RESUMEN
Understanding the molecular basis of plant responses to pathogen-associated molecular patterns (PAMPs) is an active area of research in the field of plant-microbe interactions. A growing number of plant genes involved in various steps of PAMP-triggered immunity (PTI) pathways and microbial factors involved in the elicitation or suppression of PTI have been identified. These studies have largely relied on Arabidopsis thaliana and, therefore, most of the PTI assays have been developed and optimized for that model plant system. Although PTI is a conserved feature among plants, the response spectra vary across different species. Thus, there is a need for robust PTI assays in other pathosystems, such as those involving Solanaceae plant-pathogen interactions, which include many economically important plants and their diseases. We have optimized molecular, cellular, and whole-plant methods to measure PTI responses in two widely studied solanaceous species, tomato (Solanum lycopersicum) and Nicotiana benthamiana. Here, we provide detailed protocols for measuring various PTI-associated phenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition, activation of mitogen-activated protein kinases, and a luciferase-based reporter system. These methods will facilitate limited genetic screens and detailed characterization of potential PTI-related genes in model and economically important Solanaceae spp.-pathogen interactions.
Asunto(s)
Nicotiana/inmunología , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Arabidopsis/inmunología , Arabidopsis/microbiología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/patología , Pared Celular/microbiología , Interacciones Huésped-Patógeno , Inmunidad Innata , Enfermedades de las Plantas/inmunología , Hojas de la Planta/microbiología , Protoplastos/microbiología , Nicotiana/microbiologíaRESUMEN
*The Pseudomonas syringae pv. tomato type III effector protein AvrPto has two functional domains that contribute additively to its ability to promote pathogen virulence in susceptible tomato plants and also defense responses in resistant tomato and tobacco genotypes. Here, we test the hypothesis that key amino acid residues in these two domains will be conserved even in sequence-divergent AvrPto proteins expressed by diverse P. syringae pathovars. *We cloned avrPto homologs from diverse P. syringae pathovars and characterized the four most diverse homologs from P. syringae pathovars mori, lachrymans, myricae and oryzae for their virulence activity and ability to elicit resistance in tomato and tobacco. *Key residues within the two AvrPto domains are conserved in three of the four homologs and are required for virulence activity and defense elicitation. AvrPto(oryzae), lacks conserved residues in each domain, but was found to be recognized by a previously unknown resistance gene in both tomato and tobacco. *Our results indicate that the two virulence domains of AvrPto are conserved in diverse pathovars despite the fact these domains are recognized by certain plant species. AvrPto may therefore function in pathovars infecting diverse plant species by targeting conserved host processes.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Genes de Plantas , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Pseudomonas syringae/patogenicidad , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Clonación Molecular , Genotipo , Interacciones Huésped-Patógeno/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Alineación de Secuencia , Nicotiana/genética , Nicotiana/microbiologíaRESUMEN
The type III effector protein AvrPto from Pseudomonas syringae pv. tomato is secreted into plant cells where it promotes bacterial growth and enhances symptoms of speck disease on susceptible tomato plants. The virulence activity of AvrPto is due, in part, to its interaction with components of host pattern recognition receptor complexes, which disrupts pathogen-associated molecular pattern-triggered immunity. This disruption mechanism requires a structural element of the AvrPto protein, the CD loop, which is also required for triggering Pto/Prf-mediated resistance in tomato. We have shown previously that the carboxyl-terminal domain (CTD) of AvrPto is phosphorylated and also contributes to bacterial virulence. Here we report that phosphorylation of the CTD on S147 and S149 promotes bacterial virulence in an FLS2/BAK1-independent manner, which is mechanistically distinct from the CD loop. In a striking corollary with Pto recognition of the CD loop in tomato, the tobacco species Nicotiana sylvestris and Nicotiana tabacum have a recognition mechanism that specifically detects the phosphorylation status of the CTD. Thus different species in the Solanaceae family have evolved distinct recognition mechanisms to monitor the same type III effector.
Asunto(s)
Proteínas Bacterianas/fisiología , Nicotiana/inmunología , Nicotiana/microbiología , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidad , Virulencia/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Mutagénesis Sitio-Dirigida , Fosforilación , Estructura Terciaria de Proteína , Virulencia/genéticaRESUMEN
CD1 proteins constitute a distinct lineage of antigen-presenting molecules specialized for the presentation of lipid antigens to T cells. In contrast to the extensive sequence polymorphism characteristic of classical MHC molecules, CD1 proteins exhibit limited sequence diversity. Here, we describe the identification and characterization of CD1d alleles in wild-derived mouse strains. We demonstrate that polymorphisms in CD1d affect the presentation of endogenous and exogenous ligands to CD1d-restricted T cells, including type I (Valpha14i) and type II (non-Valpha14i) natural killer T (NKT) cells. Using congenic mice, we found CD1d polymorphisms affect the thymic selection of type I NKT cells and induce allogeneic T cell responses. Collectively, results from these studies demonstrate a role for polymorphisms in influencing the development and function of CD1d-restricted T cells.