Circadian rhythms in the plant host influence rhythmicity of rhizosphere microbiota.

BACKGROUND: Recent studies demonstrated that microbiota inhabiting the plant rhizosphere exhibit diel changes in abundance. To investigate the impact of plant circadian rhythms on bacterial and fungal rhythms in the rhizosphere, we analysed temporal changes in fungal and bacterial communities in the rhizosphere of Arabidopsis plants overexpressing or lacking function of the circadian clock gene LATE ELONGATED HYPOCOTYL (LHY). RESULTS: Under diel light-dark cycles, the knock-out mutant lhy-11 and the gain-of-function mutant lhy-ox both exhibited gene expression rhythms with altered timing and amplitude compared to wild-type plants. Distinct sets of bacteria and fungi were found to display rhythmic changes in abundance in the rhizosphere of both of these mutants, suggesting that abnormal patterns of rhythmicity in the plant host caused temporal reprogramming of the rhizosphere microbiome. This was associated with changes in microbial community structure, including changes in the abundance of fungal guilds known to impact on plant health. Under constant environmental conditions, microbial rhythmicity persisted in the rhizosphere of wild-type plants, indicating control by a circadian oscillator. In contrast, loss of rhythmicity in lhy-ox plants was associated with disrupted rhythms for the majority of rhizosphere microbiota. CONCLUSIONS: These results show that aberrant function of the plant circadian clock is associated with altered rhythmicity of rhizosphere bacteria and fungi. In the long term, this leads to changes in composition of the rhizosphere microbiome, with potential consequences for plant health. Further research will be required to understand the functional implications of these changes and how they impact on plant health and productivity.

Plant circadian clock control of Medicago truncatula nodulation via regulation of nodule cysteine-rich peptides.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules, we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcription factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of nodule-specific cysteine-rich peptides (NCRs) that have the LHY-bound conserved evening element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.

Comparative Genomics across Three Ensifer Species Using a New Complete Genome Sequence of the Medicago Symbiont Sinorhizobium (Ensifer) meliloti WSM1022.

Here, we report an improved and complete genome sequence of Sinorhizobium (Ensifer) meliloti strain WSM1022, a microsymbiont of Medicago species, revealing its tripartite structure. This improved genome sequence was generated combining Illumina and Oxford nanopore sequencing technologies to better understand the symbiotic properties of the bacterium. The 6.75 Mb WSM1022 genome consists of three scaffolds, corresponding to a chromosome (3.70 Mb) and the pSymA (1.38 Mb) and pSymB (1.66 Mb) megaplasmids. The assembly has an average GC content of 62.2% and a mean coverage of 77X. Genome annotation of WSM1022 predicted 6058 protein coding sequences (CDSs), 202 pseudogenes, 9 rRNAs (3 each of 5S, 16S, and 23S), 55 tRNAs, and 4 ncRNAs. We compared the genome of WSM1022 to two other rhizobial strains, closely related Sinorhizobium (Ensifer) meliloti Sm1021 and Sinorhizobium (Ensifer) medicae WSM419. Both WSM1022 and WSM419 species are high-efficiency rhizobial strains when in symbiosis with Medicago truncatula, whereas Sm1021 is ineffective. Our findings report significant genomic differences across the three strains with some similarities between the meliloti strains and some others between the high efficiency strains WSM1022 and WSM419. The addition of this high-quality rhizobial genome sequence in conjunction with comparative analyses will help to unravel the features that make a rhizobial symbiont highly efficient for nitrogen fixation.

Tree phyllospheres are a habitat for diverse populations of CO-oxidizing bacteria.

Carbon monoxide (CO) is both a ubiquitous atmospheric trace gas and an air pollutant. While aerobic CO-degrading microorganisms in soils and oceans are estimated to remove ~370 Tg of CO per year, the presence of CO-degrading microorganisms in above-ground habitats, such as the phyllosphere, and their potential role in CO cycling remains unknown. CO-degradation by leaf washes of two common British trees, Ilex aquifolium and Crataegus monogyna, demonstrated CO uptake in all samples investigated. Based on the analyses of taxonomic and functional genes, diverse communities of candidate CO-oxidizing taxa were identified, including members of Rhizobiales and Burkholderiales which were abundant in the phyllosphere at the time of sampling. Based on predicted genomes of phyllosphere community members, an estimated 21% of phyllosphere bacteria contained CoxL, the large subunit of CO-dehydrogenase. In support of this, data mining of publicly available phyllosphere metagenomes for genes encoding CO-dehydrogenase subunits demonstrated that, on average, 25% of phyllosphere bacteria contained CO-dehydrogenase gene homologues. A CO-oxidizing Phyllobacteriaceae strain was also isolated from phyllosphere samples which contains genes encoding both CO-dehydrogenase as well as a ribulose-1,5-bisphosphate carboxylase-oxygenase. These results suggest that the phyllosphere supports diverse and potentially abundant CO-oxidizing bacteria, which are a potential sink for atmospheric CO.

Identification of microbial signatures linked to oilseed rape yield decline at the landscape scale.

