Green Fluorescent Protein Fusion Research Articles

Investigating the Interactions of the Cucumber Mosaic Virus 2b Protein with the Viral 1a Replicase Component and the Cellular RNA Silencing Factor Argonaute 1.

The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In Arabidopsis thaliana, if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.

Development of Genetically Encoded Fluorescent KSR1-Based Probes to Track Ceramides during Phagocytosis.

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe.

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.

Construction of cytomegalovirus promoter‐driven gene expression system in Laodelphax striatellus

AbstractThe small brown planthopper (SBPH, Laodelphax striatellus) is a significant rice pest, responsible for transmitting rice stripe virus (RSV) in a persistent and propagative manner. RSV is one of the most detrimental rice viruses, causing rice stripe disease, which results in considerable loss of rice grain yield. While RNA interference and gene knockout techniques have enabled gene downregulation in SBPH, no system currently exists for the overexpression of endogenous or exogenous genes. Consequently, the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function investigations. This study aimed to construct an expression vector using the promoter of the constitutive‐expressed tubulin gene of SBPH, and promoter of human cytomegalovirus (CMV). Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein (GFP) expression in SBPH, and could also facilitate the expression of a nucleocapsid protein (NP) ‐GFP fusion protein containing viral NP with comparable efficiency. Through expression vector optimization, we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV promoter. In addition, the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in SBPH. These results provide a promising technical platform for investigating gene functions in SBPH.

Side‐Chain Chemistry Governs Hierarchical Order of Charge‐Complementary β‐sheet Peptide Coassemblies

AbstractSelf‐assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient conditions. This report demonstrates how charged side‐chain chemistry affects the hierarchical co‐assembly of a family of charge‐complementary β‐sheet‐forming peptide pairs known as CATCH(X+/Y−) at physiologic pH and ionic strength in water. In a concentration‐dependent manner, the CATCH(6K+) (Ac‐KQKFKFKFKQK‐Am) and CATCH(6D−) (Ac‐DQDFDFDFDQD‐Am) pair formed either β‐sheet‐rich microspheres or β‐sheet‐rich gels with a micron‐scale plate‐like morphology, which were not observed with other CATCH(X+/Y−) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N‐ and C‐terminal amino acids in CATCH(6K+) and CATCH(6D−) to Qs, increased observed co‐assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)‐green fluorescent protein fusions could be incorporated into the β‐sheet plates and microspheres formed by the CATCH(6K+/6D−) pair, demonstrating the potential to endow functionality.

Side-Chain Chemistry Governs Hierarchical Order of Charge-Complementary β-sheet Peptide Coassemblies.

Self-assembly of proteinaceous biomolecules into functional materials with ordered structures that span length scales is common in nature yet remains a challenge with designer peptides under ambient conditions. This report demonstrates how charged side-chain chemistry affects the hierarchical co-assembly of a family of charge-complementary β-sheet-forming peptide pairs known as CATCH(X+/Y-) at physiologic pH and ionic strength in water. In a concentration-dependent manner, the CATCH(6K+) (Ac-KQKFKFKFKQK-Am) and CATCH(6D-) (Ac-DQDFDFDFDQD-Am) pair formed either β-sheet-rich microspheres or β-sheet-rich gels with a micron-scale plate-like morphology, which were not observed with other CATCH(X+/Y-) pairs. This hierarchical order was disrupted by replacing D with E, which increased fibril twisting. Replacing K with R, or mutating the N- and C-terminal amino acids in CATCH(6K+) and CATCH(6D-) to Qs, increased observed co-assembly kinetics, which also disrupted hierarchical order. Due to the ambient assembly conditions, active CATCH(6K+)-green fluorescent protein fusions could be incorporated into the β-sheet plates and microspheres formed by the CATCH(6K+/6D-) pair, demonstrating the potential to endow functionality.

The seven rice vacuolar sorting receptors localize to prevacuolar compartments

Vacuolar sorting is critically important in plants as it regulates the mobilization of proteins and plays a major role in important agricultural traits like yield and seed protein content. Vacuolar sorting receptors (VSRs) are integral membrane proteins that mediate protein trafficking from the Golgi apparatus to the vacuole via the intermediate membrane-bound prevacuolar compartment (PVC)/multivesicular body (MVB). VSR proteins, such as an 80 kD (BP-80) from pea, also serve as markers for PVC/MVB. Dissecting VSR-mediated protein trafficking pathways may provide ways to enhance agronomic traits and crop yield. Green fluorescence protein (GFP) fusions with the seven Arabidopsis (Arabidopsis thaliana) VSRs were previously shown to localize to PVCs in transgenic tobacco BY-2 cells. The Rice (Oryza sativa) genome contains seven VSRs (OsVSR1-7), but little is known about their subcellular localizations. Here we studied the subcellular localization of OsVSR1-7 b y using a reporter approach, in which GFP-OsVSR1-7 fusions containing the transmembrane domain (TMD) and cytoplasmic tail (CT) of individual OsVSR were expressed in the protoplasts of rice, transgenic tobacco BY-2 cells and transgenic rice plants. Immunofluorescent labelling studies and confocal laser scanning microscope observation demonstrated that the seven OsVSRs are localized to PVCs and form ring-like structures upon wortmannin treatment. Therefore, we have verified the subcellular localization of OsVSR1-7 in this study. The OsVSRs tagged with GFP can serve as PVCs/MVBs markers in rice for the future studies.

