Sylvia de Pater

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 approximately 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.

RAP-1 is an Arabidopsis MYC-like R protein homologue, that binds to G-box sequence motifs.

An Arabidopsis cDNA clone encoding a DNA-binding protein, RAP-1, was isolated by southwestern screening of an Escherichia coli cDNA expression library. The protein contains a bHLH DNA-binding domain and is homologous to R proteins, regulating anthocyanin biosynthesis. RAP-1 binds to the sequence CACNTG. It is encoded by a single gene, which is expressed to high levels in root and stem and to low levels in leaf and flower. No expression could be detected in siliques. Rap-1 does not correspond to one of the known loci involved in anthocyanin biosynthesis, since it is located at a different map position. In contrast to the maize R protein Lc, RAP-1 did not induce anthocyanin biosynthesis in pea cotyledons. Thus, RAP-1 is a novel member of the bHLH class of DNA-binding proteins.

ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation.

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10−3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10−3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis. To this end Arabidopsis parp1, parp2 and parp1parp2 (p1p2) mutants were isolated and functionally characterized. The p1p2 double mutant was crossed with the C-NHEJ ku80 mutant resulting in the parp1parp2ku80 (p1p2k80) triple mutant. As expected, because of their role in single strand break repair (SSBR) and base excision repair (BER), the p1p2 and p1p2k80 mutants were shown to be sensitive to treatment with the DNA damaging agent MMS. End-joining assays in cell-free leaf protein extracts of the different mutants using linear DNA substrates with different ends reflecting a variety of double strand breaks were performed. The results showed that compatible 5′-overhangs were accurately joined in all mutants, that KU80 protected the ends preventing the formation of large deletions and that PARP proteins were involved in microhomology mediated end joining (MMEJ), one of the characteristics of B-NHEJ.

Structure and expression of a light-inducible shoot-specific rice gene.

By differential screening of a cDNA library of two-week-old rice seedlings cDNA clones were obtained, corresponding to shoot-specific mRNAs. By sequence analysis two of these clones were found to be rbcS cDNA clones. The mRNA corresponding to a third cDNA clone (COS5) displayed an expression pattern similar to the expression pattern of rbcS genes. The mRNA (800 bases) was light-inducible and encoded by a single-copy gene. The genomic clone (GOS5) was isolated and the intron/exon structure was determined by comparing the nucleotide sequence of the mRNA and the genomic clone. The gene contains two introns. Transcription start sites were determined by S1-nuclease mapping and primer extension. The start site obtained by both methods is located 87 bp upstream of the translation start site and 23 bp downstream of TATA box-like sequence. In the 5′ non-coding region motifs can be found that are homologous to sequences in promoters that are light-or UV-inducible or confer leaf-specific expression. The open reading frame present in GOS5 codes for a protein (15 kDa) that contains a putative chloroplast transit peptide and does not show any significant homology to protein sequences in the NBRF protein database.

T-DNA integration in plants results from polymerase- θ -mediated DNA repair

Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown1. Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA–plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3′ end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer–template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

Transposon proliferation in an asexual parasitoid.

The widespread occurrence of sex is one of the most elusive problems in evolutionary biology. Theory predicts that asexual lineages can be driven to extinction by uncontrolled proliferation of vertically transmitted transposable elements (TEs), which accumulate because of the inefficiency of purifying selection in the absence of sex and recombination. To test this prediction, we compared genome-wide TE load between a sexual lineage of the parasitoid wasp Leptopilina clavipes and a lineage of the same species that is rendered asexual by Wolbachia-induced parthenogenesis. We obtained draft genome sequences at 15-20× coverage of both the sexual and the asexual lineages using next-generation sequencing. We identified transposons of most major classes in both lineages. Quantification of TE abundance using coverage depth showed that copy numbers in the asexual lineage exceeded those in the sexual lineage for DNA transposons, but not LTR and LINE-like elements. However, one or a small number of gypsy-like LTR elements exhibited a fourfold higher coverage in the asexual lineage. Quantitative PCR showed that high loads of this gypsy-like TE were characteristic for 11 genetically distinct asexual wasp lineages when compared to sexual lineages. We found no evidence for an overall increase in copy number for all TE types in asexuals as predicted by theory. Instead, we suggest that the expansions of specific TEs are best explained as side effects of (epi)genetic manipulations of the host genome by Wolbachia. Asexuality is achieved in a myriad of ways in nature, many of which could similarly result in TE proliferation.

Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

The SLEEPER genes: a transposase-derived angiosperm-specific gene family

BackgroundDAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for development in Arabidopsis thaliana. Little is known about the presence of DAYSLEEPER orthologs in other species, or how and when it was domesticated. We studied the presence of DAYSLEEPER orthologs in plants and propose a model for the domestication of the ancestral DAYSLEEPER gene in angiosperms.ResultsUsing specific BLAST searches in genomic and EST libraries, we found that DAYSLEEPER-like genes (hereafter called SLEEPER genes) are unique to angiosperms. Basal angiosperms as well as grasses (Poaceae) and dicotyledonous plants possess such putative orthologous genes, but SLEEPER-family genes were not found in gymnosperms, mosses and algae. Most species contain more than one SLEEPER gene. All SLEEPERs contain a C2H2 type BED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the BED-zinc finger and dimerization domain, which are hallmark features in the SLEEPER family. Although SLEEPER genes are structurally conserved between species, constructs with SLEEPER genes from grapevine and rice did not complement the daysleeper phenotype in Arabidopsis, when expressed under control of the DAYSLEEPER promoter. However these constructs did cause a dominant phenotype when expressed in Arabidopsis. Rice plant lines with an insertion in the RICESLEEPER1 or 2 locus displayed phenotypic abnormalities, indicating that these genes are functional and important for normal development in rice. We suggest a model in which we hypothesize that an ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution. Evidence is also presented for more recent retroposition events of SLEEPER genes, such as an event in the rice genome, which gave rise to the RICESLEEPER1 and 2 genes.ConclusionsWe propose the ancestral SLEEPER gene was formed after a process of retro-transposition during the evolution of the first angiosperms. It may have acquired an important function early on, as mutation of two SLEEPER genes in rice, like the daysleeper mutant in A. thaliana gave a developmental phenotype indicative of their importance for normal plant development.

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for β-glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3′ flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11 000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10–15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5′ expression control sequences.