We have explored the importance of the phyllosphere microbiome in plant resistance in the cuticle mutants bdg (BODYGUARD) or lacs2.3 (LONG CHAIN FATTY ACID SYNTHASE 2) that are strongly resistant to ...the fungal pathogen Botrytis cinerea.
The study includes infection of plants under sterile conditions, 16S ribosomal DNA sequencing of the phyllosphere microbiome, and isolation and high coverage sequencing of bacteria from the phyllosphere.
When inoculated under sterile conditions bdg became as susceptible as wild-type (WT) plants whereas lacs2.3 mutants retained the resistance. Adding washes of its phyllosphere microbiome could restore the resistance of bdg mutants, whereas the resistance of lacs2.3 results from endogenous mechanisms. The phyllosphere microbiome showed distinct populations in WT plants compared to cuticle mutants. One species identified as Pseudomonas sp isolated from the microbiome of bdg provided resistance to B. cinerea on Arabidopsis thaliana as well as on apple fruits. No direct activity was observed against B. cinerea and the action of the bacterium required the plant.
Thus, microbes present on the plant surface contribute to the resistance to B. cinerea. These results open new perspectives on the function of the leaf microbiome in the protection of plants.
In eukaryotes, cell-to-cell communication relies on the activity of small signaling peptides. In plant genomes, many hundreds of genes encode for such short peptide signals. However, only few of them ...are functionally characterized and due to the small gene size and high sequence variability, the comprehensive identification of such peptide-encoded genes is challenging. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family encodes for short peptides that have a role in plant meristem maintenance, vascular patterning and responses to environment. The full repertoire of CLE genes and the role of CLE signaling in tomato (Solanum lycopersicum)- one of the most important crop plants- has not yet been fully studied.
By using a combined approach, we performed a genome-wide identification of CLE genes using the current tomato genome version SL 4.0. We identified 52 SlCLE genes, including 37 new non annotated before. By analyzing publicly available RNAseq datasets we could confirm the expression of 28 new SlCLE genes. We found that SlCLEs are often expressed in a tissue-, organ- or condition-specific manner. Our analysis shows an interesting gene diversification within the SlCLE family that seems to be a result of gene duplication events. Finally, we could show a biological activity of selected SlCLE peptides in the root growth arrest that was SlCLV2-dependent.
Our improved combined approach revealed 37 new SlCLE genes. These findings are crucial for better understanding of the CLE signaling in tomato. Our phylogenetic analysis pinpoints the closest homologs of Arabidopsis CLE genes in tomato genome and can give a hint about the function of newly identified SlCLEs. The strategy described here can be used to identify more precisely additional short genes in plant genomes. Finally, our work suggests that the mechanism of root-active CLE peptide perception is conserved between Arabidopsis and tomato. In conclusion, our work paves the way to further research on the CLE-dependent circuits modulating tomato development and physiological responses.
Bacterial Toxin-Antitoxin systems (TAS) are involved in key biological functions including plasmid maintenance, defense against phages, persistence and virulence. They are found in nearly all phyla ...and classified into 6 different types based on the mode of inactivation of the toxin, with the type II TAS being the best characterized so far. We have herein developed a new in silico discovery pipeline named TASmania, which mines the >41K assemblies of the EnsemblBacteria database for known and uncharacterized protein components of type I to IV TAS loci. Our pipeline annotates the proteins based on a list of curated HMMs, which leads to >2.106 loci candidates, including orphan toxins and antitoxins, and organises the candidates in pseudo-operon structures in order to identify new TAS candidates based on a guilt-by-association strategy. In addition, we classify the two-component TAS with an unsupervised method on top of the pseudo-operon (pop) gene structures, leading to 1567 "popTA" models offering a more robust classification of the TAs families. These results give valuable clues in understanding the toxin/antitoxin modular structures and the TAS phylum specificities. Preliminary in vivo work confirmed six putative new hits in Mycobacterium tuberculosis as promising candidates. The TASmania database is available on the following server https://shiny.bioinformatics.unibe.ch/apps/tasmania/.
Human identification has played a prominent role in forensic science for the past two decades. Identification based on unique genetic traits is driving the field. However, this may have limitations, ...for instance, for twins. Moreover, high-throughput sequencing techniques are now available and may provide a high amount of data likely useful in forensic science.
This study investigates the potential for bacteria found in the salivary microbiome to be used to differentiate individuals. Two different targets (16S rRNA and rpoB) were chosen to maximise coverage of the salivary microbiome and when combined, they increase the power of differentiation (identification). Paired-end Illumina high-throughput sequencing was used to analyse the bacterial composition of saliva from two different people at four different time points (t = 0 and t = 28 days and then one year later at t = 0 and t = 28 days). Five major phyla dominate the samples: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria. Streptococcus, a Firmicutes, is one of the most abundant aerobic genera found in saliva and targeting Streptococcus rpoB has enabled a deeper characterisation of the different streptococci species, which cannot be differentiated using 16S rRNA alone. We have observed that samples from the same person group together regardless of time of sampling. The results indicate that it is possible to distinguish two people using the bacterial microbiota present in their saliva.
In order to thrive in constantly changing environments, animals must adaptively respond to threatening events. Noxious stimuli are not only processed according to their absolute intensity, but also ...to their context. Adaptation processes can cause animals to habituate at different rates and degrees in response to permanent or repeated stimuli. Here, we used a forward genetic approach in Caenorhabditis elegans to identify a neuropeptidergic pathway, essential to prevent fast habituation and maintain robust withdrawal responses to repeated noxious stimuli. This pathway involves the FRPR-19A and FRPR-19B G-protein coupled receptor isoforms produced from the frpr-19 gene by alternative splicing. Loss or overexpression of each or both isoforms can impair withdrawal responses caused by the optogenetic activation of the polymodal FLP nociceptor neuron. Furthermore, we identified FLP-8 and FLP-14 as FRPR-19 ligands in vitro. flp-14, but not flp-8, was essential to promote withdrawal response and is part of the same genetic pathway as frpr-19 in vivo. Expression and cell-specific rescue analyses suggest that FRPR-19 acts both in the FLP nociceptive neurons and downstream interneurons, whereas FLP-14 acts from interneurons. Importantly, genetic impairment of the FLP-14/FRPR-19 pathway accelerated the habituation to repeated FLP-specific optogenetic activation, as well as to repeated noxious heat and harsh touch stimuli. Collectively, our data suggest that well-adjusted neuromodulation via the FLP-14/FRPR-19 pathway contributes to promote nociceptive signals in C. elegans and counteracts habituation processes that otherwise tend to rapidly reduce aversive responses to repeated noxious stimuli.
Comparative genomics has seen the development of many software performing the clustering, polymorphism and gene content analysis of genomes at different phylogenetic levels (isolates, species). These ...tools rely on de novo assembly and/or multiple alignments that can be computationally intensive for large datasets. With a large number of similar genomes in particular, e.g., in surveillance and outbreak detection, assembling each genome can become a redundant and expensive step in the identification of genes potentially involved in a given clinical feature.
We have developed deltaRpkm, an R package that performs a rapid differential gene presence evaluation between two large groups of closely related genomes. Starting from a standard gene count table, deltaRpkm computes the RPKM per gene per sample, then the inter-group δRPKM values, the corresponding median δRPKM (m) for each gene and the global standard deviation value of m (s
). Genes with m > = 2 ∗ s
(standard deviation s of all the m values) are considered as "differentially present" in the reference genome group. Our simple yet effective method of differential RPKM has been successfully applied in a recent study published by our group (N = 225 genomes of Listeria monocytogenes) (Aguilar-Bultet et al. Front Cell Infect Microbiol 8:20, 2018).
To our knowledge, deltaRpkm is the first tool to propose a straightforward inter-group differential gene presence analysis with large datasets of related genomes, including non-coding genes, and to output directly a list of genes potentially involved in a phenotype.
Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of ...Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo. mRNA cleavage by the toxin occurs after the second nucleotide of the ribosomal A-site codon during translation, with a strong preference for CCA codons in vivo. Finally, we report the cryo-EM structure of the ribosome-bound TAC toxin in the presence of native M. tuberculosis cspA mRNA, revealing the specific mechanism by which the TAC toxin interacts with the ribosome and the tRNA in the P-site to cleave its mRNA target.
During eukaryotic 60S biogenesis, the 5S RNP requires a large rotational movement to achieve its mature position. Cryo-EM of the Rix1-Rea1 pre-60S particle has revealed the post-rotation stage, in ...which a gently undulating α-helix corresponding to Cgr1 becomes wedged between Rsa4 and the relocated 5S RNP, but the purpose of this insertion was unknown. Here, we show that cgr1 deletion in yeast causes a slow-growth phenotype and reversion of the pre-60S particle to the pre-rotation stage. However, spontaneous extragenic suppressors could be isolated, which restore growth and pre-60S biogenesis in the absence of Cgr1. Whole-genome sequencing reveals that the suppressor mutations map in the Rpf2-Rrs1 module and Rpl5, which together stabilize the unrotated stage of the 5S RNP. Thus, mutations in factors stabilizing the pre-rotation stage facilitate 5S RNP relocation upon deletion of Cgr1, but Cgr1 itself could stabilize the post-rotation stage.
Bacteria communicate by exchanging chemical signals, some of which are volatile and can remotely reach other organisms. HCN was one of the first volatiles discovered to severely impact exposed ...organisms by inhibiting their respiration. Using HCN-deficient mutants in two
strains, we demonstrate that HCN's impact goes beyond the sole inhibition of respiration and affects both emitting and receiving bacteria in a global way, modulating their motility, biofilm formation, and production of antimicrobial compounds. Our data suggest that bacteria could use HCN not only to control their own cellular functions, but also to remotely influence the behavior of other bacteria sharing the same environment. Since HCN emission occurs in both clinically and environmentally relevant
, these findings are important to better understand or even modulate the expression of bacterial traits involved in both virulence of opportunistic pathogens and in biocontrol efficacy of plant-beneficial strains.
The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and ...urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.
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