Biology subjects

Kalinowski, J.

Publications and source records attributed to Kalinowski, J..

At least 19 recordsLinked to original sources

bioRxiv2025

Tyrosine availability shapes Staphylococcus aureus nasal colonization and interactions with commensal communities

Only certain individuals are nasally colonized by Staphylococcus aureus, making them more susceptible to developing endogenous infections. The role of the resident microbiota in modulating S. aureus nasal colonization remains largely unclear. Using a newly assembled nasal strain collection, we demonstrate that nasal commensals can either promote or inhibit S. aureus proliferation through strain- and community-specific effects in vitro and within gnotobiotic animals. We identified nutrient-dependent interactions as a key driver of these dynamics and found that S. aureus strains auxotrophic to tyrosine rely on amino acid-providing nasal communities to thrive in the nutrient-limited environment of the nose. Large-scale screenings revealed tyrosine dependency to affect 8% of nasal S. aureus strains and to impair S. aureus colonization. Our findings uncover a crucial factor in S. aureus physiology and its interactions as a commensal nasal species, paving the way for new decolonization strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/651429v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@17b6289org.highwire.dtl.DTLVardef@182249forg.highwire.dtl.DTLVardef@1324dc1org.highwire.dtl.DTLVardef@18ec3bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Chromosomal rearrangements and instability caused by the LINE-1 retrotransposon

LINE-1 (L1) retrotransposition is widespread in many cancers, especially those with a high burden of chromosomal rearrangements. However, whether and to what degree L1 activity directly impacts genome integrity is unclear. Here, we apply whole-genome sequencing to experimental models of L1 expression to comprehensively define the spectrum of genomic changes caused by L1. Combining the analyses of experimental models of L1 induction and of cancer genomes, we demonstrate that L1 retrotransposition can directly generate reciprocal translocations, genomic DNA inversions, and foldback rearrangements resulting from illegitimate recombination of double-strand DNA ends generated by L1-encoded ORF2p. We further show that L1-induced rearrangements can produce unstable chromosomes that fuel the acquisition of complex rearrangements, large segmental copy-number alterations, and genetic heterogeneity through breakage-fusion bridge cycles or DNA fragmentation. Together, these findings suggest L1 as a potent mutagenic force capable of driving genome evolution in cancers.

genomics↗

bioRxiv2024

High-resolution mapping of Sigma Factor DNA Binding Sequences using Artificial Promoters, RNA Aptamers and Deep Sequencing

The variable sigma ({sigma}) subunit of the bacterial RNA polymerase holoenzyme determines promoter specificity and facilitate open complex formation during transcription initiation. Understanding {sigma}-factor binding sequences is therefore crucial for deciphering bacterial gene regulation. Here, we present a data-driven high-throughput approach that utilizes an extensive library of 1.54 million DNA templates providing artificial promoters and 5' UTR sequences for {sigma}-factor DNA binding motif discovery. This method combines the generation of extensive DNA libraries, in vitro transcription, RNA aptamer selection, and deep DNA and RNA sequencing. It allows direct assessment of promoter activity, identification of transcription start sites, and quantification of promoter strength based on mRNA production levels. We applied this approach to map {sigma}54 DNA binding sequences in Pseudomonas putida. Deep sequencing of the enriched RNA pool revealed 64,966 distinct {sigma}54 binding motifs, significantly expanding the known repertoire. This data-driven approach surpasses traditional methods by directly evaluating promoter function and avoiding selection bias based solely on binding affinity. This comprehensive dataset enhances our understanding of {sigma}-factor binding sequences and their regulatory roles, opening avenues for new research in biology and biotechnology.

biochemistry↗

bioRxiv2024

Role of a single MCP in evolutionary adaptation of Shewanella putrefaciens for swimming in planktonic and structured environments

