Version Of Green Fluorescent Protein Research Articles

Lineage Tracing of FOXL1+ Cells in the Tunica Muscularis Suggests Mutual Origin for Telocytes and Smooth Muscle Cells.

We recently identified a FOXL1+ intestinal subepithelial network of telocytes (TCs) without which epithelial stem and progenitor cells cannot proliferate and support regeneration. In addition to FOXL1 lineage cell distribution along the intestinal epithelium, we also observed their presence within the muscle layers. Here, we characterized FOXL1+ lineage cells along the muscle layers of the duodenum in order to understand their progeny and relation to interstitial Cajal cells (ICCs), smooth muscle cells (SMCs) and the previously reported PDGFRa+ TCs. Using a FOXL1-Cre transgenic line in conjunction with genetic lineage labeling using the Rosa26-mTmG allele, in which Cre-marked cells produce a membrane-targeted version of green fluorescent protein (GFP), we found that within the muscle layers FOXL1 lineage GFP+ cells had two main progeny; (i) elongated multinucleated SMA+ SMCs, intermingled in parallel or perpendicular to muscle fibers. (ii) TCs displaying small cell body with multiple cell processes, expressing PDGFRa and CD34. These findings may suggest a mutual origin for TCs and SMCs.

A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression

Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions.

Subcellular localization and clues for the function of the HetN factor influencing heterocyst distribution in Anabaena sp. strain PCC 7120.

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide.

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37°C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37°C are more efficiently dissociated at 2.4kbar than those produced at 47°C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69kbar) refolding step. The association of a negative temperature (−9°C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10°C to 50°C is proportional to the temperature, and a higher yield was obtained at 20°C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37°C), of their dissociation at HHP (−9°C) and of eGFP refolding (20°C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.

A Method to Evaluate the Efficiency of Transfection Reagents in an Adherent Zebrafish Cell Line

We present a simple and robust method to evaluate the transfection efficiency of commercially available transfection reagents intended to be established for use in nonmammalian cell lines. To illustrate the method, we compare the ability of four different reagents to transfect the embryonic zebrafish cell line Z3. Z3 cells were seeded in a 96-well plate and simultaneously transfected in several variations by using minimum volumes of transfection reagent and a vector DNA encoding an amplified version of green fluorescent protein (GFP). After 24 and 48 h, transfection efficiency was determined by a dual fluorescence plate reader measurement of GFP and Hoechst 33342 fluorescence, an indicator of cell density. Of the four different reagents tested, certain variations of JetPrime™ reagent and X-tremeGene™ HP reagent produced the highest fluorescence signal per cell after 24- and 48-h incubation, respectively. The simultaneous multivariate setup enables comparing different reagent/DNA combinations at different time points well, independent of cell growth variability or seeding density.

An improved short‐lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae

AbstractIdeal reporter genes for temporal transcription programmes have short half‐lives that restrict their detection to the window in which their transcripts are present and translated. In an effort to meet this criterion for reporters of transcription in individual living cells, we adapted the ubiquitin fusion strategy for programmable N‐end rule degradation to generate an N‐degron version of green fluorescent protein (GFP) with a half‐life of ~7 min. The GFP variant we used here (designated GFP*) has excellent fluorescence brightness and maturation properties, which make the destabilized reporter well suited for tracking the induction and attenuation kinetics of gene expression in living cells. These attributes are illustrated by its ability to track galactose‐ and pheromone‐induced transcription in S. cerevisiae. We further show that the fluorescence measurements using the short‐lived N‐degron GFP* reporter gene accurately predict the transient mRNA profile of the prototypical pheromone‐induced FUS1 gene. Copyright © 2012 John Wiley & Sons, Ltd.

Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K.

The green fluorescent protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan fluorescent protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.

Evaluation of different versions of the gene encoding green fluorescent protein (GFP) in plants

The main purpose of the “CDA Eucalyptus: Collaborating Center in Agriculture Defense Relative to the Biosafety of Genetically Modified (GM) Eucalypts” is to gather, assess and validate the existing information concerning GM and non-GM Eucalyptus and its derivatives in the Brazilian environment, both in laboratories and field test experiments. Accordingly, the purpose of the present research activity is to provide a binary plasmid collection containing different versions of the gene encoding green fluorescent protein (GFP), originally from Aequorea victorea, for the future generation of easily detectable GM plant phenotypes. Using commercially available plasmids encoding blue (ECFP) and yellow (EYFP) versions of GFP for expression in bacteria, the initial strategy was to transfer the coding sequences to the intermediate plasmid pSport1 (Invitrogen) and, subsequently, to pART7 plasmid, which contains the promoter and terminator sequences for gene expression in plants. Although recombinant bacteria have been obtained for both genes, DNA sequencing showed that success was achieved only with pSport1-eyfp. So far, pART7 versions were not obtained. A new cloning strategy was proposed, which involves the binary plasmid pCAMBIA1302 (Cambia). This plasmid already contains one version of the gfp gene for expression in plants. The intention is to replace the gfp gene by ecfp, eyfp and mCherryFP versions. As soon as plasmids are finished, plants will be transformed via Agrobacterium tumefaciens, and their transgenic state will be confirmed by the fluorescence of the encoded proteins. Thus, the fluorescence of the GFP different versions will be used to monitor seeds, pollen, leaves and transgenic plants as a whole in the environment. Financial support: Brazilian Ministry of Agriculture, Farming & Supply (MAPA), and The National Council for the Development of Science & Technology (CNPq), Brazilian Ministry of Science & Technology (MCT).

