RESUMO
Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.
Assuntos
Glucosiltransferases/genética , Lotus/genética , Proteínas de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Glucosiltransferases/metabolismo , Lotus/enzimologia , Lotus/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Raízes de Plantas/genéticaRESUMO
Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family. In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling. We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning. Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s).
Assuntos
Lotus , Medicago truncatula , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Lotus/genética , Lotus/metabolismo , Medicago truncatula/genética , Medicago truncatula/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Transdução de Sinais , SimbioseRESUMO
Phenomenon: Reproductive justice (RJ) is defined by women of color advocates as the right to have children, not have children and parent children while maintaining reproductive autonomy. In the United States, physicians have been complicit in multiple historical reproductive injustices, involving coercive sterilization of thousands of people of color, low income, and disabilities. Currently, reproductive injustices continue to occur; however, physicians have no formal RJ medical education to address injustices. The objective of this study was to engage leading advocates within the movement using a Delphi method to identify critical components for such a curriculum. Approach: In 2016, we invited 65 RJ advocates and leaders to participate in an expert panel to design RJ medical education. A 3-round Delphi survey was distributed electronically to identify content for inclusion in an RJ curriculum. In the next 2 survey rounds, experts offered feedback and revisions and rated agreement with including content recommendations in the final curriculum. We calculated descriptive statistics to analyze quantitative data. A team with educational expertise wrote learning outcomes based on expert content recommendations. Findings: Of the 65 RJ advocates and leaders invited, 41 participated on the expert panel of the Delphi survey. In the first survey, the expert panel recommended 58 RJ content areas through open-ended response. Over the next 2 rounds, there was consensus among the panel to include 52 of 58 of these areas in the curriculum. Recommended content fell into 11 broad domains: access, disparities, and structural competency; advocacy; approaches to reproductive healthcare; contemporary law and policy; cultural safety; historical injustices; lesbian, gay, bisexual, transgender, queer/questioning, and intersex health; oppression, power, and bias training; patient care; reproductive health; and RJ definitions. The 97 learning outcomes created from this process represented both unique and existing educational elements. Insights: A collaborative methodology infused with RJ values can bridge experts in advocacy and academics. New learning outcomes identified through this process can enhance medical education; however, it is just as important to consider education in RJ approaches to care as it is knowledge about that care. We must explore the pedagogic process of RJ medical education while considering that expertise in this area may exist outside of the medical community and thus there is a need to partner with RJ advocates. Finally, we expect to use innovative teaching methods to transform medical education and achieve an RJ focus.
Assuntos
Educação Médica , Reprodução , Justiça Social , Adulto , Técnica Delphi , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e Questionários , Estados Unidos , Adulto JovemRESUMO
During Lotus japonicus-Mesorhizobium loti symbiosis, the LOTUS HISTIDINE KINASE1 (LHK1) cytokinin receptor regulates both the initiation of nodule formation and the scope of root infection. However, the exact spatiotemporal mechanism by which this receptor exerts its symbiotic functions has remained elusive. In this study, we performed cell type-specific complementation experiments in the hyperinfected lhk1-1 mutant background, targeting LHK1 to either the root epidermis or the root cortex. We also utilized various genetic backgrounds to characterize expression of several genes regulating symbiotic infection. We show here that expression of LHK1 in the root cortex is required and sufficient to regulate both nodule formation and epidermal infections. The LHK1-dependent signalling that restricts subsequent infection events is triggered before initial cell divisions for nodule primordium formation. We also demonstrate that AHK4, the Arabidopsis orthologue of LHK1, is able to regulate M. loti infection in L. japonicus, suggesting that an endogenous cytokinin receptor could be sufficient for engineering nitrogen-fixing root nodule symbiosis in nonlegumes. Our data provide experimental evidence for the existence of an LHK1-dependent root cortex-to-epidermis feedback mechanism regulating rhizobial infection. This root-localized regulatory module functionally links with the systemic autoregulation of nodulation (AON) to maintain the homeostasis of symbiotic infection.
Assuntos
Citocininas/metabolismo , Lotus/metabolismo , Lotus/microbiologia , Mesorhizobium/fisiologia , Epiderme Vegetal/microbiologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/microbiologia , Receptores de Superfície Celular/metabolismo , Divisão Celular , Regulação da Expressão Gênica de Plantas , Lotus/genética , Modelos Biológicos , Nodulação , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/metabolismoRESUMO
Legume plants engage in intimate relationships with rhizobial bacteria to form nitrogen-fixing nodules, root-derived organs that accommodate the microsymbiont. Members of the Nuclear Factor Y (NF-Y) gene family, which have undergone significant expansion and functional diversification during plant evolution, are essential for this symbiotic liaison. Acting in a partially redundant manner, NF-Y proteins were shown, previously, to regulate bacterial infection, including selection of a superior rhizobial strain, and to mediate nodule structure formation. However, the exact mechanism by which these transcriptional factors exert their symbiotic functions has remained elusive. By carrying out detailed functional analyses of Lotus japonicus mutants, we demonstrate that LjNF-YA1 becomes indispensable downstream from the initial cortical cell divisions but prior to nodule differentiation, including cell enlargement and vascular bundle formation. Three affiliates of the SHORT INTERNODES/STYLISH transcription factor gene family, called STY1, STY2, and STY3, are demonstrated to be among likely direct targets of LjNF-YA1, and our results point to their involvement in nodule formation.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Lotus/genética , Rhizobium/fisiologia , Transcriptoma , Sequência de Aminoácidos , Fator de Ligação a CCAAT/genética , Diferenciação Celular , Mapeamento Cromossômico , Genes Reporter , Lotus/citologia , Lotus/microbiologia , Lotus/fisiologia , Mutação , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/fisiologia , Alinhamento de Sequência , Simbiose , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Leguminous plants selectively initiate primary responses to rhizobial nodulation factors (NF) that ultimately lead to symbiotic root nodule formation. Functioning downstream, cytokinin has emerged as the key endogenous plant signal for nodule differentiation, but its role in mediating rhizobial entry into the root remains obscure. Nonetheless, such a role is suggested by aberrant infection phenotypes of plant mutants with defects in cytokinin signaling. We postulate that cytokinin participates in orchestrating signaling events that promote rhizobial colonization of the root cortex and limit the extent of subsequent infection at the root epidermis, thus maintaining homeostasis of the symbiotic interaction. We further argue that cytokinin signaling must have been crucial during the evolution of plant cell predisposition for rhizobial colonization.
