RESUMO
Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.
Assuntos
Regulação da Expressão Gênica de Plantas , Nitratos , Fixação de Nitrogênio , Nódulos Radiculares de Plantas , Fatores de Transcrição , Zinco , Zinco/metabolismo , Fatores de Transcrição/metabolismo , Nitratos/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Nitrogênio/metabolismo , Medicago truncatula/metabolismo , Medicago truncatula/genética , Simbiose , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
Soil acidity is a global issue; soils with pH <4.5 are widespread in Europe. This acidity adversely affects nutrient availability to plants; pH levels <5.0 lead to aluminum (Al3+) toxicity, a significant problem that hinders root growth and nutrient uptake in faba bean (Vicia faba L.) and its symbiotic relationship with Rhizobium. However, little is known about the specific traits and tolerant genotypes among the European faba beans. This study aimed to identify response traits associated with tolerance to root zone acidity and Al3+ toxicity and potentially tolerant genotypes for future breeding efforts. Germplasm survey was conducted using 165 genotypes in a greenhouse aquaponics system. Data on the root and shoot systems were collected. Subsequently, 12 genotypes were selected for further phenotyping in peat medium, where data on physiological and morphological parameters were recorded along with biochemical responses in four selected genotypes. In the germplasm survey, about 30% of genotypes showed tolerance to acidity and approximately 10% exhibited tolerance to Al3+, while 7% showed tolerance to both. The phenotyping experiment indicated diverse morphological and physiological responses among treatments and genotypes. Acid and Al3+ increased proline concentration. Interaction between genotype and environment was observed for ascorbate peroxidase activity, malondialdehyde, and proline concentrations. Genomic markers associated with acidity and acid+Al3+-toxicity tolerances were identified using GWAS analysis. Four faba bean genotypes with varying levels of tolerance to acidity and Al3+ toxicity were identified.
Assuntos
Alumínio , Genótipo , Fenótipo , Vicia faba , Vicia faba/genética , Vicia faba/efeitos dos fármacos , Vicia faba/crescimento & desenvolvimento , Vicia faba/metabolismo , Alumínio/toxicidade , Solo/química , Concentração de Íons de Hidrogênio , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Prolina/metabolismo , Adaptação Fisiológica/genética , Adaptação Fisiológica/efeitos dos fármacos , Ácidos/metabolismoRESUMO
While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.
Assuntos
Rhizobium leguminosarum , Rhizobium , Trifolium , Trifolium/genética , Medicago/genética , Rhizobium leguminosarum/genética , Simbiose/genética , PlantasRESUMO
Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil-dwelling bacteria and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, in which the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity quantitative trait loci (QTL) to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for symbiont-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Assuntos
Bradyrhizobium , Lotus , Mesorhizobium , Rhizobium , Lotus/genética , Lotus/microbiologia , Rhizobium/genética , Mesorhizobium/genética , Bradyrhizobium/genética , NitrogênioRESUMO
Homologous recombination is expected to increase natural selection efficacy by decoupling the fate of beneficial and deleterious mutations and by readily creating new combinations of beneficial alleles. Here, we investigate how the proportion of amino acid substitutions fixed by adaptive evolution (α) depends on the recombination rate in bacteria. We analyze 3,086 core protein-coding sequences from 196 genomes belonging to five closely related species of the genus Rhizobium. These genes are found in all species and do not display any signs of introgression between species. We estimate α using the site frequency spectrum (SFS) and divergence data for all pairs of species. We evaluate the impact of recombination within each species by dividing genes into three equally sized recombination classes based on their average level of intragenic linkage disequilibrium. We find that α varies from 0.07 to 0.39 across species and is positively correlated with the level of recombination. This is both due to a higher estimated rate of adaptive evolution and a lower estimated rate of nonadaptive evolution, suggesting that recombination both increases the fixation probability of advantageous variants and decreases the probability of fixation of deleterious variants. Our results demonstrate that homologous recombination facilitates adaptive evolution measured by α in the core genome of prokaryote species in agreement with studies in eukaryotes.
