The genotype of barley cultivars influences multiple aspects of their associated microbiota via differential root exudate secretion.

Plant-associated microbes play vital roles in promoting plant growth and health, with plants secreting root exudates into the rhizosphere to attract beneficial microbes. Exudate composition defines the nature of microbial recruitment, with different plant species attracting distinct microbiota to enable optimal adaptation to the soil environment. To more closely examine the relationship between plant genotype and microbial recruitment, we analysed the rhizosphere microbiomes of landrace (Chevallier) and modern (NFC Tipple) barley (Hordeum vulgare) cultivars. Distinct differences were observed between the plant-associated microbiomes of the 2 cultivars, with the plant-growth promoting rhizobacterial genus Pseudomonas substantially more abundant in the Tipple rhizosphere. Striking differences were also observed between the phenotypes of recruited Pseudomonas populations, alongside distinct genotypic clustering by cultivar. Cultivar-driven Pseudomonas selection was driven by root exudate composition, with the greater abundance of hexose sugars secreted from Tipple roots attracting microbes better adapted to growth on these metabolites and vice versa. Cultivar-driven selection also operates at the molecular level, with both gene expression and the abundance of ecologically relevant loci differing between Tipple and Chevallier Pseudomonas isolates. Finally, cultivar-driven selection is important for plant health, with both cultivars showing a distinct preference for microbes selected by their genetic siblings in rhizosphere transplantation assays.

Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans.

Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH3) assimilation by glutamine synthetase (GS), preventing excess release of excess NH3 for plants. Diazotrophic bacteria can be engineered to excrete NH3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target plants. Here, we tested two strategies to control GS regulation and NH3 excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both PII homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH3 derived from N2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH3 excretion specifically under microaerobic conditions, the same cue that initiates N2 fixation, then deleted nifA and transferred a rhizopine nifAL94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N2 fixation and NH3 excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses" where N2 fixation and NH3 excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.

Nuclear magnetic resonance diffraction with subangstrom precision.

We have combined ultrasensitive force-based spin detection with high-fidelity spin control to achieve NMR diffraction (NMRd) measurement of ~2 million [Formula: see text]P spins in a [Formula: see text] volume of an indium-phosphide (InP) nanowire. NMRd is a technique originally proposed for studying the structure of periodic arrangements of spins, with complete access to the spectroscopic capabilities of NMR. We describe two experiments that realize NMRd detection with subangstrom precision. In the first experiment, we encode a nanometer-scale spatial modulation of the z-axis magnetization of [Formula: see text]P spins and detect the period and position of the modulation with a precision of <0.8 Å. In the second experiment, we demonstrate an interferometric technique, utilizing NMRd, to detect an angstrom-scale displacement of the InP sample with a precision of 0.07 Å. The diffraction-based techniques developed in this work extend the Fourier-encoding capabilities of NMR to the angstrom scale and demonstrate the potential of NMRd as a tool for probing the structure and dynamics of nanocrystalline materials.

Engineered plant control of associative nitrogen fixation.

Engineering N2-fixing symbioses between cereals and diazotrophic bacteria represents a promising strategy to sustainably deliver biologically fixed nitrogen (N) in agriculture. We previously developed novel transkingdom signaling between plants and bacteria, through plant production of the bacterial signal rhizopine, allowing control of bacterial gene expression in association with the plant. Here, we have developed both a homozygous rhizopine producing (RhiP) barley line and a hybrid rhizopine uptake system that conveys upon our model bacterium Azorhizobium caulinodans ORS571 (Ac) 103-fold improved sensitivity for rhizopine perception. Using this improved genetic circuitry, we established tight rhizopine-dependent transcriptional control of the nitrogenase master regulator nifA and the N metabolism σ-factor rpoN, which drove nitrogenase expression and activity in vitro and in situ by bacteria colonizing RhiP barley roots. Although in situ nitrogenase activity was suboptimally effective relative to the wild-type strain, activation was specific to RhiP barley and was not observed on the roots of wild-type plants. This work represents a key milestone toward the development of a synthetic plant-controlled symbiosis in which the bacteria fix N2 only when in contact with the desired host plant and are prevented from interaction with nontarget plant species.

The motility and chemosensory systems of Rhizobium leguminosarum, their role in symbiosis, and link to PTSNtr regulation.

