Recombinase-mediated gene stacking in cotton.

Site-specific gene stacking could reduce the number of segregating loci and expedite the introgression of transgenes from experimental lines to field lines. Recombinase-mediated site-specific gene stacking provides a flexible and efficient solution, but this approach requires a recombinase recognition site in the genome. Here, we describe several cotton (Gossypium hirsutum cv. Coker 312) target lines suitable for Mycobacteriophage Bxb1 recombinase-mediated gene stacking. Obtained through the empirical screening of random insertion events, each of these target lines contains a single intact copy of the target construct with precise sequences of RS2, lox, and attP sites that is not inserted within or close to a known gene or near a centromere and shows good expression of the reporter gene gfp. Gene stacking was tested with insertion of different combinations of three candidate genes for resistance to verticillium wilt into three cotton target lines: CTS1, CTS3, and CTS4. Nine site-specific integration events were recovered from 95 independently transformed embryogenic calluses. Southern and DNA sequence analyses of regenerated plants confirmed precise site-specific integration, and resistance to verticillium wilt was observed for plant CTS1i3, which has a single precise copy of site-specifically integrated DNA. These cotton target lines can serve as foundation lines for recombinase-mediated gene stacking to facilitate precise DNA integration and introgression to field cultivars.

Enterohemorrhagic Escherichia coli O157:H7 antigens produced in transgenic lettuce effective as an oral vaccine in mice.

KEY MESSAGE: Transgene with recombination sites to address biosafety concerns engineered into lettuce to produce EspB and γ-intimin C280 for oral vaccination against EHEC O157:H7. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen where ruminant farm animals, mainly bovine, serve as reservoirs. Bovine vaccination has been used to prevent disease outbreaks, and the current method relies on vaccines subcutaneously injected three times per year. Since EHEC O157:H7 colonizes mucosal surfaces, an oral vaccine that produces an IgA response could be more convenient. Here, we report on oral vaccination against EHEC O157:H7 in mice orally gavaged with transgenic lettuce that produces EHEC O157:H7 antigens EspB and γ-intimin C280. Younger leaves accumulated a higher concentration of antigens; and in unexpanded leaves of 30-day-old T2 plants, EspB and γ-intimin C280 were up to 32 and 51 µg/g fresh weight, respectively. Mice orally gavaged with lettuce powders containing < 3 µg antigens for 6 days showed a mucosal immune response with reduced colonization of EHEC O157:H7. This suggests that the transgenic lettuce has potential to be used for bovine vaccination. To promote the biosafety of crop plants producing medically relevant proteins, recombination sites were built into our transgenic lines that would permit optional marker removal by Cre-lox recombination, as well as transgene deletion in pollen by CinH-RS2 recombination. The ability to upgrade the transgenic lettuce by stacking additional antigen genes or replacing older genes with newer versions would also be possible through the combined use of Bxb-att and Cre-lox recombination systems.

Split-Cre mediated deletion of DNA no longer needed after site-specific integration in rice.

KEY MESSAGE: N-cre and C-cre added in separate lines reassemble functional Cre in F1 progeny to excise unnecessary DNA, including cre DNA, thereby eliminating generations needed to cross in and out cre. Crop improvement via transgenesis can benefit through efficient DNA integration strategies. As new traits are developed, new transgenes can be stacked by in planta site-specific integration near previous transgenes, thereby facilitating their introgression to field cultivars as a single segregation locus. However, as each round of integration often requires use of selectable markers, it is more convenient to reuse the selection scheme. The Cre recombinase can be used to delete away previously used selection genes, and other DNA no longer needed after transformation, but the constitutive production of this DNA scanning protein can also affect plant growth. We had previously described in Arabidopsis a split Cre protein fragment complement scheme to reassemble a functional Cre recombinase. As our goal for developing this system was to deploy its use in major crop plants, here we show that Cre protein fragment complementation works in rice with precise recombination structures confirmed by DNA sequencing. As each N-terminal and C-terminal fragment is also flanked by lox recombination sites, they can also self-excise to avoid the need to segregate away the cre DNA. Options to form F1 hybrids homozygous for one transgene, or hemizygous for two different transgenes at the same chromosome location, are discussed.

