pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

Download Full-text

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00884.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. H1647-H1654 ◽

Cited By ~ 11

Author(s):

Jean-Philippe Fortin ◽

Johanne Bouthillier ◽

François Marceau

Keyword(s):

Fluorescent Protein ◽

Cultured Cells ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Brefeldin A ◽

Metabolic Inhibitors ◽

Maximal Effect ◽

Hek 293 ◽

B1 Receptors ◽

Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

Download Full-text

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

Microscopy and Microanalysis ◽

10.1017/s1431927600007558 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 133-134 ◽

Cited By ~ 1

Author(s):

Ammasi Periasamy ◽

Richard N. Day

Keyword(s):

Transcription Factors ◽

Living Cell ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Protein Localization ◽

Resonance Energy ◽

Living Cells ◽

Specific Gene ◽

Green Fluorescent ◽

Prl Gene

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

Download Full-text

Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316018623 ◽

2016 ◽

Vol 72 (12) ◽

pp. 1298-1307 ◽

Cited By ~ 16

Author(s):

Damien Clavel ◽

Guillaume Gotthard ◽

David von Stetten ◽

Daniele De Sanctis ◽

Hélène Pasquier ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Directed Evolution ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

X Rays ◽

Visible Absorption ◽

X Ray ◽

X Ray Crystallography ◽

Green Fluorescent

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent proteinlanYFP fromBranchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures oflanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV–visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

Download Full-text

Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113510740 ◽

2014 ◽

Vol 19 (2) ◽

pp. 215-222 ◽

Cited By ~ 46

Author(s):

Simon J. Gruber ◽

Razvan L. Cornea ◽

Ji Li ◽

Kurt C. Peterson ◽

Tory M. Schaaf ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dependent Manner ◽

Chemical Library ◽

Time Resolved ◽

Endoplasmic Reticulum Calcium ◽

Green Fluorescent

We have used a “two-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Download Full-text

Engineering an efficient and bright split Corynactis californica green fluorescent protein

Scientific Reports ◽

10.1038/s41598-021-98149-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hau B. Nguyen ◽

Thomas C. Terwilliger ◽

Geoffrey S. Waldo

Keyword(s):

Green Fluorescent Protein ◽

Protein Interactions ◽

Large Scale ◽

Fluorescent Protein ◽

Protein Translocation ◽

Fluorescent Proteins ◽

Protein Crystallization ◽

Protein Solubility ◽

Sea Anemones ◽

Green Fluorescent

AbstractSplit green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein–protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

Download Full-text

CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/2008/453590 ◽

2008 ◽

Vol 2008 ◽

pp. 1-9 ◽

Cited By ~ 28

Author(s):

Rouzbeh R. Taghizadeh ◽

James L. Sherley

Keyword(s):

Stem Cells ◽

Time Scales ◽

Cell Biology ◽

Adult Stem Cells ◽

Cell Function ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Fluorescent Reporter ◽

Green Fluorescent

The stable expression of reporter genes in adult stem cells (ASCs) has important applications in stem cell biology. The ability to integrate a noncytotoxic, fluorescent reporter gene into the genome of ASCs with the capability to track ASCs and their progeny is particularly desirable for transplantation studies. The use of fluorescent proteins has greatly aided the investigations of protein and cell function on short-time scales. In contrast, the obtainment of stably expressing cell strains with low variability in expression for studies on longer-time scales is often problematic. We show that this difficulty is partly due to the cytotoxicity of a commonly used reporter, green fluorescent protein (GFP). To avoid GFP-specific toxicity effects during attempts to stably mark a rat hepatic ASC strain and, therefore, obtain stable, long-term fluorescent ASCs, we evaluated cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), in addition to GFP. Although we were unable to derive stable GFP-expressing strains, stable fluorescent clones (up to 140 doublings) expressing either CFP or YFP were established. When fluorescently marked ASCs were induced to produce differentiated progeny cells, stable fluorescence expression was maintained. This property is essential for studies that track fluorescently marked ASCs and their differentiated progeny in transplantation studies.

Download Full-text

Investigation Into the use of C- and N-terminal GFP Fusion Proteins for Subcellular Localization Studies Using Reverse Transfection Microarrays

Comparative and Functional Genomics ◽

10.1002/cfg.405 ◽

2004 ◽

Vol 5 (4) ◽

pp. 342-353 ◽

Cited By ~ 52

Author(s):

Ella Palmer ◽

Tom Freeman

Keyword(s):

Subcellular Localization ◽

High Throughput ◽

Fusion Proteins ◽

Fluorescent Protein ◽

Protein Localization ◽

Genes Encoding ◽

Reverse Transfection ◽

C And N ◽

Green Fluorescent ◽

Cellular Compartments

Reverse transfection microarrays were described recently as a high throughput method for studying gene function. We have investigated the use of this technology for determining the subcellular localization of proteins. Genes encoding 16 proteins with a variety of functions were placed in Gateway expression constructs with 3′ or 5′ green fluorescent protein (GFP) tags. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. HEK293T cells were grown over the surface of the array until confluent and GFP fluorescence visualized by confocal microscopy. All C-terminal fusion proteins localized to cellular compartments in accordance with previous studies and/or bioinformatic predictions. However, less than half of the N-terminal fusion proteins localized correctly. Of those that were not in concordance with the C-terminal tagged proteins, half did not exhibit expression and the remainder had differing subcellular localizations to the C-terminal fusion protein. This data indicates that N-terminal tagging with GFP adversely affects the protein localization in reverse transfection assays, whereas tagging with GFP at the C-terminal is generally better in preserving the localization of the native protein. We discuss these results in the context of developing high-throughput subcellular localization assays based on the reverse transfection array technology.