Bacteria can adapt to their environments by changing phenotypic traits by mutations. However, improving one trait often results in deterioration of another one, a trade-off which limits the degree of adaption. The gammaproteobacterium Shewanella putrefaciens CN-32 has an elaborate motility machinery comprising two distinct flagellar systems and an extensive chemotaxis array with 36 methyl-accepting chemotaxis sensor proteins (MCPs). In this study we performed experimental selection on S. putrefaciens for increased spreading through a porous environment. We readily obtained a mutant that showed a pronounced increase in covered distance. This phenotype was almost completely caused by a deletion of 24 bp from the chromosome, which leads to a moderately enhanced production of a single MCP. Accordingly, chemotaxis assays under planktonic conditions and cell tracking in soft agar showed that the mutation improved navigation through nutritional gradients. The study demonstrates how differences in the abundance of a single MCP can lead to an efficient upgrade of directed flagella-mediated motility in specific environments at a low expense of cellular resources. ImportanceExperimental evolution experiments have been used to determine the trade-offs occurring in specific environments. Several studies that have used the spreading behavior of bacteria in structured environments identified regulatory mutants that increase the swimming speed of the cells. While this results in a higher chemotaxis drift, the growth fitness decreases as the higher swimming speed requires substantial cellular resources. Here we show that rapid chemotaxis adaptation can also be achieved through modification of the chemotaxis signal input at a low metabolic cost for the cell.

microbiology↗

bioRxiv2024

Discovery of a pentose as a cytosine nucleobase modification in Shewanella phage Thanatos-1 genomic DNA mediating enhanced resistance towards host restriction systems

Co-evolution of bacterial defense systems and phage counter defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. To evade nuclease-based mechanisms targeting the DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits recognition by endonucleases. We found that Shewanella phage Thanatos-1 DNA is insensitive to multiple restriction enzymes and, partially, also to Cas I-Fv and Cas9 cleavage. Furthermore, the phage genome shows strongly impaired basecalling with nanopore sequencing. We characterised the phage adenine methyltransferase TH1_126 in methylase-free E. coli ER3413 and derived and confirmed its recognition motif 5-ATC-3. Moreover, the data pointed to an additional, much more substantial nucleobase modification. Using LC-MS, we identified a deoxypentose of unknown configuration attached to cytosine as a yet undiscovered phage DNA modification, which is present in Thanatos-1 genomic DNA, likely mediates the observed resistance to restriction endonucleases, as well as a strong reduction in Cas nuclease activity. To elucidate the underlying enzyme functions, we determined structural homologs of Thanatos-1 proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of phage protein TH1_063 by in vitro enzyme assays.

molecular biology↗

bioRxiv2024

5'-untranslated region sequences enhance plasmid-based protein production in Sulfolobus acidocaldarius

Sulfolobus acidocaldarius, a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of the saci_2122 promoter (Para), providing low basal activity and high pentose-dependent induction. However, available expression vectors lack a 5-terminal untranslated region (5-UTR), which is a typical element in bacterial expression vectors, usually significantly enhancing protein production in bacteria. To establish S. acidocaldarius as a production strain in biotechnology in the long-term, it is intrinsically relevant to optimize its tools and capacities to increase production efficiencies. Here we show that protein production is increased by the integration of S. acidocaldarius 5-UTRs into Para expression plasmids. Using the esterase Saci_1116 as a reporter protein, we observed a fourfold increase in soluble and active protein yield upon insertion of the saci_1322 (alba) 5-UTR. Screening of four additional 5-UTRs from other highly abundant proteins (th, slaA, slaB, saci_0330) revealed a consistent enhancement in target protein production. Additionally, site-directed mutagenesis of the Shine-Dalgarno (SD) motif within the alba 5-UTR revealed its significance for protein synthesis. Ultimately, the alba 5-UTR optimized expression vector demonstrated successful applicability in expressing various proteins, exemplified by its utilization for archaeal glycosyltransferases. Our results demonstrate that the integration of SD-motif containing 5-UTRs significantly boosted plasmid-based protein production in S. acidocaldarius. This advancement in recombinant expression not only broadens the utility of S. acidocaldarius as an archaeal expression platform but also marks a significant step toward potential biotechnological applications.

