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INTRODUCTION: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. METHODS: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. RESULTS: Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p

The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been reported, but the structural dynamics have not been fully elucidated. Herein, the crystal structure of mDsRed was determined at 2.9 Å resolution, and the molecular flexibility was analyzed. mDsRed contains a solvent-accessible hole between the β7-strand and β9-α10 loop, which is connected to the chromophore. A partial disorder was present in the electron density map of the tyrosine-ring group of the mDsRed chromophore, indicating a flexible conformation of the chromophore. The refined mDsRed chromophore displayed a cis-conformation with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the β-barrel fold of mDsRed is rigid, while the loops at the top and bottom of the β-barrel are relatively flexible. The β-barrel surface of mDsRed was closer to the native conformation compared with the previously reported Zn-bound state of mDsRed. These structural findings extend our understanding of the molecular flexibility of mDsRed. [ABSTRACT FROM AUTHOR]

Background: Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity. Methods: Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived. Results: While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible. Conclusions: Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9. [ABSTRACT FROM AUTHOR]

The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been reported, but the structural dynamics have not been fully elucidated. Herein, the crystal structure of mDsRed was determined at 2.9 Å resolution, and the molecular flexibility was analyzed. mDsRed contains a solvent-accessible hole between the β7-strand and β9-α10 loop, which is connected to the chromophore. A partial disorder was present in the electron density map of the tyrosine-ring group of the mDsRed chromophore, indicating a flexible conformation of the chromophore. The refined mDsRed chromophore displayed a cis-conformation with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the β-barrel fold of mDsRed is rigid, while the loops at the top and bottom of the β-barrel are relatively flexible. The β-barrel surface of mDsRed was closer to the native conformation compared with the previously reported Zn-bound state of mDsRed. These structural findings extend our understanding of the molecular flexibility of mDsRed.

How complex morphological patterns form is an intriguing question in developmental biology. However, the mechanisms that generate complex patterns remain largely unknown. Here, we sought to identify the genetic mechanisms that regulate the tan (t) gene in a multi-spotted pigmentation pattern on the abdomen and wings of Drosophila guttifera. Previously, we showed that yellow (y) gene expression completely prefigures the abdominal and wing pigment patterns of this species. In the current study, we demonstrate that the t gene is co-expressed with the y gene in nearly identical patterns, both transcripts foreshadowing the adult abdominal and wing melanin spot patterns. We identified cis-regulatory modules (CRMs) of t, one of which drives reporter expression in six longitudinal rows of spots on the developing pupal abdomen, while the second CRM activates the reporter gene in a spotted wing pattern. Comparing the abdominal spot CRMs of y and t, we found a similar composition of putative transcription factor binding sites that are thought to regulate the complex expression patterns of both terminal pigmentation genes y and t. In contrast, the y and t wing spots appear to be regulated by distinct upstream factors. Our results suggest that the D. guttifera abdominal and wing melanin spot patterns have been established through the co-regulation of y and t, shedding light on how complex morphological traits may be regulated through the parallel coordination of downstream target genes. [ABSTRACT FROM AUTHOR]

Abstract Background Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus. Results Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets. Conclusion A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research.

Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral Discosoma) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3′UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.

