In vivo formation of double-stranded T-DNA molecules by T-strand priming

During plant genetic transformation, Agrobacterium transfers a single-stranded DNA (T-strand) into the host cell. Increasing evidence suggests that double-stranded (ds) T-DNA, converted from T-strands, are potent substrates for integration. Nevertheless, the molecular mechanism governing T-strand conversion to dsT-DNA is unknown. Integrated T-DNA molecules typically exhibit deletions at their 3′ end as compared with their 5′ end. We hypothesize that this may result from asymmetric polymerization of T-DNA’s ends. Here we show that β-glucuronidase (GUS) expression from sense T-strands is more efficient than from antisense T-strands, supporting asymmetric conversion. Co-transfection with two partially complementary, truncated GUS-encoding T-strands results in GUS expression, which suggests functional hybridization of the T-strands via complementary annealing and supports the notion that T-strands can anneal with primers. Indeed, red fluorescent protein (RFP) expression from mutated T-strand can be restored by delivery of synthetic DNA and RNA oligonucleotides with partial wild-type RFP sequence, implying the involvement of plant DNA repair machinery.