RESUMEN
Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.
Asunto(s)
Arabidopsis/inmunología , Membrana Celular/metabolismo , Inmunidad Innata , Fosfolipasa D/metabolismo , Enfermedades de las Plantas/inmunología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Ascomicetos/patogenicidad , Brefeldino A/inmunología , Brefeldino A/metabolismo , Quitina/inmunología , Quitina/metabolismo , Ciclopentanos/metabolismo , Exocitosis/efectos de los fármacos , Exocitosis/inmunología , Peróxido de Hidrógeno/metabolismo , Inmunidad Innata/efectos de los fármacos , Oxilipinas/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/genética , Enfermedades de las Plantas/microbiología , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de Señal/inmunología , Transducción de Señal/fisiologíaRESUMEN
Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.