molecular biology↗

bioRxiv2024

Glycerol degradation in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius involves an unusual glycerol-3-phosphate dehydrogenase

Glycerol is highly abundant in nature and serve as carbon source for many organisms. Also, several Archaea have the genetic capacity to grow on glycerol but its degradation has so far only been studied Haloferax volcanii. Herein, the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius was shown to grow with glycerol as sole carbon and energy source. After uptake likely involving facilitated diffusion, glycerol is degraded via phosphorylation to glycerol-3-phosphate followed by oxidation to dihydroxyacetone phosphate (DHAP) catalyzed by glycerol kinase (GK) by an unusual quinone reducing FAD-dependent glycerol-3-phosphate dehydrogenase (G3PDH), respectively. The S. acidocaldarius genome harbors two paralogous copies of each GK and G3PDH. However, only one of these GK-G3PDH couples (Saci_2031-2033) is highly upregulated on glycerol. Deletion of the saci_2033 gene encoding GK abolished growth on glycerol and GK activity in crude extracts. In contrast, deletion of the second GK gene (saci_1117) had only minor effects indicating that only one of the two GK-G3PDH couples is essential. Biochemical characterization revealed that both isoenzymes of each, GK and G3PDH, were functionally similar. Whereas the GKs showed high similarity to known enzymes from Bacteria and Eukaryotes, the G3PDHs represent unusual homologues of the bacterial GlpA subunit of the GlpABC complex with remarkable C-terminal sequence differences and a novel type of membrane anchoring via a CoxG-like protein (Saci_2031). Further sequence analyzes discovered a higher versatility of G3PDHs in Archaea with respect to interacting proteins, electron transfer, and membrane anchoring likely reflecting tailored evolutionary solutions to meet different requirements caused by life styles and electron acceptors.

microbiology↗

bioRxiv2023

A rolling circle-replicating plasmid as an Inovirus phage satellite

Bacterial viruses (phages) are potent agents of lateral gene transfer and thus are important drivers of evolution. A group of mobile genetic elements (MGEs), referred to as phage satellites, exploit phages to disseminate their own genetic material. Here we isolated a novel member of the genus Inovirus, Shewanella phage Dolos, along with an autonomous rolling circle-replicating plasmid, pDolos. Dolos causes a chronic infection in its host Shewanella oneidensis by phage production with only minor effects on the host cell proliferation. When present, plasmid pDolos hijacks Dolos functions to be predominantly packaged into phage virions and released into the environment. pDolos can disseminate further genetic material encoding, e.g., resistances, fluorophores, and metabolically active proteins, to host cells sensitive to Dolos infection. Given the rather simple requirements of a plasmid for takeover of an inovirus, the wide distribution of phages of this group and the broad spectrum of rolling circle-replicating plasmids, we speculate that similar phage-satellite systems are common among bacteria.