Target lines for recombinase-mediated gene stacking in soybean.

KEY MESSAGE: Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking. For introgression of new transgenic traits to field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an in planta transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration. Here, we describe 5 soybean target lines from the screening of 118 Agrobacterium-mediated transgenic plants that were positive for gus expression. Each of the 5 target lines has a single copy of the transgenic DNA with precise DNA sequences of the recombinase recognition sites, located at least 1 kb away from the nearest coding region, not close to the centromere, and showed good expression of the reporter gene. We tested Bxb1 integrase-mediated integration of a gfp-containing plasmid into each of these lines and showed precise site-specific integration in bombarded calluses. For plant regeneration, we used embryonic axes of mature soybean seeds to conduct a new set of biolistic transformation with a DsRed-containing plasmid. Three integration events were regenerated into whole plants, demonstrating the principle that target lines can serve as foundation lines for the stacking of DNA to predefined locations in the soybean genome.

Root microbiome changes associated with cadmium exposure and/or overexpression of a transgene that reduces Cd content in rice.

Cadmium (Cd) is a toxic heavy metal that can accumulate in crop plants. We reported previously the engineering of a low cadmium-accumulating line (2B) of rice through overexpression of a truncated OsO3L2 gene. As expression of this transgene was highest in plant roots, amplicon and metatranscriptome sequencing were used to investigate the possibility that its expression affects root associated microbes. Based on amplicon sequencing of bacterial 16S rRNA, but less so from fungal ITS, the OTUs (operational taxonomic units) showed less diversity in soil tightly (rhizoplane) than loosely (rhizosphere) associated with plant roots. Significantly changed OTUs caused by the low-Cd accumulating plant 2B, Cd treatment or both were found, and 10 of the 13 OTUs (77%) that were enriched in Cd treated 2B samples over the wild type counterpart have been previously described as involved in tolerance to Cd or other heavy metals. Metatranscriptome sequencing of rhizosphere microbiome found that bacteria accounted for 70-75% of the microbial RNA. Photosynthesis-antenna proteins and nitrogen metabolism pathways were most active in soil microbes treated with Cd and grown with plant 2B. Correspondingly, the relative abundance of Cyanobacteria was enriched to < 1% of Cd treated rhizosphere bacteria, yet accounted for up to 13% of Cd treated 2B rhizospheric transcripts. These enriched microbes by transgene and Cd are worthy candidates for future application on reducing crop uptake of Cd.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

A C-terminal fragment of Arabidopsis OXIDATIVE STRESS 2 can play a positive role in salt tolerance.

The zinc finger transcription factor OXIDATIVE STRESS 2 (OXS2) was previously reported to be involved in oxidative stress tolerance and stress escape. Here we report that an Arabidopsis oxs2-1 mutant is also more sensitive to salt stress. Conversely, the overproduction of a C-terminal fragment of OXS2, the 'AT3' fragment, can enhance salt tolerance in Arabidopsis by upregulating the transcription of at least six salt-induced genes: COR15A, COR47, RD29B, KIN1, ACS2 and ACS6. Mutant analysis showed that the AT3-mediated salt tolerance requires MPK3, MPK6 and 14-3-3Ω. AT3 was shown to interact with MPK3 in planta, with 14-3-3Ω as a likely linker protein. AT3 can be phosphorylated by MPK3 during salt stress, upon which it relocates from the cytoplasm to the nucleus. It appears that the phosphorylation-induced nuclear localization of OXS2 contributes a positive role to the salt stress response.

Arabidopsis OXS3 family proteins repress ABA signaling through interactions with AFP1 in the regulation of ABI4 expression.

