Biology subjects

Werner, B. T.

Publications and source records attributed to Werner, B. T..

4 recordsLinked to original sources

bioRxiv2025

Cross-kingdom degradomics identifies natural plant small RNAs with disease protecting activity

RNA interference (RNAi) is a highly specific process regulating genes transcriptionally or post-transcriptionally via silencing. small regulatory RNAs (sRNAs) provide the specificity of post-transcriptional silencing by binding to complementary mRNAs leading to their degradation or translational repression. sRNAs are promising non-chemical control agents against plant pests and diseases. Their application requires, however, a deep understanding of the determinants of functional specificity and efficiency. In nature, interacting plants and pathogens follow the manipulative strategy of cross-kingdom RNAi (ckRNAi) based on the targeted transfer of sRNAs. In this study, we employed a genome-wide approach to explore the diversity of natural ck-sRNAs exchanged in the pathogenic interaction of Brachypodium distachyon with the fungal pathogen Fusarium graminearum. By deep-sequencing of the cross-kingdom sRNAome, transcriptome and degradome 258 ckRNAi-mediating sRNAs were discovered. In a simulation-based approach, specific sequence characteristics allowed categorizing sRNAs into functionality classes. Subsequent genome annotation analyses revealed the organization of plant ck-sRNAs in long non-coding RNAs. Moreover, comparative sequence analyses revealed an evolution of ck-sRNAs towards invariant mRNA target regions. Functional analyses with sRNA candidates confirmed an antipathogenic activity at exceptionally low application doses. It indicates natural sRNA as untapped resource and potential blueprint for the development of highly specific and effective plant-derived bioprotectants.

plant biology↗

bioRxiv2025

High Precision Quantification of small RNA Slicing Activity - Native Index Ligation-based Targeted Degradome Sequencing (NIL-TDS)

RNA interference (RNAi) is an effective and precise regulatory mechanism in eukaryotes in which small RNAs mediate endonucleolytic slicing of complementary target mRNAs. Despite the potential of RNAi for human therapeutics and crop bio-protection, analytical platforms to quantitatively validate the slicing activities of small RNAs remain limited. Here, we present NIL-TDS, a cost-effective method that combines RNA ligase-mediated PCR with Nanopore Sequencing for direct, rapid, and high-resolution detection and quantification of sRNA-mediated slicing events. Using NIL-TDS, we quantitatively detected minute changes in Ath-mir400 mediated slicing of PPR1 in heat and salt stressed Arabidopsis plants. We further demonstrate the broader applicability of NIL-TDS by detecting rare slicing events in a mammalian system. Of relevance for malignancy of certain cancers, and tumor progression and metastasis, NIL-TDS further confirmed miR-196-HOXB8 interactions in lung cancer cells and discovered a novel miR-7162-HOXA10-AS slicing site. These findings demonstrate the sensitivity of NIL-TDS for uncovering small RNA mediated regulatory mechanisms of gene expression and disease progression in eukaryotes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/679503v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@adef35org.highwire.dtl.DTLVardef@1339570org.highwire.dtl.DTLVardef@1980652org.highwire.dtl.DTLVardef@1c732ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Analysis of codon usage and allele frequencies reveal the double-edged nature of cross-kingdom RNAi

BackgroundIn recent years, a new class of small 21- to 24-nt-(s)RNAs has been discovered from microbial pathogens that interfere with their hosts gene expression during infection, reducing the hosts defence in a process called cross-kingdom RNA interference (ckRNAi). According to this model, microbial sRNAs should exert selection pressure on plants so that gene sequences that reduce complementarity to sRNAs are preferred. In this paper, we test this consequence of the ckRNA model by analyzing changes to target sequences considering codon usage and allele frequencies in the model system Arabidopsis thaliana (At) - Hyaloperonospora arabidopsidis (Ha) and Hordeum vulgare (Hv) - Fusarium graminearum (Fg). In both pathosystems, some selected sRNA and their corresponding target have been described and experimentally validated, while the lengthy methodology prevents the analysis of all discovered sRNAs. To expand the understanding of ckRNAi, we apply a new in silico approach that integrates the majority of sRNAs. ResultsWe calculated the probability (PCHS) that synonymous host plant codons in a predicted sRNA target region would show the same or stronger complementarity as actually observed and compared this probability to sets of virtual analogous sRNAs. For the sets of Ha and Fg sRNAs, there was a significant difference in codon usage in their plant gene target regions (for Ha: PCHS 24.9% lower than in the virtual sets; for Fg: PCHS 19.3% lower than in the virtual sets), but unexpectedly for both sets of microbial sRNA we found a tendency towards codons with an unexpectedly high complementarity. To distinguish between complementarity caused by balancing sRNA-gene coevolution and directional selection we estimated Wrights F-statistic (FST), a measurement of population structure, in which positive deviations from the background indicate directional and negative deviations balancing selection at the respective loci. We found a negative correlation between PCHS and FST (p=0.03) in the At-Ha system indicating deviations from codon usage favoring complementarity are generally directionally selected. ConclusionThe directional selection of complementary codons in host plants suggests an evolutionary pressure to facilitate silencing by exogenous microbial sRNAs, which is not consistent with the anticipated biological role of pathogen sRNAs as exclusively effectors in cross-kingdom RNAi. To resolve this conflict, we propose an extended model in which microbial sRNAs are perceived by plants via RNA interference and, via coevolution, primarily help to fine-tune plant gene expression.

plant biology↗

bioRxiv2021

Fusarium graminearum DICER-like-dependent sRNAs are required for the suppression of host immune genes and full virulence

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare Hv) and Brachypodium distachyon (Bd). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing. Significance StatementFusarium graminearum is one of the most devastating fungal pathogens in cereals, while understanding the mechanisms of fungal pathogenesis is a prerequisite for developing efficient and environmentally friendly crop protection strategies. We show exploratory data suggesting that fungal small RNAs play a critical role in Fusarium virulence by suppressing plant immunity.

microbiology↗