Plant biotechnology has a role in addressing global needs for food, fiber and fuel, by developing new crop varieties with increased pest resistance, biofortification, and abiotic stress tolerance. Publicly acceptable forms of biotechnology offer an avenue for meeting these demands [1]. Recombinase-mediated genetic engineering provides a favorable direction for enhancing the precision of biotechnological approaches. Concerns over the presence of antibiotic resistance genes in the food supply and their escape into the environment [2] can be relieved through the use of recombinase technology to excise unwanted DNA from the genome of genetically engineered (GE) crops prior to marketing or release [3, 4]. A study by Chawla and colleagues [5] documented how site-specific integration in rice exhibited stable gene expression over multiple generations. The research also demonstrated that rice with multicopy transgene inserts, initially silenced for expression, recovered expression when resolved by recombinase technology to a single genomic copy. Such studies demonstrate other potential uses for recombinase technology in the development of plant biotechnology.

Genomic engineering took a large step forward with the discovery that site-specific recombinases, a group of enzymes that are capable of precise DNA cleavage and ligation without the gain or loss of nucleotides, could facilitate conservative DNA manipulation in a heterologous host [6]. The recombinase super family is split into two fundamental groups, the tyrosine and serine enzymes. This grouping is based on the active amino acid (Y or S) within the catalytic domain of each enzyme family. The best known tyrosine recombinases are Cre, Flp and R [7]. Tyrosine recombinases utilize identical recognition sites and perform a bi-directional mode of recombination. They have been shown to be effective for excision of unwanted DNA from the genome of the host but require complex schemes for integration.

The serine enzyme group includes the phiC31, TP901-1 and Bxb1 recombinases among others [8, 9]. Members of this group recognize two non-identical recognition sites (attB and attP) and perform a uni-directional mode of recombination. While less research has been conducted on this group, it appears that the serine enzymes are well suited for precise genomic recombination due to their uni-directional catalytic activity that prevents the reversion of recombination products.

In previous studies, we identified a number of prokaryotic site-specific recombination systems that function in the eukaryote Schizosaccharomyces pombe [8, 10]. Among those, the phiC31 uni-directional recombinase was highly efficient. The system has been successfully shown capable of recombinase mediated excision, inversion and integration reactions. The phiC31-att system is derived from the broad host range Streptomyces temperate phage phiC31 [11]. The 613 amino acid phiC31 protein acts on recognition sites attB and attP that are minimally 34 bp and 39 bp, respectively [12]. Published evidence has demonstrated that the phiC31 system is functional for excision and transmission of marker-free plastids in the seed of tobacco and in the genome of Arabidopsis and wheat [13–17] but has yet to be demonstrated capable of germinal transmission of nuclear DNA in planta.

In this research, we tested the phiC31 recombination system for the capacity to germinally transmit a target sequence that has undergone site-specific excision from within the Arabidopsis genome to a subsequent generation in the absence of the recombinase gene. Plants transgenic for an attB and attP flanked target sequence were introduced with a second construct that contained the recombinase gene. The phiC31 recombinase performed excision of the target sequence from three independent plant lines (i.e. genomic locations) and generated stably excised progeny plants that carry only the recombined target DNA of interest in the absence of the recombinase gene. This demonstrates that the phiC31 recombination system is suitable for the generation of stable marker-free, recombinase-free transgenic plants.

Our interest in site-specific recombination lies in its ability to facilitate crop improvement through controlled engineering of the plant genome. Recently transgenic corn has been deregulated for the production of high lysine, a consumer directed product [22, 23]. Further, this transgenic crop was engineered with the assistance of the site-specific recombinase technology for marker removal. Deregulation in this case required extensive studies to ensure that the recombinase mediated excision event was heritably transmitted to subsequent generations in the absence of the recombinase gene [23]. Such agricultural requirements, while obviously necessary, have elicited few detailed studies on the transmission of recombined chromosome transmission to progeny plants. The recombinase systems Cre/lox, Flp/FRT, R/RS, β/six and ParA/MRS have all been shown capable of germinal transmission in planta [19, 24–30]. Therefore, our research investigated the publicly available phiC31 recombination system as a potential tool for the precise removal of plant transgenes. In order to demonstrate its utility for crop genome engineering and increase public acceptance of transgenic technology, the potential for predefined nuclear excision events and their germinal transmission was investigated. An advantage of phiC31 over existing recombinase systems is its unidirectional recombination activity, which prevents the re-insertion of the excision product into the genome. In addition, phiC31 has the ability to site-specifically integrate DNA into the host genome [8, 13] making this a versatile enzyme.