Assuntos
Recombinação Genética , Rhizobium , Evolução Molecular , Mutação , Rhizobium/genética , Seleção Genética , SoloRESUMO
Competitive and facilitative interactions influence bacterial community composition, diversity and functioning. However, the role of genetic diversity for determining interactions between coexisting strains of the same, or closely related, species remains poorly understood. Here, we investigated the type (facilitative/inhibitory) and potential underlying mechanisms of pairwise interactions between 24 genetically diverse bacterial strains belonging to three genospecies (gsA,C,E) of the Rhizobium leguminosarum species complex. Interactions were determined indirectly, based on secreted compounds in cell-free supernatants, and directly, as growth inhibition in cocultures. We found supernatants mediated both facilitative and inhibitory interactions that varied greatly between strains and genospecies. Overall, gsE strains indirectly suppressed growth of gsA strains, while their own growth was facilitated by other genospecies' supernatants. Similar genospecies-level patterns were observed in direct competition, where gsA showed the highest susceptibility and gsE the highest inhibition capacity. At the genetic level, increased gsA susceptibility was associated with a non-random distribution of quorum sensing and secondary metabolite genes across genospecies. Together, our results suggest that genetic variation is associated with facilitative and competitive interactions, which could be important ecological mechanisms explaining R. leguminosarum diversity.
Assuntos
Rhizobium leguminosarum , Rhizobium , DNA Bacteriano/genética , Variação Genética , Rhizobium/genética , Rhizobium leguminosarum/genéticaRESUMO
KEY MESSAGE: Accurate genomic prediction of yield within and across generations was achieved by estimating the genetic merit of individual white clover genotypes based on extensive genetic replication using cloned material. White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover genotypes and 170 rhizobium inocula. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.
Assuntos
Genes de Plantas , Fixação de Nitrogênio , Rhizobium leguminosarum/fisiologia , Trifolium/genética , Variação Genética , Genótipo , Modelos Genéticos , Desenvolvimento Vegetal/genética , Rhizobium leguminosarum/classificação , Rhizobium leguminosarum/isolamento & purificação , Trifolium/crescimento & desenvolvimento , Trifolium/metabolismo , Trifolium/microbiologiaRESUMO
Phosphate represents a major limiting factor for plant productivity. Plants have evolved different solutions to adapt to phosphate limitation ranging from a profound tuning of their root system architecture and metabolic profile to the evolution of widespread mutualistic interactions. Here we elucidated plant responses and their genetic basis to different phosphate levels in a plant species that is widely used as a model for AM symbiosis: Lotus japonicus. Rather than focussing on a single model strain, we measured root growth and anion content in response to different levels of phosphate in 130 Lotus natural accessions. This allowed us not only to uncover common as well as divergent responses within this species, but also enabled Genome Wide Association Studies by which we identified new genes regulating phosphate homeostasis in Lotus. Among them, we showed that insertional mutants of a cytochrome B5 reductase and a Leucine-Rich-Repeat receptor showed different phosphate concentration in plants grown under phosphate sufficient condition. Under low phosphate conditions, we found a correlation between plant biomass and the decrease of plant phosphate concentration in plant tissues, representing a dilution effect. Altogether our data of the genetic and phenotypic variation within a species capable of AM complements studies that have been conducted in Arabidopsis, and advances our understanding of the continuum of genotype by phosphate level interaction existing throughout dicot plants.
Assuntos
Estudo de Associação Genômica Ampla/métodos , Lotus/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/genética , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Citocromo-B(5) Redutase/genética , Regulação da Expressão Gênica de Plantas , Lotus/genética , Mutação , Proteínas Quinases/genética , Nódulos Radiculares de Plantas/genéticaRESUMO
Forward and reverse genetics using the model legumes Lotus japonicus and Medicago truncatula have been instrumental in identifying the essential genes governing legume-rhizobia symbiosis. However, little information is known about the effects of intraspecific variation on symbiotic signalling. Here, we use quantitative trait locus sequencing (QTL-seq) to investigate the genetic basis of the differentiated phenotypic responses shown by the Lotus accessions Gifu and MG20 to inoculation with the Mesorhizobium loti exoU mutant that produces truncated exopolysaccharides. We identified through genetic complementation the Pxy gene as a component of this differential exoU response. Lotus Pxy encodes a leucine-rich repeat receptor-like kinase similar to Arabidopsis thaliana PXY, which regulates stem vascular development. We show that Lotus pxy insertion mutants displayed defects in root and stem vascular organisation, as well as lateral root and nodule formation. Our work links Pxy to de novo organogenesis in the root, highlights the genetic overlap between regulation of lateral root and nodule formation, and demonstrates that natural variation in Pxy affects nodulation signalling.