Abstract Background Vectors based on plant viruses are important tools for functional genomics, cellular biology, plant genome engineering and molecular farming. We previously reported on the construction of PepGFP2a, a viral vector based on pepino mosaic virus (PepMV) which expressed GFP efficiently and stably in plants of its experimental host Nicotiana benthamiana, but not in its natural host tomato. We have prepared a new set of PepMV-based vectors with improved stability that are able to express a wide range of reporter genes, useful for both N. benthamiana and tomato. Results We first tested PepGFPm1 and PepGFPm2, two variants of PepGFP2a in which we progressively reduced a duplication of nucleotides encoding the N-terminal region of the coat protein. The new vectors had improved GFP expression levels and stability in N. benthamiana but not in tomato plants. Next, we replaced GFP by DsRed or mCherry in the new vectors PepDsRed and PepmCherry, respectively; while PepmCherry behaved similarly to PepGFPm2, PepDsRed expressed the reporter gene efficiently also in tomato plants. We then used PepGFPm2 and PepDsRed to study the PepMV localization in both N. benthamiana and tomato cells. Using confocal laser scanning microscopy (CLSM), we observed characteristic fluorescent bodies in PepMV-infected cells; these bodies had a cytoplasmic localization and appeared in close proximity to the cell nucleus. Already at 3 days post-agroinoculation there were fluorescent bodies in almost every cell of agroinoculated tissues of both hosts, and always one body per cell. When markers for the endoplasmic reticulum or the Golgi apparatus were co-expressed with PepGFPm2 or PepDsRed, a reorganisation of these organelles was observed, with images suggesting that both are intimately related but not the main constituents of the PepMV bodies. Altogether, this set of data suggested that the PepMV bodies are similar to the potato virus X (PVX) “X-bodies”, which have been described as the PVX viral replication complexes (VRCs). To complete the set of PepMV-based vectors, we constructed a vector expressing the BAR herbicide resistance gene, useful for massive susceptibility screenings. Conclusions We have significantly expanded the PepMV tool box by producing a set of new vectors with improved stability and efficiency in both N. benthamiana and tomato plants. By using two of these vectors, we have described characteristic cellular bodies induced by PepMV infection; these bodies are likely the PepMV VRCs.

Abstract Background In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive. Results Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library. Conclusions The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.

Background: Brassica napus is an important oilseed crop that offers a considerable amount of biomass for global vegetable oil production. The establishment of an efficient genetic transformation system with a convenient transgenic-positive screening method is of great importance for gene functional analysis and molecular breeding. However, to our knowledge, there are few of the aforementioned systems available for efficient application in B. napus. Results: Based on the well-established genetic transformation system in B. napus, five vectors carrying the red fluorescence protein encoding gene from Discosoma sp. (DsRed) were constructed and integrated into rapeseed via Agrobacterium-mediated hypocotyl transformation. An average of 59.1% tissues were marked with red fluorescence by the visual screening method in tissue culture medium, 96.1% of which, on average, were amplified with the objective genes from eight different rapeseed varieties. In addition, the final transgenic-positive efficiency of the rooted plantlets reached up to 90.7% from red fluorescence marked tissues, which was much higher than that in previous reports. Additionally, visual screening could be applicable to seedlings via integration of DsRed, including seed coats, roots, hypocotyls and cotyledons during seed germination. These results indicate that the highly efficient genetic transformation system combined with the transgenic-positive visual screening method helps to conveniently and efficiently obtain transgenic-positive rapeseed plantlets. Conclusion: A rapid, convenient and highly efficient method was developed to obtain transgenic plants, which can help to obtain the largest proportion of transgene-positive regenerated plantlets, thereby avoiding a long period of plant regeneration. The results of this study will benefit gene functional studies especially in high-throughput molecular biology research. [ABSTRACT FROM AUTHOR]

Efficient elimination of the editing machinery remains a challenge in plant biotechnology after genome editing to minimize the probability of off-target mutations, but it is also important to deliver end users with edited plants free of foreign DNA. Using the modular cloning system Golden Braid, we have included a fluorescence-dependent transgene monitoring module to the genome-editing tool box. We have tested this approach in Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana. We demonstrate that DsRED fluorescence visualization works efficiently in dry seeds as marker for the detection of the transgene in the three species allowing an efficient method for selecting transgene-free dry seeds. In the first generation of DsRED-free CRISPR/Cas9 null segregants, we detected gene editing of selected targets including homozygous mutants for the plant species tested. We demonstrate that this strategy allows rapid selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation.

Efficient elimination of the editing machinery remains a challenge in plant biotechnology after genome editing to minimize the probability of off-target mutations, but it is also important to deliver end users with edited plants free of foreign DNA. Using the modular cloning system Golden Braid, we have included a fluorescence-dependent transgene monitoring module to the genome-editing tool box. We have tested this approach in Solanum lycopersicum , Oryza sativa , and Arabidopsis thaliana. We demonstrate that DsRED fluorescence visualization works efficiently in dry seeds as marker for the detection of the transgene in the three species allowing an efficient method for selecting transgene-free dry seeds. In the first generation of DsRED-free CRISPR/Cas9 null segregants, we detected gene editing of selected targets including homozygous mutants for the plant species tested. We demonstrate that this strategy allows rapid selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation. [ABSTRACT FROM AUTHOR]

Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization.

