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.

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis.IMPORTANCEBurkholderia pseudomallei, the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as B. pseudomallei and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of B. pseudomallei isolates.

Use of Reductive Amination to Produce Capsular Polysaccharide-Based Glycoconjugates.

Reductive amination is a relatively simple and convenient strategy for coupling purified polysaccharides to carrier proteins. Following their synthesis, glycoconjugates can be used to assess the protective capacity of specific microbial polysaccharides in animal models of infection and/or to produce polyclonal antiserum and monoclonal antibodies for a variety of immune assays. Here, we describe a reproducible method for chemically activating the 6-deoxyheptan capsular polysaccharide (CPS) from Burkholderia pseudomallei and covalently linking it to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce the glycoconjugate, CPS-CRM197. Similar approaches can also be used to couple other types of polysaccharides to CRM197 with little to no modification of the protocol.

Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

Alkaline surface treatment and time-resolved reading of mn-doped nanocrystal signal transducer for enhanced bioassay sensitivity.

Recently, Mn-doped semiconductor nanocrystals (NCs) with high brightness, long lifetimes, and low-energy excitation are emerging for time-resolved luminescence biosensing/imaging. Following our previous work on Mn-doped NCs, in this work we developed poly(styrene-co-maleic anhydride) (PSMA)-encapsulated Mn-doped AgZnInS/ZnS NCs as signal transducers for immunoassay of capsular polysaccharide (CPS), a surface antigen and also a biomarker of Burkholderia pseudomallei which causes a fatal disease called melioidosis. To enhance the assay sensitivity, a surface treatment for PSMA-encapsulated NCs (NC-probes) was performed to promote the presence of carboxyl groups that help conjugate more anti-CPS antibodies to the surface of NC-probes and thus enhance bioassay signals. Meanwhile, time-resolved reading on the luminescence of NC-probes was adopted to minimize the assay background autofluorescence. Both strategies essentially enhance the assay signal-to-background ratio (or equivalently the assay sensitivity) by increasing the signal and decreasing the background, respectively. Through performing and comparing immunoassays with different NC-probes (with and without surface treatment) and different signal reading methods (time-resolved reading and non-time-resolved reading), it was proven that the immunoassay adopting surface-treated NC-probes and time-resolved reading achieved a lower limit-of-detection (LOD) than the ones adopting non-surface-treated NC-probes or non-time-resolved reading. Moreover, the achieved LOD is comparable to the LOD of immunoassay using enzyme horseradish peroxidase as a signal transducer.

Brassinosteroid coordinates cell layer interactions in plants via cell wall and tissue mechanics.

Growth coordination between cell layers is essential for development of most multicellular organisms. Coordination may be mediated by molecular signaling and/or mechanical connectivity between cells, but how genes modify mechanical interactions between layers is unknown. Here we show that genes driving brassinosteroid synthesis promote growth of internal tissue, at least in part, by reducing mechanical epidermal constraint. We identified a brassinosteroid-deficient dwarf mutant in the aquatic plant Utricularia gibba with twisted internal tissue, likely caused by mechanical constraint from a slow-growing epidermis. We tested this hypothesis by showing that a brassinosteroid mutant in Arabidopsis enhances epidermal crack formation, indicative of increased tissue stress. We propose that by remodeling cell walls, brassinosteroids reduce epidermal constraint, showing how genes can control growth coordination between layers by means of mechanics.

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

Diabetes as a risk factor for the onset of frozen shoulder: a systematic review and meta-analysis.

