TARGETING THE MAIZE T-URF13 PRODUCT INTO TOBACCO MITOCHONDRIA CONFERSMETHOMYL SENSITIVITY TO MITOCHONDRIAL RESPIRATION

The URF13 protein, which is encoded by the maize mitochondrial T-urf13 gene, is thought to be responsible for pathotoxin and methomyl sensit ivity and male sterility, We have investigated whether T-urf13 confers toxin sensitivity and male sterility when expressed in another plant species, The coding sequence of T-urf13 was fused to a mitochondrial t argeting presequence, placed under the control of the cauliflower mosa ic virus 35S promoter, and introduced into tobacco by Agrobacterium tu mefaciens-mediated transformation. Plants expressing high levels of UR F13 were methomyl sensitive, Subcellular analysis indicated that URF13 is mainly associated with the mitochondria, Adding methomyl to isolat ed mitochondria stimulated NADH-linked respiration and uncoupled oxida tive phosphorylation, indicating that URF13 was imported into the mito chondria, and conferred toxin sensitivity. Most control plants, which expressed the T-urf13 construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction, S ubcellular fractionation by sucrose gradient centrifugation showed tha t URF13 sedimented at several positions, suggesting the protein is ass ociated with various organelles, including mitochondria, No methomyl e ffect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mit ochondria. Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria, There was no correlatio n between the expression of URF13 and male sterility, suggesting eithe r that URF13 does not cause male sterility in transgenic tobacco or th at URF13 is not expressed in sufficient amounts in the appropriate ant her cells.