Blue Light-Operated CRISPR/Cas13b-Mediated mRNA Knockdown (Lockdown).

The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introduces optical control of RNA levels utilizing a blue light-dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence-specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene-expression and induce protein destabilization with blue light yields efficient triple-controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin-dependent kinase 1 (hCdk1) leads to blue light-induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.

COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling.

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these pathways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homologue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, far-red and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light signalling during early seedling development, thereby preventing an exaggerated response to light.

Optogenetic Downregulation of Protein Levels to Control Programmed Cell Death in Mammalian Cells with a Dual Blue-Light Switch.

Optogenetic approaches facilitate the study of signaling and metabolic pathways in animal cell systems. In the past 10 years, a plethora of light-regulated switches for the targeted control over the induction of gene expression, subcellular localization of proteins, membrane receptor activity, and other cellular processes have been developed and successfully implemented. However, only a few tools have been engineered toward the quantitative and spatiotemporally resolved downregulation of proteins. Here we present a protocol for reversible and rapid blue light-induced reduction of protein levels in mammalian cells. By implementing a dual-regulated optogenetic switch (Blue-OFF), both repression of gene expression and degradation of the target protein are triggered simultaneously. We apply this system for the blue light-mediated control of programmed cell death. HEK293T cells are transfected with the proapoptotic proteins PUMA and BID integrated into the Blue-OFF system. Overexpression of these proteins leads to programmed cell death, which can be prevented by irradiation with blue light. This experimental approach is very straightforward, requires just simple hardware, and therefore can be easily implemented in state-of-the-art equipped mammalian cell culture labs. The system can be used for targeted cell signaling studies and biotechnological applications.

Shade avoidance responses become more aggressive in warm environments.

When exposed to neighbour cues, competitive plants increase stem growth to reduce the degree of current or future shade. The aim of this work is to investigate the impact of weather conditions on the magnitude of shade avoidance responses in Arabidopsis thaliana. We first generated a growth rate database under controlled conditions and elaborated a model that predicts daytime hypocotyl growth as a function of the activity of the main photosensory receptors (phytochromes A and B, cryptochromes 1 and 2) in combination with light and temperature inputs. We then incorporated the action of thermal amplitude to account for its effect on selected genotypes, which correlates with the dynamics of the growth-promoting transcription factor PHYTOCHROME-INTERACTING FACTOR 4. The model predicted growth rate in the field with reasonable accuracy. Thus, we used the model in combination with a worldwide data set of current and future whether conditions. The analysis predicted enhanced shade avoidance responses as a result of higher temperatures due to the geographical location or global warming. Irradiance and thermal amplitude had no effects. These trends were also observed for our local growth rate measurements. We conclude that, if water and nutrients do not become limiting, warm environments enhance the shade avoidance response.

Erratum: Publisher Correction: Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors.

[This corrects the article DOI: 10.1038/s42003-019-0687-9.].

Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors.

Phytochrome photoreceptors mediate adaptive responses of plants to red and far-red light. These responses generally entail light-regulated association between phytochromes and other proteins, among them the phytochrome-interacting factors (PIF). The interaction with Arabidopsis thaliana phytochrome B (AtPhyB) localizes to the bipartite APB motif of the A. thaliana PIFs (AtPIF). To address a dearth of quantitative interaction data, we construct and analyze numerous AtPIF3/6 variants. Red-light-activated binding is predominantly mediated by the APB N-terminus, whereas the C-terminus modulates binding and underlies the differential affinity of AtPIF3 and AtPIF6. We identify AtPIF variants of reduced size, monomeric or homodimeric state, and with AtPhyB affinities between 10 and 700 nM. Optogenetically deployed in mammalian cells, the AtPIF variants drive light-regulated gene expression and membrane recruitment, in certain cases reducing basal activity and enhancing regulatory response. Moreover, our results provide hitherto unavailable quantitative insight into the AtPhyB:AtPIF interaction underpinning vital light-dependent responses in plants.

The Suitability of Orthogonal Hosts to Study Plant Cell Wall Biosynthesis.

Plant cells are surrounded by an extracellular matrix that consists mainly of polysaccharides. Many molecular components involved in plant cell wall polymer synthesis have been identified, but it remains largely unknown how these molecular players function together to define the length and decoration pattern of a polysaccharide. Synthetic biology can be applied to answer questions beyond individual glycosyltransferases by reconstructing entire biosynthetic machineries required to produce a complete wall polysaccharide. Recently, this approach was successful in establishing the production of heteromannan from several plant species in an orthogonal host-a yeast-illuminating the role of an auxiliary protein in the biosynthetic process. In this review we evaluate to what extent a selection of organisms from three kingdoms of life (Bacteria, Fungi and Animalia) might be suitable for the synthesis of plant cell wall polysaccharides. By identifying their key attributes for glycoengineering as well as analyzing the glycosidic linkages of their native polymers, we present a valuable comparison of their key advantages and limitations for the production of different classes of plant polysaccharides.

Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells.

Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch ('Blue-OFF'), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.