‘Functional Histology’ in Brain Ischemia Studies

Classical histology and biochemistry constitute two very different ways to study biology. Classical histology allows direct visualization of tissue, but is limited by the non-specificity of organic dyes and the need to stain fixed tissue in postmortem samples. Classical biochemistry allows direct observation of the activities of tissue constituents, however, at the expense of complete cellular disruption, and is, in a sense, ‘blind' in that kinetic or equilibrium expressions are required intermediaries between data and interpretation. But advances in microscope technology, protein engineering, and transgenic animal technology have blurred this classical distinction. One can today speak of a ‘functional histology' that goes considerably beyond that discussed by Salthouse (1976) many years ago. It is well known that brain ischemia induces morphological alterations in postischemic neurons. Studies using classical histology cataloged end-stage morphologies such as ischemic cell change (Rosenblum, 1997). However, the past decade has seen increasing application of ‘functional histology' methods that literally illustrate ischemia-induced morphological changes at earlier time periods of the injury course. Two notable examples are the Hu et al (2000) discovery of the accumulation in postischemic neurons of ubiquitin–protein clusters (Ge et al, 2007) and our own work on mRNA-containing structures (DeGracia et al, 2008), both of which occur within minutes of reperfusion. In this issue of the Journal of Cerebral Blood Flow and Metabolism, the article by Krzysztof et al (2011) advances the application of ‘functional histology' to a completely new level in the field. The authors utilized fluorescent photobleaching technology and transgenic animals bred to contain a version of green fluorescent protein fused with the calreticulin endoplasmic reticulum-targeting sequence. Using this system, the authors visualized from the cortical surface of intact whole animals the real-time dynamics of the endoplasmic reticulum of cortical neurons during the ischemic period. On the basis of the time of recovery of fluorescent signal after photobleaching, the authors elegantly and convincingly demonstrated that fragmentation of the endoplasmic reticulum occurs within minutes of the onset of global ischemia. The results are illustrated with striking three-dimensional images of fragmented endoplasmic reticulum in cortical neurons. The techniques and analysis are straightforward and unambiguous, leaving little room for controversy. To my knowledge, this study now marks the earliest observation of a clear-cut ‘functional morphological' alteration in postischemic neurons. It is also notable the authors attribute this change to activation of ‘stress responses' in the postischemic neurons. This interpretation reflects the increasing realization that postischemic neuronal death may not be because of only ischemia-induced damage but also because of a competition between ischemia-induced damage and the cell's innate ability to respond to damage (Endres et al, 2008; Lo, 2008; DeGracia, 2010). In sum, the paper by Krzysztof et al (2011) literally illustrates the power of a ‘functional histological' approach by demonstrating the very rapid response of the neuron to the ischemic perturbation. We expect the general approach used here to become increasingly prevalent, and the current paper represents an important milestone on this path.

The impact of intragenic CpG content on gene expression

The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1α). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 μg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 μg/ml, and of purified selenomethione-labeled AIA–GFP (His8 removed by treatment with TEV protease) was 172 μg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10–50 ml) cell growth and automated purification. 1H–15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA–GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 Å. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.

Optimising Blue Fluorescent Protein (BFP) for use as a mammalian reporter gene in parallel with Green Fluorescent Protein (GFP).

AbstractTo obtain an optimised form of BFP for use as a reporter gene in mammalian cells, the brightest available GFP form, EGFP, was mutated at 5 different positions, yielding 8 different mutagenised forms of BFP. The intensity of the fluorescent signals attained in mammalian cells with all these various versions of BFP was analysed by flow cytometry of transiently transfected COS 7 cells. The best mutant obtained can be detected readily both by flow cytometry and fluorescence microscopy, even when expressed together with GFP. To explore whether cellular localization could enhance the fluorescence signals any further, plasmid constructs were made to target optimised versions of GFP and BFP to the nucleus, the endoplasmic reticulum (ER) and the cell surface. Expression in the nucleus or ER increased the fluorescence signal by ca. 50%, whereas cell surface expression resulted in a five-fold decrease compared to the ER and nuclear forms. Co-expression of GFP and BFP in the same cellular compartment did not result in any significant absorption of the blue fluorescence by GFP. Thus, targeting of GFP and BFP to various cellular compartments adds even further versatility to this convenient dual reporter-gene system.

Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent Proteins

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".

