title: Reverse genetics in complex multigene operons by co-transformation of the plastid genome and its application to the open reading frame previously designated psbN.
abstract: Reverse genetics approaches have contributed enormously to the elucidation of gene functions in plastid genomes and the determination of structure-function relationships in chloroplast multiprotein complexes. Gene knock-outs are usually performed by disrupting the reading frame of interest with a selectable marker cassette. Site-directed mutagenesis is done by placing the marker into the adjacent intergenic spacer and relying on co-integration of the desired mutation by homologous recombination. These strategies are not applicable to genes residing in large multigene operons or other gene-dense genomic regions, because insertion of the marker cassette into an operon-internal gene or into the nearest intergenic spacer is likely to interfere with expression of adjacent genes in the operon or disrupt cis-elements for the expression of neighboring genes and operons. Here we have explored the possibility of using a co-transformation strategy to mutate a small gene of unknown function (psbN) that is embedded in a complex multigene operon. Although inactivation of psbN resulted in strong impairment of photosynthesis, homoplasmic knock-out lines were readily recovered by co-transformation with a selectable marker integrating >38 kb away from the targeted psbN. Our results suggest co-transformation as a suitable strategy for the functional analysis of plastid genes and operons, which allows the recovery of unselected homoplasmic mutants even if the introduced mutations entail a significant selective disadvantage. Moreover, our data provide evidence for involvement of the psbN gene product in the biogenesis of both photosystem I and photosystem II. We therefore propose to rename the gene product 'photosystem biogenesis factor 1' and the gene pbf1.
authors.affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476, Potsdam-Golm, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Krech
authors.forename: Han-Yi
authors.initials: HY
authors.lastname: Fu
authors.forename: Wolfram
authors.initials: W
authors.lastname: Thiele
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ruf
authors.forename: Mark A
authors.initials: MA
authors.lastname: Schöttler
authors.forename: Ralph
authors.initials: R
authors.lastname: Bock
mesh_terms: D055786:Gene Knockout Techniques / Q000379:methods*; D054627:Genome, Plastid; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009876:Operon / Q000235:genetics*; D010641:Phenotype; D010788:Photosynthesis / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D059386:Reverse Genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D008565:Membrane Proteins; C021261:thylakoid polypeptides
keywords: co-transformation; photosynthesis; plastid; plastid transformation; psbN; reverse genetics; technical advance