Integrative vectors for regulated expression of SARS-CoV-2 proteins implicated in RNA metabolism.

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His 8, the C-terminus with His 8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In T-REx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

Tuning Degradation to Achieve Specific and Efficient Protein Depletion.

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A ß-estradiol-inducible AID system was created, with expression of TIR1 under the control of a ß-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with ß-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate ß-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast.

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of ß-estradiol preincubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.

Nitrate reductase mutation alters potassium nutrition as well as nitric oxide-mediated control of guard cell ion channels in Arabidopsis.

Maintaining potassium (K(+) ) nutrition and a robust guard cell K(+) inward channel activity is considered critical for plants' adaptation to fluctuating and challenging growth environment. ABA induces stomatal closure through hydrogen peroxide and nitric oxide (NO) along with subsequent ion channel-mediated loss of K(+) and anions. However, the interactions of NO synthesis and signalling with K(+) nutrition and guard cell K(+) channel activities have not been fully explored in Arabidopsis. Physiological and molecular techniques were employed to dissect the interaction of nitrogen and potassium nutrition in regulating stomatal opening, CO2 assimilation and ion channel activity. These data, gene expression and ABA signalling transduction were compared in wild-type Columbia-0 (Col-0) and the nitrate reductase mutant nia1nia2. Growth and K(+) nutrition were impaired along with stomatal behaviour, membrane transport, and expression of genes associated with ABA signalling in the nia1nia2 mutant. ABA-inhibited K(+) in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for complete stomatal closure in nia1nia2. While NO is an important signalling component in ABA-induced stomatal closure in Arabidopsis, our findings demonstrate a more complex interaction associating potassium nutrition and nitrogen metabolism in the nia1nia2 mutant that affects stomatal function.

Histone deacetylase complex1 expression level titrates plant growth and abscisic acid sensitivity in Arabidopsis.

Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.

Hyperosmotic priming of Arabidopsis seedlings establishes a long-term somatic memory accompanied by specific changes of the epigenome.

BACKGROUND: In arid and semi-arid environments, drought and soil salinity usually occur at the beginning and end of a plant's life cycle, offering a natural opportunity for the priming of young plants to enhance stress tolerance in mature plants. Chromatin marks, such as histone modifications, provide a potential molecular mechanism for priming plants to environmental stresses, but whether transient exposure of seedlings to hyperosmotic stress leads to chromatin changes that are maintained throughout vegetative growth remains unclear. RESULTS: We have established an effective protocol for hyperosmotic priming in the model plant Arabidopsis, which includes a transient mild salt treatment of seedlings followed by an extensive period of growth in control conditions. Primed plants are identical to non-primed plants in growth and development, yet they display reduced salt uptake and enhanced drought tolerance after a second stress exposure. ChIP-seq analysis of four histone modifications revealed that the priming treatment altered the epigenomic landscape; the changes were small but they were specific for the treated tissue, varied in number and direction depending on the modification, and preferentially targeted transcription factors. Notably, priming leads to shortening and fractionation of H3K27me3 islands. This effect fades over time, but is still apparent after a ten day growth period in control conditions. Several genes with priming-induced differences in H3K27me3 showed altered transcriptional responsiveness to the second stress treatment. CONCLUSION: Experience of transient hyperosmotic stress by young plants is stored in a long-term somatic memory comprising differences of chromatin status, transcriptional responsiveness and whole plant physiology.

Molecular analysis of a sunflower gene encoding an homologous of the B subunit of a CAAT binding factor.

A genomic DNA fragment containing the complete LEAFY COTYLEDON1-LIKE (HaL1L) gene was retrieved by chromosome walking. Its sequence was confirmed and elongated by screening a sunflower genomic DNA BAC Library. HaL1L, whose cDNA had already been sequenced and characterized, encodes a NF-YB subunit of a CCAAT box-binding factor (NF-Y) involved in the early stages of zygotic and somatic embryogenesis in the Helianthus genus. In the HaL1L 5'-flanking region, elements specific to a putative TATA-box promoter and two "CG isles" were identified. An investigation of the methylation status of these CG rich DNA regions showed that differentially methylated cytosines were recognizable in the DNA of embryos on the fifth day after pollination in comparison to leaf DNA suggesting that during plant development epigenetic regulation of HaL1L transcription was achieved by methylating cytosine residues. We also searched the HaL1L nucleotide sequence for cis-regulatory elements able to interact with other transcription factors (TFs) involved in the HaL1L regulation. Of the elements identified, one of the most intriguing is WUSATA, the target sequence for the WUSCHEL (WUS) TF, which may be part of a complex regulation network controlling embryo development. In this article, we show that the WUSATA target site, located in the intron of HaL1L, is able to bind the TF WUS. Interestingly, we found auxin and abscisic acid responsive motifs in the HaL1L promoter region suggesting that this gene may additionally by under hormonal control. Finally, the presence of a cytoplasmic polyadenylation signal downstream to the coding region indicates that this gene may also be controlled at the translation level by a temporarily making the pre-synthesized HaL1L mRNA unavailable for protein synthesis.

Epileptic negative myoclonus in a newborn with hemimegalencephaly.

PURPOSE: We report the case of a male newborn with Ohtahara syndrome and right hemimegalencephaly who presented epileptic negative myoclonus in the first days of life. METHODS: Prolonged polygraphic studies were performed, as well as MRI and a full clinical examination. RESULTS: EEG showed a constant and nonreactive pattern of burst suppression. There were several kinds of electro-clinical seizures (generalized myoclonia, short atonias, typical spasm and tonic spasms) at the beginning of the EEG's burst. The periods of EMG silence, lasting less than 300 ms, were associated with stereotyped EEG transients. CONCLUSIONS: Epileptic negative myoclonus can be observed also in neonatal age. The short transient impairment of motor function observed in the newborn seems linked to the slow component of spike-wave discharge, but its mechanism is still not clear.