OBJECTIVE: Summarise longitudinal observational studies to determine whether diabetes (types 1 and 2) is a risk factor for frozen shoulder. DESIGN: Systematic review and meta-analysis. DATA SOURCES: MEDLINE, Embase, AMED, PsycINFO, Web of Science Core Collection, CINAHL, Epistemonikos, Trip, PEDro, OpenGrey and The Grey Literature Report were searched on January 2019 and updated in June 2021. Reference screening and emailing professional contacts were also used. ELIGIBILITY CRITERIA: Longitudinal observational studies that estimated the association between diabetes and developing frozen shoulder. DATA EXTRACTION AND SYNTHESIS: Data extraction was completed by one reviewer and independently checked by another using a predefined extraction sheet. Risk of bias was judged using the Quality In Prognosis Studies tool. For studies providing sufficient data, random-effects meta-analysis was used to derive summary estimates of the association between diabetes and the onset of frozen shoulder. RESULTS: A meta-analysis of six case-control studies including 5388 people estimated the odds of developing frozen shoulder for people with diabetes to be 3.69 (95% CI 2.99 to 4.56) times the odds for people without diabetes. Two cohort studies were identified, both suggesting diabetes was associated with frozen shoulder, with HRs of 1.32 (95% CI 1.22 to 1.42) and 1.67 (95% CI 1.46 to 1.91). Risk of bias was judged as high in seven studies and moderate in one study. CONCLUSION: People with diabetes are more likely to develop frozen shoulder. Risk of unmeasured confounding was the main limitation of this systematic review. High-quality studies are needed to confirm the strength of, and understand reasons for, the association. PROSPERO REGISTRATION NUMBER: CRD42019122963.

Functional Activities of O-Polysaccharide and Hemolysin Coregulated Protein 1 Specific Antibodies Isolated from Melioidosis Patients.

Melioidosis is a fatal tropical disease caused by the environmental Gram-negative bacterium, Burkholderia pseudomallei. This bacterium is intrinsically resistant to several antibiotics and treatment of melioidosis requires prolonged antibiotic administration. To date, there are no vaccines available for melioidosis. Previous studies have shown that humoral immunity is critical for surviving melioidosis and that O-polysaccharide (OPS) and hemolysin coregulated protein 1 (Hcp1) are important protective antigens in animal models of melioidosis. Our previous studies revealed that melioidosis patients had high levels of OPS- and Hcp1-specific antibodies and that IgG against OPS (IgG-OPS) and Hcp1 (IgG-Hcp1) were associated with patient survival. In this study, we characterized the potential function(s) of IgG-OPS and IgG-Hcp1 from melioidosis patients. IgG-OPS and IgG-Hcp1 were purified from pooled serum obtained from melioidosis patients using immuno-affinity chromatography. Antibody-dependent cellular phagocytosis assays were performed with pooled serum from melioidosis patients and compared with serum obtained from healthy controls. Serum from melioidosis patients significantly enhanced B. pseudomallei uptake into the human monocytic cell line THP-1 compared with pooled serum from healthy donors. Enhanced opsonization was observed with IgG-OPS and IgG-Hcp1 in a dose-dependent manner. Antibody-dependent complement deposition assays were performed with IgG-OPS and IgG-Hcp1 using flow cytometry and showed that there was enhanced C3b deposition on the surface of B. pseudomallei treated with IgG-OPS but to a lesser degree with IgG-Hcp1. This study provides insight into the function of IgG-OPS and IgG-Hcp1 in human melioidosis and supports that OPS and Hcp1 are potential vaccine antigens for immunization against melioidosis.

Evaluation of two different vaccine platforms for immunization against melioidosis and glanders.

Burkholderia pseudomallei and the closely related species, Burkholderia mallei, produce similar multifaceted diseases which range from rapidly fatal to protracted and chronic, and are a major cause of mortality in endemic regions. Besides causing natural infections, both microbes are Tier 1 potential biothreat agents. Antibiotic treatment is prolonged with variable results, hence effective vaccines are urgently needed. The purpose of our studies was to compare candidate vaccines that target both melioidosis and glanders to identify the most efficacious one(s) and define residual requirements for their transition to the non-human primate aerosol model. Studies were conducted in the C57BL/6 mouse model to evaluate the humoral and cell-mediated immune response and protective efficacy of three Burkholderia vaccine candidates against lethal aerosol challenges with B. pseudomallei K96243, B. pseudomallei MSHR5855, and B. mallei FMH. The recombinant vaccines generated significant immune responses to the vaccine antigens, and the live attenuated vaccine generated a greater immune response to OPS and the whole bacterial cells. Regardless of the candidate vaccine evaluated, the protection of mice was associated with a dampened cytokine response within the lungs after exposure to aerosolized bacteria. Despite being delivered by two different platforms and generating distinct immune responses, two experimental vaccines, a capsule conjugate + Hcp1 subunit vaccine and the live B. pseudomallei 668 ΔilvI strain, provided significant protection and were down-selected for further investigation and advanced development.

Layered and integrated medical countermeasures against Burkholderia pseudomallei infections in C57BL/6 mice.