Strain 212 of Penicillium rubens (PO212) is an effective fungal biological control agent against a broad spectrum of diseases of horticultural plants. A pyrimidine auxotrophic isolate of PO212, PO212_18.2, carrying an inactive pyrG gene, has been used as host for transformation by positive selection of vectors containing the gene complementing the pyrG1 mutation. Both integrative and autonomously replicating plasmids transformed PO212_18.2 with high efficiency. Novel PO212-derived strains expressed green (sGFP) and red (Ds-Red Express) fluorescent reporter proteins, driven by the A. nidulans gpdA promoter. Fluorescence microscopy revealed constitutive expression of the sGFP and Ds-Red Express proteins, homogenously distributed across fungal cells. Transformation with either type of plasmid, did not affect the growth and morphological culture characteristics, and the biocontrol efficacy of either transformed strains compared to the wild-type, PO212. Fluorescent transformants pointed the capacity of PO212 to colonize tomato roots without invading plant root tissues. This work demonstrates susceptibility of the biocontrol agent PO212 to be transformed, showing that the use of GFP and DsRed as markers for PO212 is a useful, fast, reliable and effective approach for studying plant–fungus interactions and tomato root colonization. [ABSTRACT FROM AUTHOR]

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

RNA interference (RNAi) is a powerful technology for suppressing gene function. In most studies, small interfering RNAs (siRNAs) consist of one short hairpin RNA (shRNA) and, therefore, are often unable to achieve loss-of-function of their target genes. In the current study, an RNAi vector containing three shRNAs under the control of three RNA polymerase III U6 promoters was constructed. RNAi vectors containing one or two shRNAs were generated for comparisons. A pilot study targeting exogenously expressed DsRed in the HEK293 cell line revealed promising effects and a high selectivity for the multi-shRNA RNAi vector. Akt2 is constitutively expressed in cultured SKOV3 human ovarian cancer cells, and the multi-shRNA RNAi vector showed a strong efficiency for downregulating the expression of Akt2 in these cells, with no apparent interferon response. In addition, the Akt2-3shRNA vector, containing three shRNAs targeting Akt2, showed the best effect of all the shRNA vectors in reversing paclitaxel-induced resistance in SKOV3 cells. This study developed a widely applicable resource for enhancing the efficiency of gene silencing and a novel technique for performing complex loss-of-function screens in mammalian cells. [ABSTRACT FROM AUTHOR]

Fusarium verticillioides was labeled with DsRed via Agrobacterium tumefaciens-mediated transformation to examine differences in colonization and reactions of resistant and susceptible inbred lines of maize (Zea mays L.). The extent of systemic colonization of F. verticillioides in roots from maize lines either resistant or susceptible to the fungus was studied by visualizing the red fluorescence produced by the fungus expressing DsRed. The difference in quantities of colony forming units (CFU) in roots and basal stems, production of fumonisin B1, and pH of root were determined. Although F. verticillioides colonized both resistant and susceptible lines, differences were observed in the pattern and extent of fungal colonization in the two types of maize lines. The fungus colonized the susceptible lines producing mosaic patterns by filling the individual root cells with hyphae. Such a pattern of colonization was rarely observed in resistant lines, which were less colonized by the fungus than the susceptible lines in terms of CFUs. The production of mycotoxin fumonisin B1 in roots from different lines was closely correlated with the amount of F. verticillioides colonization, rather than the pH or amylopectin concentrations in the root. The findings from this study contribute to a better understanding of the defense mechanism in resistant maize lines to F. verticillioides.

Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein.