Identification of short protein-destabilizing sequences in Arabidopsis cyclin-dependent kinase inhibitors, ICKs.

Plants have a family of cyclin-dependent kinase (CDK) inhibitors called interactors/inhibitors of CDK (ICKs) or Kip-related proteins (KRPs). ICK proteins have important functions in cell proliferation, endoreduplication, plant growth, and reproductive development, and their functions depend on the protein levels. However, understanding of how ICK protein levels are regulated is very limited. We fused Arabidopsis ICK sequences to green fluorescent protein (GFP) and determined their effects on the fusion proteins in plants, yeast, and Escherichia coli. The N-terminal regions of ICKs drastically reduced GFP fusion protein levels in Arabidopsis plants. A number of short sequences of 10-20 residues were found to decrease GFP fusion protein levels when fused at the N-terminus or C-terminus. Three of the four short sequences from ICK3 showed a similar function in yeast. Intriguingly, three short sequences from ICK1 and ICK3 caused the degradation of the fusion proteins in E. coli. In addition, computational analyses showed that ICK proteins were mostly disordered and unstructured except for the conserved C-terminal region, suggesting that ICKs are intrinsically disordered proteins. This study has identified a number of short protein-destabilizing sequences, and evidence suggests that some of them may cause protein degradation through structural disorder and instability.

A Novel High-Content Screening Assay Identified Belinostat as Protective in a FSGS-Like Zebrafish Model.

FSGS affects the complex three-dimensional morphology of podocytes, resulting in loss of filtration barrier function and the development of sclerotic lesions. Therapies to treat FSGS are limited, and podocyte-specific drugs are unavailable. To address the need for treatments to delay or stop FSGS progression, researchers are exploring the repurposing of drugs that have been approved by the US Food and Drug Administration (FDA) for other purposes. To identify drugs with potential to treat FSGS, we used a specific zebrafish screening strain to combine a high-content screening (HCS) approach with an in vivo model. This zebrafish screening strain expresses nitroreductase and the red fluorescent protein mCherry exclusively in podocytes (providing an indicator for podocyte depletion), as well as a circulating 78 kDa vitamin D-binding enhanced green fluorescent protein fusion protein (as a readout for proteinuria). To produce FSGS-like lesions in the zebrafish, we added 80 µ M metronidazole into the fish water. We used a specific screening microscope in conjunction with advanced image analysis methods to screen a library of 138 drugs and compounds (including some FDA-approved drugs) for podocyte-protective effects. Promising candidates were validated to be suitable for translational studies. After establishing this novel in vivo HCS assay, we identified seven drugs or compounds that were protective in our FSGS-like model. Validation experiments confirmed that the FDA-approved drug belinostat was protective against larval FSGS. Similar pan-histone deacetylase inhibitors also showed potential to reproduce this effect. Using an FSGS-like zebrafish model, we developed a novel in vivo HCS assay that identified belinostat and related pan-histone deacetylase inhibitors as potential candidates for treating FSGS.

Identification of novel proteins regulating lipid droplet biogenesis in filamentous fungi.

Lipid droplets (LDs) are storage organelles for neutral lipids which are critical for lipid homeostasis. Current knowledge of fungal LD biogenesis is largely limited to budding yeast, while LD regulation in multinucleated filamentous fungi which exhibit considerable metabolic activity remains unexplored. In this study, 19 LD-associated proteins were identified in the multinucleated species Aspergillus oryzae using a colocalization screening of a previously established enhanced green fluorescent protein (EGFP) fusion library. Functional screening identified 12 lipid droplet-regulating (LDR) proteins whose loss of function resulted in irregular LD biogenesis, particularly in terms of LD number and size. Bioinformatics analysis, targeted mutagenesis, and microscopy revealed four LDR proteins that localize to LD via the putative amphipathic helices (AHs). Further analysis revealed that LdrA with an Opi1 domain is essential for cytoplasmic and nuclear LD biogenesis involving a novel AH. Phylogenetic analysis demonstrated that the patterns of gene evolution were predominantly based on gene duplication. Our study identified a set of novel proteins involved in the regulation of LD biogenesis, providing unique molecular and evolutionary insights into fungal lipid storage.

Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry.

Escherichia coli is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a paraBAD expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development.

Characterization of Arabidopsis thaliana Coq9 in the CoQ Biosynthetic Pathway.

Coenzyme Q, also known as ubiquinone, is a fat-soluble isoprene quinone that serves as a cofactor for numerous enzymes across all domains of life. However, the biosynthetic pathway for this important molecule in plants has been examined in only a limited number of studies. In yeast and mammals, Coq9, an isoprenoid-lipid-binding protein, is essential for CoQ biosynthesis. Previous studies showed that Arabidopsis thaliana Coq9 failed to complement the fission yeast Schizosaccharomyces pombe coq9 null mutant, and its function in plants remains unknown. In this study, we demonstrated that expression of Arabidopsis Coq9 rescued the growth of a yeast temperature-sensitive coq9 mutant and increased CoQ content. Phylogenetic analysis revealed that Coq9 is widely present in green plants. Green fluorescent protein (GFP) fusion experiments showed that Arabidopsis Coq9 is targeted to mitochondria. Disruption of the Coq9 gene in Arabidopsis results in lower amounts of CoQ. Our work suggests that plant Coq9 is required for efficient CoQ biosynthesis. These findings provide new insights into the evolution of CoQ biosynthesis in plants. The identification of Coq9 as a key player in CoQ biosynthesis in plants opens up new avenues for understanding the regulation of this important metabolic pathway.

Genetic Engineering of Bacteriophage K1F with Human Epidermal Growth Factor to Enhance Killing of Intracellular E. coli K1.

Bacterial infections are a major cause of human morbidity and mortality on a global scale. Many bacterial pathogens, such as Escherichia coli, can cause diseases intracellularly via cell entry and avoidance of the host immune system. Antibiotic resistance has caused such infections to be problematic, which has necessitated the development of new antimicrobials. Bacteriophages are a potent alternative due to their specificity and ease of genetic modification. We have engineered phage K1F, which is specific to E. coli K1 to express an epidermal growth factor (EGF) and green fluorescent protein (GFP) fusion on the minor capsid protein. Here, we demonstrate that EGF-labeled phage K1F can be internalized more readily in human cell lines to eradicate E. coli K1 infection intracellularly. Further, we establish that K1F-GFP-EGF enters human cells primarily through endocytosis following EGF receptor (EGFR) induction, subverting the phagocytic mode of entry and permitting its accretion in the cytosol to seek out its bacterial host.

African Swine Fever Virus Gene B117L Encodes a Small Protein Endowed with Low-pH-Dependent Membrane Permeabilizing Activity.

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine in Central Europe to East Asia, resulting in economic losses for the swine industry. The virus contains a large double-stranded DNA genome that contains more than 150 genes, most with no experimentally characterized function. In this study, we evaluate the potential function of the product of ASFV gene B117L, a 115-amino-acid integral membrane protein transcribed at late times during the virus replication cycle and showing no homology to any previously published protein. Hydrophobicity distribution along B117L confirmed the presence of a single transmembrane helix, which, in combination with flanking amphipathic sequences, composes a potential membrane-associated C-terminal domain of ca. 50 amino acids. Ectopic transient cell expression of the B117L gene as a green fluorescent protein (GFP) fusion protein revealed the colocalization with markers of the endoplasmic reticulum (ER). Intracellular localization of various B117L constructs also displayed a pattern for the formation of organized smooth ER (OSER) structures compatible with the presence of a single transmembrane helix with a cytoplasmic carboxy terminus. Using partially overlapping peptides, we further demonstrated that the B117L transmembrane helix has the capacity to establish spores and ion channels in membranes at low pH. Furthermore, our evolutionary analysis showed the high conservation of the transmembrane domain during the evolution of the B117L gene, indicating that the integrity of this domain is preserved by the action of the purifying selection. Collectively our data support a viroporin-like assistant role for the B117L gene-encoded product in ASFV entry. IMPORTANCE ASFV is responsible for an extensively distributed pandemic causing important economic losses in the pork industry in Eurasia. The development of countermeasures is partially limited by the insufficient knowledge regarding the function of the majority of the more than 150 genes present on the virus genome. Here, we provide data regarding the functional experimental evaluation of a previously uncharacterized ASFV gene, B117L. Our data suggest that the B117L gene encodes a small membrane protein that assists in the permeabilization of the ER-derived envelope during ASFV infection.