Burkholderia pseudomallei, the gram-negative bacterium that causes melioidosis, is notoriously difficult to treat with antibiotics. A significant effort has focused on identifying protective vaccine strategies to prevent melioidosis. However, when used as individual medical countermeasures both antibiotic treatments (therapeutics or post-exposure prophylaxes) and experimental vaccine strategies remain partially protective. Here we demonstrate that when used in combination, current vaccine strategies (recombinant protein subunits AhpC and/or Hcp1 plus capsular polysaccharide conjugated to CRM197 or the live attenuated vaccine strain B. pseudomallei 668 ΔilvI) and co-trimoxazole regimens can result in near uniform protection in a mouse model of melioidosis due to apparent synergy associated with distinct medical countermeasures. Our results demonstrated significant improvement when examining several suboptimal antibiotic regimens (e.g., 7-day antibiotic course started early after infection or 21-day antibiotic course with delayed initiation). Importantly, this combinatorial strategy worked similarly when either protein subunit or live attenuated vaccines were evaluated. Layered and integrated medical countermeasures will provide novel treatment options for melioidosis as well as diseases caused by other pathogens that are refractory to individual strategies, particularly in the case of engineered, emerging, or re-emerging bacterial biothreat agents.

Development of Melioidosis Subunit Vaccines Using an Enzymatically Inactive Burkholderia pseudomallei AhpC.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular, Gram-negative pathogen that is highly infectious via the respiratory route and can cause severe, debilitating, and often fatal diseases in humans and animals. At present, no licensed vaccines for immunization against this CDC Tier 1 select agent exist. Studies in our lab have previously demonstrated that subunit vaccine formulations consisting of a B. pseudomallei capsular polysaccharide (CPS)-based glycoconjugate (CPS-CRM197) combined with hemolysin-coregulated protein (Hcp1) provided C57BL/6 mice with high-level protection against an acute inhalational challenge of B. pseudomallei. In this study, we evaluated the immunogenicity and protective capacity of B. pseudomallei alkyl hydroperoxide reductase subunit C (AhpC) in combination with CPS-CRM197. AhpC is a peroxiredoxin involved in oxidative stress reduction and is a potential protective antigen. To facilitate our studies and maximize safety in animals, recombinant B. pseudomallei AhpC harboring an active site mutation (AhpCC57G) was expressed in Escherichia coli and purified using tandem nickel-cobalt affinity chromatography. Immunization of C57BL/6 mice with CPS-CRM197 combined with AhpCC57G stimulated high-titer IgG responses against the CPS component of the glycoconjugate as well as stimulated high-titer IgG and robust interferon gamma (IFN-γ)-, interleukin-5 (IL-5)-, and IL-17-secreting T cell responses against AhpCC57G. When challenged via an inhalational route with a high dose (~27 50% lethal doses [LD50s]) of B. pseudomallei, 70% of the immunized mice survived 35 days postchallenge. Collectively, our findings demonstrate that AhpCC57G is a potent activator of cellular and humoral immune responses and may be a promising candidate to include in future melioidosis subunit vaccines.

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.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis.

BACKGROUND: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. CONCLUSIONS/SIGNIFICANCE: A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

A Field Evaluation of the LuciTrap and the Western Australian Trap with Three Different Baits Types for Monitoring Lucilia cuprina and Lucilia sericata in New Zealand.

Flytraps can be used on farms to monitor the populations of primary strike flies (Lucilia cuprina and Lucilia sericata) and, hence, offer a view regarding the incidence of flystrike on sheep. This study aimed to contrast the specificity and effectiveness of the LuciTrap with its combination of three chemical lures (Lucilures) and the Western Australian Trap with three bait types (LuciLure, Sheep liver with 30% sodium sulphide and squid). A mean model and rate model were fitted to the data. The mean model showed no difference (p > 0.05) in the mean weekly catch for L. cuprina between the Western Australian Trap with LuciLures and the Western Australian Trap baited with sheep liver with 30% sodium sulphide (p < 0.05). Whereas, for L. sericata, no difference (p > 0.05) was found between the Western Australian Trap with LuciLures, the Western Australian Trap baited with sheep liver with 30% sodium sulphide and the LuciTrap. The rate model illustrated that the Western Australian Trap with sheep liver with 30% sodium sulphide and LuciTrap did not differ (p > 0.05) for L. cuprina and L. sericata. Combined, these results indicate that New Zealand farmers can use either the LuciTrap or the Western Australian Trap with sheep liver with 30% sodium sulphide to monitor these target species.

