Structural dissection of two redox proteins from the shipworm symbiont Teredinibacter turnerae.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Molecular diagnostics for real-time determination of herbicide resistance in wild grasses.

The growth of resistance to multiple herbicides in grass weeds is a major threat to global cereal production and in the UK, is epitomized by the loss of control of blackgrass (Alopecurus myosuroides), causing losses in winter wheat production equating to 5% of national consumption. With an urgent need to develop new black-grass management tools, we have developed a lateral flow assay (LFA) that can predict resistance to multiple herbicides within 10 min.

Combinatorial Glycomic Analyses to Direct CAZyme Discovery for the Tailored Degradation of Canola Meal Non-Starch Dietary Polysaccharides.

Canola meal (CM), the protein-rich by-product of canola oil extraction, has shown promise as an alternative feedstuff and protein supplement in poultry diets, yet its use has been limited due to the abundance of plant cell wall fibre, specifically non-starch polysaccharides (NSP) and lignin. The addition of exogenous enzymes to promote the digestion of CM NSP in chickens has potential to increase the metabolizable energy of CM. We isolated chicken cecal bacteria from a continuous-flow mini-bioreactor system and selected for those with the ability to metabolize CM NSP. Of 100 isolates identified, Bacteroides spp. and Enterococcus spp. were the most common species with these capabilities. To identify enzymes specifically for the digestion of CM NSP, we used a combination of glycomics techniques, including enzyme-linked immunosorbent assay characterization of the plant cell wall fractions, glycosidic linkage analysis (methylation-GC-MS analysis) of CM NSP and their fractions, bacterial growth profiles using minimal media supplemented with CM NSP, and the sequencing and de novo annotation of bacterial genomes of high-efficiency CM NSP utilizing bacteria. The SACCHARIS pipeline was used to select plant cell wall active enzymes for recombinant production and characterization. This approach represents a multidisciplinary innovation platform to bioprospect endogenous CAZymes from the intestinal microbiota of herbivorous and omnivorous animals which is adaptable to a variety of applications and dietary polysaccharides.

Microbiota-directed fibre activates both targeted and secondary metabolic shifts in the distal gut.

Beneficial modulation of the gut microbiome has high-impact implications not only in humans, but also in livestock that sustain our current societal needs. In this context, we have tailored an acetylated galactoglucomannan (AcGGM) fibre to match unique enzymatic capabilities of Roseburia and Faecalibacterium species, both renowned butyrate-producing gut commensals. Here, we test the accuracy of AcGGM within the complex endogenous gut microbiome of pigs, wherein we resolve 355 metagenome-assembled genomes together with quantitative metaproteomes. In AcGGM-fed pigs, both target populations differentially express AcGGM-specific polysaccharide utilization loci, including novel, mannan-specific esterases that are critical to its deconstruction. However, AcGGM-inclusion also manifests a "butterfly effect", whereby numerous metabolic changes and interdependent cross-feeding pathways occur in neighboring non-mannanolytic populations that produce short-chain fatty acids. Our findings show how intricate structural features and acetylation patterns of dietary fibre can be customized to specific bacterial populations, with potential to create greater modulatory effects at large.

Effects of Agronomic Management and Climate on Leaf Phenolic Profiles, Disease Severity, and Grain Yield in Organic and Conventional Wheat Production Systems.

Agricultural intensification over the last 40 years has increased cereal yields, but there is very limited information on the effects of intensification practices (e.g., nondiverse rotations, mineral NPK fertilizer, and pesticides) on crop health and quality. Results from the study reported here suggest that the use of mineral NPK fertilizers reduces phenolic acid and flavonoid concentrations in leaves and increases the susceptibility of wheat to lodging and powdery mildew, when compared to composted FYM inputs. In contrast, the use of herbicides, fungicides, and growth regulators reduces lodging and foliar disease severity but had no effect on phenolic acid and flavonoid concentrations. The use of composted FYM inputs also resulted in a significant grain yield reduction and not substantially reduced the severity of opportunistic pathogens such as Septoria, which remain a major yield limiting factor unless fungicides are used and/or more Septoria resistant varieties become available.

