Genetic and transcriptomic analysis of the Bradyrhizobium T3SS-triggered nodulation in the legume Aeschynomene evenia.

Some Bradyrhizobium strains nodulate certain Aeschynomene species independently of Nod factors, but thanks to their type III secretion system (T3SS). While different T3 effectors triggering nodulation (ErnA and Sup3) have been identified, the plant signalling pathways they activate remain unknown. Here, we explored the intraspecies variability in T3SS-triggered nodulation within Aeschynomene evenia and investigated transcriptomic responses that occur during this symbiosis. Furthermore, Bradyrhizobium strains having different effector sets were tested on A. evenia mutants altered in various symbiotic signalling genes. We identified the A. evenia accession N21/PI 225551 as appropriate for deciphering the T3SS-dependent process. Comparative transcriptomic analysis of A. evenia N21 roots inoculated with ORS3257 strain and its ∆ernA mutant revealed genes differentially expressed, including some involved in plant defences and auxin signalling. In the other A. evenia accession N76, all tested strains nodulated the AeCRK mutant but not the AeNIN and AeNSP2 mutants, indicating a differential requirement of these genes for T3SS-dependent nodulation. Furthermore, the effects of AePOLLUX, AeCCaMK and AeCYCLOPS mutations differed between the strains. Notably, ORS86 nodulated these three mutant lines and required for this both ErnA and Sup3. Taken together, these results shed light on how the T3SS-dependent nodulation process is achieved in legumes.

Pathogenic nematodes exploit Achilles' heel of plant symbioses.

Cyst nematode parasites disrupt beneficial associations of crops with rhizobia and mycorrhiza. Chen et al. discovered the mechanism and demonstrated that the soybean cyst nematode Heterodera glycines secretes a chitinase that destroys key symbiotic signals from the microbial symbionts. The authors further developed a chitinase inhibitor that alleviates symbiosis inhibition.

Exploring the cellular surface polysaccharide and root nodule symbiosis characteristics of the rpoN mutants of Bradyrhizobium sp. DOA9 using synchrotron-based Fourier transform infrared microspectroscopy in conjunction with X-ray absorption spectroscopy.

The functional significance of rpoN genes that encode two sigma factors in the Bradyrhizobium sp. strain DOA9 has been reported to affect colony formation, root nodulation characteristics, and symbiotic interactions with Aeschynomene americana. rpoN mutant strains are defective in cellular surface polysaccharide (CSP) production compared with the wild-type (WT) strain, and they accordingly exhibit smaller colonies and diminished symbiotic effectiveness. To gain deeper insights into the changes in CSP composition and the nodules of rpoN mutants, we employed synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy. FTIR analysis of the CSP revealed the absence of specific components in the rpoN mutants, including lipids, carboxylic groups, polysaccharide-pyranose rings, and ß-galactopyranosyl residues. Nodules formed by DOA9WT exhibited a uniform distribution of lipids, proteins, and carbohydrates; mutant strains, particularly DOA9∆rpoNp:ΩrpoNc, exhibited decreased distribution uniformity and a lower concentration of C=O groups. Furthermore, Fe K-edge X-ray absorption near-edge structure and extended X-ray absorption fine structure analyses revealed deficiencies in the nitrogenase enzyme in the nodules of DOA9∆rpoNc and DOA9∆rpoNp:ΩrpoNc mutants; nodules from DOA9WT and DOA9∆rpoNp exhibited both leghemoglobin and the nitrogenase enzyme. IMPORTANCE This work provides valuable insights into how two rpoN genes affect the composition of cellular surface polysaccharides (CSPs) in Bradyrhizobium sp., which subsequently dictates root nodule chemical characteristics and nitrogenase production. We used advanced synchrotron methods, including synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy (XAS), for the first time in this field to analyze CSP components and reveal the biochemical changes occurring within nodules. These cutting-edge techniques confer significant advantages by providing detailed molecular information, enabling the identification of specific functional groups, chemical bonds, and biomolecule changes. This research not only contributes to our understanding of plant-microbe interactions but also establishes a foundation for future investigations and potential applications in this field. The combined use of the synchrotron-based FTIR and XAS techniques represents a significant advancement in facilitating a comprehensive exploration of bacterial CSPs and their implications in plant-microbe interactions.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Expanding Knowledge of Methylotrophic Capacity: Structure and Properties of the Rough-Type Lipopolysaccharide from Methylobacterium extorquens and Its Role on Membrane Resistance to Methanol.

The ability of Methylobacterium extorquens to grow on methanol as the sole carbon and energy source has been the object of intense research activity. Unquestionably, the bacterial cell envelope serves as a defensive barrier against such an environmental stressor, with a decisive role played by the membrane lipidome, which is crucial for stress resistance. However, the chemistry and the function of the main constituent of the M. extorquens outer membrane, the lipopolysaccharide (LPS), is still undefined. Here, we show that M. extorquens produces a rough-type LPS with an uncommon, non-phosphorylated, and extensively O-methylated core oligosaccharide, densely substituted with negatively charged residues in the inner region, including novel monosaccharide derivatives such as O-methylated Kdo/Ko units. Lipid A is composed of a non-phosphorylated trisaccharide backbone with a distinctive, low acylation pattern; indeed, the sugar skeleton was decorated with three acyl moieties and a secondary very long chain fatty acid, in turn substituted by a 3-O-acetyl-butyrate residue. Spectroscopic, conformational, and biophysical analyses on M. extorquens LPS highlighted how structural and tridimensional features impact the molecular organization of the outer membrane. Furthermore, these chemical features also impacted and improved membrane resistance in the presence of methanol, thus regulating membrane ordering and dynamics.

Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth.

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

A mutant-based analysis of the establishment of Nod-independent symbiosis in the legume Aeschynomene evenia.

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca2+/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.

The Versatile Roles of Type III Secretion Systems in Rhizobium-Legume Symbioses.

To suppress plant immunity and promote the intracellular infection required for fixing nitrogen for the benefit of their legume hosts, many rhizobia use type III secretion systems (T3SSs) that deliver effector proteins (T3Es) inside host cells. As reported for interactions between pathogens and host plants, the immune system of legume hosts and the cocktail of T3Es secreted by rhizobia determine the symbiotic outcome. If they remain undetected, T3Es may reduce plant immunity and thus promote infection of legumes by rhizobia. If one or more of the secreted T3Es are recognized by the cognate plant receptors, defense responses are triggered and rhizobial infection may abort. However, some rhizobial T3Es can also circumvent the need for nodulation (Nod) factors to trigger nodule formation. Here we review the multifaceted roles played by rhizobial T3Es during symbiotic interactions with legumes.

The Type III Effectome of the Symbiotic Bradyrhizobium vignae Strain ORS3257.

Many Bradyrhizobium strains are able to establish a Nod factor-independent symbiosis with the leguminous plant Aeschynomene indica by the use of a type III secretion system (T3SS). Recently, an important advance in the understanding of the molecular factors supporting this symbiosis has been achieved by the in silico identification and functional characterization of 27 putative T3SS effectors (T3Es) of Bradyrhizobium vignae ORS3257. In the present study, we experimentally extend this catalog of T3Es by using a multi-omics approach. Transcriptome analysis under non-inducing and inducing conditions in the ORS3257 wild-type strain and the ttsI mutant revealed that the expression of 18 out of the 27 putative effectors previously identified, is under the control of TtsI, the global transcriptional regulator of T3SS and T3Es. Quantitative shotgun proteome analysis of culture supernatant in the wild type and T3SS mutant strains confirmed that 15 of the previously determined candidate T3Es are secreted by the T3SS. Moreover, the combined approaches identified nine additional putative T3Es and one of them was experimentally validated as a novel effector. Our study underscores the power of combined proteome and transcriptome analyses to complement in silico predictions and produce nearly complete effector catalogs. The establishment of the ORS3257 effectome will form the basis for a full appraisal of the symbiotic properties of this strain during its interaction with various host legumes via different processes.