microbiology↗

bioRxiv2023

Deletion of SMC renders FtsK essential in Corynebacterium glutamicum

Structural maintenance of chromosomes (SMC) are ubiquitously distributed proteins involved in chromosome organization. Deletion of smc causes severe growth phenotypes in many organisms. Surprisingly, smc can be deleted in Corynebacterium glutamicum, a member of the Actinomycetota phylum, without any apparent growth phenotype. Earlier work has shown that SMC in C. glutamicum is loaded in a ParB-dependent fashion to the chromosome and functions in replichore cohesion. The unexpected absence of a growth phenotype in the smc mutant prompted us to screen for unknown synthetic interactions within C. glutamicum. Therefore, we generated a high-density Tn-5 library based on wild-type and smc-deleted C. glutamicum strains. The transposon sequencing (Tn-seq) data revealed that the DNA-translocase FtsK is essential in a smc deletion strain. FtsK localized to the septa and cell poles in wild type cells, however deletion of smc resulted in a decreased polar FtsK localization. Single-particle tracking analysis further suggests that prolonged FtsK complex activity is both required and sufficient to make up for the absence of SMC, thus achieving efficient chromosome segregation in C. glutamicum. Further, single molecule dynamics of FtsK is influenced, albeit indirectly, by DNA-loaded SMC. Deletion of ParB results in an increased of both SMC and FtsK mobility. While the first change agrees with previous data that show how ParB is essential for SMC loading on DNA, the latter suggests that FtsK mobility is affected in cells with defects in chromosome organization. Based on our data we propose a simple, yet efficient mechanism for efficient DNA segregation in C. glutamicum, even in absence of SMC proteins. ImportanceFaithful DNA segregation is of fundamental importance for life. Bacteria have efficient systems to coordinate chromosome compaction, DNA segregation and cell division. A key factor in DNA compaction is the SMC-complex that is found to be essential in many bacteria. In members of the Actinomycetota smc is dispensable, but the reason for this was unclear. We show here that the divisome associated DNA-pump FtsK can compensate SMC loss and the subsequent loss in correct chromosome organization. In cells with distorted chromosomes, FtsK functions for an extended period of time at the septum, until chromosomes are segregated.

microbiology↗

bioRxiv2023

Head and neck cancer cells terminally differentiate reflecting their tissue of origin: a rationale for differentiation therapies

BackgroundHuman papillomavirus-negative head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore, new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have striking tumor-targeting potential. MethodsWe modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by RNA-seq and ATAC-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. ResultsHNSCC cell differentiation was accompanied by cell adhesion, proliferation stop, diminished tumor-initiating potential in immunodeficient mice, and activation of a wound healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem-cell marker in normal mucosa, we confirm KRT17 to represent an early differentiation marker in HNSCC tissue and dysplastic mucosa. Cornification was observed to frequently surround necrotic and immune-infiltrated areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Indeed, inflammatory mediators were found to activate the HNSCC cell differentiation program. ConclusionsDistinct cell differentiation states create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon HNSCC cell differentiation, indicating that targeted differentiation approaches may be therapeutically valuable. Moreover, we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection.

cancer biology↗

bioRxiv2023

Long-read Sequencing Shows Complex Structural Variants in Tumor-stage Mycosis Fungoides

Mycosis fungoides is the most common cutaneous T-cell lymphoma. Recurrent copy-number variations are the main unifying mutations in this disease, but to date, a comprehensive analysis of occurrence and type of structural variants responsible for these copy-number variations remains elusive. In this study, we used Oxford Nanopore Technologies long-read sequencing to elucidate the highly rearranged genomic landscape of five mycosis fungoides samples. We show the occurrence of multiple classes of simple and complex SV and analyze the extend of tumor suppressor gene deletion by complex SVs. Furthermore, leveraging long-read data, we inferred the genomic structure of a chromothripsis event. Our findings highlight the potential of long-read sequencing as a powerful tool for comprehensive genomic analysis in mycosis fungoides.

genomics↗

bioRxiv2023

Histone binding of ASF1 is required for fruiting body development, but not for genome stability in the filamentous fungus Sordaria macrospora

The highly conserved eukaryotic histone chaperone ASF1 is involved in the assembly and disassembly of nucleosomes during transcription, DNA replication and repair. It was the first chaperone discovered to be involved in all three of these processes. The filamentous fungus Sordaria macrospora is one of only two multicellular organisms where asf1 deletions are viable, which makes it useful for in vivo analysis of this central regulator of eukaryotic chromatin structure. Deletion of asf1 in S. macrospora leads to sterility, a reduction of DNA methylation, and upregulation of genes that are usually weakly expressed in the wild type. Here, we focused on the functions of the highly conserved core and the divergent C-terminal tail of ASF1, studied the effects of ASF1 on histone modifications and tested its relevance for genomic stability. By Co-IP and complementation analysis we showed that substitutions of amino acid V94 or truncations of the C-terminal tail abolish histone binding and do not complement the sterile mutant phenotype. {Delta}asf1 is sensitive to the DNA damaging agent MMS, while complementation strains, even those with non-histone-binding variants, regain wild type-like resistance. To aid in subsequent ChIP-seq and Hi-C analyses, we generated a chromosome-resolved genome assembly of S. macrospora. ChIP-Seq analysis revealed a global increase of H3K27me3 in {Delta}asf1, accompanied by a global decrease of H3K56ac. By using Hi-C we detected a tandem duplication of around 600 kb on chromosome 2 in the mutant. Crossing experiments indicated linkage between the viability of {Delta}asf1 strains and the presence of the duplication. ImportanceHistone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the {Delta}asf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the {Delta}asf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

