Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.

Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.

PHYTOCHROME-INTERACTING FACTORS are involved in starch degradation adjustment via inhibition of the carbon metabolic regulator QUA-QUINE STARCH in Arabidopsis.

As sessile organisms, plants encounter dynamic and challenging environments daily, including abiotic/biotic stresses. The regulation of carbon and nitrogen allocations for the synthesis of plant proteins, carbohydrates, and lipids is fundamental for plant growth and adaption to its surroundings. Light, one of the essential environmental signals, exerts a substantial impact on plant metabolism and resource partitioning (i.e., starch). However, it is not fully understood how light signaling affects carbohydrate production and allocation in plant growth and development. An orphan gene unique to Arabidopsis thaliana, named QUA-QUINE STARCH (QQS) is involved in the metabolic processes for partitioning of carbon and nitrogen among proteins and carbohydrates, thus influencing leaf, seed composition, and plant defense in Arabidopsis. In this study, we show that PHYTOCHROME-INTERACTING bHLH TRANSCRIPTION FACTORS (PIFs), including PIF4, are required to suppress QQS during the period at dawn, thus preventing overconsumption of starch reserves. QQS expression is significantly de-repressed in pif4 and pifQ, while repressed by overexpression of PIF4, suggesting that PIF4 and its close homologs (PIF1, PIF3, and PIF5) act as negative regulators of QQS expression. In addition, we show that the evening complex, including ELF3 is required for active expression of QQS, thus playing a positive role in starch catabolism during night-time. Furthermore, QQS is epigenetically suppressed by DNA methylation machinery, whereas histone H3 K4 methyltransferases (e.g., ATX1, ATX2, and ATXR7) and H3 acetyltransferases (e.g., HAC1 and HAC5) are involved in the expression of QQS. This study demonstrates that PIF light signaling factors help plants utilize optimal amounts of starch during the night and prevent overconsumption of starch before its biosynthesis during the upcoming day.

Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach.

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

SPAs promote thermomorphogenesis by regulating the phyB-PIF4 module in Arabidopsis.

High ambient temperature attributable to global warming has a profound influence on plant growth and development at all stages of the life cycle. The response of plants to high ambient temperature, termed thermomorphogenesis, is characterized by hypocotyl and petiole elongation and hyponastic growth at the seedling stage. However, our understanding of the molecular mechanism of thermomorphogenesis is still rudimentary. Here, we show that a set of four SUPPRESSOR OF PHYA-105 (SPA) genes is required for thermomorphogenesis. Consistently, SPAs are necessary for global changes in gene expression in response to high ambient temperature. In the spaQ mutant at high ambient temperature, the level of SPA1 is unaffected, whereas the thermosensor phytochrome B (phyB) is stabilized. Furthermore, in the absence of four SPA genes, the pivotal transcription factor PIF4 fails to accumulate, indicating a role of SPAs in regulating the phyB-PIF4 module at high ambient temperature. SPA1 directly phosphorylates PIF4 in vitro, and a mutant SPA1 affecting the kinase activity fails to rescue the PIF4 level in addition to the thermo-insensitive phenotype of spaQ, suggesting that the SPA1 kinase activity is necessary for thermomorphogenesis. Taken together, these data suggest that SPAs are new components that integrate light and temperature signaling by fine-tuning the phyB-PIF4 module.

Plant photoreceptors: Multi-functional sensory proteins and their signaling networks.