Abscisic acid (ABA) and the AP2/ERF (APETALA2/ETHYLENE-RESPONSIVE FACTOR)-type transcription factor called ABA INSENSITIVE 4 (ABI4) play pivotal roles in plant growth responses to environmental stress. An analysis of seedling development in Arabidopsis ABA hypersensitive mutants suggested that OXS3 (OXIDATIVE STRESS 3), OXS3b, O3L3 (OXS3 LIKE 3), O3L4, and O3L6 were negative regulators of ABI4 expression. We therefore characterized the roles of the OXS3 family members in ABA signaling. All the above five OXS3 proteins were found to interact with AFP1 (ABI FIVE BINDING PROTEIN 1) in yeast two hybrid assays. Seven OXS3 family members including two other members O3L1 and O3L5 were found to interact with histone H2A.X, although OXS3b, O3L3, and O3L5 showed weaker interactions. ChIP-qPCR analysis showed that the absence of some of these OXS3 family proteins was associated with increased occupancy of histone γ-H2A.X at the ABI4 promoter, which also corresponded with de-repression of ABI4 expression. Repression of ABI4 expression, however, required both AFP1 and OXS3, OXS3b or O3L6. We conclude that in the absence of stress, OXS3 family proteins regulate γ-H2A.X deposition at the ABI4 promoter and that together with AFP1, OXS3 family proteins function to prevent ABA-induced growth arrest by co-repressing ABI4 through decreased promoter occupancy of histone γ-H2A.X.

SnRK1 regulates chromatin-associated OXS3 family proteins localization through phosphorylation in Arabidopsis thaliana.

In plants, SNF1-related protein kinase 1 (SnRK1) senses nutrient and energy status and transduces this information into appropriate responses. Oxidative Stress 3 (OXS3) and family members share a highly conserved putative N-acetyltransferase catalytic domain (ACD). Here, we describe that the ACD contains two candidate SnRK1 recognition motifs and that SnRK1 can interact with most of the OXS3 family proteins. In vitro, SnRK1.1 can phosphorylate OXS3, OXS3b and O3L4, and in vivo promote the translocation of OXS3, OXS3b and O3L6 from the nucleus to the cytoplasm. Phosphorylation sites within the OXS3 ACD affect OXS3 cytoplasmic accumulation, as well as their interactions with SnRK1.1. This suggests that signal transduction from SnRK1 to OXS3 family proteins, and that SnRK1 can control their activities through phosphorylation-induced nuclear exclusion.

Overproduction of plant nuclear export signals enhances diamide tolerance in Schizosaccharomyces pombe.

The nuclear export signal (NES) endows a protein nuclear export ability. Surprisingly, our previous study shows that just the NES peptide of Schizosaccharomyces pombe Oxs1 (SpOxs1NES) can confer diamide tolerance by competing with transcription factor Pap1 for nuclear transport. This finding intrigued us to test the function of NESs from heterologous organisms. The Arabidopsis thaliana zinc finger transcription factor OXIDATIVE STRESS 2 (AtOXS2) is a nucleocytoplasmic shuttling protein and nearly all OXS2 members from maize and rice contain an NES. In this study, we find that the plant OXS2 members and their C-terminus (AT3 peptide) can confer diamide tolerance due to their NESs, and amino acids in non-conserved as well as conserved positions are necessary for the diamide tolerance. As in SpOxs1NES, the enhanced tolerance to diamide in fission yeast depends on Pap1. Like SpOxs1NES, OXS2 family NESs appear to compete for nuclear transport of the Pap1-like Arabidopsis protein bZIP10, as when overproduced in Arabidopsis protoplasts, bZIP10 is retained in the nucleus.

Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation.

As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.

Nucleocytoplasmic OXIDATIVE STRESS 2 can relocate FLOWERING LOCUS T.

