ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium -mediated floral dip transformation. A T-DNA containing the target site for a ZFN pair, that was shown to be active in yeast, was integrated in the Arabidopsis genome. Subsequently, the corresponding pair of ZFN genes was stably integrated in the Arabidopsis genome and ZFN activity was determined by PCR and sequence analysis of the target site. Footprints were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1 and 200 bp and insertions ranging between 1 and 14 bp. We did not observe any toxicity from expression of the ZFNs. In order to obtain ZFN-induced gene-targeting (GT), Arabidopsis plants containing the target site and expressing the ZFN pair were transformed with a T-DNA GT construct. Three GT plants were obtained from ∼3000 transformants. Two of these represent heritable true GT events, as determined by PCR, Southern blot analysis and sequencing of the resulting recombined locus. The third plant showed an ectopic GT event. No GT plants were obtained in a comparable number of transformants that did not contain the ZFNs. Our results demonstrate that ZFNs enhance site-specific mutagenesis and gene-targeting of Agrobacterium T-DNA constructs delivered through floral dip transformation.     

Efficient in planta gene targeting in Arabidopsis using egg cell‐specific expression of the Cas9 nuclease of Staphylococcus aureus

Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a double‐stranded break (DSB) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single‐stranded break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5′ or 3′ overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell‐specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will.     

Multiple host‐cell recombination pathways act in Agrobacterium‐mediated transformation of plant cells

Using floral‐dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non‐homologous and homologous recombination pathways in Agrobacterium‐mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium‐mediated transformation. Although integration of T–DNA into the plant genome is severely compromised in the absence of known DNA double‐strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T–DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Agrobacterium‐mediated transformation of reed (Phragmites communis Trinius) using mature seed‐derived calli

Reed (Phragmites communis) is a potential bioenergy plant. We report on its first Agrobacterium‐mediated transformation using mature seed‐derived calli. The Agrobacterium strains LBA4404, EHA105, and GV3101, each harboring the binary vector pIG121Hm, were used to optimize T‐DNA delivery into the reed genome. Bacterial strain, cocultivation period and acetosyringone concentration significantly influenced the T‐DNA transfer. About 48% transient expression and 3.5% stable transformation were achieved when calli were infected with strain EHA105 for 10 min under 800 mbar negative pressure and cocultivated for 3 days in 200 μm acetosyringone containing medium. Putative transformants were selected in 25 mg l^?1 hygromycin B. PCR, and Southern blot analysis confirmed the presence of the transgenes and their stable integration. Independent transgenic lines contained one to three copies of the transgene. Transgene expression was validated by RT‐PCR and GUS staining of stems and leaves.     

Nuclear gene targeting in Chlamydomonas using engineered zinc‐finger nucleases

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc‐finger nucleases (ZFNs). Our approach includes (i) design of gene‐specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light‐activated ion channel channelrhodopsin‐1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non‐functional aminoglycoside 3′‐phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co‐transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin‐resistant (Pm‐R) clones with expressing nucleases. Of these Pm‐R clones, 1% also contained a modified COP3 locus. In cases where cells were co‐transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.     

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

An efficient Agrobacterium‐mediated site‐specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development.     

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

Zinc‐finger nucleases (ZFNs) are engineered nucleases that induce DNA double‐strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non‐homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non‐homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP ‐ZFN mRNAs into the embryos of an EGFP ‐transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP ‐ZFNs at off‐target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on‐target site with less effect on the off‐target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.     

CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens

The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.     

Gene targeting in polymerase theta-deficient Arabidopsis thaliana

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.     

Precise marker excision system using an animal‐derived piggyBac transposon in plants

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth‐derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase‐expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T₀ plants without concomitant re‐integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.     

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T₁ progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.     

Extended TAQing system for large‐scale plant genome reorganization

We previously developed a large‐scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double‐strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5′‐TCGA‐3′ sequences in the genome at higher temperatures (37–42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT‐rich regions. To overcome such problems we developed an extended TAQing (Ex‐TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant‐growing temperatures. We established the Ex‐TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5′‐TTAA‐3′ sequence which is highly abundant in the Arabidopsis genome. A synthetic intron‐spanning MseI gene, which was placed downstream of a heat‐shock‐inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end‐joining at MseI cleavage sites. The Ex‐TAQing system could induce large‐scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex‐TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization.     

Spike‐dip transformation of Setaria viridis

Traditional method of Agrobacterium‐mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium‐mediated genetic transformation of S. viridis using spike dip. Pre‐anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the β‐glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5‐day‐old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike‐dip medium supplemented with 0.025% Silwet L‐77 and 200 μm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β‐glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron‐interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high‐throughput transformation and potentially facilitates translational research in a monocot model plant.     

Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize

In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants. Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides. Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure. Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene. To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection. The high frequency of A. tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil.     

Pollinator‐mediated assemblage processes in California wildflowers

Community assembly is the result of multiple ecological and evolutionary forces that influence species coexistence. For flowering plants, pollinators are often essential for plant reproduction and establishment, and pollinator‐mediated interactions may influence plant community composition. Here, we use null models and community phylogenetic analyses of co‐occurrence patterns to determine the role of pollinator‐mediated processes in structuring plant communities dominated by congeners. We surveyed three species‐rich genera (Limnanthes, Mimulus and Clarkia) with centres of diversity in the Sierra Nevada of California. Each genus contains species that co‐flower and share pollinators, and each has a robust phylogeny. Within each genus, we surveyed 44–48 communities at three spatial scales, measured floral and vegetative traits and tested for segregation or aggregation of: (i) species, (ii) floral traits (which are likely to be influenced by pollinators), and (iii) vegetative traits (which are likely affected by other environmental factors). We detected both aggregation and segregation of floral traits that were uncorrelated with vegetative trait patterns; we infer that pollinators have shaped the community assembly although the mechanisms may be varied (competition, facilitation, or filtering). We also found that mating system differences may play an important role in allowing species co‐occurrence. Together, it appears that pollinators influence community assemblage in these three clades.     

Agrobacterium‐produced and exogenous cytokinin‐modulated Agrobacterium‐mediated plant transformation

Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called ‘crown gall’, via the transfer and integration of transferred DNA (T‐DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)‐induced tumour‐inducing (Ti) plasmid gene, tzs (trans‐zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline‐type A. tumefaciens strains. The loss of Tzs protein expression and trans‐zeatin secretions by the tzs frameshift (tzs‐fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs‐fs mutant with a wild‐type tzs gene restored wild‐type levels of trans‐zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium‐produced cytokinin. Interestingly, although the tzs‐fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans‐zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.