Tissue-specific regulation of lipid polyester synthesis genes controlling oxygen permeation into Lotus japonicus nodules.

Legumes establish endosymbiotic associations with nitrogen-fixing rhizobia, which they host inside root nodules. Here, specific physiological and morphological adaptations, such as the production of oxygen-binding leghemoglobin proteins and the formation of an oxygen diffusion barrier in the nodule periphery, are essential to protect the oxygen-labile bacterial nitrogenase enzyme. The molecular basis of the latter process remains elusive as the identification of required genes is limited by the epistatic effect of nodule organogenesis over nodule infection and rhizobia accommodation. We overcame this by exploring the phenotypic diversity of Lotus japonicus accessions that uncouple nodule organogenesis from nodule infection when inoculated with a subcompatible Rhizobium strain. Using comparative transcriptomics, we identified genes with functions associated with oxygen homeostasis and deposition of lipid polyesters on cell walls to be specifically up-regulated in infected compared to noninfected nodules. As hydrophobic modification of cell walls is pivotal for creating diffusion barriers like the root endodermis, we focused on two Fatty acyl-CoA Reductase genes that were specifically activated in the root and/or in the nodule endodermis. Mutant lines in a Fatty acyl-CoA Reductase gene expressed exclusively in the nodule endodermis had decreased deposition of polyesters on this cell layer and increased nodule permeability compared to wild-type plants. Oxygen concentrations were significantly increased in the inner cortex of mutant nodules, which correlated with reduced nitrogenase activity, and impaired shoot growth. These results provide the first genetic evidence for the formation of the nodule oxygen diffusion barrier, a key adaptation enabling nitrogen fixation in legume nodules.

A Promiscuity Locus Confers Lotus burttii Nodulation with Rhizobia from Five Different Genera.

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.

Microbiome profiling reveals that Pseudomonas antagonises parasitic nodule colonisation of cheater rhizobia in Lotus.

Nodule microbiota are dominated by symbiotic nitrogen-fixing rhizobia, however, other non-rhizobial bacteria also colonise this niche. Although many of these bacteria harbour plant-growth-promoting functions, it is not clear whether these less abundant nodule colonisers impact root-nodule symbiosis. We assessed the relationship between the nodule microbiome and nodulation as influenced by the soil microbiome, by using a metabarcoding approach to characterise the communities inside nodules of healthy and starved Lotus species. A machine learning algorithm and network analyses were used to identify nodule bacteria of interest, which were re-inoculated onto plants in controlled conditions to observe their potential functionality. The nodule microbiome of all tested species differed according to inoculum, but only that of Lotus burttii varied with plant health. Amplicon sequence variants representative of Pseudomonas species were the most indicative non-rhizobial signatures inside healthy L. burttii nodules and negatively correlated with Rhizobium sequences. A representative Pseudomonas isolate co-colonised nodules infected with a beneficial Mesorhizobium, but not with an ineffective Rhizobium isolate and another even reduced the number of ineffective nodules induced on Lotus japonicus. Our results show that nodule endophytes influence the overall outcome of the root-nodule symbiosis, albeit in a plant host-specific manner.

Bradyrhizobium hipponense sp. nov., isolated from Lupinus angustifolius growing in the northern region of Tunisia.

Strain aSej3T was isolated from a root nodule of a Lupinus angustifolius plant growing in Bizerte, Tunisia. 16S rRNA gene analysis placed this strain within the genus Bradyrhizobium. Multilocus sequence analysis (MLSA) including three housekeeping genes (glnII, gyrB and recA) grouped aSej3T together with Bradyrhizobium rifense CTAW71T, Bradyrhizobium cytisi CTAW11T, Bradyrhizobium ganzhouense RITF806T, Bradyrhizobium lupini USDA 3051T and Bradyrhizobium canariense BTA-1T. MLSA with five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed that this strain shares less than 93.5 % nucleotide identity with other type strains. Genome sequencing and inspection revealed a genome size of 8.83 Mbp with a G+C content of 62.8 mol%. Genome-wide average nucleotide identity and digital DNA-DNA hybridization values were below 87.5 and 36.2 %, respectively, when compared to described Bradyrhizobium species. Strain aSej3T nodulated L. angustifolius plants under axenic conditions and its nodC gene clustered within the genistearum symbiovar. Altogether, the phylogenetic data and the chemotaxonomic characteristics of this strain support that aSej3T represents a new species for which we propose the name Bradyrhizobium hipponense sp. nov. with the type strain aSej3T (=DSM 108913T=LMG 31020T).

Mesorhizobium norvegicum sp. nov., a rhizobium isolated from a Lotus corniculatus root nodule in Norway.

Strain 10.2.2T was isolated from a root nodule of a Lotus corniculatus plant growing near Skammestein (Norway). Phenotypic and chemotaxonomic characterization revealed that colonies grown on yeast-mannitol broth agar were circular, convex and slimy. Growth occurred at 28 °C in 0-1 % NaCl and in a pH range from above 4 to 10. Cells were resistant to kanamycin and phosphomycin. They could assimilate carbon sources such as l-lysine, d-mannose, d-mannitol, and l-alanine. Major fatty acids found in the organism were 11-methyl C18  :  1ω7c, C16  :  0, C18  :  1ω7c, C18  :  0 and C19  :  0 cyclo ω8c. Genome sequencing and characterization of the genome revealed its size to be 8.27 Mbp with a G+C content of 62.4 mol%. Phylogenetic analyses based on the 16S rRNA gene and housekeeping gene alignments placed this strain within the genus Mesorhizobium. Pairwise genome-wide average nucleotide identity values supported that strain 10.2.2T represents a new species, for which we propose the name Mesorhizobium norvegicum sp. nov. with the type strain 10.2.2T (=DSM 108834T=LMG 31153T).

Draft Genome Sequence of Phyllobacterium endophythicum mTS5, Isolated from Lupinus micranthus in Tunisia.

We report here the draft genome sequence of Phyllobacterium endophyticum mTS5, isolated from a Lupinus micranthus root nodule. The genome consists of 5,454,168 bp, with a GC content of 57%, and contains 5,676 protein-coding sequences.

A subcompatible rhizobium strain reveals infection duality in Lotus.

Lotus species develop infection threads to guide rhizobia into nodule cells. However, there is evidence that some species have a genetic repertoire to allow other modes of infection. By conducting confocal and electron microscopy, quantification of marker gene expression, and phenotypic analysis of transgenic roots infected with mutant rhizobia, we elucidated the infection mechanism used by Rhizobium leguminosarum Norway to colonize Lotus burttii. Rhizobium leguminosarum Norway induces a distinct host transcriptional response compared with Mesorhizobium loti. It infects L. burttii utilizing an epidermal and transcellular infection thread-independent mechanism at high frequency. The entry into plant cells occurs directly from the apoplast and is primarily mediated by 'peg'-like structures, the formation of which is dependent on the production of Nod factor by the rhizobia. These results demonstrate that Lotus species can exhibit duality in their infection mechanisms depending on the rhizobial strain that they encounter. This is especially relevant in the context of interactions in the rhizosphere where legumes do not encounter single strains, but complex rhizobial communities. Additionally, our findings support a perception mechanism at the nodule cell entry interface, reinforcing the idea that there are successive checkpoints during rhizobial infection.

Complete genome of Rhizobium leguminosarum Norway, an ineffective Lotus micro-symbiont.

