RESUMO
Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.
Assuntos
Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Phaseolus/microbiologia , Rhizobium tropici/fisiologia , Proteínas de Bactérias/genética , Cobre/toxicidade , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mutagênese Insercional , Mutação , Phaseolus/efeitos dos fármacos , Phaseolus/crescimento & desenvolvimento , Phaseolus/metabolismo , Nodulação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Rhizobium tropici/genética , Rhizobium tropici/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , SimbioseRESUMO
The plant xylem is a preferred niche for some important bacterial phytopathogens, some of them encoding expansin proteins, which bind plant cell walls. Yet, the identity of the substrate for bacterial expansins within the plant cell wall and the nature of its interaction with it are poorly known. Here, we determined the localization of two bacterial expansins with differing isoelectric points (and with differing binding patterns to cell wall extracts) on plant tissue through in vitro fluorophore labeling and confocal imaging. Differential localization was observed, in which Exl1 from Pectobacterium carotovorum located into the intercellular spaces between xylem vessels and adjacent cells of the plant xylem, whereas EXLX1 from Bacillus subtilis bound cell walls of most cell types. In isolated vascular tissue, however, both PcExl1 and BsEXLX1 preferentially bound to tracheary elements over the xylem fibers, even though both are composed of secondary cell walls. Fluorescence correlation spectroscopy, employed to analyze the interaction of expansins with isolated xylem, indicates that binding is governed by more than one factor, which could include interaction with more than one type of polymer in the fibers, such as cellulose and hemicellulose or pectin. Binding to different polysaccharides could explain the observed reduction of cellulolytic and xylanolytic activities in the presence of expansin, possibly because of competition for the substrate. Our findings are relevant for the comprehensive understanding of the pathogenesis by P. carotovorum during xylem invasion, a process in which Exl1 might be involved.
RESUMO
Root hair curling is an early and essential morphological change required for the success of the symbiotic interaction between legumes and rhizobia. At this stage rhizobia grow as an infection thread within root hairs and are internalized into the plant cells by endocytosis, where the PI3K enzyme plays important roles. Previous observations show that stress conditions affect early stages of the symbiotic interaction, from 2 to 30 min post-inoculation, which we term as very early host responses, and affect symbiosis establishment. Herein, we demonstrated the relevance of the very early host responses for the symbiotic interaction. PI3K and the NADPH oxidase complex are found to have key roles in the microsymbiont recognition response, modulating the apoplastic and intracellular/endosomal ROS induction in root hairs. Interestingly, compared with soybean mutant plants that do not perceive the symbiont, we demonstrated that the very early symbiont perception under sublethal saline stress conditions induced root hair death. Together, these results highlight not only the importance of the very early host-responses on later stages of the symbiont interaction, but also suggest that they act as a mechanism for local control of nodulation capacity, prior to the abortion of the infection thread, preventing the allocation of resources/energy for nodule formation under unfavorable environmental conditions.
Assuntos
Bradyrhizobium/fisiologia , Glycine max/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas de Plantas/metabolismo , Nodulação , Simbiose , Interações Hospedeiro-Patógeno , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinase/genética , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Glycine max/genética , Glycine max/microbiologia , Glycine max/fisiologiaRESUMO
Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.
RESUMO
The symbiotic interaction of legumes and rhizobia results in the formation of nitrogen-fixing nodules. Nodulation depends on the finely coordinated expression of a battery of genes involved in the infection and the organogenesis processes. After Nod factor perception, symbiosis receptor kinase (SymRK) receptor triggers a signal transduction cascade essential for nodulation leading to cortical cell divisions, infection thread (IT) formation and final release of rhizobia to the intracellular space, forming the symbiosome. Herein, the participation of SymRK receptor during the nodule organogenesis in Phaseolus vulgaris is addressed. Our findings indicate that besides its expression in the nodule epidermis, in IT, and in uninfected cells of the infection zone, PvSymRK immunolocalizes in the root and nodule vascular system. On the other hand, knockdown expression of PvSymRK led to the formation of scarce and defective nodules, which presented alterations in both IT/symbiosome formation and vascular system.
Assuntos
Phaseolus/genética , Proteínas de Plantas/metabolismo , Nodulação , Proteínas Serina-Treonina Quinases/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Phaseolus/enzimologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Proteínas Serina-Treonina Quinases/genética , Nódulos Radiculares de Plantas/enzimologia , Transdução de Sinais , SimbioseRESUMO
Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar ß-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.