Light is a crucial environmental cue not only for photosynthetic energy production but also for plant growth and development. Plants employ sophisticated methods to detect and interpret information from incoming light. Five classes of photoreceptors have been discovered in the model plant Arabidopsis thaliana. These photoreceptors act either distinctly and/or redundantly in fine-tuning many aspects of plant life cycle. Unlike mobile animals, sessile plants have developed an enormous plasticity to adapt and survive in changing environment. By monitoring different information arising from ambient light, plants precisely regulate downstream signaling pathways to adapt accordingly. Given that changes in the light environment is typically synchronized with other environmental cues such as temperature, abiotic stresses, and seasonal changes, it is not surprising that light signaling pathways are interconnected with multiple pathways to regulate plant physiology and development. Indeed, recent advances in plant photobiology revealed a large network of co-regulation among different photoreceptor signaling pathways as well as other internal signaling pathways (e.g., hormone signaling). In addition, some photoreceptors are directly involved in perception of non-light stimuli (e.g., temperature). Therefore, understanding highly inter-connected signaling networks is essential to explore the photoreceptor functions in plants. Here, we summarize how plants co-ordinate multiple photoreceptors and their internal signaling pathways to regulate a myriad of downstream responses at molecular and physiological levels.

Direct phosphorylation of HY5 by SPA kinases to regulate photomorphogenesis in Arabidopsis.

Elongated hypocotyl5 (HY5) is a key transcription factor that promotes photomorphogenesis. Constitutive photomorphogenic1 (COP1)-Suppressor of phytochrome A-105 (SPA) E3 ubiquitin ligase complex promotes ubiquitination and degradation of HY5 to repress photomorphogenesis in darkness. HY5 is also regulated by phosphorylation at serine 36 residue. However, the kinase responsible for phosphorylation of HY5 remains unknown. Here, using extensive in vitro and in vivo biochemical, genetic, and photobiological techniques, we have identified a new kinase that phosphorylates HY5 and demonstrated the significance of phosphorylation of HY5 in Arabidopsis thaliana. We show that SPA proteins are the missing kinases necessary for HY5 phosphorylation. SPAs can directly phosphorylate HY5 in vitro, and the phosphorylated HY5 is absent in the spaQ background in vivo. We also demonstrate that the unphosphorylated HY5 interacts strongly with both COP1 and SPA1 and is the preferred substrate for degradation, whereas the phosphorylated HY5 is more stable in the dark. In addition, the unphosphorylated HY5 actively binds to the target promoters and is the physiologically more active form. Consistently, the transgenic plants expressing the unphosphorylated form of HY5 display enhanced photomorphogenesis. Collectively, our study revealed the missing kinase responsible for direct phosphorylation of HY5 that fine-tunes its stability and activity to regulate photomorphogenesis.

Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana.

Growth plasticity is a key mechanism by which plants adapt to the ever-changing environmental conditions. Since growth is a high-energy-demanding and irreversible process, it is expected to be regulated by the integration of endogenous energy status as well as environmental conditions. Here, we show that trehalose-6-phosphate (T6P) functions as a sugar signaling molecule that coordinates thermoresponsive hypocotyl growth with endogenous sugar availability. We found that the loss of T6P SYNTHASE 1 (TPS1) in Arabidopsis thaliana impaired high-temperature-mediated hypocotyl growth. Consistently, the activity of PIF4, a transcription factor that positively regulates hypocotyl growth, was compromised in the tps1 mutant. We further show that, in the tps1 mutant, a sugar signaling kinase KIN10 directly phosphorylates and destabilizes PIF4. T6P inhibits KIN10 activity in a GRIK-dependent manner, allowing PIF4 to promote hypocotyl growth at high temperatures. Together, our results demonstrate that T6P determines thermoresponsive growth through the KIN10-PIF4 signaling module. Such regulation of PIF4 by T6P integrates the temperature-signaling pathway with the endogenous sugar status, thus optimizing plant growth response to environmental stresses.

One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/µL of input viral genomic RNA.

Characterization of Phytochrome Interacting Factors from the Moss Physcomitrella patens Illustrates Conservation of Phytochrome Signaling Modules in Land Plants.