Rhizobia bacteria engage in nitrogen-fixing root nodule symbiosis, a mutualistic interaction with legume plants in which a bidirectional nutrient exchange takes place. Occasionally, this interaction is suboptimal resulting in the formation of ineffective nodules in which little or no atmospheric nitrogen fixation occurs. Rhizobium leguminosarum Norway induces ineffective nodules in a wide range of Lotus hosts. To investigate the basis of this phenotype, we sequenced the complete genome of Rl Norway and compared it to the genome of the closely related strain R. leguminosarum bv. viciae 3841. The genome comprises 7,788,085 bp, distributed on a circular chromosome containing 63% of the genomic information and five large circular plasmids. The functionally classified bacterial gene set is distributed evenly among all replicons. All symbiotic genes (nod, fix, nif) are located on the pRLN3 plasmid. Whole genome comparisons revealed differences in the metabolic repertoire and in protein secretion systems, but not in classical symbiotic genes.

Symbiotic root infections in Medicago truncatula require remorin-mediated receptor stabilization in membrane nanodomains.

Plant cell infection is tightly controlled by cell surface receptor-like kinases (RLKs). Like other RLKs, the Medicago truncatula entry receptor LYK3 laterally segregates into membrane nanodomains in a stimulus-dependent manner. Although nanodomain localization arises as a generic feature of plant membrane proteins, the molecular mechanisms underlying such dynamic transitions and their functional relevance have remained poorly understood. Here we demonstrate that actin and the flotillin protein FLOT4 form the primary and indispensable core of a specific nanodomain. Infection-dependent induction of the remorin protein and secondary molecular scaffold SYMREM1 results in subsequent recruitment of ligand-activated LYK3 and its stabilization within these membrane subcompartments. Reciprocally, the majority of this LYK3 receptor pool is destabilized at the plasma membrane and undergoes rapid endocytosis in symrem1 mutants on rhizobial inoculation, resulting in premature abortion of host cell infections. These data reveal that receptor recruitment into nanodomains is indispensable for their function during host cell infection.

S-acylation anchors remorin proteins to the plasma membrane but does not primarily determine their localization in membrane microdomains.

Remorins are well-established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post-translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S-acylation on defined residues in these proteins. S-acylation of cysteine residues in a C-terminal hydrophobic core contributes to membrane association of most remorin proteins. While S-acylation patterns differ between members of this multi-gene family, initial membrane association is mediated by protein-protein or protein-lipid interactions. However, S-acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane-resident proteins may be involved in the recruitment of remorins into membrane domains. S-acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S-acylation is a reversible post-translational modification, stimulus-dependent intracellular trafficking of these proteins can be envisioned.

Intrinsic disorder in pathogen effectors: protein flexibility as an evolutionary hallmark in a molecular arms race.

Effector proteins represent a refined mechanism of bacterial pathogens to overcome plants' innate immune systems. These modular proteins often manipulate host physiology by directly interfering with immune signaling of plant cells. Even if host cells have developed efficient strategies to perceive the presence of pathogenic microbes and to recognize intracellular effector activity, it remains an open question why only few effectors are recognized directly by plant resistance proteins. Based on in-silico genome-wide surveys and a reevaluation of published structural data, we estimated that bacterial effectors of phytopathogens are highly enriched in long-disordered regions (>50 residues). These structurally flexible segments have no secondary structure under physiological conditions but can fold in a stimulus-dependent manner (e.g., during protein-protein interactions). The high abundance of intrinsic disorder in effectors strongly suggests positive evolutionary selection of this structural feature and highlights the dynamic nature of these proteins. We postulate that such structural flexibility may be essential for (1) effector translocation, (2) evasion of the innate immune system, and (3) host function mimicry. The study of these dynamical regions will greatly complement current structural approaches to understand the molecular mechanisms of these proteins and may help in the prediction of new effectors.

Microcin e492 amyloid formation is retarded by posttranslational modification.

Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.

The intrinsically disordered N-terminal region of AtREM1.3 remorin protein mediates protein-protein interactions.

