Isolation of precise plastid deletion mutants by homology‐based excision: a resource for site‐directed mutagenesis, multi‐gene changes and high‐throughput plastid transformation

Abstract

SummaryWe describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aadAbased transformation to duplicate a 649 bp region of plastid DNA corresponding to the atpB promoter region. Efficient recombination between atpB repeats deletes the intervening foreign genes and 1984 bp of plastid DNA (co-ordinates 57 424-59 317) containing the rbcL gene. Only five foreign bases are present in DrbcL plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbcL-related sequences in DrbcL plants indicated an extra-plastidic origin. Mutant DrbcL plants were heterotrophic, palegreen and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aadA-based transformation with the wild-type rbcL gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aadA genes. Rescue of the DrbcL mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbcL deletion mutant isolated here is readily transformed with the efficient aadA marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.

Transformation of Tobacco cpDNA with FusionE7GGG/GUSGene and Homologous Recombination Mediated Elimination of the Marker Gene

“…Other strategies include excision by phage site-specific recombinases (11), transient co-integration of the marker gene (12) or co-transformation followed by segregation (3). Different variants of the homology-based marker excision strategy were evolved lately (7,14) but the original procedure that we used in this study, seems to be the simplest and highly efficient approach. Rt-PcR (reverse transcription PcR) assays showed ( fig.…”

Study of Plastid Genome Stability in Tobacco Reveals That the Loss of Marker Genes Is More Likely by Gene Conversion Than by Recombination between 34-bp loxP Repeats

“…To obtain reasonably frequent deletions, 649-bp repeats were used because a 418-bp sequence seldom yields deletions (Iamtham and Day, 2000;Kode et al, 2006). In this laboratory, the bar au gene flanked by an 84-bp promoter repeat was lost in 0.6% of the seed progeny (10 events in 1,584 selfed seed progeny; Lutz et al, 2007).…”

“…Deletion of ptDNA sequences between direct repeats by the plastid's homologous recombination machinery has been exploited for plastid marker gene excision (Iamtham and Day, 2000;Kode et al, 2006). The CRE recombinase target sites are identical 34-bp directly oriented loxP sequences.…”

A Guide to Choosing Vectors for Transformation of the Plastid Genome of Higher Plants

“…D, Larger variegated seedling among smaller aurea siblings. apart barely yielded any deletions (Iamtham and Day, 2000;Kode et al, 2006). We now only recommend vectors targeting the trnV-3#rps12 intergenic region, in which the Prrn promoter is in an inverted orientation relative to the native rRNA operon.…”