Assuntos
Citocininas/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Rhizobium/fisiologia , Etilenos/metabolismo , Transdução de Sinais , SimbioseRESUMO
A symbiotic mutant of Lotus japonicus, called sunergos1-1 (suner1-1), originated from a har1-1 suppressor screen. suner1-1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild-type nodule numbers are reached by suner1-1 at a later point after infection. Using an approach that combined map-based cloning and next-generation sequencing we have identified the causative mutation and show that the suner1-1 phenotype is determined by a weak recessive allele, with the corresponding wild-type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen-fixing nodules.
Assuntos
Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Lotus/enzimologia , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/metabolismo , Simbiose/fisiologia , Proteínas Arqueais/genética , DNA Topoisomerases Tipo II/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Nódulos Radiculares de Plantas/genética , Simbiose/genéticaRESUMO
Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.
Assuntos
Citocininas/metabolismo , Lotus/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Alelos , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Citocininas/farmacologia , Escherichia coli , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lotus/efeitos dos fármacos , Lotus/genética , Lotus/microbiologia , Mesorhizobium , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Mutação/genética , Organogênese/efeitos dos fármacos , Organogênese/genética , Filogenia , Proteínas de Plantas/química , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/química , Nódulos Radiculares de Plantas/efeitos dos fármacos , Nódulos Radiculares de Plantas/microbiologia , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nck-associated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.
Assuntos
Lotus/metabolismo , Mesorhizobium/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Loci Gênicos , Lotus/genética , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Mutação , Micorrizas/crescimento & desenvolvimento , Fenótipo , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Sementes/genética , Sementes/metabolismo , SimbioseRESUMO
One of the earliest responses of legumes to symbiotic signalling is oscillation of the calcium concentration in the nucleoplasm of root epidermal cells. Integration and decoding of the calcium-spiking signal involve a calcium- and calmodulin-dependent protein kinase (CCaMK) and its phosphorylation substrates, such as CYCLOPS. Here we describe the Lotus japonicus ccamk-14 mutant that originated from a har1-1 suppressor screen. The ccamk-14 mutation causes a serine to asparagine substitution at position 337 located within the calmodulin binding site, which we determined to be an in vitro phosphorylation site in CCaMK. We show that ccamk-14 exerts cell-specific effects on symbiosis. The mutant is characterized by an increased frequency of epidermal infections and significantly compromised cortical infections by Mesorhizobium loti and also the arbuscular mycorrhiza fungus Rhizophagus irregularis. The S337 residue is conserved across angiosperm CCaMKs, and testing discrete substitutions at this site showed that it participates in a negative regulation of CCaMK activity, which is required for the cell-type-specific integration of symbiotic signalling.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lotus/enzimologia , Simbiose , Alelos , Substituição de Aminoácidos , Asparagina/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Mapeamento Cromossômico , Ativação Enzimática , Lotus/genética , Lotus/microbiologia , Mesorhizobium/crescimento & desenvolvimento , Mutagênese Sítio-Dirigida , Mutação , Micorrizas/crescimento & desenvolvimento , Fenótipo , Fosforilação , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Raízes de Plantas/microbiologia , Serina/metabolismoRESUMO
Morlin (7-ethoxy-4-methyl chromen-2-one) was discovered in a screen of 20,000 compounds for small molecules that cause altered cell morphology resulting in swollen root phenotype in Arabidopsis. Live-cell imaging of fluorescently labeled cellulose synthase (CESA) and microtubules showed that morlin acts on the cortical microtubules and alters the movement of CESA. Morlin caused a novel syndrome of cytoskeletal defects, characterized by cortical array reorientation and compromised rates of both microtubule elongation and shrinking. Formation of shorter and more bundled microtubules and detachment from the cell membrane resulted when GFP::MAP4-MBP was used to visualize microtubules during morlin treatment. Cytoskeletal effects were accompanied by a reduction in the velocity and redistribution of CESA complexes labeled with YFP::CESA6 at the cell cortex. Morlin caused no inhibition of mouse myoblast, bacterial or fungal cell proliferation at concentrations that inhibit plant cell growth. By contrast, morlin stimulated microtubule disassembly in cultured hippocampal neurons but had no significant effect on cell viability. Thus, morlin appears to be a useful new probe of the mechanisms that regulate microtubule cortical array organization and its functional interaction with CESA.
Assuntos
Arabidopsis/citologia , Cumarínicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/metabolismo , Microtúbulos/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Cumarínicos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Proteínas de Fluorescência Verde/metabolismo , Cinética , Microscopia Confocal , Microscopia de Vídeo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Modelos Químicos , Estrutura MolecularRESUMO
Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.