The longstanding structure-function paradigm, which states that a protein only serves a biological function in a structured state, had to be substantially revised with the description of intrinsic disorder in proteins. Intrinsically disordered regions that undergo a stimulus-dependent disorder-to-order transition are common to a large number of signaling proteins. However, little is known about the functionality of intrinsically disordered regions in plant proteins. Here we investigated intrinsic disorder in a plant-specific remorin protein that has been described as a signaling component in plant-microbe interactions. Using bioinformatic, biochemical, and biophysical approaches, we characterized the highly abundant remorin AtREM1.3, showing that its N-terminal region is intrinsically disordered. Although only the AtREM1.3 C-terminal domain is essential for stable homo-oligomerization, the N-terminal region facilitates this interaction. Furthermore, we confirmed the stable interaction between AtREM1.3 and four isoforms of the importin α protein family in a yeast two-hybrid system and by an in planta bimolecular fluorescent complementation assay. Phosphorylation of Ser-66 in the intrinsically disordered N-terminal region decreases the interaction strength with the importin α proteins. Hence, the N-terminal region may constitute a regulatory domain, stabilizing these interactions.

Phosphorylation of intrinsically disordered regions in remorin proteins.

Plant-specific remorin proteins reside in subdomains of plasma membranes, originally termed membrane rafts. They probably facilitate cellular signal transduction by direct interaction with signaling proteins such as receptor-like kinases and may dynamically modulate their lateral segregation within plasma membranes. Recent evidence suggests such functions of remorins during plant-microbe interactions and innate immune responses, where differential phosphorylation of some of these proteins has been described to be dependent on the perception of the microbe-associated molecular pattern (MAMP) flg22 and the presence of the NBS-LRR resistance protein RPM1. A number of specifically phosphorylated residues in their highly variable and intrinsically disordered N-terminal regions have been identified. Sequence diversity of these evolutionary distinct domains suggests that remorins may serve a wide range of biological functions. Here, we describe patterns and features of intrinsic disorder in remorin protein and discuss possible functional implications of phosphorylation within these rapidly evolving domains.

Degradation of 2,3-dihydroxybenzoate by a novel meta-cleavage pathway.

2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via meta-cleavage of the aromatic ring. The dhb cluster of Pseudomonas reinekei MT1 encodes a chimeric meta-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-p-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of P. reinekei MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the dhB gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-p-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate.

Modified 3-oxoadipate pathway for the biodegradation of methylaromatics in Pseudomonas reinekei MT1.

Catechols are central intermediates in the metabolism of aromatic compounds. Degradation of 4-methylcatechol via intradiol cleavage usually leads to the formation of 4-methylmuconolactone (4-ML) as a dead-end metabolite. Only a few microorganisms are known to mineralize 4-ML. The mml gene cluster of Pseudomonas reinekei MT1, which encodes enzymes involved in the metabolism of 4-ML, is shown here to encode 10 genes found in a 9.4-kb chromosomal region. Reverse transcription assays revealed that these genes form a single operon, where their expression is controlled by two promoters. Promoter fusion assays identified 4-methyl-3-oxoadipate as an inducer. Mineralization of 4-ML is initiated by the 4-methylmuconolactone methylisomerase encoded by mmlI. This reaction produces 3-ML and is followed by a rearrangement of the double bond catalyzed by the methylmuconolactone isomerase encoded by mmlJ. Deletion of mmlL, encoding a protein of the metallo-beta-lactamase superfamily, resulted in a loss of the capability of the strain MT1 to open the lactone ring, suggesting its function as a 4-methyl-3-oxoadipate enol-lactone hydrolase. Further metabolism can be assumed to occur by analogy with reactions known from the 3-oxoadipate pathway. mmlF and mmlG probably encode a 4-methyl-3-oxoadipyl-coenzyme A (CoA) transferase, and the mmlC gene product functions as a thiolase, transforming 4-methyl-3-oxoadipyl-CoA into methylsuccinyl-CoA and acetyl-CoA, as indicated by the accumulation of 4-methyl-3-oxoadipate in the respective deletion mutant. Accumulation of methylsuccinate by an mmlK deletion mutant indicates that the encoded acetyl-CoA hydrolase/transferase is crucial for channeling methylsuccinate into the central metabolism.

