In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria.

Over the past decades, crop yields have risen in parallel with increasing use of fossil fuel-derived nitrogen (N) fertilizers but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic, and endophytic plant growth-promoting bacteria (PGPB) are known to stimulate plant growth. However, compared with the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix+) or a fix- mutant strain (SmR54) compared with uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole-root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in-situ laser ablation electrospray ionization mass spectrometry to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants, while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix- strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix- strain, perhaps reflecting a plant defense response.

Proteomic and Metabolomic Analysis of Azospirillum brasilensentrC Mutant under High and Low Nitrogen Conditions.

Azospirillum brasilense is a diazotrophic microorganism capable of associating with roots of important grasses and cereals, promoting plant growth and increasing crop yields. Nitrogen levels and the Ntr regulatory system control the nitrogen metabolism in A. brasilense. This system comprises the nitrogen regulatory proteins GlnD, which is capable of adding uridylyl groups to the PII proteins, GlnB (PII-1) and GlnZ (PII-2), under limiting nitrogen levels. Under such conditions, the histidine kinase NtrB (nitrogen regulatory protein B) cannot interact with GlnB and phosphorylate NtrC (nitrogen regulatory protein C). The phosphorylated form of NtrC acts as a transcriptional activator of genes involved in the metabolism of alternative nitrogen sources. Considering the key role of NtrC in nitrogen metabolism in A. brasilense, in this work we evaluated the proteomic and metabolomic profiles of the wild-type FP2 strain and its mutant ntrC grown under high and low nitrogen. Analysis of the integrated data identifies novel NtrC targets, including proteins involved in the response against oxidative stress (i.e., glutathione S-transferase and hydroperoxide resistance protein), underlining the importance of NtrC to bacterial survival under oxidative stress conditions.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

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.

Differential growth responses of Brachypodium distachyon genotypes to inoculation with plant growth promoting rhizobacteria.

Plant growth promoting rhizobacteria (PGPR) can associate and enhance the growth of important crop grasses. However, in most cases, the molecular mechanisms responsible for growth promotion are not known. Such research could benefit by the adoption of a grass model species that showed a positive response to bacterial inoculation and was amenable to genetic and molecular research methods. In this work we inoculated different genotypes of the model grass Brachypodium distachyon with two, well-characterized PGPR bacteria, Azospirillum brasilense and Herbaspirillum seropedicae, and evaluated the growth response. Plants were grown in soil under no nitrogen or with low nitrogen (i.e., 0.5 mM KNO3). A variety of growth parameters (e.g., shoot height, root length, number of lateral roots, fresh and dry weight) were measured 35 days after inoculation. The data indicate that plant genotype plays a very important role in determining the plant response to PGPR inoculation. A positive growth response was observed with only four genotypes grown under no nitrogen and three genotypes tested under low nitrogen. However, in contrast, relatively good root colonization was seen with most genotypes, as measured by drop plate counting and direct, microscopic examination of roots. In particular, the endophytic bacteria H. seropedicae showed strong epiphytic and endophytic colonization of roots.

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.