No plant or cryptogam exists in nature without microorganisms associated with its tissues. Plants as microbial hosts are puzzles of different microhabitats, each of them colonized by specifically adapted microbiomes. The interactions with such microorganisms have drastic effects on the host fitness. Since the last 20 years, the combination of microscopic tools and molecular approaches contributed to new insights into microbe-host interactions. Particularly, confocal laser scanning microscopy (CLSM) facilitated the exploration of microbial habitats and allowed the observation of host-associated microorganisms in situ with an unprecedented accuracy. Here I present an overview of the progresses made in the study of the interactions between microorganisms and plants or plant-like organisms, focusing on the role of CLSM for the understanding of their significance. I critically discuss risks of misinterpretation when procedures of CLSM are not properly optimized. I also review approaches for quantitative and statistical analyses of CLSM images, the combination with other molecular and microscopic methods, and suggest the re-evaluation of natural autofluorescence. In this review, technical aspects were coupled with scientific outcomes, to facilitate the readers in identifying possible CLSM applications in their research or to expand their existing potential. The scope of this review is to highlight the importance of confocal microscopy in the study of plant-microbe interactions and also to be an inspiration for integrating microscopy with molecular techniques in future researches of microbial ecology.

Potential enhancement of mycoparasitic efficacy of Coniothyrium minitans and Microsphaeropsis ochracea through concomitant colonization of Sclerotinia sclerotiorum sclerotia was investigated, following observation that the two mycoparasites did not exhibit any mutual antagonism in dual culture assays. Simultaneous application of both mycoparasites increased sclerotia mortality in a temperature range from 16 to 26°C compared to single application, indicating a predominantly additive interaction. With increasing temperature the efficacy of M. ochracea decreased, but C. minitans was unaffected. Degradation of sclerotia by C. minitans proceeded slightly faster than with M. ochracea. Simultaneous colonization of sclerotia was studied at the histopathological level with mycoparasite strains transformed via Agrobacterium tumefaciens-mediated transformation ( ATMT) with reporter genes encoding for DsRed and GFP. Sclerotia colonization followed by fluorescence microscopy revealed effective penetration of the sclerotial rind, growth and formation of pycnidia in the cortex and medulla by both antagonists, resulting in complete degradation of sclerotia within 25 days after single inoculation. Upon simultaneous inoculation, both antagonists concomitantly colonized the sclerotial tissue and independently formed pycnidia in the sclerotial medulla and on the sclerotial rind, demonstrating their ability to co-colonize the same host fungus. Although the individual growth of the two mycoparasites in dual inoculations was slightly delayed, the sclerotia degrading effects were additive, suggesting a complementary antagonistic interaction. The combined application of two different species of mycoparasites cooperating on the same host fungus and differing in temperature requirements may be advantageous for making biocontrol applications in the field less sensitive to varying environmental and host conditions. [ABSTRACT FROM AUTHOR]

Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1) strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

It has been proposed that transgenic mosquitoes can be used as a "flying syringe" for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect. [ABSTRACT FROM AUTHOR]

Summary Heavy metal accumulation in the environment poses great risks to flora and fauna. However, monitoring sites prone to accumulation poses scale and economic challenges. In this study, we present and test a method for monitoring these sites using fluorescent resonance energy transfer (FRET) change in response to zinc (Zn) accumulation in plants as a proxy for environmental health. We modified a plant Zn transport protein by adding flanking fluorescent proteins (FPs) and deploying the construct into two different species. In Arabidopsis thaliana, FRET was monitored by a confocal microscope and had a 1.4-fold increase in intensity as the metal concentration increased. This led to a 16.7% overall error-rate when discriminating between a control (1 μ m Zn) and high (10 m m Zn) treatment after 96 h. The second host plant ( Populus tremula × Populu salba) also had greater FRET values (1.3-fold increase) when exposed to the higher concentration of Zn, while overall error-rates were greater at 22.4%. These results indicate that as plants accumulate Zn, protein conformational changes occur in response to Zn causing differing interaction between FPs. This results in greater FRET values when exposed to greater amounts of Zn and monitored with appropriate light sources and filters. We also demonstrate how this construct can be moved into different host plants effectively including one tree species. This chimeric protein potentially offers a method for monitoring large areas of land for Zn accumulation, is transferable among species, and could be modified to monitor other specific heavy metals that pose environmental risks. [ABSTRACT FROM AUTHOR]