Melioidosis Patient Survival Correlates With Strong IFN-γ Secreting T Cell Responses Against Hcp1 and TssM.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines.

Disrupting hierarchical control of nitrogen fixation enables carbon-dependent regulation of ammonia excretion in soil diazotrophs.

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.

The branched-chain amino acid aminotransferase TaBCAT1 modulates amino acid metabolism and positively regulates wheat rust susceptibility.

Plant pathogens suppress defense responses to evade recognition and promote successful colonization. Although identifying the genes essential for pathogen ingress has traditionally relied on screening mutant populations, the post-genomic era provides an opportunity to develop novel approaches that accelerate identification. Here, RNA-seq analysis of 68 pathogen-infected bread wheat (Triticum aestivum) varieties, including three (Oakley, Solstice and Santiago) with variable levels of susceptibility, uncovered a branched-chain amino acid aminotransferase (termed TaBCAT1) as a positive regulator of wheat rust susceptibility. We show that TaBCAT1 is required for yellow and stem rust infection and likely functions in branched-chain amino acid (BCAA) metabolism, as TaBCAT1 disruption mutants had elevated BCAA levels. TaBCAT1 mutants also exhibited increased levels of salicylic acid (SA) and enhanced expression of associated defense genes, indicating that BCAA regulation, via TaBCAT1, has a key role in SA-dependent defense activation. We also identified an association between the levels of BCAAs and resistance to yellow rust infection in wheat. These findings provide insight into SA-mediated defense responses in wheat and highlight the role of BCAA metabolism in the defense response. Furthermore, TaBCAT1 could be manipulated to potentially provide resistance to two of the most economically damaging diseases of wheat worldwide.

Diverting tyrosine: Data from untargeted metabolic analysis of tomato fruit accumulating L-DOPA.

L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, is synthesised in plants from the amino acid tyrosine, through oxidation. Conversion of tyrosine to L-DOPA constitues the first step of betalain biosynthesis in plants. Recently, the gene responsible for this step was identified in beetroot, BvCYP76AD6, that is the source of yellow and purple betalain pigments. Overexpression of this gene, specifically in tomato fruit, led to accumulation of L-DOPA that otherwise is not detectable [1]. Co-expression of the Arabidopsis transcription factor, AtMYB12, in fruit, increased L-DOPA levels further. To study the metabolic changes in these fruit, we performed untargeted metabolite analysis of ripe fruit: GC-MS was performed to identify changes in primary metabolites, LC-MS analysis was used to identify alterations in specialised metabolites. These data can be used to study the impact of diversion of tyrosine in fruit, accompanied by the accumulation of L-DOPA in planta and to identify new biological roles associated with the accumulation of these metabolites.

Metabolic engineering of tomato fruit enriched in L-DOPA.

L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, is a non-standard amino acid, and the gold standard drug for the treatment for Parkinson's Disease (PD). Recently, a gene encoding the enzyme that is responsible for its synthesis, as a precursor of the coloured pigment group betalains, was identified in beetroot, BvCYP76AD6. We have engineered tomato fruit enriched in L-DOPA through overexpression of BvCYP76AD6 in a fruit specific manner. Analysis of the transgenic fruit revealed the feasibility of accumulating L-DOPA in a non-naturally betalain-producing plant. Fruit accumulating L-DOPA also showed major effects on the fruit metabolome. Some of these changes included elevation of amino acids levels, changes in the levels of intermediates of the TCA and glycolysis pathways and reductions in the levels of phenolic compounds and nitrogen-containing specialised metabolites. Furthermore, we were able to increase the L-DOPA levels further by elevating the expression of the metabolic master regulator, MYB12, specifically in tomato fruit, together with BvCYP76AD6. Our study elucidated new roles for L-DOPA in plants, because it impacted fruit quality parameters including antioxidant capacity and firmness. The L-DOPA levels achieved in tomato fruit were comparable to the levels in other non-seed organs of L-DOPA - accumulating plants, offering an opportunity to develop new biological sources of L-DOPA by widening the repertoire of L-DOPA-accumulating plants. These tomato fruit could be used as an alternative source of this important pharmaceutical.