Motility and chemotaxis are crucial processes for soil bacteria and plant-microbe interactions. This applies to the symbiotic bacterium Rhizobium leguminosarum, where motility is driven by flagella rotation controlled by two chemotaxis systems, Che1 and Che2. The Che1 cluster is particularly important in free-living motility prior to the establishment of the symbiosis, with a che1 mutant delayed in nodulation and reduced in nodulation competitiveness. The Che2 system alters bacteroid development and nodule maturation. In this work, we also identified 27 putative chemoreceptors encoded in the R. leguminosarum bv. viciae 3841 genome and characterized its motility in different growth conditions. We describe a metabolism-based taxis system in rhizobia that acts at high concentrations of dicarboxylates to halt motility independent of chemotaxis. Finally, we show how PTSNtr influences cell motility, with PTSNtr mutants exhibiting reduced swimming in different media. Motility is restored by the active forms of the PTSNtr output regulatory proteins, unphosphorylated ManX and phosphorylated PtsN. Overall, this work shows how rhizobia typify soil bacteria by having a high number of chemoreceptors and highlights the importance of the motility and chemotaxis mechanisms in a free-living cell in the rhizosphere, and at different stages of the symbiosis.

Aspartate aminotransferase of Rhizobium leguminosarum has extended substrate specificity and metabolizes aspartate to enable N2 fixation in pea nodules.

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 fixation in pea nodules.

Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

Performance of quantum-dash mode-locked lasers (QD-MLLDs) for high-capacity coherent optical communications.

We investigate the capabilities and limitations of quantum-dash mode-locked lasers (QD-MLLDs) as optical frequency comb sources in coherent optical communication systems. We demonstrate that QD-MLLDs are on par with conventional single-wavelength narrow linewidth laser sources and can support high symbol rates and modulation formats. We manage to transmit 64 quadrature amplitude modulation (QAM) signals up to 80 GBd over 80 km of standard single-mode fiber (SSMF), which highlights the distinctive phase noise performance of the QD-MLLD. Using a 38.5 GHz (6 dB bandwidth) silicon photonic (SiP) modulator, we achieve a maximum symbol rate of 104 GBd with 16QAM signaling and a maximum net rate of 416 Gb/s per carrier in a single polarization setup and after 80 km-SSMF transmission. We also compare QD-MLLD performance with commercial narrow-linewidth integrable tunable laser assemblies (ITLAs) and explore their potential for use as local oscillators (LOs) and signal carriers. The QD-MLLD has 45 comb lines usable for transmission at a frequency spacing of 25 GHz, and an RF linewidth of 35 kHz.

Instantaneous frequency measurement system based on quantum dash mode-locked laser.

We present the theory and experimental results of a microwave photonic (MWP) filter based instantaneous frequency measurement system. A quantum dash mode-locked laser is used as an optical frequency comb source. With up to 41 flat comb lines and a real-time feedback loop for comb shaping, a set of MWP filters with linear frequency responses for either linear unit or dB unit are experimentally demonstrated. The maximum measurement frequency can be up to 20 GHz limited by the available test-and-measurement instruments. By using one MWP filter, the root-mean-square error is 51∼66 MHz, which can be improved to 42.2 MHz for linear unit, and 30.7 MHz for dB unit by using two MWP filters together.

Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis.

Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include fixNOQP, encoding the high-affinity cbb3-type terminal oxidase used in symbiosis. In free-living Rlv3841, the hFixL-FxkR-FixK pathway was active at 1% O2, and confocal microscopy showed hFixL-FxkR-FixK activity in the earliest stages of Rlv3841 differentiation in nodules (zones I and II). Work on Rlv3841 inside and outside nodules showed that the hFixL-FxkR-FixK pathway also induces transcription of fnrN at 1% O2 and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in fixNOQP expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III and IV of pea nodules. Quantification of fixNOQP expression in nodules showed this was driven primarily by FnrN, with minimal direct hFixL-FxkR-FixK induction. Thus, FnrN is key for full symbiotic expression of fixNOQP. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK pathway effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In zone III of mature nodules, near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required for wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O2 sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O2 concentration. Multi-sensor O2 regulation is prevalent in rhizobia, suggesting the fine-tuned control this enables is common and maximizes the effectiveness of the symbioses.

Conditional sanctioning in a legume-Rhizobium mutualism.

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nodule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later developmental stages, sanctioned nodules contained fewer viable bacteria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners.

Position-Controlled Telecom Single Photon Emitters Operating at Elevated Temperatures.

A key resource in quantum-secured communication protocols are single photon emitters. For long-haul optical networks, it is imperative to use photons at wavelengths compatible with telecom single mode fibers. We demonstrate high purity single photon emission at 1.31 µm using deterministically positioned InP photonic waveguide nanowires containing single InAsP quantum dot-in-a-rod structures. At excitation rates that saturate the emission, we obtain a single photon collection efficiency at first lens of 27.6% and a probability of multiphoton emission of g(2)(0) = 0.021. We have also evaluated the performance of the source as a function of temperature. Multiphoton emission probability increases with temperature with values of 0.11, 0.34, and 0.57 at 77, 220 and 300 K, respectively, which is attributed to an overlap of temperature-broadened excitonic emission lines. These results are a promising step toward scalably fabricating telecom single photon emitters that operate under relaxed cooling requirements.

