Jonah Nolf

The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Transgenic loci obtained after Agrobacterium tumefaciens-mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6-30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20-36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4-6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24-31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells.

Constitutively active UVR8 photoreceptor variant in Arabidopsis

Significance Sunlight is an essential environmental factor for photosynthetic plants and ultimately for life on Earth, which is sustained through plants as fundamental source of food. However, plants have a love/hate relationship with sunlight and must be protected from potentially harmful UV-B radiation. The UV-B photoreceptor UVR8 is of great importance in mounting UV-protective responses and thus for survival in sunlight. Based on our understanding of UVR8 signaling, we have engineered a UVR8 variant that is constitutively active in transgenic plants. The generation of a constitutively active photoreceptor variant is an important step in understanding the molecular signaling mechanism and may hold opportunities for crop improvement. Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) is a UV-B photoreceptor that initiates photomorphogenic responses underlying acclimation and UV-B tolerance in plants. UVR8 is a homodimer in its ground state, and UV-B exposure results in its instantaneous monomerization followed by interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), a major factor in UV-B signaling. UV-B photoreception by UVR8 is based on intrinsic tryptophan aromatic amino acid residues, with tryptophan-285 as the main chromophore. We generated transgenic plants expressing UVR8 with a single amino acid change of tryptophan-285 to alanine. UVR8W285A appears monomeric and shows UV-B–independent interaction with COP1. Phenotypically, the plants expressing UVR8W285A exhibit constitutive photomorphogenesis associated with constitutive activation of target genes, elevated levels of anthocyanins, and enhanced, acclimation-independent UV-B tolerance. Moreover, we have identified COP1, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 (RUP1 and RUP2), and the SUPPRESSOR OF PHYA-105 (SPA) family as proteins copurifying with UVR8W285A. Whereas COP1, RUP1, and RUP2 are known to directly interact with UVR8, we show that SPA1 interacts with UVR8 indirectly through COP1. We conclude that UVR8W285A is a constitutively active UVR8 photoreceptor variant in Arabidopsis, as is consistent with the crucial importance of monomer formation and COP1 binding for UVR8 activity.

Generation of Single-Copy T-DNA Transformants in Arabidopsis by the CRE/loxP Recombination-Mediated Resolution System

We investigated whether complex T-DNA loci, often resulting in low transgene expression, can be resolved efficiently into single copies by CRE/loxP-mediated recombination. An SB-loxP T-DNA, containing two invertedly oriented loxP sequences located inside and immediately adjacent to the T-DNA border ends, was constructed. Regardless of the orientation and number of SB-loxP-derived T-DNAs integrated at one locus, recombination between the outermost loxP sequences in direct orientation should resolve multiple copies into a single T-DNA copy. Seven transformants with a complex SB-loxP locus were crossed with a CRE-expressing plant. In three hybrids, the complex T-DNA locus was reduced efficiently to a single-copy locus. Upon segregation of the CRE recombinase gene, only the simplified T-DNA locus was found in the progeny, demonstrating DNA had been excised efficiently in the progenitor cells of the gametes. In the two transformants with an inverted T-DNA repeat, the T-DNA resolution was accompanied by at least a 10-fold enhanced transgene expression. Therefore, the resolution of complex loci to a single-copy T-DNA insert by the CRE/loxP recombination system can become a valuable method for the production of elite transgenic Arabidopsis thaliana plants that are less prone to gene silencing.

Fusion of an Fc chain to a VHH boosts the accumulation levels in Arabidopsis seeds

Nanobodies® (VHHs) provide powerful tools in therapeutic and biotechnological applications. Nevertheless, for some applications, bivalent antibodies perform much better, and for this, an Fc chain can be fused to the VHH domain, resulting in a bivalent homodimeric VHH-Fc complex. However, the production of bivalent antibodies in Escherichia coli is rather inefficient. Therefore, we compared the production of VHH7 and VHH7-Fc as antibodies of interest in Arabidopsis seeds for detecting prostate-specific antigen (PSA), a well-known biomarker for prostate cancer in the early stages of tumour development. The influence of the signal sequence (camel versus plant) and that of the Fc chain origin (human, mouse or pig) were evaluated. The accumulation levels of VHHs were very low, with a maximum of 0.13% VHH of total soluble protein (TSP) in homozygous T3 seeds, while VHH-Fc accumulation levels were at least 10- to 100-fold higher, with a maximum of 16.25% VHH-Fc of TSP. Both the camel and plant signal peptides were efficiently cleaved off and did not affect the accumulation levels. However, the Fc chain origin strongly affected the degree of proteolysis, but only had a slight influence on the accumulation level. Analysis of the mRNA levels suggested that the low amount of VHHs produced in Arabidopsis seeds was not due to a failure in transcription, but rather to translation inefficiency, protein instability and/or degradation. Most importantly, the plant-produced VHH7 and VHH7-Fc antibodies were functional in detecting PSA and could thus be used for diagnostic applications.