Across the plant kingdom, phytochrome (PHY) photoreceptors play an important role during adaptive and developmental responses to light. In Arabidopsis thaliana, light-activated PHYs accumulate in the nucleus, where they regulate downstream signaling components, such as phytochrome interacting factors (PIFs). PIFs are transcription factors that act as repressors of photomorphogenesis; their inhibition by PHYs leads to substantial changes in gene expression. The nuclear function of PHYs, however, has so far been investigated in only a few non-seed plants. Here, we identified putative target genes of PHY signaling in the moss Physcomitrella patens and found light-regulated genes that are putative orthologs of PIF-controlled genes in Arabidopsis. Phylogenetic analyses revealed that an ancestral PIF-like gene was already present in streptophyte algae, i.e., before the water-to-land transition of plants. The PIF homologs in the genome of P. patens resemble Arabidopsis PIFs in their protein domain structure, molecular properties, and physiological effects, albeit with notable differences in the motif-dependent PHY interaction. Our results suggest that P. patens PIFs are involved in PHY signaling. The PHY-PIF signaling node that relays light signals to target genes has been largely conserved during land plant evolution, with evidence of lineage-specific diversification.

Genomic evidence reveals SPA-regulated developmental and metabolic pathways in dark-grown Arabidopsis seedlings.

Photomorphogenesis is repressed in the dark mainly by an E3 ubiquitin ligase complex comprising CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and four homologous proteins called SUPPRESSOR OF PHYA-105 (SPA1-SPA4) in Arabidopsis. This complex induces the ubiquitination and subsequent degradation of positively acting transcription factors (TFs; e.g. ELONGATED HYPOCOTYL (HY5), LONG HYPOCOTYL IN FAR-RED 1 (HFR1), PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) and others] in the dark to repress photomorphogenesis. Genomic evidence showed a large number of genes regulated by COP1 in the dark, of which many are direct targets of HY5. However, the genomic basis for the constitute photomorphogenic phenotype of spaQ remains unknown. Here, we show that >7200 genes are differentially expressed in the spaQ background compared to wild-type in the dark. Comparison of the RNA sequencing (RNA-Seq) data between cop1 and spaQ revealed a large overlapping set of genes regulated by the COP1-SPA complex. In addition, many of the genes coordinately regulated by the COP1-SPA complex are also regulated by HY5 directly and indirectly. Taken together, our data reveal that SPA proteins repress photomorphogenesis by controlling gene expression in concert with COP1, likely through regulating the abundance of downstream TFs in light signaling pathways. Moreover, SPA proteins may function both in a COP1-dependent and -independent manner in regulating many biological processes and developmental pathways in Arabidopsis.

PHYTOCHROME INTERACTING FACTORS mediate metabolic control of the circadian system in Arabidopsis.

The circadian (c. 24 h) system has a central role in regulating the timing and coordination of photosynthesis, and in turn photosynthesis and photosynthetic products which are controlled by the circadian clock feedback to affect the circadian oscillator that generates rhythms. However, little is known about the mechanism(s) by which this feedback occurs. One group of likely candidates for signal transduction to the circadian clock are the PHYTOCHROME INTERACTING FACTOR (PIF) family of transcription factors which have been shown to be involved in numerous signaling pathways in Arabidopsis. Yet despite evidence that some PIF genes are under circadian control and bind promoter motifs present in circadian genes, until now PIFs have not been shown to affect the circadian system. Using a range of techniques, we have examined how circadian rhythms are affected in higher order pif mutants and the mechanisms by which PIFs regulate signaling to the circadian clock. We show that PIFs mediate metabolic signals to the circadian oscillator and that sucrose directly affects PIF binding to the promoters of key circadian oscillator genes in vivo that may entrain the oscillator. Our results provide a basis for understanding the mechanism for metabolic signaling to the circadian system in Arabidopsis.

PHYTOCHROME INTERACTING FACTOR1 Enhances the E3 Ligase Activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to Synergistically Repress Photomorphogenesis in Arabidopsis.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat-containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYTOCHROME A (SPA) proteins, and these complexes degrade positively acting transcription factors in the dark to repress photomorphogenesis. Phytochrome-interacting basic helix-loop-helix transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces the rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we present genetic evidence that the pif and cop1/spa Arabidopsis thaliana mutants synergistically promote photomorphogenesis in the dark. LONG HYPOCOTYL5 (HY5) is stabilized in the cop1 pif1, spa123 pif1, and pif double, triple, and quadruple mutants in the dark. Moreover, the hy5 mutant suppresses the constitutive photomorphogenic phenotypes of the pifq mutant in the dark. PIF1 forms complexes with COP1, HY5, and SPA1 and enhances the substrate recruitment and autoubiquitylation and transubiquitylation activities of COP1. These data uncover a novel function of PIFs as the potential cofactors of COP1 and provide a genetic and biochemical model of how PIFs and COP1-SPA complexes synergistically repress photomorphogenesis in the dark.

