RESUMEN
Immunity cell-surface receptors Ve1 and Ve2 protect against fungi of the genus Verticillium causing early dying, a worldwide disease in many crops. Characterization of microbe-associated molecular pattern immunity receptors has advanced our understanding of disease resistance but signal amplification remains elusive. Here, we report that transgenic plants expressing Ve1 and Ve2 together, reduced pathogen titres by a further 90% compared to plants expressing only Ve1 or Ve2. Confocal and immunoprecipitation confirm that the two receptors associate to form heteromeric complexes in the absence of the ligand and positively regulate signaling. Bioassays show that the Ve1Ve2 complex activates race-specific amplified immunity to the pathogen through a rapid burst of reactive oxygen species (ROS). These results indicate a mechanism by which the composition of a cell-surface receptor heterocomplex may be optimized to increase immunity against devastating plant diseases.
Asunto(s)
Resistencia a la Enfermedad , Solanum lycopersicum , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/genética , Transducción de SeñalRESUMEN
Nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) regulate innate immunity by activating inflammatory responses in a variety of biological systems following the recognition of pathogen- or disease-associated molecular patterns. NLRs are characterized by a central nucleotide-binding and oligomerization (NACHT) domain found in P-loop NTPases. In this review, we detail the functional and structural properties of the NACHT domain of a subfamily of NLRs, the NLRPs (NLR containing a pyrin domain), based on previous studies, sequence analysis, homology modeling, and structure predictions. Several NLRPs have been found to regulate inflammatory responses through the assembly of oligomeric caspase 1-activating platforms known as inflammasomes, the 3-dimensional structure of the NLRP NACHT domain has still not been solved. Homology modeling suggests that sequence variability within the NACHT domains of different NLRP family members may alter the topology of the ATP-binding pocket. Based on this finding, we discuss the potential therapeutic prospects aligned with the NACHT domain and the development of selective inhibitors of inflammasome activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis/química , Inmunidad Innata , Inflamasomas/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Humanos , Inflamasomas/antagonistas & inhibidores , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Proteínas NLR , Nucleósido-Trifosfatasa/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Opium poppy (Papaver somniferum) remains the sole commercial source for several pharmaceutical alkaloids including the narcotic analgesics codeine and morphine, and the semi-synthetic drugs oxycodone, buprenorphine and naltrexone. Although most of the biosynthetic genes have been identified, the post-transcriptional regulation of the morphinan alkaloid pathway has not been determined. We have used virus-induced gene silencing (VIGS) as a functional genomics tool to investigate the regulation of morphine biosynthesis via a systematic reduction in enzyme levels responsible for the final six steps in the pathway. Specific gene silencing was confirmed at the transcript level by real-time quantitative PCR (polymerase chain reaction), and at the protein level by immunoblot analysis using antibodies raised against salutaridine synthase (SalSyn), salutaridine reductase (SalR), salutaridine 7-O-acetyltransferase (SalAT), thebaine 6-O-demethylase (T6ODM), codeinone reductase (COR), and codeine O-demethylase (CODM). In some cases, silencing a specific biosynthetic gene resulted in a predictable accumulation of the substrate for the corresponding enzyme. Reduced SalSyn, SalR, T6ODM and CODM protein levels correlated with lower morphine levels and a substantial increase in the accumulation of reticuline, salutaridine, thebaine and codeine, respectively. In contrast, the silencing of genes encoding SalAT and COR resulted in the accumulation of salutaridine and reticuline, respectively, which are not the corresponding enzymatic substrates. The silencing of alkaloid biosynthetic genes using VIGS confirms the physiological function of enzymes previously characterized in vitro, provides insight into the biochemical regulation of morphine biosynthesis, and demonstrates the immense potential for metabolic engineering in opium poppy.