Production of Camel-Like Antibodies in Plants

Transgenic plants for the production of high-value recombinant complex and/or glycosylated proteins are a promising alternative for conventional systems, such as mammalian cells and bacteria. Many groups use plants as production platform for antibodies and antibody fragments. Here, we describe how bivalent camel-like antibodies can be produced in leaves and seeds. Camel-like antibodies are fusions of the antigen-binding domain of heavy chain camel antibodies (VHH) with an Fc fragment of choice. Transient expression in Nicotiana benthamiana leaves allows the production of VHH-Fc antibodies within a few days after the expression plasmid has been obtained. Generation of stable Arabidopsis thaliana transformants allows production of scalable amounts of VHH-Fc antibodies in seeds within a year. Further, we describe how the in planta-produced VHH-Fc antibodies can be quantified by Western blot analysis with Fc-specific antibodies.

Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase

Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants.

Transitive RNA silencing signals induce cytosine methylation of a transgenic but not an endogenous target.

Post-transcriptional gene silencing of a primary target gene in plants can coincide with the production of secondary small interfering RNAs (siRNAs) of coding sequences adjacent to the target region and with de novo RNA-directed DNA methylation (RdDM) thereof. Here, we analyzed the susceptibility of transgenic and endogenous targets to RdDM induced by primary and secondary silencing signals. In three different configurations, primary silencing signals were able to direct in trans methylation of chimeric transgenes and the CATALASE2 (CAT2) endogene; however, extensive spreading of methylation occurred only in the transgene, resulting in the methylation of the flanking CAT2 sequence, whereas methylation of the CAT2 endogene was restricted to the target region and the enclosed introns. The secondary silencing signals arising from this transgenic primary target simultaneously silenced a secondary transgene target and the CAT2 endogene, but were only capable of directing RdDM to the transgene. Our data indicate that RdDM is correlated with the in situ generation of secondary siRNAs, occurring in P35S-driven transgenes but not in most endogenes. We conclude that although both endogenes and transgenes are equally sensitive to transitive silencing, differences exist in their susceptibility to undergo secondary RdDM.

The Efficiency of Arabidopsis thaliana Floral Dip Transformation Is Determined Not Only by the Agrobacterium Strain Used but Also by the Physiology and the Ecotype of the Dipped Plant

To evaluate the chromosomal background of different Agrobacterium strains on floral dip transformation frequency, eight wild-type Agrobacterium strains, provided by Laboratorium voor Microbiologie Gent (LMG) and classified in different genomic groups, were compared with the commonly used Agrobacterium strains C58C1 Rif(r) (pMP90) and LBA4404 in Arabidopsis thaliana Columbia (Col-0) and C24 ecotypes. The C58C1 Rif(r) chromosomal background in combination with the pMP90 virulence plasmid showed high Col-0 floral dip transformation frequencies (0.76 to 1.57%). LMG201, which is genetically close to the Agrobacterium C58 strain, with the same virulence plasmid showed comparable or even higher transformation frequencies (1.22 to 2.28%), whereas the LBA4404 strain displayed reproducibly lower transformation frequencies (<0.2%). All other tested LMG Agrobacterium chromosomal backgrounds had transformation frequencies between those of the C58C1 Rif(r) (pMP90) and LBA4404 reference strains. None of the strains could transform the C24 ecotype with a frequency higher than 0.1%. Strikingly, all Arabidopsis Col-0 floral dip transformation experiments showed a high transformation variability from plant to plant (even more than 50-fold) within and across the performed biological repeats for all analyzed Agrobacterium strains. Therefore, the physiology of the plant and, probably, the availability of competent flowers to be transformed determine, to a large extent, floral dip transformation frequencies.

The Mitochondrial DNA (mtDNA)-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination

SWIB5 can associate with DNA in mitochondria, where its proposed role in DNA repair and architecture might influence cell proliferation during development, suggesting that these processes are linked. In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

High frequency of single-copy T-DNA transformants produced after floral dip in CRE-expressing Arabidopsis plants

Transgenic plants that harbor a single copy of the introduced transgene are preferable to those with multiple transgene copies because multiple T-DNA copies correlate with expression variability and susceptibility to silencing. Especially after the commonly used floral-dip Agrobacterium-mediated transformation method, the frequency of single-copy transformants is low. The CRE/loxP recombinase-based strategy to resolve complex T-DNA loci has proven to be successful to efficiently obtain single-copy T-DNA transformants by directly transforming loxP-containing T-DNA vectors in CRE-expressing Arabidopsis thaliana plants. This chapter describes in detail how to transform three available loxP-containing T-DNA vectors into CRE-producing Arabidopsis C24 plants and subsequently how to analyze the transgenic plants for the T-DNA locus structure.