This study aimed at defining the role of a basic helix—loop—helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root ...nodules produced upon infection by rhizobia bacteria. We used MtbHLH1 promoter::GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics. We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor. Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots.
A plant–microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont ...Sinorhizobium meliloti.
Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts.
Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor.
The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.
Cellulose is the world’s most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. ...Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold—the “cortical belt”—which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing β- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.
A Gram-negative, strictly aerobic bacterial strain was isolated from asymptomatic leaf tissue of a wild yam plant. Optimal growth was observed at 28 °C and pH 7, and catalase and oxidase activities ...were detected. Polyphasic taxonomic and comparative genomics revealed that strain LMG 33091 T represents a novel species of Pseudomonas . The nearest phylogenetic neighbours of strain LMG 33091 T were Pseudomonas putida NBRC 14164 T (with 99.79 % 16S rRNA sequence identity), Pseudomonas alkylphenolica KL28 T (99.28 %) and Pseudomonas asplenii (99.07 %) ATCC 23835 T . MALDI-TOF MS analysis yielded distinct profiles for strain LMG 33091 T and the nearest phylogenetic neighbours. Average nucleotide identity analyses between the whole genome sequence of strain LMG 33091 T and of the type strains of its nearest-neighbour taxa yielded values below the species delineation threshold and thus confirmed that the strain represented a novel Pseudomonas species, for which we propose the name Pseudomonas fortuita sp. nov., with strain LMG 33091 T (=GMI12077 T = CFBP 9143 T ) as the type strain.
Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. ...Although bacterial cellulose (BC) was first described in the 19
century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing β- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.
Summary
Next‐generation sequencing technologies allow an almost exhaustive survey of the transcriptome, even in species with no available genome sequence. To produce a Unigene set representing most ...of the expressed genes of pea, 20 cDNA libraries produced from various plant tissues harvested at various developmental stages from plants grown under contrasting nitrogen conditions were sequenced. Around one billion reads and 100 Gb of sequence were de novo assembled. Following several steps of redundancy reduction, 46 099 contigs with N50 length of 1667 nt were identified. These constitute the ‘Caméor’ Unigene set. The high depth of sequencing allowed identification of rare transcripts and detected expression for approximately 80% of contigs in each library. The Unigene set is now available online (http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi), allowing (i) searches for pea orthologs of candidate genes based on gene sequences from other species, or based on annotation, (ii) determination of transcript expression patterns using various metrics, (iii) identification of uncharacterized genes with interesting patterns of expression, and (iv) comparison of gene ontology pathways between tissues. This resource has allowed identification of the pea orthologs of major nodulation genes characterized in recent years in model species, as a major step towards deciphering unresolved pea nodulation phenotypes. In addition to a remarkable conservation of the early transcriptome nodulation apparatus between pea and Medicago truncatula, some specific features were highlighted. The resource provides a reference for the pea exome, and will facilitate transcriptome and proteome approaches as well as SNP discovery in pea.
Significance Statement
From Mendel's discovery of the laws of genetics up to the advent of molecular biology, pea has been a valuable model for genetics and physiology. We present a comprehensive inventory of the expressed genes of pea in a readily searchable format. This resource strengthens pea as a model species and will facilitate searches for candidate gene sequences, microarray design, large‐scale proteomics studies, and identification of major genes in available mutant populations.
Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. ...Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (ERF) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the RESPONSE REGULATOR 4 (MtRR4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a nonlegume homolog of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralog MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with downregulation of a set of genes, including notably NUCLEAR FACTOR-YA1 (MtNF-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We, therefore, conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.
Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of ...genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies
, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.
Mechanisms regulating legume root nodule development are still poorly understood, and very few regulatory genes have been cloned and characterized. Here, we describe EFD (for ethylene response factor ...required for nodule differentiation), a gene that is upregulated during nodulation in Medicago truncatula. The EFD transcription factor belongs to the ethylene response factor (ERF) group V, which contains ERN1, 2, and 3, three ERFs involved in Nod factor signaling. The role of EFD in the regulation of nodulation was examined through the characterization of a null deletion mutant (efd-1), RNA interference, and overexpression studies. These studies revealed that EFD is a negative regulator of root nodulation and infection by Rhizobium and that EFD is required for the formation of functional nitrogen-fixing nodules. EFD appears to be involved in the plant and bacteroid differentiation processes taking place beneath the nodule meristem. We also showed that EFD activated Mt RR4, a cytokinin primary response gene that encodes a type-A response regulator. We propose that EFD induction of Mt RR4 leads to the inhibition of cytokinin signaling, with two consequences: the suppression of new nodule initiation and the activation of differentiation as cells leave the nodule meristem. Our work thus reveals a key regulator linking early and late stages of nodulation and suggests that the regulation of the cytokinin pathway is important both for nodule initiation and development.
LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a ...putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box-dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant-microbe interactions and development through interacting with receptor-like kinases is discussed.
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