Biology subjects

Wacker, T.

Publications and source records attributed to Wacker, T..

3 recordsLinked to original sources

bioRxiv2025

DiPPER2 - a user-friendly pipeline for picking and evaluating taxon-specific PCR primers

Rapid, specific and sensitive detection is essential for pathogenic microorganisms in contexts ranging from phytosanitary-regulated diseases in agriculture to sepsis management in a clinical setting. Well-established, cost-effective, genomic-sequence-based methods are replacing or complementing laborious, slow and costly culture-based diagnostics. These methods include: end-point PCR and quantitative real-time PCR (qPCR), as well as digital PCR (dPCR) and droplet digital PCR (ddPCR). All these methods require oligonucleotide primers whose nucleotide sequences confer high specificity and sensitivity for the intended target. The most challenging steps in designing these assays include finding genomic sequences that are unique to the target pathogen and absent from off-target organisms, as well as designing primers for those sequences. Here, we present user-friendly, reliable and comprehensive tool DiPPER2 (Diagnostic Primer Picking and Evaluation pipeline for Reproducibility and Reliability), which finds unique diagnostic targets, designs end-point or qPCR primers for them, evaluates the primers in silico for specificity and sensitivity, characterizes the diagnostic target and produces a comprehensive and intuitive report. The pipeline has been developed according to FAIR (findable, accessible, interoperable and reproducible) principles and allows for flexible adjustment of primer parameters. It designs primers compatible with qPCR-, dPCR- and ddPCR. DiPPER2 can also evaluate previously designed primer sequences in silico for specificity and sensitivity. We use Pseudomonas amygdali pv. morsprunorum as an example to show that a clade-based, phylogeny-informed approach to diagnostic primer design ensures effective target discovery and specificity.

bioinformatics↗

bioRxiv2025

Advancing RNAi-Based Strategies Against Downy Mildews: Insights Into dsRNA Uptake and Gene Silencing

Downy mildew (DMs) dieases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. In this study, we evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana, pea and lettuce DM pathogens, Hyaloperonospora arabidopsidis (Hpa), Peronospora viciae f. sp. pisi (Pvp) and Bremia lactucae (Bl), respectively. We specifically targeted the cellulose synthase 3 (CesA3) and the beta tubulin (BTUB) genes. Silencing CesA3 impaired spore germination and infection across multiple species, while BTUB silencing reinforced the potential of dsRNA-mediated inhibition. Reduction in gene expression levels correlated well with the sporulation assays confirming the effectiveness of dsRNA-mediated gene silencing. We used dsRNAs that were chemically synthesized, in vitro transcribed (IVT) or produced in E. coli. We found that the length and concentration of these dsRNAs significantly affected uptake efficiency, spore germination, and sporulation, with higher concentrations enhancing inhibitory effects. Confocal microscopy using Cy-5-labelled short-synthesized dsRNA (SS-dsRNA) provided direct evidence of spore uptake, confirming the potential of SS-dsRNA for pathogen control. However, species-specific sequence variations influenced dsRNA efficacy, underscoring the importance of target sequence design. Multiplexed RNAi impacted silencing synergisticly, further reducing germination and sporulation in Hpa. Additionally, we demonstrated that SS-dsRNA-mediated gene silencing is sustained over time, with a significant reduction in gene expression level at 4, 7, 10 and 11dpi. This indicates the durability and efficacy of this approach. Taken together, these findings demonstrate the potential of dsRNA-mediated gene silencing as a precision tool for managing DM pathogens.

plant biology↗

bioRxiv2021

Two-speed genome expansion drives the evolution of pathogenicity in animal fungal pathogens

The origins of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. Bsal has the most repeat-rich genome, comprising 40.9% repetitive elements, which has expanded to more than 3X the length of its conspecific Bd. M36 metalloprotease virulence factors are highly expanded in Bsal and 53% of the 177 unique genes are flanked by transposable elements, suggesting repeat-driven expansion. The largest M36 sub-family are mostly (84%) flanked upstream by a novel LINE element, a repeat superfamily implicated with gene copy number variations. We find that Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes occur in gene-rich/repeat-poor compartments. This is a hallmark of two-speed genome evolution. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting they are a cradle for chytrid pathogenicity evolution, and Bd also has a two-speed genome. This is the first evidence of two-speed genomes in any animal pathogen, and sheds new light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

genomics↗