The Coiled-Coil Domain of EHD2 Mediates Inhibition of LeEix2 Endocytosis and Signaling

Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis

The impact of plant biotechnology on food allergy

Concerns about food allergy and its societal growth are intertwined with the growing advances in plant biotechnology. The knowledge of plant genes and protein structures provides the key foundation to understanding biochemical processes that produce food allergy. Biotechnology offers the prospect of producing low-allergen or allergen null plants that could mitigate the allergic response. Modified low-IgE binding variants of allergens could be used as a vaccine to build immunotolerance in sensitive individuals. The potential to introduce new allergens into the food supply by biotechnology products is a regulatory concern. Introduction Food allergies are a growing concern in the industrialized countries where the percentage of the population that exhibit clinical food allergies has increased rapidly over the past few decades Plant biotechnology has had a major role in defining the problems of food allergy. Modifying food plants presents the potential to provide a means to address the problems of sensitization and management of food allergies. As plant biotechnology is used as production platforms to produce altered food and feed as well as industrial products there is potential that this will inadvertently produce potent food allergies is a risk, but how to define that risk is a continuing problem. Using the plant biotechnology tool kit and its implications for food defining allergy Biotechnology has revolutionized our understanding of which proteins are food allergens, how these proteins are related and often closely related to other members of the same family that are not known to be allergens. Using the sequence databases many food allergen proteins have had maps of antigenic sites produced and these antigenic sites have been placed on crystal structures. [5,6,7 ]. The interpretation from this is that only a small fraction of proteins, perhaps 2%, are allergens however this should be viewed with caution as the vast majority of proteins produced by any cell are accumulated at levels that are below the threshold for sensitization or hypersensitive Seed storage proteins including 2S albumins, 7 S vicilins, and 11 S legumin family of proteins include the most potent of the plant allergens responsible for most plantsource induced anaphylaxis deaths. The 2S storage proteins of tree nuts as well as the 2S proteins of sunflower and peanut result in instances of anaphylaxis death. Other seeds have abundant 2S proteins including the Brassicas and the Cucurbit squashes are much more rarely allergenic. Similar broad allergenic responses have demonstrated with the lipid transfer proteins (LTP) with many examples from seed and from vegetative parts of the plant such as tap-roots and fruit being dominant allergens [11 ]. LTP examples include carrot, peach, apple, beet-root as well as seeds including tree nuts and peanuts with some sensitive people broadly reactive to the LTPs of diverse species. Even in closely related plants such legume seeds where the 7S proteins of peanut and soybean are significant allergens while the homologous 7S proteins of the common green bean appears to be rarely allergenic. Altering plants and their allergens to mitigate food allegenicity Attempts have been made to reduce allergenicity by producing allergen-reduced or allergen-null plants by biotechnology or by selection as a proof of concept. These experiments have demonstrated that it is feasible to completely eliminate specific allergens from food plants. Beginning with the first attempts to partially silence the rice allergen [12,13], to completely eliminating a major allergen of soybean [14,15] [25 ,26 ,27 ]. This approach has been used for peanut and has resulted in identifying peanuts lines null for Ara h 2 the major demonstrated allergen [28,29 ]. One of the difficulties in using genetic modification or nulls to create low-allergen or hypoallergenic seeds is that for many seeds the allergenic proteins account for the dominant portion of the seed proteome A further complication in silencing part of a seed's protein content is that seeds generally appear to compensate for a shortfall of a major protein by accumulating other seed proteins to maintain a relatively constant protein content. Soybeans with silenced b-conglycinin storage protein the protein content was compensated by increased accumulation of glycinin storage protein that maintains the normal 39% protein level [14]. b-conglycinin is an established IgE binding protein so silencing removes one allergen replaced by glycinin also an IgE binding protein. Whether this is a net loss or gain of allergenicity has not been tested on sensitized people. This observation was one of the first of what is emerging to be a broader potential problem and opportunity with strategies to alter seed allergenicity by producing nulls of major seed allergens. Because each event of silencing a major protein, The IgE binding proteins of many allergenic seeds comprise the large majority of the seed's protein content. Soybean has 16 described allergens of which 7 (italics) are illustrated on the two dimensional gel of the total seed proteins. The pie chart shows the relative abundance of the 7 allergens determined by spot volume analysis that together are in excess of 60% of the total proteins. This illustrates the problem of modifying seeds to create allergen nulls with most of the protein content and valued nutritional composition being the seed allergens. Current Opinion in Biotechnology 2011, 22:224-230 www.sciencedirect.com Immunotherapy is a promising treatment approach for pre-existing food allergy where small and increasing doses of an antigen is given to a sensitive individual to build up immunotolerance ([37 ], for review). While many clinical tests of immunotherapy have been conducted with extracts of the whole allergenic source, peanut, for example, there is a biotechnological variant of this approach that may prove effective and suitable to standardize as a vaccine. With comprehensive transcript and genomic sequences the entire gene families encoding allergenic proteins have been determined. These sequences are used to produce comprehensive peptide maps of the allergen then the IgE binding sites for the gene family members can be determined (e.g. [8][9][10] Good gut health may have an important role in impeding the acquisition of food allergy. Gut health and general health can be improved by micronutrients and there many projects underway to produce functional foods with enhanced nutrient content ([37 ] for review). Among these micronutrients b-carotene produced in plants will on demand be cleaved to produce vitamin A. There are many projects underway to improve b-carotene content in plant foods of which the 'golden rice' project Food allergy and the deployment of plant biotechnology Two biotechnology-generated events catapulted the awareness of the potential for biotechnology to increased allergenic risk as a consequence of introducing new traits into plants. Most seeds do not possess an optimum balance of amino acids, fatty acids, and other constituents for use as food and feed. Among the earliest goals of plant biotechnology were efforts to rebalance essential amino acid content to be more aligned with food and feed needs. Among the strategies tested was the expression of highsulfur content 2S storage protein genes derived from the Brazil nut in soybean. The use of this strategy was aborted when it was recognized that the 2S storage proteins from tree nuts are potent allergens and correlated with potentially lethal anaphylaxis Biotechnology offers the prospect to express and accumulate essentially any protein from any source in plants. Laboratory-level studies have produced antibodies, vaccines, enzymes, food/feed proteins, and many other potential products in diverse plants. Plant-based production offers the economic advantages of mass protein production using the efficient multiplier of agricultural production. The continuing use, and need, for conventional crop plants as production platforms will require protocols to evaluate the potential for novel proteins including synthetic proteins to be food allergens. The impact of plant biotechnology on food allergy Herman and Burks 227 Conclusion

