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Small regulatory RNAs (sRNAs) finely control gene expression in prokaryotes and synthetic sRNA has become a useful high-throughput approach to tackle current challenges in metabolic engineering because of its many advantages compared to conventional gene knockouts. In this review, we first focus on the modular structures of sRNAs and rational design strategies of synthetic sRNAs on the basis of their modular structures. The wide applications of synthetic sRNAs in bacterial metabolic engineering, with or without the aid of heterogeneously expressed Hfq protein, were also covered. In addition, we give attention to the improvements in implementing synthetic sRNAs, which make the synthetic sRNA strategy universally applicable in metabolic engineering and synthetic biology.Key points• Synthetic sRNAs can be rationally designed based on modular structures of natural sRNAs.• Synthetic sRNAs were widely used for metabolic engineering in various microorganisms.• Several technological improvements made the synthetic sRNA strategy more applicable.

Botrytis cinerea Pers. Fr. (teleomorph: Botryotinia fuckeliana) is a necrotrophic fungal pathogen that attacks a wide range of plants. This updated pathogen profile explores the extensive genetic diversity of B. cinerea, highlights the progress in genome sequencing, and provides current knowledge of genetic and molecular mechanisms employed by the fungus to attack its hosts. In addition, we also discuss recent innovative strategies to combat B. cinerea. Taxonomy Kingdom: Fungi, phylum: Ascomycota, subphylum: Pezizomycotina, class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae, genus: Botrytis, species: cinerea. Host range B. cinerea infects almost all of the plant groups (angiosperms, gymnosperms, pteridophytes, and bryophytes). To date, 1606 plant species have been identified as hosts of B. cinerea. Genetic diversity This polyphagous necrotroph has extensive genetic diversity at all population levels shaped by climate, geography, and plant host variation. Pathogenicity Genetic architecture of virulence and host specificity is polygenic using multiple weapons to target hosts, including secretory proteins, complex signal transduction pathways, metabolites, and mobile small RNA. Disease control strategies Efforts to control B. cinerea, being a high‐diversity generalist pathogen, are complicated. However, integrated disease management strategies that combine cultural practices, chemical and biological controls, and the use of appropriate crop varieties will lessen yield losses. Recently, studies conducted worldwide have explored the potential of small RNA as an efficient and environmentally friendly approach for combating grey mould. However, additional research is necessary, especially on risk assessment and regulatory frameworks, to fully harness the potential of this technology. Botrytis cinerea is a generalist fungal phytopathogen with high genetic diversity that utilizes diverse signalling cascades to infect a wide range of hosts.

Understanding the role of short-interfering RNA (siRNA) in diverse biological processes is of current interest and often approached through small RNA sequencing. However, analysis of these datasets is difficult due to the complexity of biological RNA processing pathways, which differ between species. Several properties like strand specificity, length distribution, and distribution of soft-clipped bases are few parameters known to guide researchers in understanding the role of siRNAs. We present RAPID, a generic eukaryotic siRNA analysis pipeline, which captures information inherent in the datasets and automatically produces numerous visualizations as user-friendly HTML reports, covering multiple categories required for siRNA analysis. RAPID also facilitates an automated comparison of multiple datasets, with one of the normalization techniques dedicated for siRNA knockdown analysis, and integrates differential expression analysis using DESeq2.

The aim of this study was to access the effect of HmsA, a 65-nt small regulatory RNA encoded by the pPCP1 plasmid, on virulence. Survival and the competition index were determined in mice infected with wild-type  and an deletion mutant. RNA-seq was used to identify HmsA-regulated genes. HmsA deletion enhanced virulence. However, there was no overlap between 18 upregulated genes associated with pathogenicity and potential direct HmsA targets, based on gene expression screening after HmsA-pulse overexpression. HmsA inhibits virulence, but this effect may be mediated by indirect effects on pathogenesis, iron homeostasis and/or other cellular processes.

The aim of this study was to access the effect of HmsA, a 65-nt small regulatory RNA encoded by the pPCP1 plasmid, on virulence. Survival and the competition index were determined in mice infected with wild-type  and an deletion mutant. RNA-seq was used to identify HmsA-regulated genes. HmsA deletion enhanced virulence. However, there was no overlap between 18 upregulated genes associated with pathogenicity and potential direct HmsA targets, based on gene expression screening after HmsA-pulse overexpression. HmsA inhibits virulence, but this effect may be mediated by indirect effects on pathogenesis, iron homeostasis and/or other cellular processes.