Scalable Generation and Detection of on-Demand W States in Nanophotonic Circuits.

Quantum physics phenomena, entanglement and coherence, are crucial for quantum information protocols, but understanding these in systems with more than two parts is challenging due to increasing complexity. The W state, a multipartite entangled state, is notable for its robustness and benefits in quantum communication. Here, we generate eight-mode on-demand single-photon W states, using nanowire quantum dots and a silicon nitride photonic chip. We demonstrate a reliable and scalable technique for reconstructing the W state in photonic circuits using Fourier and real-space imaging, supported by the Gerchberg-Saxton phase retrieval algorithm. Additionally, we utilize an entanglement witness to distinguish between mixed and entangled states, thereby affirming the entangled nature of our generated state. The study provides a new imaging approach of assessing multipartite entanglement in W states, paving the way for further progress in image processing and Fourier-space analysis techniques for complex quantum systems.

Rhizopine biosensors for plant-dependent control of bacterial gene expression.

Engineering signalling between plants and microbes could be exploited to establish host-specificity between plant-growth-promoting bacteria and target crops in the environment. We previously engineered rhizopine-signalling circuitry facilitating exclusive signalling between rhizopine-producing (RhiP) plants and model bacterial strains. Here, we conduct an in-depth analysis of rhizopine-inducible expression in bacteria. We characterize two rhizopine-inducible promoters and explore the bacterial host-range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild-type parent strain. This work provides advancement towards establishing more robust plant-dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.

Photonic beamforming using a quantum-dash optical frequency comb source.

We demonstrate photonic beamforming using a quantum-dash (QD) optical frequency comb (OFC) source. Thanks to the 25 GHz free spectral range (FSR) and up to 40 comb lines available from the QD OFC, we can implement phased antenna arrays (PAAs) with directional radiation and scanning. We consider two types of PAAs: a uniform linear array (ULA) and a uniform planar array (UPA). By selecting different comb lines with a programmable optical filter, we can tune the FSR of the OFC source and realize a discrete scanning function. We evaluate the beam squint of the ULAs, and the results show that we can achieve broadband operation. Finally, we show that we can achieve both directional radiation and scanning simultaneously using the UPA.

Optimizing Rhizobium-legume symbioses by simultaneous measurement of rhizobial competitiveness and N2 fixation in nodules.

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N2) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N2 fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium-legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.

Global control of bacterial nitrogen and carbon metabolism by a PTSNtr-regulated switch.

The nitrogen-related phosphotransferase system (PTSNtr) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTSNtr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTSNtr is widely conserved in proteobacteria, highlighting its global importance.

Lifestyle adaptations of Rhizobium from rhizosphere to symbiosis.

By analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2 Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.

Nodulation and nitrogen fixation in Medicago truncatula strongly alters the abundance of its root microbiota and subtly affects its structure.

The plant common symbiosis signalling (SYM) pathway has shared function between interactions with rhizobia and arbuscular mycorrhizal fungi, the two most important symbiotic interactions between plants and microorganisms that are crucial in plant and agricultural yields. Here, we determine the role of the plant SYM pathway in the structure and abundance of the microbiota in the model legume Medicago truncatula and whether this is controlled by the nitrogen or phosphorus status of the plant. We show that SYM mutants (dmi3) differ substantially from the wild type (WT) in the absolute abundance of the root microbiota, especially under nitrogen limitation. Changes in the structure of the microbiota were less pronounced and depended on both plant genotype and nutrient status. Thus, the SYM pathway has a major impact on microbial abundance in M. truncatula and also subtly alters the composition of the microbiota.

Robust gene coexpression networks using signed distance correlation.

MOTIVATION: Even within well studied organisms, many genes lack useful functional annotations. One way to generate such functional information is to infer biological relationships between genes/proteins, using a network of gene coexpression data that includes functional annotations. However, the lack of trustworthy functional annotations can impede the validation of such networks. Hence, there is a need for a principled method to construct gene coexpression networks that capture biological information and are structurally stable even in the absence of functional information. RESULTS: We introduce the concept of signed distance correlation as a measure of dependency between two variables, and apply it to generate gene coexpression networks. Distance correlation offers a more intuitive approach to network construction than commonly used methods such as Pearson correlation and mutual information. We propose a framework to generate self-consistent networks using signed distance correlation purely from gene expression data, with no additional information. We analyse data from three different organisms to illustrate how networks generated with our method are more stable and capture more biological information compared to networks obtained from Pearson correlation or mutual information. SUPPLEMENTARY INFORMATION: Supplementary Information and code are available at Bioinformatics and https://github.com/javier-pardodiaz/sdcorGCN online.