Background: The production of transgenic plants, either for the overproduction of the protein of interest, for promoter: reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming. Results: We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1: DsRed marker and a CaMV:: GUS cassette within the T-DNA and is useful for the production of promoter: GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1: DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia. Conclusions: The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest. [ABSTRACT FROM AUTHOR]

Abstract: Red fluorescent protein (RFP) DsRed and its variants are widely applied in live-cell imaging experiments. However, a major factor that restricts the application of DsRed is its cytotoxicity. Here, we report that DsRed and its variant DsRed-Express2 inhibit the expression of B-cell lymphoma-extra large (Bcl-xL) in HeLa cells by translational regulation. Over-expression of Bcl-xL can reduce the cytotoxicity of DsRed. Meanwhile, Turbo RFP, a mutant RFP from Entacmaea quadricolor, does not affect Bcl-xL expression, suggesting that cytotoxic mechanisms of RFP from different species may be varied. Our results reveal a possible mechanism for the cytotoxicity of DsRed, providing a potential strategy to improve the application of DsRed and its variants. [Copyright &y& Elsevier]

To develop effective biocontrol strategies, basic knowledge of plant growth promotion (PGP) and root colonization by antagonists is essential. The survival and colonization patterns of five different biocontrol agents against Rhizoctonia solani AG2-2IIIB in the rhizosphere of greenhouse-grown sugar beet plants were analysed in single and combined treatments. The study included bacteria ( Pseudomonas fluorescens L13-6-12, Pseudomonas trivialis RE*1-1-14, Serratia plymuthica 3Re4-18) as well as fungi ( Trichoderma gamsii AT1-2-4, Trichoderma velutinum G1/8). Microscopic analysis by confocal laser scanning microscopy revealed different colonization patterns for each DsRed2/green fluorescent protein-labelled strain. Bacteria and T. velutinum G1/8 colonized the root surface and the endorhiza in single and co-culture, while for T. gamsii AT1-2-4, only the transfer of spores was observed. Whereas Pseudomonas strains formed large microcolonies consisting of hundreds of cells, S. plymuthica was arranged in small endophytic clusters or clouds around the entire root system. In co-culture, each strain showed its typical pattern and occupied specific niches on the root, without clear evidence of morphological interactions. PGP was only observed for four strains with rhizosphere competence and not for T. gamsii AT1-2-4. The results provide useful information on which combination of strains to test in larger biocontrol experiments directed to applications. [ABSTRACT FROM AUTHOR]

The expression of the fluorescent protein, DsRed, facilitates the optimization of protein production in orally-infected whole larvae. Trichoplusia ni was shown to be a much better host for recombinant AcMNPV compared to four other noctuid Lepidopteran species achieving 100% infectivity at the minimal tested dose. The highest density of marker protein was found in endothelial and tracheal cells, fat body, and hemocytes. Trichoplusia ni larvae possessed visually detected color over sequential passages of oral infection until the sixth round. Western blot analysis confirmed the progressive decrease of both tetramer and monomer forms of DsRed. The intact DsRed gene and promoter region was present in late passages, but viral population carrying the heterologous gene had dropped more than 2-logs after the fifth round while the amount of total viral DNA remained unchanged over sequential passages. [ABSTRACT FROM AUTHOR]

Abstract: Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2–3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters. [Copyright &y& Elsevier]

Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. Here, a transient assay system in microprojectile-bombarded barley leaves is described that allows the functional testing of dehydration stress-related candidate genes by RNA interference (RNAi) or overexpression. Cellular stress or damage in dedydrated leaves is reported by a reduced accumulation of slowly maturing, native red-fluorescing protein DsRed that is known to be sensitive to denaturing conditions. After a dehydration-stress period of 4 d during which the relative fresh weight of leaves was kept at 60–66% of initial fresh weight, a reproducible reduction of normalized DsRed fluorescence was observed. In order to obtain proof of concept, a number of barley mRNAs homologous to drought response genes were selected and targeted by transient induced gene silencing (TIGS). TIGS of four tested genes resulted in a significantly stronger decrease of normalized DsRed fluorescence in dehydration-stressed leaves, whereas they had no effect in fully turgescent control leaves. These genes encode barley drought-responsive factor HvDRF1 (DREB2-like), dehydrin 6, late embryogenesis-abundant protein HVA1, and the vacuolar sodium/proton antiporter HvHNX1. The four targeted transcripts were also found to accumulate rapidly in dehydration-stressed barley leaf segments. The results suggest a value of the TIGS system for functional pre-screening of larger numbers of drought or dehydration stress-related candidate genes in barley. [ABSTRACT FROM PUBLISHER]