Checkpoint Genes at the Cancer Side of the Immunological Synapse in Bladder Cancer.

Immune checkpoint inhibitors have revolutionized cancer therapy, but not all cancers respond to the currently available drugs, and even within cancers considered responsive to such modality, response rates range between 15 and 40%, depending on the cancer type, the line of treatment, and yet unknown clinical/molecular factors. Coordinated expression of checkpoint proteins was shown to occur on T cells, probably allowing fine-tuning of the signal transmitted to the cell. We performed a bioinformatic analysis of the expression of putative checkpoint mRNAs at the cancer side of the immunological synapse from the bladder cancer tumorgenome atlas (TCGA) database. Fifteen mRNAs, corresponding to both coinhibitory and costimulatory checkpoints, were shown to be expressed above a designated threshold. Of these, seven mRNAs were found to be coexpressed: CD277, PD-1L, CD48, CD86, galectin-9, TNFRSF14 (HVEM), and CD40. The expression of 2 of these mRNAs-BTN3A1 (CD277) and TNFRSF14 (HVEM)-was positively correlated with overall survival in the TCGA database. All these seven mRNA share putative binding sites of a few transcription factors (TFs). Of these, the expression of the TF BACH-2 was positively correlated with the expression of checkpoint mRNAs from the network. This suggests a joint transcriptional regulation on the expression of checkpoint mRNAs at the bladder tumor side of the immunological synapse