Our strategy began with the assumption that we could use gusA expression as a reporter for site-specific recombination. The pattern of GUS enzyme activity would reveal genomic excision of the target sequence and any tissue specificity in recombination. This strategy, however, failed to perform as expected with initial excised plants being either weak or completely devoid of GUS activity. Subsequent analysis of the original TR₁-phiC31 progeny confirmed that use of reporter enzyme activity was an unreliable indicator of excision. We had also observed this phenomenon with other constructs used in both Arabidopsis and S. pombe [8, 19]. It is possible that the 54 bp attB/P hybrid sequence present within the transcript leader sequence of the gusA gene may cause poor expression due to methylation or by some other mechanism that inhibits gene expression. Due to this circumstance, the analysis and scoring of site-specific excision was performed using PCR.

Site-specific excision was detected in all TR₁-phiC31.22, TR₁-phiC31.31 and TR₁-phiC31.34 plants. The majority (72%) of the TR₁-phiC31.22 and TR₁-phiC31.31 plants that demonstrated the presence of the excision product did not yield the PCR amplified unexcised target band. This indicates that the phiC31-mediated genomic excision reaction was complete, or nearly so, within many of these TR₁ plants. The exception was line TR₁-phiC31.34. Only 12% of the TR₁-phiC31.34 plants were positive for the 0.44 kb excision band in the absence of the 1.26 kb unexcised target band. This may be due to unfavorable placement of the target construct within the Arabidopsis genome. Indeed, although the TR-phiC31.34 lines generated lower levels of recombinase-mediated excision than either the TR-phiC31.22 or TR-phiC31.31 lines, when segregants (derived from TR-phiC31.34) containing only the phiC31 expression cassette were manually crossed with TA-phiC31.22 target plants, 92% of the progeny generated only the 0.44 kb excised target PCR product. This indicates that phiC31 functions well in these plants, despite performing less efficiently on the TA-phiC31.34 target. The simplest explanation is that the TA-phiC31.34 genomic location or structure was unfavorable to recombination in the germinal tissue.

From analysis of the BC₁ plants, 85.6% (352 of 411) of those derived from the three TR₁-phiC31 lines showed excision, while in a previous line of research 77.3% and 99.6% of the BC₁ plants of the TR₁-ParA and TR₁-Cre lines exhibited excision, respectively [19]. By this measure, it appears that the phiC31 recombinase mediated excision efficiency is more effective than ParA and approaching that of the Cre-lox system. Although, the majority of the BC₁ lines displayed excised genomic target, it is difficult to give a precise quantitative assessment of the phiC31 activity since only a modest number of different target locations were thoroughly characterized. Variability in copy number and chromosome locations of the phiC31 gene can affect the amount of recombinase protein produced and thus impact the efficiency of the excision reaction observed, making a direct comparison difficult. Other excision strategies for the phiC31 recombinase are being investigated. These include the use of inducible or tissue specific promoters for controllable expression [31] use of self-deleting designs [32] and use of viral inoculation or Agrobacterium-infiltration for immediate but transient expression [33, 34].

As an alternative method of recombinase introduction into the plant target lines, our lab tested hand pollination between phiC31 recombinase expressing plants and pN3-phiC31 target plants. PCR analysis of the manually crossed MC₁ progeny demonstrated that this is a viable method for the generation of individuals with genomic target excision (Fig. 6). However, it was observed that like secondary Agrobacterium transformation with the recombinase expression cassette, the genomic excision results varied between lines (Table 3). Use of a demonstrated recombinase expression line such as phiC31.31.83 (Table 3) enabled sufficient recombinase mediated excision events to fully excise all target DNA when crossed together. It was also observed that segregation of the secondary Agrobacterium transformed TR₁ lines, without benefit of backcrossing, produced excised target and recombinase expression-only T-DNA lines in the TR₂ and TR₃ generations (data not shown). This indicates that the phiC31 expression T-DNA in these lines was at a single locus or a low number of loci within the genome and that expression was sufficient to facilitate recombination allowing segregation by self- pollination.

