RESUMEN
Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ascomicetos/fisiología , Enfermedades de las Plantas/inmunología , Proteínas R-SNARE/metabolismo , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Resistencia a la Enfermedad , Genes Reporteros , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Transporte de Proteínas , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas Recombinantes de FusiónRESUMEN
Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus. Exogenous application of biologically active GA, GA(3), inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus, SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus.
Asunto(s)
Giberelinas/metabolismo , Lotus/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Secuencia de Aminoácidos , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Giberelinas/farmacología , Lotus/efectos de los fármacos , Lotus/genética , Datos de Secuencia Molecular , Mutación , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Nódulos de las Raíces de las Plantas/efectos de los fármacos , Nódulos de las Raíces de las Plantas/genética , Transducción de Señal , Triazoles/farmacologíaRESUMEN
In the model leguminous plant Lotus japonicus, the reception of a symbiotic signal called Nod factor (NF), which is secreted by the symbiont bacterium Mesorhizobium loti, induces wavy shaped root hairs. This is called root hair deformation. To dissect the root hair deformation process, we studied symbiosis- deficient mutants of L. japonicus, castor, nup85, ccamk and nsp2. The CASTOR, NUP85, and CCaMK genes are also required for mycorrhizal infection and thus called common symbiotic genes. On the global application of NF, all the mutants except nsp2 exhibited extensive branching of root hairs. The actin cytoskeleton was also observed as a marker for NF-dependent responses in mutant root hairs. At 2 hours of NF treatment, the ccamk mutant showed exaggerated swelling compared with the other mutants, indicating CCaMK to be required to terminate the swelling. In the nsp2 mutant, two hours of NF treatment remarkably induced swelling at root hair tips, although root hair deformation was not apparent at 24 hours of NF treatment. These results showed that common symbiotic components are involved in root hair deformation, which is regulated by a fine tuning mechanism early in the symbiosis between leguminous plants and rhizobia.
RESUMEN
The brush mutant of Lotus japonicus exhibits a temperature-dependent impairment in nodule, root, and shoot development. At 26 degrees C, brush formed fewer nodules, most of which were not colonized by rhizobia bacteria. Primary root growth was retarded and the anatomy of the brush root apical meristem revealed distorted cellular organization and reduced cell expansion. Reciprocal grafting of brush with wild-type plants indicated that this genotype only affected the root and that the shoot phenotype was a secondary effect. The root and nodulation phenotype cosegregated as a single Mendelian trait and the BRUSH gene could be mapped to the short arm of chromosome 2. At 18 degrees C, the brush root anatomy was rescued and similar to the wild type, and primary root length, number of infection threads, and nodule formation were partially rescued. Superficially, the brush root phenotype resembled the ethylene-related thick short root syndrome. However, treatment with ethylene inhibitor did not recover the observed phenotypes, although brush primary roots were slightly longer. The defects of brush in root architecture and infection thread development, together with intact nodule architecture and complete absence of symptoms from shoots, suggest that BRUSH affects cellular differentiation in a tissue-dependent way.