Phytochrome regulates translation of mRNA in the cytosol.

An array of photoreceptors including cryptochromes, phototropin, and phytochromes regulates various light responses in plants. Among these photoreceptors, phytochromes perceive red and far-red light by switching between two interconvertible spectral forms (Pr and Pfr). The Pfr form promotes light responses partly by destabilizing negatively acting, phytochrome-interacting basic helix-loop-helix transcription factors (PIFs), thus modulating transcription in the nucleus. The Pfr form is also present in the cytosol. However, the role of phytochromes in the cytosol is not well understood. Here we show that the Pfr form interacts with the cytosolic protein PENTA1 (PNT1) and inhibits the translation of protochlorophyllide reductase (PORA) mRNA. PNT1 possesses five C3H-type zinc finger domains and displays similarity to various RNA binding proteins including Tristetraprolin, which regulates stabilities of mRNAs such as TNF-α mRNA in humans. Consistent with its function as an RNA binding protein, PNT1 directly binds to mRNA of a key chlorophyll biosynthetic gene, protochlorophyllide reductase in vivo and inhibits the translation of PORA mRNA in the presence of phytochromes. The present results demonstrate that phytochromes transmit light signals to regulate not only transcription in the nucleus through PIFs, but also translation in the cytosol through PNT1.

Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions.

The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.

Preparation and Use of Cellular Reagents: A Low-resource Molecular Biology Reagent Platform.

Protein reagents are indispensable for most molecular and synthetic biology procedures. Most conventional protocols rely on highly purified protein reagents that require considerable expertise, time, and infrastructure to produce. In consequence, most proteins are acquired from commercial sources, reagent expense is often high, and accessibility may be hampered by shipping delays, customs barriers, geopolitical constraints, and the need for a constant cold chain. Such limitations to the widespread availability of protein reagents, in turn, limit the expansion and adoption of molecular biology methods in research, education, and technology development and application. Here, we describe protocols for producing a low-resource and locally sustainable reagent delivery system, termed "cellular reagents," in which bacteria engineered to overexpress proteins of interest are dried and can then be used directly as reagent packets in numerous molecular biology reactions, without the need for protein purification or a constant cold chain. As an example of their application, we describe the execution of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) using cellular reagents, detailing how to replace pure protein reagents with optimal amounts of rehydrated cellular reagents. We additionally describe a do-it-yourself fluorescence visualization device for using these cellular reagents in common molecular biology applications. The methods presented in this article can be used for low-cost, on-site production of commonly used molecular biology reagents (including DNA and RNA polymerases, reverse transcriptases, and ligases) with minimal instrumentation and expertise, and without the need for protein purification. Consequently, these methods should generally make molecular biology reagents more affordable and accessible. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cellular reagents Alternate Protocol 1: Preparation of lyophilized cellular reagents Alternate Protocol 2: Evaluation of bacterial culture growth via comparison to McFarland turbidity standards Support Protocol 1: SDS-PAGE for protein expression analysis of cellular reagents Basic Protocol 2: Using Taq DNA polymerase cellular reagents for PCR Basic Protocol 3: Using Br512 DNA polymerase cellular reagents for loop-mediated isothermal amplification (LAMP) Support Protocol 2: Building a fluorescence visualization device.

Phytochrome Signaling Networks.

