Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation.

Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.

From Intracellular Bacteria to Differentiated Bacteroids: Transcriptome and Metabolome Analysis in Aeschynomene Nodules Using the Bradyrhizobium sp. Strain ORS285 bclA Mutant.

Soil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells, where they differentiate into nitrogen-fixing bacteroids. Several legume lineages force their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process in Bradyrhizobium sp. strain ORS285, a symbiont of Aeschynomene spp. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells, but they cannot differentiate into enlarged polyploid and functional bacteroids. Thus, the bclA nodule bacteria constitute an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied by a first transcriptome switch involving several hundred upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving fewer genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a nonsymbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process, fueling the biogeochemical nitrogen cycle with reduced nitrogen. It also represents a promising strategy to reduce the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs, where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis of Bradyrhizobium wild-type and bclA mutant bacteria in culture and in symbiosis with Aeschynomene host plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.

Comparative cytology, physiology and transcriptomics of Burkholderia insecticola in symbiosis with the bean bug Riptortus pedestris and in culture.

In the symbiosis of the bean bug Riptortus pedestris with Burkholderia insecticola, the bacteria occupy an exclusive niche in the insect midgut and favor insect development and reproduction. In order to understand how the symbiotic bacteria stably colonize the midgut crypts and which services they provide to the host, we compared the cytology, physiology, and transcriptomics of free-living and midgut-colonizing B. insecticola. The analyses revealed that midgut-colonizing bacteria were smaller in size and had lower DNA content, they had increased stress sensitivity, lost motility, and an altered cell surface. Transcriptomics revealed what kinds of nutrients are provided by the bean bug to the Burkholderia symbiont. Transporters and metabolic pathways of diverse sugars such as rhamnose and ribose, and sulfur compounds like sulfate and taurine were upregulated in the midgut-colonizing symbionts. Moreover, pathways enabling the assimilation of insect nitrogen wastes, i.e. allantoin and urea, were also upregulated. The data further suggested that the midgut-colonizing symbionts produced all essential amino acids and B vitamins, some of which are scarce in the soybean food of the host insect. Together, these findings suggest that the Burkholderia symbiont is fed with specific nutrients and also recycles host metabolic wastes in the insect gut, and in return, the bacterial symbiont provides the host with essential nutrients limited in the insect food, contributing to the rapid growth and enhanced reproduction of the bean bug host.

Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components.

Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.

Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis.

Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward Escherichia coli and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis in vitro and in vivo Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on E. coli ribosomes but a poor inhibitor on Thermus thermophilus ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.

Analysis of DNAs associated with coconut foliar decay disease implicates a unique single-stranded DNA virus representing a new taxon.

The unique ecology, pathology and undefined taxonomy of coconut foliar decay virus (CFDV), found associated with coconut foliar decay disease (CFD) in 1986, prompted analyses of old virus samples by modern methods. Rolling circle amplification and deep sequencing applied to nucleic acid extracts from virion preparations and CFD-affected palms identified twelve distinct circular DNAs, eleven of which had a size of about 1.3 kb and one of 641 nt. Mass spectrometry-based protein identification proved that a 24 kDa protein encoded by two 1.3 kb DNAs is the virus capsid protein with highest sequence similarity to that of grabloviruses (family Geminiviridae), even though CFDV particles are not geminate. The nine other 1.3 kb DNAs represent alphasatellites coding for replication initiator proteins that differ clearly from those encoded by nanovirid DNA-R. The 641 nt DNA-gamma is unique and may encode a movement protein. Three DNAs, alphasatellite CFDAR, capsid protein encoding CFDV DNA-S.1 and DNA-gamma share sequence motifs near their replication origins and were consistently present in all samples analysed. These DNAs appear to be integral components of a possibly tripartite CFDV genome, different from those of any Geminiviridae or Nanoviridae family member, implicating CFDV as representative of a new genus and family.

The complete genome sequence of Ensifer meliloti strain CCMM B554 (FSM-MA), a highly effective nitrogen-fixing microsymbiont of Medicago truncatula Gaertn.

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA.

