Does Host Plant Drive Variation in Microbial Gut Communities in a Recently Shifted Pest?

Biotic interactions can modulate the responses of organisms to environmental stresses, including diet changes. Gut microbes have substantial effects on diverse ecological and evolutionary traits of their hosts, and microbial communities can be highly dynamic within and between individuals in space and time. Modulations of the gut microbiome composition and their potential role in the success of a species to maintain itself in a new environment have been poorly studied to date. Here we examine this question in a large wood-boring beetle Cacosceles newmannii (Cerambycidae), that was recently found thriving on a newly colonized host plant. Using 16S metabarcoding, we assessed the gut bacterial community composition of larvae collected in an infested field and in "common garden" conditions, fed under laboratory-controlled conditions on four either suspected or known hosts (sugarcane, tea tree, wattle, and eucalyptus). We analysed microbiome variation (i.e. diversity and differentiation), measured fitness-related larval growth, and studied host plant lignin and cellulose contents, since their degradation is especially challenging for wood-boring insects. We show that sugarcane seems to be a much more favourable host for larval growth. Bacterial diversity level was the highest in field-collected larvae, whereas lab-reared larvae fed on sugarcane showed a relatively low level of diversity but very specific bacterial variants. Bacterial communities were mainly dominated by Proteobacteria, but were significantly different between sugarcane-fed lab-reared larvae and any other hosts or field-collected larvae. We identified changes in the gut microbiome associated with different hosts over a short time frame, which support the hypothesis of a role of the microbiome in host switches.

Evolutionary analysis and quality assessment of ζ-carbonic anhydrase sequences from environmental microbiome.

Carbonic anhydrase (CA) is one of the most vital enzymes in living cells. This study has been performed due to the significance of this metalloenzyme for life and the novelty of some CA families like ζ-CA to evaluate evolutionary processes and quality check their sequences. In this study, bioinformatics methods revealed the presence of ζ-CA in some eukaryotic and prokaryotic microorganisms. Notably, it has not been previously reported in prokaryotes. The coexistence of ß- and ζ-CAs in some microorganisms is also a novel finding as well. Also, our analysis identified several CA proteins with 6-14 amino acid intervals between histidine and cysteine in the second highly conserved motif, which can be classified as the novel ζ-CA subfamily members that emerged under the Zn deficiency of aquatic ecosystems and selection pressure in these environments. There is also a possibility that the achieved results are rooted in the contamination of samples from the environmental microbiome genome with genomes of diatom species and the occurrence of errors was observed in the DNA sequencing outcomes. Combining of all results from evolutionary analysis to quality control of ζ-CA DNA sequences is the incentive motivation to explore more the hidden aspects of ζ-CAs.

Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry.

Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome.

MAIN CONCLUSION: Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

Mutation of M13 Bacteriophage Major Coat Protein for Increased Conjugation to Exogenous Compounds.

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.

Visualizing calcium-dependent signaling networks in plants.

Calcium-dependent protein kinases (CDPKs) are a multigene protein kinase family that have key regulatory roles in plants. However, imaging CDPK signals in plant cells remains challenging. The recently developed genetically encoded CDPK-Förster resonance energy transfer (FRET) reporter developed by Liese et al. allows visualization of calcium (Ca2+)-dependent conformational changes during activation or inactivation of CDPKs, providing a powerful tool for real-time monitoring of calcium decoding in plants.

Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting.

Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.

Virus-induced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduces drought stress tolerance in Nicotiana benthamiana.

The role of pyrophosphate in primary metabolism is poorly understood. Here, we report on the transient down-regulation of plastid-targeted soluble inorganic pyrophosphatase in Nicotiana benthamiana source leaves. Physiological and metabolic perturbations were particularly evident in chloroplastic central metabolism, which is reliant on fast and efficient pyrophosphate dissipation. Plants lacking plastidial soluble inorganic pyrophosphatase (psPPase) were characterized by increased pyrophosphate levels, decreased starch content, and alterations in chlorophyll and carotenoid biosynthesis, while constituents like amino acids (except for histidine, serine, and tryptophan) and soluble sugars and organic acids (except for malate and citrate) remained invariable from the control. Furthermore, translation of Rubisco was significantly affected, as observed for the amounts of the respective subunits as well as total soluble protein content. These changes were concurrent with the fact that plants with reduced psPPase were unable to assimilate carbon to the same extent as the controls. Furthermore, plants with lowered psPPase exposed to mild drought stress showed a moderate wilting phenotype and reduced vitality, which could be correlated to reduced abscisic acid levels limiting stomatal closure. Taken together, the results suggest that plastidial pyrophosphate dissipation through psPPase is indispensable for vital plant processes.