The perception of light signals by the phytochrome family of photoreceptors has a crucial influence on almost all aspects of growth and development throughout a plant's life cycle. The holistic regulatory networks orchestrated by phytochromes, including conformational switching, subcellular localization, direct protein-protein interactions, transcriptional and posttranscriptional regulations, and translational and posttranslational controls to promote photomorphogenesis, are highly coordinated and regulated at multiple levels. During the past decade, advances using innovative approaches have substantially broadened our understanding of the sophisticated mechanisms underlying the phytochrome-mediated light signaling pathways. This review discusses and summarizes these discoveries of the role of the modular structure of phytochromes, phytochrome-interacting proteins, and their functions; the reciprocal modulation of both positive and negative regulators in phytochrome signaling; the regulatory roles of phytochromes in transcriptional activities, alternative splicing, and translational regulation; and the kinases and E3 ligases that modulate PHYTOCHROME INTERACTING FACTORs to optimize photomorphogenesis.

Genetic transformation technologies for the common dandelion, Taraxacum officinale.

BACKGROUND: Taraxacum officinale, or the common dandelion, is a widespread perennial species recognized worldwide as a common lawn and garden weed. Common dandelion is also cultivated for use in teas, as edible greens, and for use in traditional medicine. It produces latex and is closely related to the Russian dandelion, T. kok-saghyz, which is being developed as a rubber crop. Additionally, the vast majority of extant common dandelions reproduce asexually through apomictically derived seeds- an important goal for many major crops in modern agriculture. As such, there is increasing interest in the molecular control of important pathways as well as basic molecular biology and reproduction of common dandelion. RESULTS: Here we present an improved Agrobacterium-based genetic transformation and regeneration protocol, a protocol for generation and transformation of protoplasts using free DNA, and a protocol for leaf Agrobacterium infiltration for transient gene expression. These protocols use easily obtainable leaf explants from soil-grown plants and reagents common to most molecular plant laboratories. We show that common markers used in many plant transformation systems function as expected in common dandelion including fluorescent proteins, GUS, and anthocyanin regulation, as well as resistance to kanamycin, Basta, and hygromycin. CONCLUSION: Reproducible, stable and transient transformation methods are presented that will allow for needed molecular structure and function studies of genes and proteins in T. officinale.

Producing molecular biology reagents without purification.

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

Rapid Examination of Phytochrome-Phytochrome Interacting Factor (PIF) Interaction by In Vitro Coimmunoprecipitation Assay.

Phytochromes are red/far-red light receptors that perceive and transduce light signals to regulate various physiological responses. A large number of phytochrome interacting proteins have been identified using genetic and biochemical approaches. A direct interaction between phytochrome and its interacting proteins, therefore, defines one of the critical steps to initiate light signaling cascades in plants. Thus it is important to thoroughly examine the light-dependent interaction between phytochromes and putative phytochrome-interacting proteins. In this chapter, a protocol for rapid and simple light dependent in vitro coimmunoprecipitation between phytochromes and phytochrome interacting factors is described. In principle, this protocol can be adapted for other putative phytochrome interacting proteins.

A phyB-PIF1-SPA1 kinase regulatory complex promotes photomorphogenesis in Arabidopsis.

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) is a highly conserved E3 ubiquitin ligase from plants to animals and acts as a central repressor of photomorphogenesis in plants. SUPPRESSOR OF PHYA-105 1 family members (SPA1-SPA4) directly interact with COP1 and enhance COP1 activity. Despite the presence of a kinase domain at the N-terminus, no COP1-independent role of SPA proteins has been reported. Here we show that SPA1 acts as a serine/threonine kinase and directly phosphorylates PIF1 in vitro and in vivo. SPAs are necessary for the light-induced phosphorylation, ubiquitination and subsequent degradation of PIF1. Moreover, the red/far-red light photoreceptor phyB interacts with SPA1 through its C-terminus and enhances the recruitment of PIF1 for phosphorylation. These data provide a mechanistic view on how the COP1-SPA complexes serve as an example of a cognate kinase-E3 ligase complex that selectively triggers rapid phosphorylation and removal of its substrates, and how phyB modulates this process to promote photomorphogenesis.