Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Human CD8+ T-cells Recognizing Peptides from Mycobacterium tuberculosis (Mtb) Presented by HLA-E Have an Unorthodox Th2-like,Multifunctional, Mtb Inhibitory Phenotype and Represent a Novel Human T-cell Subset

Mycobacterial antigens are not exclusively presented to T-cells by classical HLA-class Ia and HLA-class II molecules, but also through alternative antigen presentation molecules such as CD1a/b/c, MR1 and HLA-E. We recently described mycobacterial peptides that are presented in HLA-E and recognized by CD8⁺ T-cells. Using T-cell cloning, phenotyping, microbiological, functional and RNA-expression analyses, we report here that these T-cells can exert cytolytic or suppressive functions, inhibit mycobacterial growth, yet express GATA3, produce Th2 cytokines (IL-4,-5,-10,-13) and activate B-cells via IL-4. In TB patients, Mtb specific cells were detectable by peptide-HLA-E tetramers, and IL-4 and IL-13 were produced following peptide stimulation. These results identify a novel human T-cell subset with an unorthodox, multifunctional Th2 like phenotype and cytolytic or regulatory capacities, which is involved in the human immune response to mycobacteria and demonstrable in active TB patients’ blood. The results challenge the current dogma that only Th1 cells are able to inhibit Mtb growth and clearly show that Th2 like cells can strongly inhibit outgrowth of Mtb from human macrophages. These insights significantly expand our understanding of the immune response in infectious disease.     

Contrast Enhanced Abdominal Ultrasound in the Assessment of Ileal Inflammation in Crohn’s Disease: A Comparison with MR Enterography

Background and Aims

To prospectively examine the feasibility and accuracy of Contrast Enhanced Ultrasound (CEUS) in the assessment of Crohn’s disease (CD) activity in the terminal ileum in comparison to Magnetic Resonance Enterography (MRE), using endoscopy as a reference standard.

Methods

105 consecutive patients with alleged clinically active CD were assessed by MRE and CEUS. CEUS of the terminal ileum was performed using an intravenous microbubble contrast enhancer. Accuracy values of CEUS and MRE for the presence of active terminal ileitis were evaluated using the Receiver Operating Characteristic method, using endoscopic findings as a reference standard. Sensitivity and specificity values of MRE and CEUS were compared by the McNemar test.

Results

CEUS was feasible in 98% of patients, MRE in all. Optimal diagnostic accuracy in CEUS was obtained at a peak intensity value of 10%, showing 100% sensitivity, 92% specificity and an accuracy of 99% in demonstrating ileal mucosal inflammation. For MRE, overall sensitivity, specificity and accuracy were, 87%, 100%, and 88%, respectively. CEUS and MRE were highly correlated in assessing length and wall thickness of the terminal ileum. CEUS identified 11 of 16 MRE-detected strictures, but no fistulae.

Conclusion

The accuracy of CEUS is comparable to that of MRE in the assessment of active, uncomplicated terminal ileal CD and therefore a valuable bedside alternative to MRE in the follow-up of these patients.     

Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.     

Focus on Metabolism: Spatially Resolved Plant Metabolomics: Some Potentials and Limitations of Laser-Ablation Electrospray Ionization Mass Spectrometry Metabolite Imaging