molecular biology↗

bioRxiv2023

Enhanced protein secretion in reduced genome strains of Streptomyces lividans

S. lividans TK24 is a popular host for the production of small molecules and for the secretion of heterologous proteins. TK24 has a large genome with at least 29 secondary metabolite gene clusters that are non-essential for viability and undergo complex regulation. To optimize heterologous protein secretion, we previously constructed ten chassis strains that are devoid of several secondary metabolite gene clusters. Genome reduction was aimed at reducing carbon flow to secondary metabolites and pigmentation in the spent growth medium and improving colony morphology. Strains RG1.0-RG1.10 contain various deletion combinations of the blue actinorhodin cluster (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red) and coelimycin A (cpk) clusters, the melanin cluster (mel), the matAB genes that affect mycelial aggregation and the non-essential sigma factor hrdD that controls the transcription of Act and Red regulatory proteins. Two derivative strains, RG1.5 and 1.9, showed a [~]15% reduction in growth rate, >2-fold increase in the total mass yield of their native secretome and altered abundance of several specific proteins compared with TK24. Metabolomics and RNAseq analysis revealed that genome reduction led to rapid cessation of growth due to aminoacid depletion and caused both redox and cell envelope stresses, upregulation of the Sec-pathway components secDF and chaperones and a cell envelope two component regulator. RG1.9 maintained elevated heterologous secretion of mRFP and mTNF by 12-70%. An integrated model is presented linking genome reduction and enhanced secretion.

systems biology↗

bioRxiv2022

Gene losses resulting in specialized metabolism in the earliest divergent symbiotic Frankia clade can be linked to its low saprotrophic capabilities

Frankia cluster-2 are diazotrophs that engage in root nodule symbiosis with host plants of the Cucurbitales and the Rosales. They are rarely found in the soil in the absence of their hosts. Previous studies have shown that an assimilated nitrogen source, presumable arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), but not in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported by cluster-2 strains, and which nitrogen source would then be exported to C. thyrsiflorus, gene expression levels, metabolite profiles and enzyme activities were analysed. We found that the export of assimilated nitrogen in symbiosis is a common feature for Frankia cluster-2 strains, but which source is host-plant dependent. We also identified several gene losses. The ammonium assimilation via the GS/GOGAT cycle for export to the host, entails a high demand of 2-oxoglutarate from the TCA cycle. This specialised metabolism seems to have led to genome reduction: we show that Frankia cluster-2 strains have lost the glyoxylate shunt and succinate semialdehyde dehydrogenase, leading to a linearization of the TCA cycle. This could explain the low saprotrophic potential of Frankia cluster-2.

plant biology↗

bioRxiv2021

Uncoupling of the Diurnal Growth Program by Artificial Genome Relaxation in Synechocystis sp. PCC 6803

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knock-down of gyrase subunits and overexpression of topoisomerase I (TopoI), and characterized the phenotypes. Cell division was blocked, most likely due to inhibition of genomic but not plasmid DNA replication. Cell growth continued to 4-5x of the wildtype cell volume, and metabolic flux was redirected towards glycogen in the TopoI overexpression strain. TopoI induction initially lead to down-regulation of GC-rich and up-regulation of AT-rich genes. The response quickly bifurcated and four diurnal co-expression cohorts (dawn, noon, dusk and night) all responded differently, in part with a circadian ({approx} 24 h) pattern. A GC-rich region - 50 bp of transcription start sites is differentially enriched in these four cohorts. We suggest a model where energy- and gyrase-gated transcription of growth genes at the dark/light transition (dawn) generates DNA supercoiling which then facilitates DNA replication and initiates the diurnal transcriptome program.