Abstract: A recombinant red fluorescent protein, DsRed, was chosen as a model protein to prepare a protein-encapsulated bioaerogel, DsRed-SAG. It was prepared using sol–gel polymerization of tetraethyl orthosilicate (TEOS) with an ionic liquid as the solvent and pore-forming agent. The DsRed-SAG bioaerogel was characterized by Fourier transformation infrared, scanning electron microscopy and Brunauer–Emmett-Teller measurements. It was found that the as-prepared bioaerogel had high porosity, and the silica network exhibited little shrinkage during the drying process. The stability of the bioaerogel was monitored by fluorescence spectroscopy and confirmed by confocal laser scanning microscopy. In addition, the protection of the encapsulated proteins by the silica network was further investigated using the degradation test by a protease. The results indicated that the as-prepared protein was quite stable during formation of the protein-containing wet gel and extraction of the ionic liquid, demonstrating that the new method can be extended to prepare other protein-encapsulated bioaerogels. [Copyright &y& Elsevier]

This paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using Pseudomonas putida KT2442 cells bearing the TOL (pWWO) plasmid. The methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. Expression of reporter proteins (GFP and DsRed) allowed in situ monitoring of donor cell attachment and plasmid transfer to the recipient cells using confocal laser scanning microscopy. Concomitant with donor integration and transconjugant proliferation in the granules, a significant increase in degradation of benzyl alcohol (used as sole substrate) was observed in the augmented microcosms. In contrast, control microcosms (with non-augmented granules) did not show any noticeable increase in the degradation of the substrate. This study shows that bioaugmentation of aerobic granular sludge via donor colonization and plasmid transfer is feasible for enhanced biodegradation. [Copyright &y& Elsevier]

• Here, arbuscular mycorrhizal (AM) fungi were monitored in vivo introducing the fluorescent reporters DsRed and GFP (green fluorescent protein) in Glomus intraradices using a biolistic approach and Agrobacterium tumefaciens-mediated transformation. • Both reporter genes were fused to the nuclear localization signal of the Aspergillus nidulans transcription factor StuA to target fluorescence to nuclei. Expression of DsRed was driven by two Glomus mosseae promoters highly expressed during early symbiosis, GmPMA1 and GmFOX2, while expression of GFP was driven by the A. nidulans gpd promoter. • All promoters worked in G. intraradices as well as in A. nidulans. Red and green fluorescence was localized to nuclei of G. intraradices spores and hyphae 3 d after bombardment. However, expression was transient. The efficiency of the Agrobacterium-mediated transformation was very low. • These results indicate that the biolistic method allows the expression of foreign DNA into G. intraradices with high frequency, but it is insufficient to render stable transformants. DsRed vs GFP is a more appropriate living reporter to be used in G. intraradices because of the lower autofluorescence in the red channel but targeted to the nucleus both marker genes can be visualized. This is the first report of fluorescent marker expression in an AM fungus driven by arbuscular mycorrhizal promoters. [ABSTRACT FROM AUTHOR]

Abstract: DsRed1 is a red fluorescent protein that can be used as a fusion partner with other proteins to determine their subcellular localization, similarly to the popular green fluorescent proteins (GFP). Here, we report that fusion of DsRed1 to estrogen receptor α (ERα) renders the transcription factor immobile within the nucleus. Furthermore, we show that the immobilization is dependent on DNA interaction and that the binding to the DNA can be direct as well as indirect for DsRed to immobilize with its fusion partners. This observation could provide a new tool to be used for the identification of target genes containing low affinity binding sites for several transcription factors including ERα. In addition, it could be employed for studies on protein–DNA interactions as well as protein–protein interactions during protein complex formation on chromatin in the event of transcription initiation and regulation. [Copyright &y& Elsevier]

