Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

Comparison of VHH-Fc antibody production in Arabidopsis thaliana, Nicotiana benthamiana and Pichia pastoris.

VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH-Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH-Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH-Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N-glycans were expected for KDEL-tagged VHH-Fcs, several VHH-Fcs with an intact KDEL-tag carried complex-type N-glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH-Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.

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.

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.

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.

Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds.

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway.