Involvement of Rad52 in T‐DNA circle formation during Agrobacterium tumefaciens‐mediated transformation of Saccharomyces cerevisiae

Abstract

SummaryAgrobacterium tumefaciens cells carrying a tumour inducing plasmid (Ti-plasmid) can transfer a defined region of transfer DNA (T-DNA) to plant cells as well as to yeast. This process of Agrobacterium-mediated transformation (AMT) eventually results in the incorporation of the T-DNA in the genomic DNA of the recipient cells. All available evidence indicates that T-strand transfer closely resembles conjugal DNA transfer as found between Gram-negative bacteria. However, where conjugal plasmid DNA transfer starts via relaxase-mediated processing of a single origin of transfer (oriT), the T-DNA is flanked by two imperfect direct border repeats which are both substrates for the Ti-plasmid encoded relaxase VirD2. Yeast was used as a model system to investigate the requirements of the recipient cell for the formation of T-DNA circles after AMT. It was found that, despite the absence of selfhomology on the T-DNA, the homologous repair proteins Rad52 and Rad51 are involved in T-DNA circle formation. A model is presented involving the formation of T-DNA concatemers derived from T-strands by a process of strand-transfer catalysed by VirD2. These concatemers are then resolved into T-DNA circles by homologous recombination in the recipient cell.

DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA

“…Rolloos et al . [ 13 ] showed that the T-DNA circles are formed in recipient yeast cells when they used some T-DNA constructs that contained ARS/CEN sequence but not telomere sequences.…”

“…This observation is consistent with a mutant phenotype that is defective in HR. Rolloos et al [ 13 ] and Bundock et al [ 29 ] reported T-DNA circle structures being present in wild-type and rad52Δ strains. Our results showing the perfect border fusions and the transfer of the whole plasmid are consistent with these previous reports.…”

“…[ 12 ] for the transfer of T-DNA having sequences identical with yeast chromosomal genes, and by Rolloos et al . [ 13 ] for the transfer of T-DNA containing autonomously replicating sequences. In contrast, the other mutants defective in DSBR through HR, such as the mutant of the recA homolog gene RAD51, exhibited only 50 % defect in AMT, as shown in Fig.…”

“…Their studies revealed that T-DNA integration into the recipient genome requires the DNA repair pathway of either NHEJ or homologous recombination (HR). The linear T-DNA is a substrate for integration into the recipient genome, while it is also a substrate for ligation between T-DNA molecules or itself in plant and yeast cells [ 13 ]. They also demonstrated that the formation of circularized structures involves the HR pathway in yeast.…”

Functional genomics of lipid metabolism in the oleaginous yeastRhodosporidium toruloides

“…The remainder of sequenced barcodes could not be mapped for several reasons (Figure 1 – figure supplement 2A). T-DNA is often inserted in concatemeric repeats (6-8), in which case only RB-TDNAseq reads from the terminal repeat provides mapping information. If the terminal repeat is truncated (9), or if it abuts genomic sequence that is recalcitrant to sequencing for any reason, then we are able to detect the barcode at junctions between T-DNA repeats, but not at junctions with the genome.…”