Survival of a species depends on reproductive fitness and a plant's floral transition is controlled by developmental and environmental signals. In Arabidopsis, the floral integrators SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) and FT (FLOWERING LOCUS T) sense various pathway signals to activate floral meristem identity genes. At high stress intensity, greater nuclear accumulation of the zinc-finger transcription factor OXS2 (OXIDATIVE STRESS 2) activates an early-flowering stress-escape response. Curiously, accumulation of OXS2 in the cytoplasm can delay flowering, prompting the hypothesis that in absence of stress, OXS2 helps to maintain vegetative growth. While the mechanism of stress-escape was identified as the OXS2-mediated transcription of SOC1, how cytoplasmic OXS2 delays flowering was unknown. Here, we report that OXS2 can interact indirectly with florigen FT and transcription factor FD (FLOWERING LOCUS D), the two proteins known to induce floral transition. By using 14-3-3Ω as a bridge linker, OXS2 can alter the subcellular distribution of FT. This lead to a speculation on how cytoplasmic OXS2 is able to prevent early flowering, by keeping FT from the nucleus.

OXIDATIVE STRESS 3 regulates drought-induced flowering through APETALA 1.

Stress-induced regulation of flowering time insures evolutionary fitness. Stress-induced late flowering is thought to result from a plant evoking tolerance mechanism to wait out the stress before initiating reproduction. Stress-induced early flowering, on the other hand, is thought to be a stress-escape response. By shortening their life cycle to produce seeds before severe stress leads to death, this insures survival of the species at the cost of lower seed yield. Previously, we reported that overexpression of OXS3 (OXIDATIVE STRESS 3) could enhance tolerance to cadmium and oxidizing agents in Arabidopsis whereas an oxs3 null mutant was slightly more sensitive to these chemicals. In this study, we found that the absence of OXS3 also causes early flowering under a mild drought stress treatment. This contrasts with the behavior of wild type Ws4 and Col ecotypes that responded to the same condition by delaying flowering time. We tested the hypothesis that OXS3 might ordinarily exert a negative regulatory role on flowering during drought stress, which in its absence, would lead to stress-induced early flowering. In a search of whether OXS3 could interfere with regulators that activate flowering, we found that OXS3 could bind SOC1 in vitro and in vivo. Overexpression of OXS3 in a transient expression assay was found to repress the AP1 promoter, and the full repression effect required SOC1. It is possible that the OXS3/SOC1 interaction serves to prevent precocious flower development and prevent low seed set from a premature stress-induced flowering response.

A Pap1-Oxs1 signaling pathway for disulfide stress in Schizosaccharomyces pombe.

We describe a Pap1-Oxs1 pathway for diamide-induced disulfide stress in Schizosaccharomyces pombe, where the nucleocytoplasmic HMG protein Oxs1 acts cooperatively with Pap1 to regulate transcription. Oxs1 and Pap1 form a complex when cells are exposed to diamide or Cd that causes disulfide stress. When examined for promoters up-regulated by diamide, effective Pap1 binding to these targets requires Oxs1, and vice versa. With some genes, each protein alone enhances transcription, but the presence of both exerts an additive positive effect. In other genes, although transcription is induced by diamide, Oxs1 or Pap1 plays a negative role with full de-repression requiring loss of both proteins. In a third class of genes, Oxs1 positively regulates expression, but in its absence, Pap1 plays a negative role. The Oxs1-Pap1 regulatory interaction appears evolutionarily conserved, as heterologous (human, mouse and Arabidopsis) Oxs1 and Pap1-homologues can bind interchangeably with each other in vitro, and at least in the fission yeast, heterologous Oxs1 and Pap1-homologues can substitute for S. pombe Oxs1 and Pap1 to enhance stress tolerance.

A new location to split Cre recombinase for protein fragment complementation.