Isolation of FLOWERING LOCUS C and Preliminary Characterization in the Floral Transition of Xinjiang Precocious Walnut

Walnut (Juglans regia L.) plants typically flower after eight to ten years of juvenile growth. Precocious germplasm, also known as early-flowering or early-mature genotypes, have shortened juvenile phases of one to two years and are therefore crucial for enhancing breeding efficiency. However, such precocious germplasms are very limited. Here, we isolated and characterized the key flowering-time gene FLOWERING LOCUS C (FLC) in the precocious walnuts of the Xinjiang Uygur Autonomous Region. Sequence alignment showed that Juglans regia FLC (JrFLC)contained a conserved MINICHROMOSOME MAINTENANCE 1 (MCM1), AGAMOUS (AG), DEFICIENS (DEF), and SERUM RESPONSE FACTOR (SRF) (MADS)-box domain. Analysis of an FLC–green fluorescent protein (GFP) fusion protein revealed that JrFLC was localized to the nucleus. Gene expression analysis showed that JrFLC was specifically expressed during the bud dormancy stage of precocious walnut, and that expression levels gradually decreased as the ambient temperature warmed. Exogenous JrFLC overexpression in Arabidopsis thaliana delayed flowering and increased the total leaf number, suggesting a similar function of JrFLC as a floral repressor in walnut and in other plants. Together, these results showed that JrFLC played an important role in regulating the floral transition of Xinjiang precocious walnut. Further studies, including a detailed characterization of JrFLC, are expected to validate JrFLC as a strong target for genetic improvement in flowering time in walnut.

A new Drosophila melanogaster fly that expresses GFP-tagged Orco

Insects perceive countless odors with the help of odorant receptors (ORs) expressed by olfactory sensory neurons (OSNs). OR complexes are composed of odor-specific proteins OrX and olfactory co-receptor proteins Orco, with both extracellular C-terminus and intracellular N-terminus. Here, we generated a genetically modified Drosophila melanogaster fly line that expresses Orco proteins with an N-terminally fused green fluorescent protein (GFP). We generated four genotypes (homozygous and heterozygous) for which we performed antennal cryosectioning and immunolabeling to determine which genotype would show high GFP labeling intensity in the dendritic areas of OSNs. We found that fluorescent intensity was the highest in homozygous flies. As Orco is known to guide OrX proteins to the OSN dendrites, we checked whether the fusion of GFP to the Orco N-terminus may affect such trafficking and thereby OR function. Using single sensillum recording (SSR), we found no difference in the olfactory capability of homozygous flies compared with wild-type flies. As future application, we want to use this transgenic fly line to resolve the structure of OR complexes using Cryo electron microscopy (cryo-EM).

Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus.

Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.

Arabinogalactan Structures of Repetitive Serine-Hydroxyproline Glycomodule Expressed by Arabidopsis Cell Suspension Cultures.

Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily. They are heavily glycosylated with arabinogalactans, which are usually composed of a β-1,3-linked galactan backbone with 6-O-linked galactosyl, oligo-1,6-galactosyl, or 1,6-galactan side chains that are further decorated with arabinosyl, glucuronosyl, rhamnosyl, and/or fucosyl residues. Here, our work with Hyp-O-polysaccharides isolated from (Ser-Hyp)32-EGFP (enhanced green fluorescent protein) fusion glycoproteins overexpressed in transgenic Arabidopsis suspension culture is consistent with the common structural features of AGPs isolated from tobacco. In addition, this work confirms the presence of β-1,6-linkage on the galactan backbone identified previously in AGP fusion glycoproteins expressed in tobacco suspension culture. Furthermore, the AGPs expressed in Arabidopsis suspension culture lack terminal-rhamnosyl residues and have a much lower level of glucuronosylation compared with those expressed in tobacco suspension culture. These differences not only suggest the presence of distinct glycosyl transferases for AGP glycosylation in the two systems, but also indicate the existence of minimum AG structures for type II AG functional features.

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

Arabidopsis translation factor eEF1Bγ impacts plant development and is associated with heat-induced cytoplasmic foci.

The important role of translational control for maintenance of proteostasis is well documented in plants, but the exact mechanisms that coordinate translation rates during plant development and stress response are not well understood. In Arabidopsis, the translation elongation complex eEF1B consists of three subunits: eEF1Bα, eEF1Bβ, and eEF1Bγ. While eEF1Bα and eEF1Bβ have a conserved GDP/GTP exchange function, the function of eEF1Bγ is still unknown. By generating Arabidopsis mutants with strongly reduced eEF1Bγ levels, we revealed its essential role during plant growth and development and analysed its impact on translation. To explore the function of the eEF1B subunits under high temperature stress, we analysed their dynamic localization as green fluorescent protein fusions under control and heat stress conditions. Each of these fusion proteins accumulated in heat-induced cytoplasmic foci and co-localized with the stress granule marker poly(A)-binding protein 8-mCherry. Protein-protein interaction studies and co-expression analyses indicated that eEF1Bβ physically interacted with both of the other subunits and promoted their recruitment to cytoplasmic foci. These data provide new insights into the mechanisms allowing for rapid adaptation of translation rates during heat stress response.