Isolation and characterization of a mutant protoporphyrinogen oxidase genefrom Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxida se (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive p rotoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment enco ding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a h igh frequency of herbicide-resistant transformants, consistent with frequen t non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfr agment (Xho3.4) yields rare herbicide-resistant transformants, suggestive o f homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized th e rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% ident ity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment rev ealed a G --> A change at 291 in the first putative exon, resulting in a Va l --> Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fra gments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G -->A change at the equivalent position (5751) within exon 10.