Biology subjects

Busche, T.

Publications and source records attributed to Busche, T..

18 recordsLinked to original sources

bioRxiv2026

The diadenosine tetraphosphate hydrolase YqeK controls fitness, biofilm formation, staphyloxanthin production and virulence in Staphylococcus aureus

Diadenosine tetraphosphate (Ap4A) is a nucleotide metabolite, which is degraded by the YqeK hydrolase in Staphylococcus aureus in vitro. In this study, we analyzed the phenotypes of the yqeK mutant under stress, antibiotics, biofilm and macrophage infection conditions to investigate the functions of Ap4A in S. aureus COL. Using nucleotide metabolomics, we confirmed that Ap4A levels are 105-fold higher in the yqeK mutant, accompanied by lower adenylate and guanylate nucleotide pools. The yqeK mutant showed a delayed growth in LB, TSB and RPMI medium, and decreased survival under lethal oxidative and quinone stress. Transcriptome analysis revealed the upregulation of the sspABC operon and the CodY, T-box Met and G-box regulons indicating increased amino acids and GTP biosynthesis, whereas the AgrA, Fur, PurR, and T-box Cys regulons were downregulated in the yqeK mutant. These gene expression changes could be restored to WT level in the yqeK complemented strain, resulting also in a DNA damage response as revealed by the induction of LexA regulon members and mobile genetic elements (pathogenicity island SAPI3 and prophage L54a). Moreover, the yqeK mutant showed enhanced biofilm formation, higher intracellular iron levels and lower staphyloxanthin levels. Using infection assays, we demonstrated a decreased survival of the yqeK mutant inside J774A.1 murine macrophages, supporting a link between Ap4A and pathogenicity regulation via Agr-controlled virulence factors in S. aureus. Future research should be directed to understand how Ap4A regulates nucleotide, iron and amino acid metabolism as well as biofilm and virulence phenotypes in S. aureus. IMPORTANCE: S. aureus is an important human pathogen, which can cause life-threatening infections especially in immunocompromised patients. Due to the prevalence of multidrug resistant strains, the search for new drug targets is an urgent goal. Diadenosine tetraphosphate (Ap4A) has been shown to contribute to stress responses, antibiotic resistance, biofilm development and virulence in bacteria. In this work, we showed that Ap4A is upregulated upon deletion of yqeK encoding the Ap4A hydrolase in S. aureus COL. Moreover, the yqeK mutant was impaired in growth and survival during oxidative stress and after infection of murine macrophages, indicating that Ap4A contributes to the host-pathogen interactions in S. aureus. Transcriptome analyses revealed alterations of the nucleotide, amino acid and iron metabolism as well as the downregulation of Agr-controlled cytotoxins, contributing to the lower virulence of the yqeK mutant. Altogether, our results provide leads for the design of inhibitors against YqeK to combat S. aureus infections.

microbiology↗

bioRxiv2026

Molecular sources of monoterpenoid chemodiversity in the Asteraceae Tanacetum vulgare suggest a new model for the evolution of specialized metabolism

Highly diversified specialized metabolism enables plant communication with pollinators, herbivores, and protectors1-4. Its chemodiversity, under which evenness, richness and variation is summarized5-7, includes many compounds without known function1-4 and presents an evolutionary conundrum about how and what is selected for8,9. Due to its complexity, it is frequently unknown how it is encoded in genomes. To produce population level chemodiversity, the traits need to allow for highly chemodiverse and highly specific individuals in the same population. Here we use metabolomics, transcriptomics, and genomics combined with field analyses and functional assays of monoterpene synthases in the Asteraceae Tanacetum vulgare (tansy) and identify forces which produce high population level chemodiversity: selection for product specificity in enzymes, loss-of-expression alleles, absence variation, and specialized metabolism islands drive individuals towards low chemodiversity while unlinked enzyme loci, expression variation alleles, presence variation, and de novo enzyme evolution enable high individual chemodiversity. Since the molecular data suggests selection for mechanisms that increase chemodiversity itself at the population level, the screening hypothesis which posited plants produce a reservoir of diverse chemicals prior to selection8 should be replaced by a chemodiversity selection hypothesis. The results demonstrate that, in addition to plant protection via individual chemicals with known targeting mechanisms for predators, being different from your neighbors even if you are closely related is likely an important element in plant protection.