Laser-ablation electrospray ionization (LAESI)-mass spectrometry imaging has been applied to contrasting plant organs to assess its potential as a procedure for performing in vivo metabolomics in plants. In a proof-of-concept experiment, purple/white segmented Phalaenopsis spp. petals were first analyzed using standard liquid chromatography-mass spectrometry analyses of separate extracts made specifically from the purple and white regions. Discriminatory compounds were defined and putatively annotated. LAESI analyses were then performed on living tissues, and these metabolites were then relocalized within the LAESI-generated data sets of similar tissues. Maps were made to illustrate their locations across the petals. Results revealed that, as expected, anthocyanins always mapped to the purple regions. Certain other (nonvisible) polyphenols were observed to colocalize with the anthocyanins, whereas others were found specifically within the white tissues. In a contrasting example, control and Cladosporium fulvum-infected tomato (Solanum lycopersicum) leaves were subjected to the same procedures, and it could be observed that the alkaloid tomatine has clear heterogeneous distribution across the tomato leaf lamina. Furthermore, LAESI analyses revealed perturbations in alkaloid content following pathogen infection. These results show the clear potential of LAESI-based imaging approaches as a convenient and rapid way to perform metabolomics analyses on living tissues. However, a range of limitations and factors have also been identified that must be taken into consideration when interpreting LAESI-derived data. Such aspects deserve further evaluation before this approach can be applied in a routine manner.Plants are a tremendously rich source of a myriad of structurally and chemically diverse metabolites (Rao and Ravishankar, 2002; D’Auria and Gershenzon, 2005). Many of these metabolites have a (partly) known function in the plant, although our knowledge of the vast majority of plant secondary metabolites is still sparse, or even nonexistent (Rao and Ravishankar, 2002; D’Auria and Gershenzon, 2005; Fernie, 2007). Plant metabolites are also of considerable importance in a crop context. Indeed, most plant species that have undergone domestication have become crops specifically because they provide us with a source of chemicals. This is not only true for all of our food crops, but also for many other species of genera such as Pyrethrum (insecticides), Jasminium and Santalum (perfumes), Hevea (rubber), Nicotiana and Cannabis (drugs), Linum (oils), Artemisia and Taxus (pharmaceuticals), Cinnamomum (flavors), etc. However, despite the importance of plants as a source of exploitable and essential biochemicals, we often still have remarkably limited knowledge of the relevant biosynthetic pathways, the genetics behind the key enzymes, and indeed when, why, and where these metabolites are produced and stored within the plant in question (Fernie, 2007; Sumner et al., 2011; Kueger et al., 2012).The field of plant metabolomics has grown tremendously since its recent inception earlier this century (Fiehn et al., 2000; Fiehn, 2002). As an untargeted approach to gain a broad overview of the complexity of plant metabolic composition, the technology has, in a short time, made significant inroads into helping expand our knowledge of plant biochemistry (Kueger et al., 2012; Etalo et al., 2013; Hunerdosse and Nomura, 2014; Meret et al., 2014). Typically, rich metabolomics data sets already provide us with a valuable means to generate hypotheses relating to plant metabolism, which then become the focus of further, more direct investigation (Quanbeck et al., 2012). New technologies are being developed, and especially, new data-mining strategies are being designed to allow us to look deep into plant metabolism without having first to rely on preconceptions. However, there are significant limitations to the application of the technology, which still remain the topic of much research effort.Robust sampling approaches for plant biochemical analysis generally entail taking reliably measurable amounts of plant material that will yield detectable levels of the chemical components. Although for metabolomics analyses, samples of just 50 mg can often suffice, obtaining a reliable sample with minimum biological variation generally requires an initial pooling of materials from which a representative sample is then taken. We therefore treat plant tissue as being homogeneous, but this is clearly a gross oversimplification (Fernie, 2007). Plants have been considered to be composed of roughly 40 different cell types, and a plant organ such as a leaf will generally contain up to 15 different cell types (Martin et al., 2001). Different morphologies also parallel different biochemical composition. Even directly neighboring cells within an organ, for example, a leaf epidermis that often comprises pavement, guard, trichome, and glandular hair cells, are formed from cells already known to have distinctly different biochemistries. Making an extract, for any kind of metabolomics or standard biochemical analysis, therefore entails that we immediately lose most intercellular and intertissue resolution. However, our knowledge is growing in that, in addition to known or expected biochemical differences between cell types, metabolite accumulation across organs can be far from uniform; indeed, islands of higher and lower concentrations of particular metabolites have been observed. This is of course immediately visible when the metabolites concerned can be seen by the naked eye; anthocyanins, for example, are often found to be heterogeneously distributed across leaves, fruits, and flower petals, creating clear phenotypic patterns. The same may also be true of other compounds that are invisible to the human eye but that, in contrast, may still be detectable by insects (e.g. through their fluorescence capacity; see http://www.naturfotograf.com/UV_flowers_list.html; Gronquist et al., 2001).In an ideal situation, we would like to be able to look directly into a plant tissue and be able to analyze the biochemical composition at the single cell level. Some so-called metabolite imaging technologies, usually based on mass spectrometric detection (mass spectrometry imaging [MSI]), have recently been introduced as a step toward this optimistic goal. Included here are matrix-assisted laser desorption/ionization (MALDI)-MSI, direct analysis in real time, and desorption electrospray ionization approaches (Cody et al., 2005; Cornett et al., 2007; Ifa et al., 2010). Early examples of MALDI-MSI have shown not only how primary metabolites such as sugars can be strongly localized within plant organs (Rolletschek et al., 2011), but also how the heterogeneous distribution of glucosinolates in Arabidopsis (Arabidopsis thaliana) can potentially determine grazing behavior of caterpillars (Shroff et al., 2008). This technology continues to improve, and recent exciting developments have resulted in cellular and subcellular imaging of metabolites at a resolution of 5 to 9 µm using MALDI (Korte et al., 2015). However, some key practical limitations of MALDI-based approaches are centered around the need to initially have to pretreat/dehydrate the tissue prior to applying the required matrix solution and the requirement of applying a vacuum during the biochemical analysis. Recently, a new technology has been introduced, laser ablation electrospray ionization (LAESI), which can potentially overcome some of these limitations, given that measurements can be made on fresh, living tissue without the need for a vacuum, thus creating the potential for high-resolution in vivo metabolomics.Here, we report on a set of experiments performed to assess both the potential and limitations of using LAESI-based MSI approaches to perform metabolic mapping on living plant tissues. While identifying a number of technological challenges that still need to be tackled, we were able to show that it is possible to use LAESI-MSI to map metabolites directly onto their known location (in this case, by exploiting the visibility of anthocyanins) as well as localize invisible metabolites in the same tissue. Results have revealed that in plants, for both petal and leaf tissue, the distribution of metabolites can be highly heterogeneous, and that this heterogeneity is of potential relevance to our gaining a broader, more detailed understanding of the overall molecular organization and phenotypic features of plant tissues. Furthermore, knowledge of the nature and extent of this heterogeneity has particular relevance and importance when trying to understand how a plant functions as a system, interacting with its environment. We predict that a higher resolution understanding of plant biochemistry will lead to an increasingly discriminatory capacity in our ability to define more accurately the spatial complexity of plant molecular organization.     