Integrated roles of BclA and DD-carboxypeptidase 1 in Bradyrhizobium differentiation within NCR-producing and NCR-lacking root nodules.

Legumes harbor in their symbiotic nodule organs nitrogen fixing rhizobium bacteria called bacteroids. Some legumes produce Nodule-specific Cysteine-Rich (NCR) peptides in the nodule cells to control the intracellular bacterial population. NCR peptides have antimicrobial activity and drive bacteroids toward terminal differentiation. Other legumes do not produce NCR peptides and their bacteroids are not differentiated. Bradyrhizobia, infecting NCR-producing Aeschynomene plants, require the peptide uptake transporter BclA to cope with the NCR peptides as well as a specific peptidoglycan-modifying DD-carboxypeptidase, DD-CPase1. We show that Bradyrhizobium diazoefficiens strain USDA110 forms undifferentiated bacteroids in NCR-lacking soybean nodules. Unexpectedly, in Aeschynomene afraspera nodules the nitrogen fixing USDA110 bacteroids are hardly differentiated despite the fact that this host produces NCR peptides, suggesting that USDA110 is insensitive to the host peptide effectors and that nitrogen fixation can be uncoupled from differentiation. In agreement with the absence of bacteroid differentiation, USDA110 does not require its bclA gene for nitrogen fixing symbiosis with these two host plants. Furthermore, we show that the BclA and DD-CPase1 act independently in the NCR-induced morphological differentiation of bacteroids. Our results suggest that BclA is required to protect the rhizobia against the NCR stress but not to induce the terminal differentiation pathway.

Specific Host-Responsive Associations Between Medicago truncatula Accessions and Sinorhizobium Strains.

Legume plants interact with rhizobia to form nitrogen-fixing root nodules. Legume-rhizobium interactions are specific and only compatible rhizobia and plant species will lead to nodule formation. Even within compatible interactions, the genotype of both the plant and the bacterial symbiont will impact on the efficiency of nodule functioning and nitrogen-fixation activity. The model legume Medicago truncatula forms nodules with several species of the Sinorhizobium genus. However, the efficiency of these bacterial strains is highly variable. In this study, we compared the symbiotic efficiency of Sinorhizobium meliloti strains Sm1021, 102F34, and FSM-MA, and Sinorhizobium medicae strain WSM419 on the two widely used M. truncatula accessions A17 and R108. The efficiency of the interactions was determined by multiple parameters. We found a high effectiveness of the FSM-MA strain with both M. truncatula accessions. In contrast, specific highly efficient interactions were obtained for the A17-WSM419 and R108-102F34 combinations. Remarkably, the widely used Sm1021 strain performed weakly on both hosts. We showed that Sm1021 efficiently induced nodule organogenesis but cannot fully activate the differentiation of the symbiotic nodule cells, explaining its weaker performance. These results will be informative for the selection of appropriate rhizobium strains in functional studies on symbiosis using these M. truncatula accessions, particularly for research focusing on late stages of the nodulation process.

Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides.

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

Bradyrhizobium BclA Is a Peptide Transporter Required for Bacterial Differentiation in Symbiosis with Aeschynomene Legumes.

Nodules of legume plants are highly integrated symbiotic systems shaped by millions of years of evolution. They harbor nitrogen-fixing rhizobium bacteria called bacteroids. Several legume species produce peptides called nodule-specific cysteine-rich (NCR) peptides in the symbiotic nodule cells which house the bacteroids. NCR peptides are related to antimicrobial peptides of innate immunity. They induce the endosymbionts into a differentiated, enlarged, and polyploid state. The bacterial symbionts, on their side, evolved functions for the response to the NCR peptides. Here, we identified the bclA gene of Bradyrhizobium sp. strains ORS278 and ORS285, which is required for the formation of differentiated and functional bacteroids in the nodules of the NCR peptide-producing Aeschynomene legumes. The BclA ABC transporter promotes the import of NCR peptides and provides protection against the antimicrobial activity of these peptides. Moreover, BclA can complement the role of the related BacA transporter of Sinorhizobium meliloti, which has a similar symbiotic function in the interaction with Medicago legumes.

Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida.

Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar.