Philip S. Poole

The plant microbiome.

Plant genomes contribute to the structure and function of the plant microbiome, a key determinant of plant health and productivity. High-throughput technologies are revealing interactions between these complex communities and their hosts in unprecedented detail.

Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants

Plant–microbe interactions in the rhizosphere have important roles in biogeochemical cycling, and maintenance of plant health and productivity, yet remain poorly understood. Using RNA-based metatranscriptomics, the global active microbiomes were analysed in soil and rhizospheres of wheat, oat, pea and an oat mutant (sad1) deficient in production of anti-fungal avenacins. Rhizosphere microbiomes differed from bulk soil and between plant species. Pea (a legume) had a much stronger effect on the rhizosphere than wheat and oat (cereals), resulting in a dramatically different rhizosphere community. The relative abundance of eukaryotes in the oat and pea rhizospheres was more than fivefold higher than in the wheat rhizosphere or bulk soil. Nematodes and bacterivorous protozoa were enriched in all rhizospheres, whereas the pea rhizosphere was highly enriched for fungi. Metabolic capabilities for rhizosphere colonisation were selected, including cellulose degradation (cereals), H2 oxidation (pea) and methylotrophy (all plants). Avenacins had little effect on the prokaryotic community of oat, but the eukaryotic community was strongly altered in the sad1 mutant, suggesting that avenacins have a broader role than protecting from fungal pathogens. Profiling microbial communities with metatranscriptomics allows comparison of relative abundance, from multiple samples, across all domains of life, without polymerase chain reaction bias. This revealed profound differences in the rhizosphere microbiome, particularly at the kingdom level between plants.

Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics

BackgroundThe rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biospheres productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere.ResultsCarbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere.ConclusionsComparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.

What determines the efficiency of N2-fixing rhizobium-legume symbioses?

Biological nitrogen fixation is vital to nutrient cycling in the biosphere and is the major route by which atmospheric dinitrogen (N(2)) is reduced to ammonia. The largest single contribution to biological N(2) fixation is carried out by rhizobia, which include a large group of both alpha and beta-proteobacteria, almost exclusively in association with legumes. Rhizobia must compete to infect roots of legumes and initiate a signaling dialog with host plants that leads to nodule formation. The most common form of infection involves the growth of rhizobia down infection threads which are laid down by the host plant. Legumes form either indeterminate or determinate types of nodules, with these groups differing widely in nodule morphology and often in the developmental program by which rhizobia form N(2) fixing bacteroids. In particular, indeterminate legumes from the inverted repeat-lacking clade (IRLC) (e.g., peas, vetch, alfalfa, medics) produce a cocktail of antimicrobial peptides which cause endoreduplication of the bacterial genome and force rhizobia into a nongrowing state. Bacteroids often become dependent on the plant for provision of key cofactors, such as homocitrate needed for nitrogenase activity or for branched chain amino acids. This has led to the suggestion that bacteroids at least from the IRLC can be considered as ammoniaplasts, where they are effectively facultative plant organelles. A low O(2) tension is critical both to induction of genes needed for N(2) fixation and to the subsequent exchange of nutrient between plants and bacteroids. To achieve high rates of N(2) fixation, the legume host and Rhizobium must be closely matched not only for infection, but for optimum development, nutrient exchange, and N(2) fixation. In this review, we consider the multiple steps of selection and bacteroid development and how these alter the overall efficiency of N(2) fixation.

What determines the efficiency of N(2)-fixing Rhizobium-legume symbioses?

Biological nitrogen fixation is vital to nutrient cycling in the biosphere and is the major route by which atmospheric dinitrogen (N(2)) is reduced to ammonia. The largest single contribution to biological N(2) fixation is carried out by rhizobia, which include a large group of both alpha and beta-proteobacteria, almost exclusively in association with legumes. Rhizobia must compete to infect roots of legumes and initiate a signaling dialog with host plants that leads to nodule formation. The most common form of infection involves the growth of rhizobia down infection threads which are laid down by the host plant. Legumes form either indeterminate or determinate types of nodules, with these groups differing widely in nodule morphology and often in the developmental program by which rhizobia form N(2) fixing bacteroids. In particular, indeterminate legumes from the inverted repeat-lacking clade (IRLC) (e.g., peas, vetch, alfalfa, medics) produce a cocktail of antimicrobial peptides which cause endoreduplication of the bacterial genome and force rhizobia into a nongrowing state. Bacteroids often become dependent on the plant for provision of key cofactors, such as homocitrate needed for nitrogenase activity or for branched chain amino acids. This has led to the suggestion that bacteroids at least from the IRLC can be considered as ammoniaplasts, where they are effectively facultative plant organelles. A low O(2) tension is critical both to induction of genes needed for N(2) fixation and to the subsequent exchange of nutrient between plants and bacteroids. To achieve high rates of N(2) fixation, the legume host and Rhizobium must be closely matched not only for infection, but for optimum development, nutrient exchange, and N(2) fixation. In this review, we consider the multiple steps of selection and bacteroid development and how these alter the overall efficiency of N(2) fixation.