Download Full-text

A Gene in the Pseudomonas syringae pv. tomato Hrp Pathogenicity Island Conserved Effector Locus, hopPtoA1, Contributes to Efficient Formation of Bacterial Colonies in Planta and Is Duplicated Elsewhere in the Genome

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.10.1014 ◽

2002 ◽

Vol 15 (10) ◽

pp. 1014-1024 ◽

Cited By ~ 33

Author(s):

J. L. Badel ◽

A. O. Charkowski ◽

W.-L. Deng ◽

A. Collmer

Keyword(s):

Pseudomonas Syringae ◽

Laser Scanning ◽

Fluorescent Protein ◽

Pathogenicity Island ◽

Effector Proteins ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

In Planta ◽

Tomato Dc3000 ◽

Green Fluorescent

The ability of Pseudomonas syringae to grow in planta is thought to be dependent upon the Hrp (type III secretion) system and multiple effector proteins that this system injects into plant cells. ORF5 in the conserved effector locus of the P. syringae pv. tomato DC3000 Hrp pathogenicity island was shown to encode a Hrp-secreted protein and to have a similarly secreted homolog encoded in an effector-rich pathogenicity island located elsewhere in the genome. These putative effector genes were designated hopPtoA1 and hopPtoA2, respectively. DNA gel blot analysis revealed that sequences hybridizing with hopPtoA1 were widespread among P. syringae pathovars, and some strains, like DC3000, appear to have two copies of the gene. uidA transcriptional fusions revealed that expression of hopPtoA1 and hopPtoA2 can be activated by the HrpL alternative sigma factor. hopPtoA1 and hopPtoA1/hopPtoA2 double mutants were not obviously different from wild-type P. syringae pv. tomato DC3000 in their ability to produce symptoms or to increase their total population size in host tomato and Arabidopsis leaves. However, confocal laser-scanning microscopy of GFP (green fluorescent protein)-labeled bacteria in Arabidopsis leaves 2 days after inoculation revealed that the frequency of undeveloped individual colonies was higher in the hopPtoA1 mutant and even higher in the hopPtoA1/hopPtoA2 double mutant. These results suggest that hopPtoA1 and hopPtoA2 contribute redundantly to the formation of P. syringae pv. tomato DC3000 colonies in Arabidopsis leaves.

Download Full-text

Development of an Efficient and Stable Transformation System for Aspergillus Oryzae Based on the pyrG Gene

10.21203/rs.3.rs-26135/v1 ◽

2020 ◽

Author(s):

Caixia Zhou ◽

Yujun Wan ◽

Huipeng Yao ◽

Hui Chen ◽

Yirong Xiao ◽

...

Keyword(s):

Aspergillus Oryzae ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Transformation Method ◽

Stable Transformation ◽

Fungal Transformation ◽

Transformation System ◽

Efficient Expression ◽

The Stability ◽

Green Fluorescent

Abstract Background Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. An efficient and stable transformation system is the key to the successful expression of the gene of interest in A. oryzae.Results To improve the expression efficiency of the gene of interest in A. oryzae, we constructed the uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG by Ultraviolet (UV) mutagenesis of pyrG gene deletion which would be used as a host for further transformation. In addition, a novel and efficient expression vector pBC-hygro.4 was constructed, including the pyrG cassette gene, His-Tag, amyB promoter and terminator,and green fluorescent protein GFP marker. pBC-hygro.4 transformed A. oryzae RIB40ΔpyrG efficiently via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was tested by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, we successfully obtained a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG. At the same time, the developed vectors are fully functional for heterologous expression of the GFP fluorescent proteins in the A. oryzae RIB40ΔpyrG.Conclusion Our work provides a new method that can be applied to other filamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.

Download Full-text

Beyond identity: Understanding the contribution of the 5’ nucleotide of the antisense strand to RNAi activity

PLoS ONE ◽

10.1371/journal.pone.0256863 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256863

Author(s):

Peizhen Yang ◽

Ericka Havecker ◽

Matthew Bauer ◽

Carl Diehl ◽

Bill Hendrix ◽

...

Keyword(s):

Gene Expression ◽

Fluorescent Protein ◽

Effector Proteins ◽

Antisense Strand ◽

Homologous Region ◽

In Planta ◽

Sirna Duplex ◽

Magnesium Chelatase ◽

Sense Strand ◽

Green Fluorescent

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene’s mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5’ nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5’ nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5’ nt identities and different pairing states between the 5’ antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5’ nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5’ thermodynamic rules governing topical siRNA efficacy across plants and animals.

Download Full-text