plant biology↗

bioRxiv2026

Tuning into xanthan: A conserved yet flexible polysaccharide utilization system in Microbacterium

Bacteria encounter structurally complex extracellular polysaccharides in natural environments, yet the regulatory and evolutionary basis of their utilization remains poorly understood. Here, we isolated a soil-derived Microbacterium strain, named Microbacterium xanthanicum UB-LE1, that grows on xanthan as the sole carbon source. We dissected the genetic and regulatory architecture underlying this capability. Genome sequencing combined with transcriptomic and proteomic profiling uncovered a discrete, strongly inducible regulon associated with xanthan utilization, encoding 23 proteins with five secreted proteins and three candidate transcriptional regulators. DNA-affinity purification sequencing confirmed two regulators binding to operons within the xanthan utilization locus. Comparative genomics across the Microbacteriaceae revealed conserved and lineage-specific features of this system and supports recent acquisition and modular integration of the locus, with at least two predominant architectural variants possibly shaped by substrate availability and ecological specialization. Coordinated induction at both the transcript and protein levels, together with two experimentally validated regulators, points to tight regulatory control of complex polysaccharide degradation in Microbacterium xanthanicum UB-LE1. Together, these findings provide mechanistic and evolutionary insight into how bacteria adapt to complex extracellular carbohydrates, expand current knowledge of xanthan turnover in microbial ecosystems, and establish a framework for exploring the emergence and diversification of specialized polysaccharide utilization pathways across bacterial taxa. IMPORTANCEMicroorganisms are central drivers of carbon turnover in soils and other terrestrial ecosystems, determining the availability of nutrients and shaping microbial community structure. A significant portion of soil carbon is contained in extracellular polysaccharides, yet the pathways by which microorganisms degrade these complex polymers remain poorly understood. Xanthan, a structurally complex and widely produced microbial exopolysaccharide, represents a persistent and largely overlooked carbon pool. By dissecting the genetic, regulatory, and evolutionary basis of xanthan utilization in Microbacterium xanthanicum UB-LE1, this study advances our understanding of how soil bacteria adapt to complex extracellular carbohydrates and how substrate availability shapes the emergence and diversification of specialized metabolic pathways. Importantly, the identification of additional xanthan-active enzymes and regulatory components in M. xanthanicum UB-LE1 opens opportunities for targeted modification of xanthan structure and properties, paving the way for new biotechnological applications in food, materials, and industrial biotechnology, while linking microbial ecology to functional innovation.

microbiology↗

bioRxiv2026

An SPFH Protein Couples Membrane Stress to Differentiation in Bacillus subtilis

Bacillus subtilis adapts to fluctuating environmental stress, such as membrane perturbation or alkaline conditions, using membrane-associated regulatory complexes. Here, we rename the previously termed pspA-ydjGHI operon to pspA-samGHI (for starvation and motility) to reflect its functional roles in membrane envelope stress signalling. The SamG-SamH membrane proteins recruit SamI, a cytosolic SPFH protein, which stabilizes focal membrane localization and recruitment of PspA, an ESCRT-III homolog. Under normal conditions, this system transiently assembles at the membrane, stabilizing it and allowing proper motility, secretion, and biofilm formation. Loss of SamI ({Delta}samI/{Delta}ydjI) leads to unbalanced SamG-SamH activity leading to a constitutive stress signalling, and global transcriptional changes reminiscent of starvation situations. This, in turn, blocks secretion of the matrix protein BslA, preventing biofilm formation, and reducing motility. Deletion of samH in combination with {Delta}samI restores biofilm formation, while {Delta}pspA mutants form biofilms normally, indicating that PspA is dispensable for the developmental phenotype. Our findings reveal that beside membrane integrity SamGHI coordinates transcriptional homeostasis and multicellular development through formation of a membrane integral stress sensor complex.

microbiology↗

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

Systems-level analysis provides insights on methanol-based production of L-glutamate and its decarboxylation product γ-aminobutyric acid by Bacillus methanolicus

BackgroundBacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of various value-added chemicals. The wild-type strain B. methanolicus MGA3 naturally overproduces O_SCPLOWLC_SCPLOW-glutamate in methanol-based fed-batch fermentations. ResultsHere we generated, by induced mutagenesis, an evolved B. methanolicus strain exhibiting enhanced O_SCPLOWLC_SCPLOW-glutamate production capability (>150%). To showcase the potential of this evolved strain, further metabolic engineering enabled the production of {gamma}-aminobutyric acid (GABA) directly from O_SCPLOWLC_SCPLOW-glutamate, with a yield of 13.2 g/L from methanol during fed-batch fermentations. By using a systems-level analysis, encompassing whole-genome sequencing, RNA sequencing, fluxome analysis and genome-scale metabolic modelling, we were able to elucidate the metabolic and regulatory adaptations that sustain the biosynthesis of these products. The metabolism of the mutant strain evolved to prioritize energy conservation and efficient carbon utilization. Key metabolic shifts include the downregulation of energy-intensive processes such as flagellation and motility and the rerouting of carbon fluxes towards -ketoglutarate and its derivative, O_SCPLOWLC_SCPLOW-glutamate. Moreover, we observed that transformation of the evolved strain with a GABA biosynthesis plasmid had a positive effect on O_SCPLOWLC_SCPLOW-glutamate production, likely due to an upregulation of various transaminases involved in the O_SCPLOWLC_SCPLOW-glutamate biosynthesis from -ketoglutarate. ConclusionsThese results and insights provide a foundation for further rational metabolic engineering and bioprocess optimization, enhancing the industrial viability of B. methanolicus for sustainable production of O_SCPLOWLC_SCPLOW-glutamate and its derivatives.

systems biology↗

bioRxiv2024

Tracing active bugs in microbial communities by BONCAT and click chemistry-based enrichment of newly synthesised proteins

A comprehensive understanding of microbial community dynamics is fundamental to the advancement of environmental microbiology, human health, and biotechnology. Metaproteomics, i.e. the analysis of all proteins in a microbial community, provides insights into these complex systems. Microbial adaptation and activity depend to an important extent on newly synthesized proteins (nP), however, the distinction between nP and bulk proteins is challenging. The application of bioorthogonal non-canonical amino acid tagging (BONCAT) with click chemistry has demonstrated efficacy in the enrichment of nP in pure cultures. However, the transfer of this technique to microbial communities has proven challenging and has therefore not been used on microbial communities before. To address this, a new workflow with efficient and specific nP enrichment was developed using a laboratory-scale mixture of labelled E. coli and unlabelled yeast. This workflow was successfully applied to an anaerobic microbial community with initially low BONCAT efficiency. A substrate shift from glucose to ethanol selectively enriched nP with minimal background. The identification of bifunctional alcohol dehydrogenase and a syntrophic interaction between an ethanol-utilizing bacterium and two methanogens (hydrogenotrophic and acetoclastic) demonstrates the potential of metaproteomics targeting nP to trace microbial activity in complex microbial communities.

microbiology↗

bioRxiv2024

HPV-positive head and neck squamous cell carcinoma cells lose viability during triggered myocyte lineage differentiation

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease, and death rates have remained at approximately 50% for decades. New tumor-targeting treatment strategies are desperately needed. Using patient-derived tumor cells, we created an HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions, including irregularly enlarged cell morphology, cell cycle arrest with downregulation of Ki67, and reduced cell viability. RNA-seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly, including TPM1, TAGLN, and ACTA1. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. A recent sarcoma study was able to turn rhabdomyosarcoma into muscle-like cells. We are the first to report that carcinoma cells can undergo a triggered myocyte differentiation. Our study suggests that the targeted myo-differentiation of tumor cells might be therapeutically valuable in HPV+ HNSCCs.

cancer 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

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

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↗

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↗

bioRxiv2021

Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles and their role in RNAi-based crop protection

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce gene silencing of pathogenic targets was groundbreaking. However, future field applications will require fundamental mechanistic knowledge on dsRNA uptake, processing, and its transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. Here, we examined the role of EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to the target fungus Fusarium graminearum. We found barley EVs with 156 nm in size containing predominantly 21 and 19 nucleotide (nt) siRNAs starting with a 5-terminal Adenine. Notably, barley EVs contain less siRNA compared to EVs isolated from transgenic HIGS Arabidopsis plants. Together our results further underpin mechanistic differences between HIGS and SIGS applications and a minor role of EVs in SIGS.

