Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

An improved protocol for efficient transformation and regeneration of Setaria italica.

KEY MESSAGE: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.

Importance of Poly-3-Hydroxybutyrate Metabolism to the Ability of Herbaspirillum seropedicae To Promote Plant Growth.

Herbaspirillum seropedicae is an endophytic bacterium that establishes an association with a variety of plants, such as rice, corn, and sugarcane, and can significantly increase plant growth. H. seropedicae produces polyhydroxybutyrate (PHB), stored in the form of insoluble granules. Little information is available on the possible role of PHB in bacterial root colonization or in plant growth promotion. To investigate whether PHB is important for the association of H. seropedicae with plants, we inoculated roots of Setaria viridis with H. seropedicae strain SmR1 and mutants defective in PHB production (ΔphaP1, ΔphaP1 ΔphaP2, ΔphaC1, and ΔphaR) or mobilization (ΔphaZ1 ΔphaZ2). The strains producing large amounts of PHB colonized roots, significantly increasing root area and the number of lateral roots compared to those of PHB-negative strains. H. seropedicae grows under microaerobic conditions, which can be found in the rhizosphere. When grown under low-oxygen conditions, only the parental strain and ΔphaP2 mutant exhibited normal growth. The lack of normal growth under low oxygen correlated with the inability to stimulate plant growth, although there was no effect on the level of root colonization. The data suggest that PHB is produced in the root rhizosphere and plays a role in maintaining normal metabolism under microaerobic conditions. To confirm this, we screened for green fluorescent protein (GFP) expression under the control of the H. seropedicae promoters of the PHA synthase and PHA depolymerase genes in the rhizosphere. PHB synthesis is active on the root surface and later PHB depolymerase expression is activated.IMPORTANCE The application of bacteria as plant growth promoters is a sustainable alternative to mitigate the use of chemical fertilization in agriculture, reducing negative economic and environmental impacts. Several plant growth-promoting bacteria synthesize and accumulate the intracellular polymer polyhydroxybutyrate (PHB). However, the role of PHB in plant-bacterium interactions is poorly understood. In this study, applying the C4 model grass Setaria viridis and several mutants in the PHB metabolism of the endophyte Herbaspirillum seropedicae yielded new findings on the importance of PHB for bacterial colonization of S. viridis roots. Taken together, the results show that deletion of genes involved in the synthesis and degradation of PHB reduced the ability of the bacteria to enhance plant growth but with little effect on overall root colonization. The data suggest that PHB metabolism likely plays an important role in supporting specific metabolic routes utilized by the bacteria to stimulate plant growth.

The oil-contaminated soil diazotroph Azoarcus olearius DQS-4T is genetically and phenotypically similar to the model grass endophyte Azoarcus sp. BH72.

The genome of Azoarcus olearius DQS-4T , a N2 -fixing Betaproteobacterium isolated from oil-contaminated soil in Taiwan, was sequenced and compared with other Azoarcus strains. The genome sequence showed high synteny with Azoarcus sp. BH72, a model endophytic diazotroph, but low synteny with five non-plant-associated strains (Azoarcus CIB, Azoarcus EBN1, Azoarcus KH32C, A. toluclasticus MF63T and Azoarcus PA01). Average Nucleotide Identity (ANI) revealed that DQS-4T shares 98.98% identity with Azoarcus BH72, which should now be included in the species A. olearius. The genome of DQS-4T contained several genes related to plant colonization and plant growth promotion, such as nitrogen fixation, plant adhesion and root surface colonization. In accordance with the presence of these genes, DQS-4T colonized rice (Oryza sativa) and Setaria viridis, where it was observed within the intercellular spaces and aerenchyma mainly of the roots. Although they promote the growth of grasses, the mechanism(s) of plant growth promotion by A. olearius strains is unknown, as the genomes of DQS-4T and BH72 do not contain genes for indole acetic acid (IAA) synthesis nor phosphate solubilization. In spite of its original source, both the genome and behaviour of DQS-4T suggest that it has the capacity to be an endophytic, nitrogen-fixing plant growth-promoting bacterium.

Robust biological nitrogen fixation in a model grass-bacterial association.

Nitrogen-fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen-13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen-limiting conditions when inoculated with an ammonium-excreting strain of Azospirillum brasilense. (11)C-labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen-starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen-sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.