Changes in the proteome of the problem weed blackgrass correlating with multiple-herbicide resistance.

Herbicide resistance in grass weeds is now one of the greatest threats to sustainable cereal production in Northern Europe. Multiple-herbicide resistance (MHR), a poorly understood multigenic and quantitative trait, is particularly problematic as it provides tolerance to most classes of chemistries currently used for post-emergence weed control. Using a combination of transcriptomics and proteomics, the evolution of MHR in populations of the weed blackgrass (Alopecurus myosuroides) has been investigated. While over 4500 genes showed perturbation in their expression in MHR versus herbicide sensitive (HS) plants, only a small group of proteins showed >2-fold changes in abundance, with a mere eight proteins consistently associated with this class of resistance. Of the eight, orthologues of three of these proteins are also known to be associated with multiple drug resistance (MDR) in humans, suggesting a cross-phyla conservation in evolved tolerance to chemical agents. Proteomics revealed that MHR could be classified into three sub-types based on the association with resistance to herbicides with differing modes of action (MoA), being either global, specific to diverse chemistries acting on one MoA, or herbicide specific. Furthermore, the proteome of MHR plants were distinct from that of HS plants exposed to a range of biotic (insect feeding, plant-microbe interaction) and abiotic (N-limitation, osmotic, heat, herbicide safening) challenges commonly encountered in the field. It was concluded that MHR in blackgrass is a uniquely evolving trait(s), associated with changes in the proteome that are distinct from responses to conventional plant stresses, but sharing common features with MDR in humans.

Potential roles for microbial endophytes in herbicide tolerance in plants.

Herbicide tolerance in crops and weeds is considered to be monotrophic, i.e. determined by the relative susceptibility of the physiological process targeted and the plant's ability to metabolise and detoxify the agrochemical. A growing body of evidence now suggests that endophytes, microbes that inhabit plant tissues and provide a range of growth, health and defence enhancements, can contribute to other types of abiotic and biotic stress tolerance. The current evidence for herbicide tolerance being bitrophic, with both free-living and plant-associated endophytes contributing to tolerance in the host plant, has been reviewed. We propose that endophytes can directly contribute to herbicide detoxification through their ability to metabolise xenobiotics. In addition, we explore the paradigm that microbes can 'prime' resistance mechanisms in plants such that they enhance herbicide tolerance by inducing the host's stress responses to withstand the downstream toxicity caused by herbicides. This latter mechanism has the potential to contribute to the growth of non-target-site-based herbicide resistance in weeds. Microbial endophytes already contribute to herbicide detoxification in planta, and there is now significant scope to extend these interactions using synthetic biology approaches to engineer new chemical tolerance traits into crops via microbial engineering.

Modelling pathways to Rubisco degradation: a structural equation network modelling approach.

'Omics analysis (transcriptomics, proteomics) quantifies changes in gene/protein expression, providing a snapshot of changes in biochemical pathways over time. Although tools such as modelling that are needed to investigate the relationships between genes/proteins already exist, they are rarely utilised. We consider the potential for using Structural Equation Modelling to investigate protein-protein interactions in a proposed Rubisco protein degradation pathway using previously published data from 2D electrophoresis and mass spectrometry proteome analysis. These informed the development of a prior model that hypothesised a pathway of Rubisco Large Subunit and Small Subunit degradation, producing both primary and secondary degradation products. While some of the putative pathways were confirmed by the modelling approach, the model also demonstrated features that had not been originally hypothesised. We used Bayesian analysis based on Markov Chain Monte Carlo simulation to generate output statistics suggesting that the model had replicated the variation in the observed data due to protein-protein interactions. This study represents an early step in the development of approaches that seek to enable the full utilisation of information regarding the dynamics of biochemical pathways contained within proteomics data. As these approaches gain attention, they will guide the design and conduct of experiments that enable 'Omics modelling to become a common place practice within molecular biology.

Identification of plant quantitative trait loci modulating a rhizobacteria-aphid indirect effect.