Since PCR assays of genomic DNA from leaf tissue only indicates that excision has occurred in somatic cells, we utilized Southern blot analysis to ascertain whether target sequence removal had occurred in the germline. As long as phiC31 DNA was present in the genome, or the phiC31 protein was present in the germline cells, the possibility that recombination was generated de novo could not be ruled out. Hence, BC₁ plants were screened by PCR for the absence of the phiC31 recombinase gene, and the following generation (S₁ plants) was confirmed by Southern blot hybridization. As is clearly shown in Fig. 5 lanes #1 - 5, germinal transmission of the genomic excision event in the absence of the phiC31 recombinase gene occurred, illustrating that the production of stable lines with the unwanted DNA removed can be achieved.

Controlled targeted integration with recombinase technology allows the application of more sophisticated recombinase strategies [35]. This technology enables the production of precisely engineered transgenic plants through genome specific transgene integration and has been reported to function in Arabidopsis, tobacco and rice [5, 36–44] with Cre, Flp and R recombinase systems. The phiC31 recombinase with its uni-directional catalytic activity presents a novel way to facilitate stable site-specific integration events without the elaborate strategies required by the bi-directional systems. Peer-reviewed literature reported that phiC31 is capable of mammalian genome targeting [45, 46] and targeted integration into the plastid genome of tobacco [13]. Utilization of phiC31 for genome modification has been facilitated in mammalian species through the identification of cryptic attB or attP sites as potential locations for transgene introduction [46]. To this end our lab investigated, in silico, the presence of sequences similar to the phiC31 att sites within the Arabidopsis thaliana genome. We used a BLASTn search to investigate whether the Arabidopsis genome contains sequences similar to the minimal 34 bp attB and 39 bp attP sites [12]. The genomic sequences with the highest similarity to the att sites exhibited >60% overall nucleotide identity. A total of seven sequences had 21-23 (61.8-67.7%) of the 34 nucleotides conserved with the minimal attB sequence, while 14 native sequences had 24-27 (61.5-69.2%) nucleotides in common with the 39 bp attP sequence (Fig. 7). While most of the sequences including the best matches for attP did contain the conserved core domain presumably essential for phiC31-mediated recombination, only three of the attB-like sequences contained the core sequence (Fig. 1d; Fig. 7). It is possible that some of these att-like sequences could potentially be used as a native target site for phiC31 mediated integration in Arabidopsis. Pseudo phiC31 attP sequences in the mouse, bovine and human genomes have been reported and some of them have been shown suitable for integration of introduced DNA [47–49].

Although unlikely, the potential for genomic excision, inversion and translocation mediated by these cryptic att sequences in Arabidopsis is possible. For excision, Arabidopsis chromosomes 3 and 5 carry both attB and attP-like sequences in direct orientation (Fig. 7). The closest correctly oriented sites are located >500 kb apart on chromosome 3, but the cryptic attB does not contain a conserved core domain. Although it is theoretically possible that genomic recombination could occur via endogenous att-like sequences, the OXS3 promoter-phiC31 plants did not exhibit compromised viability, morphological or growth defects. This differs from earlier observations using a 35S-phiC31 construct where Arabidopsis plants with crinkled leaves were common [C. Day and D.W. Ow, unpublished data]. Hence, this underscores the importance in controlling expression of the recombinase gene through appropriate use of promoters.

The purpose of the research was to provide proof-of-concept that the phiC31 recombinase can mediate site-specific genome modification in the plant germline tissue without affecting fecundity. The research established that the excision event was passed to subsequent generations in the absence of phiC31 and that the excision of attB and attP-flanked DNA from the plant genome was a conservative site-specific event. In a majority of the phiC31 lines examined (11 out of 15), at least one BC₁ segregant was recovered that contained a germinally transmitted excision event lacking the phiC31 gene. These results were validated with Southern blot hybridization and demonstrate that the secondary transformation strategy used in this study is feasible for the production of marker-free transgenic plants. This approach may prove particularly useful in those species where cross pollination is not possible or undesirable. We further demonstrate that an alternative approach to marker removal where the recombinase is introduced into the excision test target plants with cross pollination is also a viable strategy. Molecular analysis confirmed that the genomic excision was site-specific and conservative. Therefore, taken together the results clearly establish that the phiC31 system performs genomic excision, generating stable transgenic recombinase-free Arabidopsis plants with unwanted DNA removed.