The response of peatlands to climate warming: A review

Peatlands hold a large portion of the Earth’s terrestrial organic carbon and serve as important pools in the global carbon cycle. Due to their strong feedbacks, peatlands are one of the most important ecosystems with respect to climate warming. This paper reviews the effects of climate warming on peatland ecosystems. Climate warming will shift the point in time when vascular peatland plants flower and reach maximum biomass to an earlier date. Flower production for some plants will increase, but how the phenology of peatland bryophytes will react is still unknown. Climate warming may increase productivity of peatlands, especially ombrotrophic Sphagnum bogs, but in the long run the negative effects from decreased water availability may prevail. Climate warming will change the basic characteristics of peatlands: their wetness and the related cold environment and nutrient shortage. By increased mineralization and nitrogen and phosphorus availability, climate warming will facilitate the growth of vascular plants. This will suppress endangered plant species (which usually grow in low-productive, phosphorus-limited habitats) and lead to a change in vegetation composition and a decrease in peatland biodiversity. Climate warming will change the competitive balance between bryophytes and between Sphagnum and vascular plants. Climate warming in the Late Pleistocene facilitated the initiation of peatland formation, but most current experiments show an obvious tendency for climate warming to drive many peatlands to regressive succession with a shift in dominance from Sphagnum to vascular plants. This change in vegetation will increase the flux of CH₄ and possibly also CO₂. The effect of accelerated peat decay as a result of climate warming will vary between types of peatlands. Since climate warming will generally enhance peat respiration more than net primary production, more and more peatlands will become carbon sources rather than carbon sinks, which will aggravate climate warming by positive feedback. Finally, this paper addresses some problems with current manipulative experimental studies on peatland response to climate warming and makes suggestions for further studies.     

Population studies of the wild tomato species Solanum chilense reveal geographically structured major gene-mediated pathogen resistance

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato Solanum chilense to investigate natural resistance against Cladosporium fulvum, a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of S. chilense differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a homologues of Cladosporium resistance (Hcr) locus, underlying the recognition of C. fulvum, in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.