microbiology↗

bioRxiv2020

The genome of a persistent giant algal virus encodes an unprecedented number of genes involved in energy metabolism

Viruses have long been viewed as entities possessing extremely limited metabolic capacities. Over the last decade, however, this view has been challenged, as metabolic genes have been identified in viruses possessing large genomes and virions--the synthesis of which is energetically demanding. Here, we unveil peculiar phenotypic and genomic features of Prymnesium kappa virus RF01 (PkV RF01), a giant virus of the Mimiviridae family. We found that this virus encodes an unprecedented number of proteins involved in energy metabolism, such as all four succinate dehydrogenase (SDH) subunits (A-D) as well as key enzymes in the {beta}-oxidation pathway. The SDHA gene was transcribed upon infection, indicating that the viral SDH is actively used by the virus-- potentially to modulate its hosts energy metabolism. We detected orthologous SDHA and SDHB genes in numerous genome fragments from uncultivated marine Mimiviridae viruses, which suggests that the viral SDH is widespread in oceans. PkV RF01 was less virulent compared with other cultured prymnesioviruses, a phenomenon possibly linked to the metabolic capacity of this virus and suggestive of relatively long co-evolution with its hosts. It also has a unique morphology, compared to other characterized viruses in the Mimiviridae family. Finally, we found that PkV RF01 is the only alga-infecting Mimiviridae virus encoding two aminoacyl-tRNA synthetases and enzymes corresponding to an entire base-excision repair pathway, as seen in heterotroph-infecting Mimiviridae. These Mimiviridae encoded-enzymes were found to be monophyletic and branching at the root of the eukaryotic tree of life. This placement suggests that the last common ancestor of Mimiviridae was endowed with a large, complex genome prior to the divergence of known extant eukaryotes. ImportanceViruses on Earth are tremendously diverse in terms of morphology, functionality, and genomic composition. Over the last decade, the conceptual gap separating viruses and cellular life has tightened because of the detection of metabolic genes in viral genomes that express complex virus phenotypes upon infection. Here, we describe Prymnesium kappa virus RF01, a large alga-infecting virus with a unique morphology, an atypical infection profile, and an unprecedented number of genes involved in energy metabolism (such as the tricarboxylic (TCA) cycle and the {beta}-oxidation pathway). Moreover, we show that the gene corresponding to one of these enzymes (the succinate dehydrogenase subunit A) is transcribed during infection and is widespread among marine viruses. This discovery provides evidence that a virus has the potential to actively regulate energy metabolism with its own gene.

microbiology↗

bioRxiv2020

Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles

Small (s)RNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through expression in transgenic plants and targeted gene silencing (host-induced gene silencing, HIGS). While HIGS strategies proved to be effective, the mechanism of RNA transfer at the plant - pathogen interface is widely unknown. Here we show that extracellular vesicles (EVs) purified from Arabidopsis thaliana plants expressing CYP3RNA, a dsRNA originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. These transgene specific siRNAs had a length of 21 and 22 nucleotides with a bias towards 5-uracil (U) and 5-adenine (A). Notably, stringent protease and RNase treated EV fractions contained >70% less CYP3RNA-derived siRNAs, suggesting the presence of co-purified extravesicular nucleoprotein complexes stabilizing siRNAs outside of EVs. In addition, mutants of the ESCRT-III complex showed a loss of HIGS-mediated disease resistance and EVs isolated from these mutants were free of CYP3RNA-derived siRNAs. Together, these findings support the view that endosomal vesicle trafficking is required for HIGS mediating the transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells probably in an EV-independent manner.

plant biology↗

bioRxiv2019

Nanopore sequencing of native adeno-associated virus single-stranded DNA using a transposase-based rapid protocol

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene-therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, while additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with qPCR results demonstrated the techniques future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.

genomics↗