Background Mycoviruses are viruses that naturally infect and replicate in fungi. Aspergillus fumigatus, an opportunistic pathogen causing fungal lung diseases in humans and animals, was recently shown to harbour several different types of mycoviruses. A well-characterised defence against virus infection is RNA silencing. The A. fumigatus genome encodes essential components of the RNA silencing machinery, including Dicer, Argonaute and RNA-dependent RNA polymerase (RdRP) homologues. Active silencing of double-stranded (ds)RNA and the generation of small RNAs (sRNAs) has been shown for several mycoviruses and it is anticipated that a similar mechanism will be activated in A. fumigatus isolates infected with mycoviruses. Results To investigate the existence and nature of A. fumigatus sRNAs, sRNA-seq libraries of virus-free and virus-infected isolates were created using Scriptminer adapters and compared. Three dsRNA viruses were investigated: Aspergillus fumigatus partitivirus-1 (AfuPV-1, PV), Aspergillus fumigatus chrysovirus (AfuCV, CV) and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1, NK) which were selected because they induce phenotypic changes such as coloration and sectoring. The dsRNAs of all three viruses, which included two conventionally encapsidated ones PV and CV and one unencapsidated example NK, were silenced and yielded characteristic vsiRNAs together with co-incidental silencing of host fungal genes which shared sequence homology with the viral genomes. Conclusions Virus-derived sRNAs were detected and characterised in the presence of virus infection. Differentially expressed A. fumigatus microRNA-like (miRNA-like) sRNAs and small interfering RNAs (siRNAs) were detected and validated. Host sRNA loci which were differentially expressed as a result of virus infection were also identified. To our knowledge, this is the first study reporting the sRNA profiles of A. fumigatus isolates.

Background: Staphylococcus aureus is a major pathogen responsible for a variety of infections. It expresses a wide range of factors to precisely coordinate gene expression in response to the ever-changing conditions. Among them, regulatory RNAs appear as key players of post-transcriptional and translational regulations. Here, we investigated the role of Srn₉342, a sRNA candidate previously identified in a cluster of five srna genes in Newman strain.Results: We showed that Srn₉342 is expressed under two isoforms of different lengths (Srn₉342S and Srn₉342L) whose transcript levels are divergent as a function of growth phase with Srn₉342S being expressed at low cell-density, then being substituted by Srn₉342L at high cell-density. Using MS2-Affinity Purification Coupled with RNA Sequencing, we searched for RNA molecular partners of both Srn₉342S and Srn₉342L. Interestingly, we found that Srn₉342S was mainly bound to sRNAs whereas the expression of Srn₉342L led to the enrichment of mRNAs often linked with transport and metabolism. Among the sRNAs identified, the master regulator of virulence RNAIII appeared as an attractive partner. Using various constructs, we showed that the 5’ end of Srn₉342 specifically binds the 3’ end of RNAIII with high affinity in vitro, and that Srn₉342 mitigate RNAIII transcript level and stability. Finally, we report that the deletion of srn₉342 modulates the expression of the RNAIII encoded toxin δ-hemolysin and hemolytic activity, suggesting that the binding of Srn₉342 onto RNAIII may induce structural changes of RNAIII, and hence expression of the toxin.Conclusions: Overall, we showed that Srn₉342 has an unusual pattern of expression and that uncovering its targetome suggests a potential role in S. aureus virulence.

To explore the complete gene networks regulated by small RNA SprC and its targets in . The isobaric tags for relative and absolute quantitation and bioinformatic methods were utilized to identify and analyze the target proteins affected by SprC in N315. Proteomic analysis showed that the expression of 44 proteins was modulated by SprC. Further, bioinformatic analysis displayed that these affected proteins mainly associated with metabolic and cellular process, biological regulation and catalytic activity. Our data provide a rich resource of SprC targets in , although the mechanism of regulation by SprC is yet to be elucidated.

Abstract Small RNAs (sRNAs) are important gene regulators in bacteria, but it is unclear how new sRNAs originate and become part of regulatory networks that coordinate bacterial response to environmental stimuli. Using a covariance modeling-based approach, we analyzed the presence of hundreds of sRNAs in more than a thousand genomes across Enterobacterales, a bacterial order with a confluence of factors that allows robust genome-scale sRNA analyses: several well-studied organisms with fairly conserved genome structures, an established phylogeny, and substantial nucleotide diversity within a narrow evolutionary space. We discovered that a majority of sRNAs arose recently, and uncovered protein-coding genes as a potential source from which new sRNAs arise. A detailed investigation of the emergence of OxyS, a peroxide-responding sRNA, revealed that it evolved from a fragment of a peroxidase messenger RNA. Importantly, although it replaced the ancestral peroxidase, OxyS continues to be part of the ancestral peroxide-response regulon, indicating that an sRNA that arises from a protein-coding gene would inherently be part of the parental protein’s regulatory network. This new insight provides a fresh framework for understanding sRNA origin and regulatory integration in bacteria.

MicroRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) play vital regulatory roles in plant growth and development. Little is known about these small RNAs in litchi (Litchi chinensis), an economically important fruit crop widely cultivated in Southeast Asia. We profiled the litchi small RNA population with various deep-sequencing techniques and in-depth bioinformatic analyses. The genome-wide identification of miRNAs, their target genes, and phasiRNA-generating (PHAS) genes/loci showed that the function of miR482/2118 has expanded, relative to its canonical function. We also discovered that, for 29 PHAS loci, miRNA-mediated phasiRNA production was coupled with alternative splicing (AS) and alternative polyadenylation (APA). Most of these loci encoded long noncoding RNAs. An miR482/2118 targeted locus gave rise to four main transcript isoforms through AS/APA, and diverse phasiRNAs generated from these isoforms appeared to target long terminal repeat (LTR) retrotransposons and other unrelated genes. This coupling enables phasiRNA production from different exons of noncoding PHAS genes and yields diverse phasiRNA populations, both broadening and altering the range of downstream phasiRNA-regulated genes. Our results reveal the diversity of miRNA and phasiRNA in litchi, and demonstrate AS/APA as a new layer of regulation in small RNA-mediated gene silencing.