Abstract: A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781-bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin. [Copyright &y& Elsevier]

A type I transmembrane protein, Mat-8 (FXYD-3), was tagged with fluorescent protein, Discosoma red fluorescent protein, at the carboxyl terminal cytoplasmic tail, and stably expressed in Chinese Hamster Ovary (CHO)-K1 cells. The fluorescence signal was distributed in intracellular membranes, being not only detected around the nuclear envelope but also partly overlapping with an endoplasmic reticulum marker. Subcellular fractionation by density gradient centrifugation supported this partial overlapping. The spherical structures observed were not colocalized with markers for lysosomes, endosomes, and Golgi bodies, suggesting that Mat-8 is distributed in a distinct endoplasmic reticulum region and the nuclear envelope after synthesis on membrane-bound ribosomes. [ABSTRACT FROM AUTHOR]

Abstract: The translation start site, immediately downstream from the start codon, is a dominant factor for gene expression in Escherichia coli. At present, no method exists to improve the expression level of cloned genes, since it remains difficult to find the best codon combination within the region. We determined the expression parameters that correspond to all sense codons within the first four codons using GFPuv which encodes a derivative of green fluorescent protein. Using a genetic algorithm (GA)-based computer program, these parameters were incorporated in a simple, static model for the prediction of translation efficiency, and optimized to the expression level for 137 randomly isolated GFPuv genes. The calculated initial translation index (ITI), also proven for the DsRed2 gene that encodes a red fluorescent protein, should provide a solution to overcome the gene expression problem in cloned genes whose expression is often inherently blocked at the translation process. The proposed method facilitates heterologous protein production in E. coli, the most commonly used host in biological and industrial fields. [Copyright &y& Elsevier]

We have used the tetrameric nature of the fluorescent protein DsRed to cross-link F1Fo-ATPase complexes incorporating a subunit γ-DsRed fusion protein in vivo. Cells expressing such a fusion protein have impaired growth relative to control cells. Strikingly, fluorescence microscopy of these cells revealed aberrant mitochondrial morphology. Electron microscopy of cell sections revealed the absence of cristae and multiple layers of unfolded inner mitochondrial membrane. Complexes recovered from detergent lysates of mitochondria were present largely as tetramers. Co-expression of &lrsquo;free’ DsRed targeted to the mitochondria reduced F1Fo-ATPase oligomerisation and partially reversed the impaired growth and abnormal mitochondrial morphology. We conclude that the correct arrangement of F1Fo-ATPase complexes within the mitochondrial inner membrane is crucial for the genesis and/or maintenance of mitochondrial cristae and morphology. Our findings further suggest that F1Fo-ATPase can exist in oligomeric associations within the membrane during respiratory growth. [ABSTRACT FROM AUTHOR]

A rapidly maturing variant of the red fluorescent protein DsRed was optimized for bacterial expression by random mutagenesis. The brightest variant contains six mutations, two of which (S4T and a silent mutation in codon 2) explain most of the fluorescence enhancement. The novel variants are expressed at 9–60-fold higher levels in Escherichia coli compared to DsRed.T3, but are not superior fluorophores on a per molecule basis. In contrast to previously available DsRed variants, DsRed.T3_S4T is sufficiently bright to monitor Salmonella gene expression in infected animals using flow cytometry. However, no fluorescence enhancement was observed in Leishmania or HeLa cells, indicating that these novel variants are specifically useful for bacteria. [Copyright &y& Elsevier]

Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, nonfluorescent pocilloporin to 2.2 A˚ resolution and a genetically engineered fluorescent variant to 2.4 A˚ resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gln-Tyr-Gly) being identical; the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment. [Copyright &y& Elsevier]

Summary We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co-localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus , a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co-localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al ., 2001). This locale appears to be distinct from the nucleolus as indicated by co-expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co-localization and interactions of proteins in a variety of experimental dicotyledonous hosts. [ABSTRACT FROM AUTHOR]

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645–656. [Copyright &y& Elsevier]