Stability and succession of the rhizosphere microbiota depends upon plant type and soil composition

We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alternative community compositions rather than a single distinctive community. Organisms selected in rhizospheres can have positive or negative effects. Two abundant bacteria are shown to promote plant growth, but in Brassica the pathogen Olpidium brassicae came to dominate the fungal community. So plants exert strong selection on the rhizosphere microbiota but soil composition is critical to its stability. microbial succession/ plant–microbe interactions/rhizosphere microbiota/selection.

Role of root microbiota in plant productivity

The growing human population requires increasing amounts of food, but modern agriculture has limited possibilities for increasing yields. New crop varieties may be bred to have increased yields and be more resistant to environmental stress and pests. However, they still require fertilization to supplement essential nutrients that are normally limited in the soil. Soil microorganisms present an opportunity to reduce the requirement for inorganic fertilization in agriculture. Microorganisms, due to their enormous genetic pool, are also a potential source of biochemical reactions that recycle essential nutrients for plant growth. Microbes that associate with plants can be considered to be part of the plants pan-genome. Therefore, it is essential for us to understand microbial community structure and their metagenome and how it is influenced by different soil types and crop varieties. In the future we may be able to modify and better utilize the soil microbiota potential for promoting plant growth.

Role of Symbiotic Auxotrophy in the Rhizobium-Legume Symbioses

Background Rhizobium leguminosarum bv. viciae mutants unable to transport branched-chain amino acids via the two main amino acid ABC transport complexes AapJQMP and BraDEFGC produce a nitrogen starvation phenotype when inoculated on pea (Pisum sativum) plants [1], [2]. Bacteroids in indeterminate pea nodules have reduced abundance and a lower chromosome number. They reduce transcription of pathways for branched-chain amino acid biosynthesis and become dependent on their provision by the host. This has been called “symbiotic auxotrophy”. Methodology/Principal Findings A region important in solute specificity was identified in AapQ and changing P144D in this region reduced branched-chain amino acid transport to a very low rate. Strains carrying P144D were still fully effective for N2 fixation on peas demonstrating that a low rate of branched amino acid transport in R. leguminosarum bv. viciae supports wild-type rates of nitrogen fixation. The importance of branched-chain amino acid transport was then examined in other legume-Rhizobium symbioses. An aap bra mutant of R. leguminosarum bv. phaseoli also showed nitrogen starvation symptoms when inoculated on French bean (Phaseolus vulgaris), a plant producing determinate nodules. The phenotype is different from that observed on pea and is accompanied by reduced nodule numbers and nitrogen fixation per nodule. However, an aap bra double mutant of Sinorhizobium meliloti 2011 showed no phenotype on alfalfa (Medicago sativa). Conclusions/Significance Symbiotic auxotrophy occurs in both determinate pea and indeterminate bean nodules demonstrating its importance for bacteroid formation and nodule function in legumes with different developmental programmes. However, only small quantities of branched chain amino acids are needed and symbiotic auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants inoculated on different legumes probably reflects altered timing of amino acid availability, development of symbiotic auxotrophy and nodule developmental programmes.

Mutation of GOGAT prevents pea bacteroid formation and N2 fixation by globally downregulating transport of organic nitrogen sources

Mutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Glnu2003:u2003Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post‐translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute‐binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem‐loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N2 was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N2 on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

The PTSNtr system globally regulates ATP‐dependent transporters in Rhizobium leguminosarum

Mutation of ptsP encoding EINtr of the PTSNtr system in Rhizobium leguminosarum strain Rlv3841 caused a pleiotropic phenotype as observed with many bacteria. The mutant formed dry colonies and grew poorly on organic nitrogen or dicarboxylates. Most strikingly the ptsP mutant had low activity of a broad range of ATP‐dependent ABC transporters. This lack of activation, which occurred post‐translationally, may explain many of the pleiotropic effects. In contrast proton‐coupled transport systems were not inhibited in a ptsP mutant. Regulation by PtsP also involves two copies of ptsN that code for EIIANtr, resulting in a phosphorylation cascade. As in Escherichia coli, the Rlv3841 PTSNtr system also regulates K+ homeostasis by transcriptional activation of the high‐affinity ATP‐dependent K+ transporter KdpABC. This involves direct interaction of a two‐component sensor regulator pair KdpDE with unphosphorylated EIIANtr. Critically, ptsP mutants, which cannot phosphorylate PtsN1 or PtsN2, had a fully activated KdpABC transporter. This is the opposite pattern from that observed with ABC transporters which apparently require phosphorylation of PtsN. These results suggest that ATP‐dependent transport might be regulated via PTSNtr responding to the cellular energy charge. ABC transport may be inactivated at low energy charge, conserving ATP for essential processes including K+ homeostasis.