PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome

Abstract

Background: The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe.

Results: In this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. The phiC31 recombinase excised the attB and attP-flanked DNA, and the excision event was detected in subsequent generations in the absence of the phiC31 gene, indicating germinal transmission was possible. We further verified that the genomic excision was conservative and that introduction of a functional recombinase can be achieved through secondary transformation as well as manual crossing.

Conclusion: The phiC31 system performs site-specific recombination in germinal tissue, a prerequisite for generating stable lines with unwanted DNA removed. The precise site-specific deletion by phiC31 in planta demonstrates that the recombinase can be used to remove selectable markers or other introduced transgenes that are no longer desired and therefore can be a useful tool for genome engineering in plants.

Figure 1

T-DNA structures. (not to scale) from a) pN3-phiC31; b) pCOXS3-phiC31; and c) predicted single copy T-DNA structures after excision of stuffer by phiC31-att recombination. PCR primers shown as e, f, g, h; att sites as grey arrowheads; hybridization probes as grey rectangles. Abbreviations: B, BamHI; E, EcoRI; V, EcoRV; X, XhoI; RB, T-DNA right border; LB, T-DNA left border. Length in kb of PCR products (dotted lines) and DNA fragments (dashed lines). d) Sequence of the 54 bp attB and 57 bp attP phiC31 recognition sites, where the minimal required sequence is underlined and the 2 nucleotide 'AA' core region of crossover is in bold. e) sequence of a PCR product detecting a conservative site-specific excision event. Not shown are gene terminators and promoters for hptII (hygromycin phosphotransferase II) and nptII (neomycin phosphotransferase II) and the gene terminator for gusA (β-glucuronidase).

Figure 2

Strategy for generating site-specific excision plant lines.

Figure 3

PCR analysis for site-specific recombination and the presence of the phiC31 gene in the TR₁ and BC₁ generations. PCR reactions (a, c) with primers e and f (Fig. 1) or (b, d) with primers g and h (Fig. 1) on representative plant DNAs. a, b) retransformed TR₁- phiC31.22 lines. c, d) Back Crossed line BC₁-phiC31.22.3. Unlined numbers represent excision or recombinase only plants lines. Control lanes are B (blank, no DNA); E (excision, pN3-phiC31_exc); N (no excision, pN3-phiC31); P (recombinase, pCOXS3- phiC31).

Figure 4

PCR analysis for site-specific recombination and the presence of the phiC31 gene in the S₁ generation. PCR reactions (a, c) with primers e and f (Fig. 1) or (b, d) with primers g and h (Fig. 1) on representative plant DNAs. a, b) (lanes 1, 2) Self fertilized -Excision only target lines S₁-phiC31. 22.3.18.1, 22.29.7.1; (lanes 3, 4) S1-phiC31.31.1.1, 31.31.13.1; (lane 5) S₁-phiC31.34.2.10.1. c, d) Self fertilized - Recombinase only expression lines (lanes 1, 2) S₁-phiC31.22.3.5.1, 22.15.5.1; (lanes 3, 4) S₁-phiC31.31.23.10.1, 31.31.36.2; (lane 5) S₁-phiC31.34.9.2.1. Control lanes are B (blank, no DNA); E (excision, pN3-phiC31_exc); N (no excision, pN3-phiC31); P (recombinase, pCOXS3-phiC31).

Figure 5

S₁ plants examined by Southern blot analysis for excision and segregation of phiC31 gene. a) Genomic DNA cleaved with EcoRV hybridized with a ³²P-labeled GUS1350 probe (Fig. 1). b) Genomic DNA digested with XhoI and hybridized with a ³²P-labeled NPT690 probe. Plant lines (lanes 1, 2) S1-phiC31.22.3.18.1, 22.29.7.1; (lanes 3, 4) S₁- phiC31.31.31.1.1, 31.31.13.1; (lane 5, 6) S₁-phiC31.34.2.10.1; 34.9.20.1. Control lanes are wt (wild type Arabidopsis genomic DNA), TA₃-phiC31.22, (target lines), TR₁- phiC31.22.23 (phiC31 recombinase expression line).

Figure 6

PCR analysis for site-specific recombination and the presence of the phiC31 gene in the MC₁ generation. a) PCR reactions with primers e and f (Fig. 1) or b) with primers g and h (Fig. 1) on manually crossed lines; (lanes 1, 2) MC₁-phiC31.22.3, 22.15; (lanes 3, 4) MC₁-phiC31.31.40, 31.83; (lane 5, 6) MC₁-phiC31.34.9, 34.20. Control lanes are B (blank, no DNA); E (excision, pN3-phiC31_exc); N (no excision, pN3-phiC31); P (recombinase, pCOXS3-phiC31).

Figure 7

Arabidopsis genomic DNA sequences with >60% similarity to phiC31 attP and attB sites. a) Alignment of the 39 bp attP site with 14 sequences from the Arabidopsis genome. b) Alignment of the 34 bp attB site with seven sequences from the Arabidopsis genome. Nucleotides identical to the att site are highlighted with white text and blue backshading. A conserved core domain is highlighted in red text. The chromosomal location coordinates of each sequence are shown on the left, the percent identity and nucleotide match is shown on the right. c) The position and orientation of the 21 att-like sequences are displayed on a diagram of the five Arabidopsis chromosomes.