plant biology↗

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

Phloem-mediated spreading of SIGS-derived non-coding RNAs in Hordeum vulgare

Small (s)RNA molecules are crucial factors in the communication between hosts and their interacting pathogens/pests that can modulate both host defense and microbial virulence/pathogenicity known as cross-kingdom RNA interference (ckRNAi). Consistent with this, sRNAs and their double-stranded (ds)RNA precursors have been adopted to control plant diseases through exogenously applied RNA biopesticides, known as spray-induced gene silencing (SIGS). While RNA spray proved to be effective, the mechanisms underlying the transfer and uptake of SIGS-associated RNAs are inadequately understood. Moreover, the use of the SIGS-technology as a biopesticide will require the systemic spreading of dsRNA/siRNA signals. Our results strongly support the notion of phloem-mediated long-distance movement of SIGS-associated dsRNA and/or siRNA. These findings are significant contributions to our mechanistic understanding of RNA spray technology, as our previous data indicate that SIGS requires the processing of dsRNAs by the fungal RNAi machinery. In summary, our findings support the model that SIGS involves: (i) uptake of sprayed dsRNA by the plant (via stomata); (ii) transfer of apoplastic dsRNAs into the symplast (DCL processing into siRNAs); (iii) systemic translocation of siRNA or unprocessed dsRNA via the vascular system (phloem/xylem); (iv) uptake of apoplastic dsRNA or symplastic dsRNA/siRNA depending on the lifestyle/feeding behavior of the pathogen/pest.

plant biology↗

bioRxiv2019

Discovery of interaction-related sRNAs and their targets in the Brachypodium distachyon and Magnaporthe oryzae pathosystem

Microbial pathogens secrete small RNA (sRNA) effectors into plant hosts to aid infection by silencing transcripts of immunity and signaling-related genes through RNA interference (RNAi). Similarly, sRNAs from plant hosts have been shown to contribute to plant defense against microbial pathogens by targeting transcripts involved in virulence. This phenomenon is called bidirectional RNA communication or cross kingdom RNAi (ckRNAi). How far this RNAi-mediated mechanism is evolutionarily conserved is the subject of controversial discussions. We examined the bidirectional accumulation of sRNAs in the interaction of the hemibiotrophic rice blast fungus Magnaporthe oryzae (Mo) with the grass model plant Brachypodium distachyon (Bd). By comparative deep sequencing of sRNAs and mRNAs from axenic fungal cultures and infected leaves and roots, we found a wide range of fungal sRNAs that accumulated exclusively in infected tissues. Amongst those, 20-21 nt candidate sRNA effectors were predicted in silico by selecting those Mo reads that had complementary mRNA targets in Bd. Many of those mRNAs predicted to be targeted by Mo sRNAs were differentially expressed, particularly in the necrotrophic infection phase, including gene transcripts involved in plant defense responses and signaling. Vice versa, by applying the same strategy to identify Bd sRNA effectors, we found that Bd produced sRNAs targeting a variety of fungal transcripts, encoding fungal cell wall components, virulence genes and transcription factors. Consistent with function as effectors of these Bd sRNAs, their predicted fungal targets were significantly down-regulated in the infected tissues compared to axenic cultures, and deletion mutants for some of these target genes showed heavily impaired virulence phenotypes. Overall, this study provides the first experimentally-based evidence for bidirectional ckRNAi in a grass-fungal pathosystem, paving the way for further validation of identified sRNA-target duplexes and contributing to the emerging research on naturally occurring cross-kingdom communication and its implications for agriculture on staple crops.\n\nAuthor SummaryIn the present work, we provide first experimental evidence for bidirectional RNA communication in a grass-fungal pathosystem. We deployed the monocotyledonous plant Brachypodium distachyon, which is a genetic model for the staple crops wheat and rice, to investigate the interaction-related sRNAs for their role in RNA communication. By applying a previously published bioinformatics pipeline for the detection of sRNA effectors we identified potential plant targets for fungal sRNAs and vice versa, fungal targets for plant sRNAs. Inspection of the respective targets confirmed their downregulation in infected relative to uninfected tissues and fungal axenic cultures, respectively. By focusing on potential fungal targets, we identified several genes encoding fungal cell wall components, virulence proteins and transcription factors. The deletion of those fungal targets has already been shown to produce disordered virulence phenotypes. Our findings establish the basis for further validation of identified sRNA-mRNA target duplexes and contribute to the emerging research on naturally occurring cross-kingdom communication and its implications for agriculture.

plant biology↗