Assuntos
Lotus , Mesorhizobium , Regulação da Expressão Gênica de Plantas , Lotus/genética , Lotus/metabolismo , Mesorhizobium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/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
During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 (Rhizobial Infection Receptor-like Kinase1), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception (NIN), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection.
Assuntos
Lotus/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/microbiologiaRESUMO
The NOOT-BOP-COCH-LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE-ON-PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen-fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference-mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild-type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.
Assuntos
Flores/crescimento & desenvolvimento , Lotus/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Meristema/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimentoRESUMO
Establishment of symbiotic nitrogen-fixation in legumes is regulated by the plant hormone ethylene, but it has remained unclear whether and how its biosynthesis is regulated by the symbiotic pathway. We established a sensitive ethylene detection system for Lotus japonicus and found that ethylene production increased as early as 6 hours after inoculation with Mesorhizobium loti This ethylene response was dependent on Nod factor production by compatible rhizobia. Analyses of nodulation mutants showed that perception of Nod factor was required for ethylene emission, while downstream transcription factors including CYCLOPS, NIN, and ERN1 were not required for this response. Activation of the nodulation signaling pathway in spontaneously nodulating mutants was also sufficient to elevate ethylene production. Ethylene signaling is controlled by EIN2, which is duplicated in L. japonicus We obtained a L. japonicus Ljein2a Ljein2b double mutant that exhibits complete ethylene insensitivity and confirms that these two genes act redundantly in ethylene signaling. Consistent with this redundancy, both LjEin2a and LjEin2b are required for negative regulation of nodulation and Ljein2a Ljein2b double mutants are hypernodulating and hyperinfected. We also identified an unexpected role for ethylene in the onset of nitrogen fixation, with the Ljein2a Ljein2b double mutant showing severely reduced nitrogen fixation. These results demonstrate that ethylene production is an early and sustained nodulation response that acts at multiple stages to regulate infection, nodule organogenesis, and nitrogen fixation in L. japonicus.
Assuntos
Etilenos/análise , Lotus/fisiologia , Mesorhizobium/fisiologia , Fixação de Nitrogênio , Reguladores de Crescimento de Plantas/análise , Transdução de Sinais , Etilenos/metabolismo , Lotus/microbiologia , Mutação , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rhizobium/fisiologia , Plântula/microbiologia , Plântula/fisiologia , Simbiose , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.
Assuntos
Lotus/genética , Proteínas de Plantas/metabolismo , Retroelementos/genética , Sequências Repetidas Terminais/genética , Metilação de DNA/genética , Mutagênese Insercional , Mutação/genética , Proteínas de Plantas/genéticaRESUMO
Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.
Assuntos
Lotus/microbiologia , Polissacarídeos Bacterianos/metabolismo , Sinorhizobium fredii/fisiologia , Simbiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Especificidade de Hospedeiro , Lotus/citologia , Lotus/fisiologia , Mutação , Fenótipo , Nodulação , Raízes de Plantas/citologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Polissacarídeos Bacterianos/química , Purinas/metabolismo , Pirimidinas/metabolismo , Sinorhizobium fredii/citologia , Sinorhizobium fredii/genéticaRESUMO
The classic phytohormones cytokinin and auxin play essential roles in the maintenance of stem-cell systems embedded in shoot and root meristems, and exhibit complex functional interactions. Here we show that the activity of both hormones directly converges on the promoters of two A-type ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, ARR7 and ARR15, which are negative regulators of cytokinin signalling and have important meristematic functions. Whereas ARR7 and ARR15 expression in the shoot apical meristem (SAM) is induced by cytokinin, auxin has a negative effect, which is, at least in part, mediated by the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP) transcription factor. Our results provide a mechanistic framework for hormonal control of the apical stem-cell niche and demonstrate how root and shoot stem-cell systems differ in their response to phytohormones.
Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Brotos de Planta/citologia , Nicho de Células-Tronco/citologia , Nicho de Células-Tronco/metabolismo , Células-Tronco/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citocininas/metabolismo , Citocininas/farmacologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Meristema/citologia , Meristema/efeitos dos fármacos , Meristema/genética , Meristema/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Legumes interact with rhizobial bacteria to form nitrogen-fixing root nodules. Host signalling following mutual recognition ensures a specific response, but is only partially understood. Focusing on the stage of epidermal infection with Mesorhizobium loti, we analysed endogenous small RNAs (sRNAs) of the model legume Lotus japonicus to investigate their involvement in host response regulation. We used Illumina sequencing to annotate the L. japonicus sRNA-ome and isolate infection-responsive sRNAs, followed by candidate-based functional characterization. Sequences from four libraries revealed 219 novel L. japonicus micro RNAs (miRNAs) from 114 newly assigned families, and 76 infection-responsive sRNAs. Unlike infection-associated coding genes such as NODULE INCEPTION (NIN), a micro RNA 172 (miR172) isoform showed strong accumulation in dependency of both Nodulation (Nod) factor and compatible rhizobia. The genetics of miR172 induction support the existence of distinct epidermal and cortical signalling events. MIR172a promoter activity followed a previously unseen pattern preceding infection thread progression in epidermal and cortical cells. Nodule-associated miR172a expression was infection-independent, representing the second of two genetically separable activity waves. The combined data provide a valuable resource for further study, and identify miR172 as an sRNA marking successful epidermal infection. We show that miR172 acts upstream of several APETALA2-type (AP2) transcription factors, and suggest that it has a role in fine-tuning AP2 levels during bacterial symbiosis.
Assuntos
Regulação da Expressão Gênica de Plantas , Lotus/genética , Mesorhizobium/crescimento & desenvolvimento , MicroRNAs/metabolismo , Epiderme Vegetal/microbiologia , Nódulos Radiculares de Plantas/microbiologia , Simbiose , Endófitos/crescimento & desenvolvimento , Genes de Plantas , Lotus/metabolismo , Lotus/microbiologia , Fenótipo , Epiderme Vegetal/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Regiões Promotoras Genéticas , Rhizobium , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Legume symbiosis with rhizobia results in the formation of a specialized organ, the root nodule, where atmospheric dinitrogen is reduced to ammonia. In Lotus japonicus (Lotus), several genes involved in nodule development or nodule function have been defined using biochemistry, genetic approaches, and high-throughput transcriptomics. We have employed proteomics to further understand nodule development. Two developmental stages representing nodules prior to nitrogen fixation (white) and mature nitrogen fixing nodules (red) were compared with roots. In addition, the proteome of a spontaneous nodule formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7%. Interestingly, an indication of more pronounced PTMs in nodules than in roots was determined. Between the two nodule developmental stages, higher levels of pathogen-related 10 proteins, HSPs, and proteins involved in redox processes were found in white nodules, suggesting a higher stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous presence of several nitrilases, ascorbate metabolic enzymes, and putative rhizobial effectors.
Assuntos
Lotus/fisiologia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Raízes de Plantas/fisiologia , Nódulos Radiculares de Plantas/fisiologia , Regulação da Expressão Gênica de Plantas , Lotus/química , Lotus/genética , Lotus/microbiologia , Mutação , Fixação de Nitrogênio , Proteínas de Plantas/genética , Raízes de Plantas/química , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Proteoma/análise , Proteoma/genética , Proteoma/metabolismo , Proteômica , Nódulos Radiculares de Plantas/química , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Transdução de Sinais , SimbioseRESUMO
Plant-microbe symbioses require intense interaction and genetic coordination to successfully establish in specific cell types of the host and symbiont. Traditional RNA-seq methodologies lack the cellular resolution to fully capture these complexities, but single-cell and spatial transcriptomics (ST) are now allowing scientists to probe symbiotic interactions at an unprecedented level of detail. Here, we discuss the advantages that novel spatial and single-cell transcriptomic technologies provide in studying plant-microbe endosymbioses and highlight key recent studies. Finally, we consider the remaining limitations of applying these approaches to symbiosis research, which are mainly related to the simultaneous capture of both plant and microbial transcripts within the same cells.
RESUMO
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.