Expression of murine and viral interleukin-10 in tobacco for immunomodulation of galt for the prevention of autoimmune diseases

L\u2019interleuchina-10 (IL-10) e\u2019 una citochina immunosoppressiva con potenziale applicazione terapeutica in diverse malattie autoimmuni e infiammatorie. La somministrazione orale di questa citochina, da sola o in combinazione con autoantigeni associati alla malattia, potrebbe conferire protezione dall\u2019insorgenza di specifiche malattie autoimmuni attraverso l\u2019induzione di tolleranza orale. Un valido sistema alternativo per la produzione di proteine ricombinanti di interesse farmaceutico \ue8 rappresentato dalle piante transgeniche, data la possibilit\ue0 di estendere la produzione su larga scala a bassi costi, e i minimi requisiti che richiedono per il loro mantenimento. Le piante, inoltre, possono essere assunte direttamente come alimento e potrebbero diventare esse stesse veicoli per la somministrazione delle proteine ricombinanti eliminando la necessit\ue0 di purificarle. Ci si \ue8 proposti di valutare se le piante di tabacco fossero in grado di produrre alti livelli di IL-10 virale e murina biologicamente attiva. Per ottenere alti livelli di espressione dei transgeni, sono state effettuate sia la trasformazione dei plastidi, sia la trasformazione nucleare, valutando diverse strategie di localizzazione delle proteine ricombinanti. La trasformazione dei cloroplasti \ue8 risultato un approccio non attuabile per la produzione di IL-10 virale o murina ricombinante, in quanto i livelli di accumulo sono risultati decisamente insoddisfacenti per entrambi i transgeni. Invece, per quanto riguarda la trasformazione nucleare, sono state valutate tre diverse strategie di localizzazione sub-cellulare, per dirigere la proteina ricombinante nel reticolo endoplasmico (RE), nel citosol e nell\u2019apoplasto, dapprima in esperimenti di espressione transiente e, successivamente, sono state generate piante transgeniche stabili con il costrutto che aveva fornito i pi\uf9 alti livelli di accumulo indirizzando la proteina nel RE. Le proteine ricombinanti sono state purificate da materiale fogliare transgenico e sono state caratterizzate rispetto alla composizione degli N-glicani, alla dimerizzazione, alla loro stabilit\ue0 e attivit\ue0 biologica in saggi in vitro. Entrambe le molecole prodotte in pianta formano dimeri stabili, e sono in grado di attivare la cascata di trasduzione del segnale di IL-10 e di indurre risposte anti-infiammatorie specifiche nella linea cellulare di macrofagi murini J774. E\u2019 stato dimostrato che le piante di tabacco sono in grado di processare correttamente l\u2019IL-10 virale e murina in dimeri biologicamente attivi, e che quindi rappresentano una valida piattaforma per la produzione di queste citochine. Inoltre, i livelli di accumulo ottenuti sono sufficientemente elevati da permettere di somministrare una dose immunologicamente rilevante di IL-10 in una ragionevole quantit\ue0 di materiale fogliare, senza richiedere laboriose purificazioni. Questo lavoro apre la strada alla realizzazione di studi di somministrazione orale in modelli murini di malattie autoimmuni, che permetteranno di effettuare una valutazione comparativa delle propriet\ue0 immunomodulatorie nonch\ue9 dell\u2019efficacia delle IL-10 virale e murina nell\u2019indurre tolleranza orale.Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine, alone or in combination with disease-associated autoantigens, could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. The ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10 was investigated. To reach high accumulation levels of the transgenes, plastid transformation of the IL-10 genes as well as different targeting strategies of the nuclear encoded recombinant proteins were investigated. Chloroplast transformation turned out not to be a feasible approach for the recombinant production of IL-10, as unsatisfactory accumulation levels were obtained upon expression of both transgenes. For tobacco nuclear transformation, three different subcellular targeting strategies, directing the recombinant protein into the endoplasmic reticulum (ER), cytosol and apoplast, were first assessed in transient expression experiments, and stable transgenic plants were then generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the ER. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization, stability and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells. It was therefore demonstrated that tobacco plants are able to correctly process viral and murine IL- 10 into biologically active dimers, representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow evaluation of the immunomodulatory properties and effectiveness of the viral and murine IL-10 in inducing oral tolerance

Russell-like bodies in plant seeds share common features with prolamin bodies and occur upon recombinant protein production

Although many recombinant proteins have been produced in seeds at high yields without adverse effects on the plant, endoplasmic reticulum (ER) stress and aberrant localization of endogenous or recombinant proteins have also been reported. The production of murine interleukin-10 (mIL-10) in Arabidopsis thaliana seeds resulted in the de novo formation of ER-derived structures containing a large fraction of the recombinant protein in an insoluble form. These bodies containing mIL-10 were morphologically similar to Russell bodies found in mammalian cells. We confirmed that the compartment containing mIL-10 was enclosed by ER membranes, and 3D electron microscopy revealed that these structures have a spheroidal shape. Another feature shared with Russell bodies is the continued viability of the cells that generate these organelles. To investigate similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds, we crossed plants containing ectopic ER-derived prolamin protein bodies with a line accumulating mIL-10 in Russell-like bodies. This resulted in seeds containing only one population of protein bodies in which mIL-10 inclusions formed a central core surrounded by the prolamin-containing matrix, suggesting that both types of protein aggregates are together removed from the secretory pathway by a common mechanism. We propose that, like mammalian cells, plant cells are able to form Russell-like bodies as a self-protection mechanism, when they are overloaded with a partially transport-incompetent protein, and we discuss the resulting challenges for recombinant protein production

General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species

<p>Abstract</p> <p>Background</p> <p>Downy mildew is a destructive grapevine disease caused by <it>Plasmopara viticola </it>(Berk. and Curt.) Berl. and de Toni, which can only be controlled by intensive fungicide treatments. Natural sources of resistance from wild grapevine (<it>Vitis</it>) species are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood.</p> <p>Results</p> <p>Early transcriptional changes associated with <it>P. viticola </it>infection in susceptible <it>V. vinifera </it>and resistant <it>V. riparia </it>plants were analyzed using the Combimatrix microarray platform. Transcript levels were measured 12 and 24 h post-inoculation, reflecting the time points immediately preceding the onset of resistance in <it>V. riparia</it>, as determined by microscopic analysis. Our data indicate that resistance in <it>V. riparia </it>is induced after infection, and is not based on differences in basal gene expression between the two species. The strong and rapid transcriptional reprogramming involves the induction of pathogenesis-related proteins and enzymes required for the synthesis of phenylpropanoid-derived compounds, many of which are also induced, albeit to a lesser extent, in <it>V. vinifera</it>. More interestingly, resistance in <it>V. riparia </it>also involves the specific modulation of numerous transcripts encoding components of signal transduction cascades, hypersensitive reaction markers and genes involved in jasmonate biosynthesis. The limited transcriptional modulation in <it>V. vinifera </it>represents a weak attempted defense response rather than the activation of compatibility-specific pathways.</p> <p>Conclusions</p> <p>Several candidate resistance genes were identified that could be exploited in future biotechnological approaches to increase disease resistance in susceptible grapevine species. Measurements of jasmonic acid and methyl jasmonate in infected leaves suggest that this hormone may also be involved in <it>V. riparia </it>resistance to <it>P. viticola</it>.</p

The CRISPR/Cas9 system for plant genome editing and beyond

Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently developed tool for the introduction of site-specific double-stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments