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Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

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In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of >multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1-STAP1 and dTALE2-STAP2—can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Agriculture is by far the biggest water consumer on our planet, accounting for 70 percent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently (‘more crop per drop’). Water‐use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here we describe a combinatorial engineering approach to optimize signaling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium‐dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water‐limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water‐limited conditions. Our work provides an efficient strategy for engineering complex signaling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function.

Hypericin is a molecule of high pharmaceutical importance that is synthesized and stored in dark glands (DGs) of St. John's wort (Hypericum perforatum). Understanding which genes are involved in dark gland development and hypericin biosynthesis is important for the development of new Hypericum extracts that are highly demanded for medical applications. We identified two transcription factors, whose expression is strictly synchronized with the differentiation of DGs. We correlated the content of hypericin, pseudohypericin, endocrocin, skyrin glycosides and several flavonoids with gene expression and DG development to obtain a revised model for hypericin biosynthesis. Here we report for the first‐time genotypes which are polymorphic for the presence/total‐absence (G+/G‐) of DGs in their placental tissues (PTs). DG development was characterized in PTs using several microscopy techniques. Fourier‐transformed infrared microscopy was established as a novel method to precisely locate polyaromatic compounds, such as hypericin, in plant tissues. In addition, we obtained transcriptome and metabolome profiles of unprecedented resolution in Hypericum. This study addresses for the first time the development of dark glands and identifies genes that constitute strong building blocks for the further elucidation of hypericin synthesis, its manipulation in plants, its engineering in microbial systems, and its applications in medical research.

Amyloid-β (Aβ) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer’s disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aβ and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches.Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aβ deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAβ were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAβ together with endogenous mAβ. Furthermore, the cellular response to Aβ deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.

Alzheimer’s disease primarily occurs as sporadic disease and is accompanied with vast socio-economic problems. The mandatory basic research relies on robust and reliable disease models to overcome increasing incidence and emerging social challenges. Rodent models are most efficient, versatile, and predominantly used in research. However, only highly artificial and mostly genetically modified models are available. As these ‘engineered’ models reproduce only isolated features, researchers demand more suitable models of sporadic neurodegenerative diseases. One very promising animal model was the South American rodent Octodon degus, which was repeatedly described as natural ‘sporadic Alzheimer’s disease model’ with ‘Alzheimer’s disease-like neuropathology’. To unveil advantages over the ‘artificial’ mouse models, we re-evaluated the age-dependent, neurohistological changes in young and aged Octodon degus (1 to 5-years-old) bred in a wild-type colony in Germany. In our hands, extensive neuropathological analyses of young and aged animals revealed normal age-related cortical changes without obvious signs for extensive degeneration as seen in patients with dementia. Neither significant neuronal loss nor enhanced microglial activation were observed in aged animals. Silver impregnation methods, conventional, and immunohistological stains as well as biochemical fractionations revealed neither amyloid accumulation nor tangle formation. Phosphoepitope-specific antibodies against tau species displayed similar intraneuronal reactivity in both, young and aged Octodon degus.In contrast to previous results, our study suggests that Octodon degus born and bred in captivity do not inevitably develop cortical amyloidosis, tangle formation or neuronal loss as seen in Alzheimer’s disease patients or transgenic disease models.

IntroductionAlzheimer’s disease (AD) is associated with the accumulation of β-amyloid (Aβ) as senile plaques in the brain, thus leading to neurodegeneration and cognitive impairment. Plaque formation depends not merely on the amount of generated Aβ peptides, but more importantly on their effective removal. Chronic infections with neurotropic pathogens, most prominently the parasite Toxoplasma (T.) gondii, are frequent in the elderly, and it has been suggested that the resulting neuroinflammation may influence the course of AD. In the present study, we investigated how chronic T. gondii infection and resulting neuroinflammation affect plaque deposition and removal in a mouse model of AD.ResultsChronic infection with T. gondii was associated with reduced Aβ and plaque load in 5xFAD mice. Upon infection, myeloid-derived CCR2hi Ly6Chi monocytes, CCR2+ Ly6Cint, and CCR2+ Ly6Clow mononuclear cells were recruited to the brain of mice. Compared to microglia, these recruited mononuclear cells showed highly increased phagocytic capacity of Aβ ex vivo. The F4/80+ Ly6Clow macrophages expressed high levels of Triggering Receptor Expressed on Myeloid cells 2 (TREM2), CD36, and Scavenger Receptor A1 (SCARA1), indicating phagocytic activity. Importantly, selective ablation of CCR2+ Ly6Chi monocytes resulted in an increased amount of Aβ in infected mice. Elevated insulin-degrading enzyme (IDE), matrix metalloproteinase 9 (MMP9), as well as immunoproteasome subunits β1i/LMP2, β2i/MECL-1, and β5i/LMP7 mRNA levels in the infected brains indicated increased proteolytic Aβ degradation. Particularly, LMP7 was highly expressed by the recruited mononuclear cells in the brain, suggesting a novel mechanism of Aβ clearance.ConclusionsOur results indicate that chronic Toxoplasma infection ameliorates β-amyloidosis in a murine model of AD by activation of the immune system, specifically by recruitment of Ly6Chi monocytes and by enhancement of phagocytosis and degradation of soluble Aβ. Our findings provide evidence for a modulatory role of inflammation-induced Aβ phagocytosis and degradation by newly recruited peripheral immune cells in the pathophysiology of AD.

Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co‐expression of BChE with a novel gene‐stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N‐glycans. The N‐glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma‐derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER‐typical oligomannosidic structures. Biochemical analyses and live‐cell imaging experiments indicated that impaired N‐glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic‐reticulum‐derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in‐depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.