BACKGROUND: The plant microbiome plays a vital role in determining host health and productivity. However, we lack real-world comparative understanding of the factors which shape assembly of its diverse biota, and crucially relationships between microbiota composition and plant health. Here we investigated landscape scale rhizosphere microbial assembly processes in oilseed rape (OSR), the UK's third most cultivated crop by area and the world's third largest source of vegetable oil, which suffers from yield decline associated with the frequency it is grown in rotations. By including 37 conventional farmers' fields with varying OSR rotation frequencies, we present an innovative approach to identify microbial signatures characteristic of microbiomes which are beneficial and harmful to the host. RESULTS: We show that OSR yield decline is linked to rotation frequency in real-world agricultural systems. We demonstrate fundamental differences in the environmental and agronomic drivers of protist, bacterial and fungal communities between root, rhizosphere soil and bulk soil compartments. We further discovered that the assembly of fungi, but neither bacteria nor protists, was influenced by OSR rotation frequency. However, there were individual abundant bacterial OTUs that correlated with either yield or rotation frequency. A variety of fungal and protist pathogens were detected in roots and rhizosphere soil of OSR, and several increased relative abundance in root or rhizosphere compartments as OSR rotation frequency increased. Importantly, the relative abundance of the fungal pathogen Olpidium brassicae both increased with short rotations and was significantly associated with low yield. In contrast, the root endophyte Tetracladium spp. showed the reverse associations with both rotation frequency and yield to O. brassicae, suggesting that they are signatures of a microbiome which benefits the host. We also identified a variety of novel protist and fungal clades which are highly connected within the microbiome and could play a role in determining microbiome composition. CONCLUSIONS: We show that at the landscape scale, OSR crop yield is governed by interplay between complex communities of both pathogens and beneficial biota which is modulated by rotation frequency. Our comprehensive study has identified signatures of dysbiosis within the OSR microbiome, grown in real-world agricultural systems, which could be used in strategies to promote crop yield. Video abstract.

Gene Expression Associated with Early and Late Chronotypes in Drosophila melanogaster.

The circadian clock provides the temporal framework for rhythmic behavioral and metabolic functions. In the modern era of industrialization, work, and social pressures, clock function is jeopardized, and can result in adverse and chronic effects on health. Understanding circadian clock function, particularly individual variation in diurnal phase preference (chronotype), and the molecular mechanisms underlying such chronotypes may lead to interventions that could abrogate clock dysfunction and improve human (and animal) health and welfare. Our preliminary studies suggested that fruit-flies, like humans, can be classified as early rising "larks" or late rising "owls," providing a convenient model system for these types of studies. We have identified strains of flies showing increased preference for morning emergence (Early or E) from the pupal case, or more pronounced preference for evening emergence (Late or L). We have sampled pupae the day before eclosion (fourth day after pupariation) at 4 h intervals in the E and L strains, and examined differences in gene expression by RNA-seq. We have identified differentially expressed transcripts between the E and L strains, which provide candidate genes for subsequent studies of Drosophila chronotypes and their human orthologs.

Evolutionary analysis of regulatory sequences (EARS) in plants.

Identification of regulatory sequences within non-coding regions of DNA is an essential step towards elucidation of gene networks. This approach constitutes a major challenge, however, as only a very small fraction of non-coding DNA is thought to contribute to gene regulation. The mapping of regulatory regions traditionally involves the laborious construction of promoter deletion series which are then fused to reporter genes and assayed in transgenic organisms. Bioinformatic methods can be used to scan sequences for matches for known regulatory motifs, however these methods are currently hampered by the relatively small amount of such motifs and by a high false-discovery rate. Here, we demonstrate a robust and highly sensitive, in silico method to identify evolutionarily conserved regions within non-coding DNA. Sequence conservation within these regions is taken as evidence for evolutionary pressure against mutations, which is suggestive of functional importance. We test this method on a small set of well characterised promoters, and show that it successfully identifies known regulatory regions. We further show that these evolutionarily conserved sequences contain clusters of transcription binding sites, often described as regulatory modules. A version of the tool optimised for the analysis of plant promoters is available online at http://wsbc.warwick.ac.uk/ears/main.php.

Evolutionarily conserved regulatory motifs in the promoter of the Arabidopsis clock gene LATE ELONGATED HYPOCOTYL.

The transcriptional regulation of the LATE ELONGATED HYPOCOTYL (LHY) gene is key to the structure of the circadian oscillator, integrating information from multiple regulatory pathways. We identified a minimal region of the LHY promoter that was sufficient for rhythmic expression. Another upstream sequence was also required for appropriate waveform of transcription and for maximum amplitude of oscillations under both diurnal and free-running conditions. We showed that two classes of protein complexes interact with a G-box and with novel 5A motifs; mutation of these sites reduced the amplitude of oscillation and broadened the peak of expression. A genome-wide bioinformatic analysis showed that these sites were enriched in phase-specific clusters of rhythmically expressed genes. Comparative genomic analyses showed that these motifs were conserved in orthologous promoters from several species. A position-specific scoring matrix for the 5A sites suggested similarity to CArG boxes, which are recognized by MADS box transcription factors. In support of this, the FLOWERING LOCUS C (FLC) protein was shown to interact with the LHY promoter in planta. This suggests a mechanism by which FLC might affect circadian period.