Electrical Modelling of In-Vivo Impedance Spectroscopy of Nicotiana tabacum Plants

Electrical impedance spectroscopy has been suggested as a sensing method for plants. Here, a theoretical approach for electrical conduction via the plant stem is presented and validated, linking its living electrical characteristics to its internal structure. An electrical model for the alternating current conduction and the associated impedance in a live plant stem is presented. The model accounts for biological and geometrical attributes. It uses the electrically prevalent coupled transmission line model approach for a simplified description of the complicated vessel structure. It considers the electrode coupling to the plant stem (either Galvanic or Faradic), and accounts for the different interactions of the setup. Then the model is simplified using the lumped element approach. The model is then validated using a four-point probe impedance spectroscopy method, where the probes are galvanically coupled to the stem of Nicotiana tabacum plants. The electrical impedance data was collected continuously and the results exhibit an excellent fitting to the theoretical model, with a fitting error of less than 1.5% for data collected on various days and plants. A parametric evaluation of the fitting corresponds to the proposed physically based model, therefore providing a baseline for future plant sensor design

Towards Optimal Green Plant Irrigation: Watering and Body Electrical Impedance

With the growth of world population and food demand, it is crucial to optimize water consumption for agriculture cultivation. Here we propose a method to monitor plant status, relating the measured parameters to the watering or drying situation of a single plant. Plant trunk electrical impedance measurements and environmental parameters were analyzed with a statistical approach. Correlation and causality among the data are showed and analyzed. In this way, it was possible to easily obtain the needed information about plant status

The Receptor for the Fungal Elicitor Ethylene-Inducing Xylanase Is a Member of a Resistance-Like Gene Family in Tomato

An ethylene-inducing xylanase (EIX) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). The LeEix locus in tomatoes was characterized by map-based cloning, which led to the identification of a novel gene cluster from which two members (LeEix1 and LeEix2) were isolated. Similar to the tomato Ve resistance genes in tomato plants, the deduced amino acid sequences encoded by LeEix1 and LeEix2 contain a Leu zipper, an extracellular Leu-rich repeat domain with glycosylation signals, a transmembrane domain, and a C-terminal domain with a mammalian endocytosis signal. Silencing expression of the LeEix genes prevented the binding of EIX to cells of an EIX-responsive plant and thus inhibited the hypersensitive response. Overexpression of either LeEix1 or LeEix2 genes in EIX-nonresponsive tobacco plants enabled the binding of EIX, although only LeEix2 could transmit the signal that induced the hypersensitive response. Overexpressing LeEix2 in mammalian COS-7 cells enables binding of EIX, indicating physical interaction between the EIX elicitor and LeEix2 gene product. Structural analysis of the LeEix proteins suggests that they belong to a class of cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Mutating the endocytosis signal in LeEix2 (Tyr 993 to Ala) abolished its ability to induce the hypersensitive response, suggesting that endocytosis plays a key role in the signal transduction pathway

Translational research: exploring and creating genetic giversity

International audienceThe crop selection process has created a genetic bottleneck ultimately restricting breeding output. Wild relatives of major crops as well as the so-called 'neglected plant' species represent a reservoir of genetic diversity that remains underutilized. These species could be used as a tool to discover new alleles of agronomic interest or could be the target of breeding programs. Targeted induced local lesions in the genome (TILLING) can be used to translate in neglected crops what has been discovered in major crops and reciprocally. However, random mutagenesis, used in TILLING approaches, provides only a limited density of mutational events at a defined target locus. Alternatively, clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) fused to a cytidine deaminase could serve as a localized mutagenic agent to produce high-density mutant populations. Artificial evolution is at hand