We have previously described a recombinase-mediated gene stacking system in which the Cre recombinase is used to remove lox-site flanked DNA no longer needed after each round of Bxb1 integrase-mediated site-specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. However, maintaining an efficient cre-expressing line over many generations can be a problem, as high production of this DNA-binding protein might interfere with normal chromosome activities. To counter this selection against high Cre activity, we considered a split-cre approach, in which Cre activity is reconstituted after separate parts of Cre are brought into the same genome by hybridization. To insure that the recombinase-mediated gene stacking system retains its freedom to operate, we tested for new locations to split Cre into complementing fragments. In this study, we describe testing four new locations for splitting the Cre recombinase for protein fragment complementation and show that the two fragments of Cre split between Lys244 and Asn245 can reconstitute activity that is comparable to that of wild-type Cre.

Maize OXIDATIVE STRESS2 Homologs Enhance Cadmium Tolerance in Arabidopsis through Activation of a Putative SAM-Dependent Methyltransferase Gene.

Previously the Arabidopsis (Arabidopsis thaliana) zinc finger protein OXIDATIVE STRESS2 (AtOXS2) and four OXS2-like (AtO2L) family members were described to play a role in stress tolerance and stress escape. For stress escape, SOC1 was a target of AtOXS2. However, for stress tolerance, the downstream targets were not identified. We cloned two OXS2 homolog genes from sweet corn, ZmOXS2b and ZmO2L1 Both genes are transiently inducible by Cd treatment. When expressed in Arabidopsis, each enhances tolerance against cadmium. Further analysis showed that ZmOXS2b and ZmO2L1 proteins enhance Cd tolerance in Arabidopsis by activating at least one target gene, that encoding a putative S-adenosyl-l-Met-dependent methyltransferase superfamily protein (AT5G37990), which we named CIMT1 This activation involves the in vivo interaction with a segment of the CIMT1 promoter that contains a BOXS2 motif previously identified as the binding element for AtOXS2. More importantly, CIMT1 is induced by Cd treatment, and overexpression of this gene alone was sufficient to enhance Cd tolerance in Arabidopsis. The connection of ZmOXS2b and ZmO2L1 to Arabidopsis CIMT1 suggests a similar network may exist in maize (Zea mays) and may provide a clue to possibly using a CIMT1 maize homolog to engineer stress tolerance in a major crop.

The long road to recombinase-mediated plant transformation.

The use of site-specific recombinases to manipulate eukaryotic genomes began nearly three decades ago. Although seemingly parallel efforts were being made in animal and plant systems, the motivation for its development in plants was unique to, at least at the time, crop bioengineering issues. The impetus behind site-specific deletion in plants was to remove antibiotic resistance genes used during transformation but unnecessary in commercial products. Site-specific integration in plants was more than academic curiosity of position effects on gene expression, but a necessary step towards developing the serial stacking of DNA to the same chromosome locus - to insure that bioengineered crops can be improved over time through transgene additions without inflating the number of segregating loci. This article is not a review of the literature on site-specific recombination, but a first person account of the series of events leading to the development of a gene stacking transformation system in plants.

Stress tolerance to stress escape in plants: role of the OXS2 zinc-finger transcription factor family.

During dire conditions, the channelling of resources into reproduction ensures species preservation. This strategy of survival through the next generation is particularly important for plants that are unable to escape their environment but can produce hardy seeds. Here, we describe the multiple roles of OXIDATIVE STRESS 2 (OXS2) in maintaining vegetative growth, activating stress tolerance, or entering into stress-induced reproduction. In the absence of stress, OXS2 is cytoplasmic and is needed for vegetative growth; in its absence, the plant flowers earlier. Upon stress, OXS2 is nuclear and is needed for stress tolerance; in its absence, the plant is stress sensitive. OXS2 can activate its own gene and those of floral integrator genes, with direct binding to the floral integrator promoter SOC1. Stress-induced SOC1 expression and stress-induced flowering are impaired in mutants with defects in OXS2 and three of the four OXS2-like paralogues. The autoactivation of OXS2 may be a commensurate response to the stress intensity, stepping up from a strategy based on tolerating the effects of stress to one of escaping the stress via reproduction.

Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis.

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.