase gene. Transgenic tobacco lines expressing ORF 1 are resistant to PVX infection when inoculated with either PVX or PVX RNA. Analyses of lines containing various portions of the ORF 1 gene demonstrated that resistance is conferred to plants by expressing approximately the first half of the ORF 1 gene. One line expressing the untranslated leader and first 674 codons of ORF 1 is highly resistant to PVX infection. Conversely, lines expressing either approximately the third or fourth quarter of the ORF 1 gene, which contain the conserved nucleotide triphosphate (NTP) binding motif and Gly-Asp-Asp (GDD) motif, respectively, are not protected from PVX infection. In the resistant full-length and amino-terminal lines, lower numbers of local lesions were observed, and the virus accumulation in the inoculated and upper leaves was reduced when compared with the nontransformed control. When the performance of the most resistant ORF 1 line was compared with the most resistant coat protein (CP) line in a resistance test, the best ORF 1 line was more resistant to PVX infection than the best transgenic line expressing the PVX CP gene. These findings define a promising new approach for controlling plant viral infection.

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13.

Expression of the maize mitochondrial T-urf73 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urfl3 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URFl3 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein.Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13.Binding analysis in E. coli reveals cooperative toxin binding. A low leve1 of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URFl3 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URFl3 important in toxin binding. A third toxin-insensitive URFl3 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URFl3.