Two short sequences from amaranth 11S globulin are sufficient to target green fluorescent protein and beta-glucuronidase to vacuoles in Arabidopsis cells

Vacuolar sorting of seed storage proteins is a very complex process since several sorting pathways and interactions among proteins of different classes have been reported. In addition, although the C-terminus of several 7S proteins is important for vacuolar delivery, other signals seem also to be involved in this process. In this work, the ability of two sequences of the Amaranthus hypochondriacus 11S globulin (amaranthin) to target reporter proteins to vacuoles was studied. We show that the C-terminal pentapeptide (KISIA) and the GNIFRGF internal sequence fused at the C terminal region of genes encoding secretory versions of green fluorescent protein (GFP) and GFP-β-glucuronidase (GFP-GUS) were sufficient to redirect these reporter proteins to the vacuole of Arabidopsis cells. According to the three-dimensional structure of 7S and 11S storage globulins, this internal vacuolar sorting sequence corresponds to the alpha helical region involved in trimer formation, and is conserved within these families. In addition, these sequences were able to interact in vitro, in a calcium dependent manner, with the sunflower vacuolar sorting receptor homolog to pea BP-80/AtVSR1/pumpkin PV72. This work shows for the first time the role of a short internal sequence conserved among 7S and 11S proteins in vacuolar sorting.

Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids.

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.

Double-Hexahistidine Tag with High-Affinity Binding for Protein Immobilization, Purification, and Detection on Ni−Nitrilotriacetic Acid Surfaces

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine (His6) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni-NTA-modified surfaces than a conventional single-His6 tag or two single-His6 tags at N- and C-termini. Using, as a model, tagged versions of green fluorescent protein (GFP), stable and tight binding of the double-His6 tag/Ni-NTA interaction was demonstrated by competitive elution from Ni-NTA agarose beads, surface plasmon resonance on a Ni-NTA chip, and ELISA in Ni-NTA microwell plates. Protein purification by Ni-NTA chromatography was improved by a 6-8-fold increase in imidazole concentration required for elution, while the dissociation rate of double-His6 GFP from Ni-NTA chips in SPR (BIAcore) was 10 times slower than for single-His6-tagged proteins. ELISA assays and protein microarrays constructed with double-His6 GFP demonstrated greater detection sensitivity with anti-His antibodies and Ni-NTA conjugates. Moreover, the double-His6 tag could serve simultaneously both for protein immobilization and for detection on surfaces. The double-His6 peptide has the potential to be a universal tag for protein immobilization and detection on arrays and single-step purification of proteins from crude mixtures.

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy.

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.

Engineering and characterization of a superfolder green fluorescent protein

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.

A Causative Role of Stromelysin-3 in Extracellular Matrix Remodeling and Epithelial Apoptosis during Intestinal Metamorphosis in Xenopus laevis

The matrix metalloproteinases are a family of proteases capable of degrading various components of the extracellular matrix. Expression studies have implicated the involvement of the matrix metalloproteinase stromelysin-3 (ST3) in tissue remodeling and pathogenesis. However, the in vivo role of ST3 has been difficult to study because of a lack of good animal models. Here we used intestinal remodeling during thyroid hormone-dependent metamorphosis of Xenopus laevis as a model to investigate in vivo the role of ST3 during postembryonic organ development in vertebrates. We generated transgenic tadpoles expressing ST3 under control of a heat shock-inducible promoter. We showed for the first time in vivo that wild type ST3 but not a catalytically inactive mutant was sufficient to induce larval epithelial cell death and fibroblast activation, events that normally occur only in the presence of thyroid hormone. We further demonstrated that these changes in cell fate are associated with altered gene expression in the intestine and remodeling of the intestinal basal lamina. These results thus suggest that ST3 regulates cell fate and tissue morphogenesis through direct or indirect ECM remodeling.

Monitoring Host Nuclear Migration and Degradation with Green Fluorescent Protein during Compatible and Incompatible Interactions of Nicotiana tabacum with Colletotrichum Species

AbstractRecent evidence has emerged suggesting that nuclei sense and migrate towards infection sites in plants, and a novel approach to examine the dynamics of nuclei is described utilizing transgenic plants expressing a version of green fluorescent protein (GFP) that specifically labels plant nuclei. Nicotiana tabacum with GFP‐labelled nuclei were inoculated with GFP‐labelled strains of the hemibiotrophic fungi, Colletotrichum destructivum and C. graminicola. The nucleus in an epidermal host cell migrated to just underneath the appressorium of the compatible fungus, C. destructivum, but then migrated away from the developing fungus once it had penetrated and started to grow biotrophically. As the necrotrophic phase developed, the nuclei appeared to shrink and eventually their green fluorescence was no longer visible. The interaction of C. graminicola with N. tabacum was considered to show non‐host incompatibility. The host nuclei in the epidermal cells also migrated underneath the appressoria. Once fungal penetration had failed, the nuclei then migrated back towards locations typically observed in epidermal cells of uninoculated plants. The use of both plant structures and a fungus that are labelled with a readily detectable fluorescent marker provides significant advantages as it permits direct observation of changes in living host and pathogen cells during a plant–fungal interaction.