Novel metal-binding site of Pseudomonas reinekei MT1 trans-dienelactone hydrolase.

Pseudomonasreinekei MT1 is capable of growing on 4- and 5-chlorosalicylate as the sole carbon source involving a pathway with trans-dienelactone hydrolase as the key enzyme. This enzyme transforms 4-chloromuconolactone to maleylacetate and thereby avoids the spontaneous formation of toxic protoanemonin. trans-Dienelactone hydrolase is a Zn(2+)-dependent hydrolase where activity can be modulated by the exchange of Zn(2+) by Mn(2+) in at least two of the three metal-binding sites. Site directed variants of conserved residues of the Q(101)XXXQ(105)XD(107)XXXH(111) motif and of H281 and E294 exhibit a two order of magnitude decrease in activity and a strong decrease in metal-binding capability. As none of the variants exhibited a change in secondary structure, the analyzed amino acid residues can be assumed to be involved in metal binding, forming a novel trinuclear metal-binding motif.

Crystal structure and catalytic mechanism of 4-methylmuconolactone methylisomerase.

When methyl-substituted aromatic compounds are degraded via ortho (intradiol)-cleavage of 4-methylcatechol, the dead-end metabolite 4-methylmuconolactone (4-ML) is formed. Degradation of 4-ML has only been described in few bacterial species, including Pseudomonas reinekei MT1. The isomerization of 4-ML to 3-methylmuconolactone (3-ML) is the first step required for the mineralization of 4-ML and is catalyzed by an enzyme termed 4-methylmuconolactone methylisomerase (MLMI). We identified the gene encoding MLMI in P. reinekei MT1 and solved the crystal structures of MLMI in complex with 3-ML at 1.4-A resolution, with 4-ML at 1.9-A resolution and with a MES buffer molecule at 1.45-A resolution. MLMI exhibits a ferredoxin-like fold and assembles as a tight functional homodimeric complex. We were able to assign the active site clefts of MLMI from P. reinekei MT1 and of the homologous MLMI from Cupriavidus necator JMP134, which has previously been crystallized in a structural genomics project. Kinetic and structural analysis of wild-type MLMI and variants created by site-directed mutagenesis indicate Tyr-39 and His-26 to be the most probable catalytic residues. The previously proposed involvement of Cys-67 in covalent catalysis can now be excluded. Residue His-52 was found to be important for substrate affinity, with only marginal effect on catalytic activity. Based on these results, a novel catalytic mechanism for the isomerization of 4-ML to 3-ML by MLMI, involving a bislactonic intermediate, is proposed. This broadens the knowledge about the diverse group of proteins exhibiting a ferredoxin-like fold.

trans-Dienelactone hydrolase from Pseudomonas reinekei MT1, a novel zinc-dependent hydrolase.

Pseudomonas reinekei MT1 is capable of growing on 4- and 5-chlorosalicylate, involving a pathway with trans-dienelactone hydrolase (trans-DLH) as a key enzyme. It acts on 4-chloromuconolactone formed during cycloisomerization of 3-chloromuconate by hydrolyzing it to maleylacetate. The gene encoding this activity was localized, sequenced and expressed in Escherichia coli. Inductively coupled plasma mass spectrometry showed that both the wild-type as well as recombinant enzymes contained 2 moles of zinc but variable amounts of manganese/mol of protein subunit. The inactive metal-free apoenzyme could be reactivated by Zn(2+) or Mn(2+). Thus, trans-DLH is a Zn(2+)-dependent hydrolase using halosubstituted muconolactones and trans-dienelactone as substrates, where Mn(2+) can substitute for Zn(2+). It is the first member of COG1878 and PF04199 for which a direct physiological function has been reported.