Plants simultaneously interact with a plethora of species both belowground and aboveground, which can result in indirect effects mediated by plants. Studies incorporating plant genetic variation indicate that indirect effects mediated by plants may be a significant factor influencing the ecology and evolution of species within a community. Here, we present findings of a Quantitative Trait Locus (QTL) mapping study, where we mapped a rhizobacteria-aphid indirect effect onto the barley genome. We measured the size of aphid populations on barley when the barley rhizosphere either was or was not supplemented with a rhizobacterial species. Using a QTL mapping subset, we located five regions of the barley genome associated with the rhizobacteria-aphid indirect effect. Rhizobacterial supplementation led to an increase in aphid population size (mapped to three barley QTL), or a decrease in aphid population size (mapped to two barley QTL). One QTL associated with plant resistance to aphids was affected by a significant QTL-by-environment interaction, because it was not expressed when rhizobacteria was supplemented. Our results indicated that rhizobacterial supplementation of barley roots led to either increased or reduced aphid population size depending on plant genotype at five barley QTL. This indicates that the direction of a rhizobacteria-aphid indirect effect could influence the selection pressure on plants, when considering species that affect plant fitness. Further research may build on the findings presented here, to identify genes within QTL regions that are involved in the indirect interaction.

Effect of organic and conventional crop rotation, fertilization, and crop protection practices on metal contents in wheat (Triticum aestivum).

The effects of organic versus conventional crop management practices (crop rotation, crop protection, and fertility management strategies) on wheat yields and grain metal (Al, Cd, Cu, Ni, Pb, and Zn) concentrations were investigated in a long-term field trial. The interactions between crop management practices and the season that the crop was grown were investigated using univariate and redundancy analysis approaches. Grain yields were highest where conventional fertility management and crop protection practices were used, but growing wheat after a previous crop of grass/clover was shown to partially compensate for yield reductions due to the use of organic fertility management. All metals except for Pb were significantly affected by crop management practices and the year that the wheat was grown. Grain Cd and Cu levels were higher on average when conventional fertility management practices were used. Al and Cu were higher on average when conventional crop protection practices were used. The results demonstrate that there is potential to manage metal concentrations in the diet by adopting specific crop management practices shown to affect crop uptake of metals.

Community genetic interactions mediate indirect ecological effects between a parasitoid wasp and rhizobacteria.

Indirect ecological effects (IEEs) clearly influence species dynamics and abundance, yet relatively little is known about how they influence the evolution of species involved. While genetic variation in the species causing and responding to the IEE has obvious effects, the influence of genetic variation in intermediate species remains unexamined. Given the often counterintuitive responses of populations to IEEs this seems a significant omission. Following a community genetics approach, we used a model tetra-trophic system (parasitoid wasp, aphid, barley, and rhizobacteria) to investigate the effect of genetic interactions within the two linking species (aphids and barley) on the IEE of rhizobacteria on wasps. We show that 12.4% of the variation in wasp size, a proxy for fitness, is explained by higher-order interactions between aphid genotype (A), barley genotype (B), and presence or absence of rhizobacteria (R) (Genotype[B] x Genotype[A] x Environment[R]). Thus, the IEE of rhizobacteria on the parasitoid wasp is influenced by the specific combination of aphid and barley genotypes that mediate the interactions. In some cases changes in the genotypes of the intermediate species completely reverse the effect of rhizobacteria on wasp size. Our work demonstrates that within-species genetic variation is important in shaping IEEs in communities, an essential component of community evolutionary processes.

Genotype-by-genotype interactions modified by a third species in a plant-insect system.

Community genetics examines how genotypic variation within a species influences the associated ecological community. The inclusion of additional environmental and genotypic factors is a natural extension of the current community genetics framework. However, the extent to which the presence of and genetic variation in associated species influences interspecific interactions (i.e., genotype x genotype x environment [G x G x E] interactions) has been largely ignored. We used a community genetics approach to study the interaction of barley and aphids in the absence and presence of rhizosphere bacteria. We designed a matrix of aphid genotype and barley genotype combinations and found a significant G x G x E interaction, indicating that the barley-aphid interaction is dependent on the genotypes of the interacting species as well as the biotic environment. We discuss the consequences of the strong G x G x E interaction found in our study in relation to its impact on the study of species interactions in a community context.