Genetic manipulation of trehalose-6-phosphate synthase results in changes in the soluble sugar profile in transgenic sugarcane stems.

Trehalose is a non-reducing disaccharide widely distributed in nature. The trehalose biosynthetic intermediate, trehalose 6-phosphate (Tre6P) is an essential regulatory and signaling molecule involved in both regulation of carbon metabolism and photosynthesis. To investigate the effect of altered trehalose synthesis on sucrose accumulation in sugarcane (Saccharum spp. hybrid), we independently overexpressed the Escherichia coli otsA (trehalose-6-phosphate synthase; TPS) and otsB (trehalose-6-phosphate phosphatase; TPP) genes and additionally partially silenced native TPS expression. In mature cane, sucrose levels in the otsA transgenic plants were lowered, whereas sucrose levels in the otsB transgenic plants were increased. Partial silencing of TPS expression in sugarcane transformed with a TPS-targeted microRNA recombinant construct was confirmed in leaf and mature internode tissue of transgenic plants. Most of the silencing transgenic lines accumulated trehalose at lower levels than the wild-type (WT) plants. The immature stalk tissue of these transgenic lines had lower levels of glucose and fructose, whereas the mature internode tissue had lower sucrose and glucose levels, when compared with the WT. Furthermore, various minor metabolites and sugars were detected in the sugarcane plants, which mostly decreased as the stalk tissue of the cane matured. The results demonstrate that manipulation of Tre6P/trehalose metabolism has the potential to modify the profile of soluble sugars accumulated in sugarcane stems.

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato.

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.

Mutations in Glucan, Water Dikinase Affect Starch Degradation and Gametophore Development in the Moss Physcomitrella patens.

The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.

Starch Trek: The Search for Yield.

Starch is a plant storage polyglucan that accumulates in plastids. It is composed of two polymers, amylose and amylopectin, with different structures and plays several roles in helping to determine plant yield. In leaves, it acts as a buffer for night time carbon starvation. Genetically altered plants that cannot synthesize or degrade starch efficiently often grow poorly. There have been a number of successful approaches to manipulate leaf starch metabolism that has resulted in increased growth and yield. Its degradation is also a source of sugars that can help alleviate abiotic stress. In edible parts of plants, starch often makes up the majority of the dry weight constituting much of the calorific value of food and feed. Increasing starch in these organs can increase this as well as increasing yield. Enzymes involved in starch metabolism are well known, and there has been much research analyzing their functions in starch synthesis and degradation, as well as genetic and posttranslational regulatory mechanisms affecting them. In this mini review, we examine work on this topic and discuss future directions that could be used to manipulate this metabolite for improved yield.

Modification of Cassava Root Starch Phosphorylation Enhances Starch Functional Properties.

Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.

Repression of Sex4 and Like Sex Four2 Orthologs in Potato Increases Tuber Starch Bound Phosphate With Concomitant Alterations in Starch Physical Properties.

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.

Leaf starch degradation comes out of the shadows.

During the day, plants accumulate starch in their leaves as an energy source for the coming night. Based on recent findings, the prevailing view of how the transitory starch is remobilized needs considerable revision. Analyses of transgenic and mutant plants demonstrate that plastidic glucan phosphorylase is not required for normal starch breakdown and cast doubt on the presumed essential role of alpha-amylase but do show that beta-amylase is important. Repression of the activity of a plastidic beta-amylase, the export of its product (maltose) or further metabolism of maltose by a newly identified transglucosidase impairs starch degradation. Breakdown of particulate starch also depends on the activity of glucan-water dikinase, which phosphorylates glucosyl residues within the polymer.

Analysis of genes involved in glycogen degradation in Escherichia coli.

Escherichia coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly, analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen.