Biology subjects

Gruden, K.

Publications and source records attributed to Gruden, K..

At least 19 recordsLinked to original sources

bioRxiv2026

BoolDog: integrated Boolean and semi-quantitative network modelling in Python

SummaryBoolean and logic-based modeling approaches are well suited for the analysis of complex biological systems, particularly when detailed biochemical and kinetic information is unavailable. In such settings, biological pathways are represented as networks capturing system components and their interactions, providing a simplified yet informative abstraction of system behavior. While the structural topology of these networks is often well characterized, the absence of mechanistic detail limits the applicability of parameter-dependent modeling frameworks. To address this, we present BoolDog, a Python package for the construction, simulation, and analysis of Boolean and semi-quantitative Boolean networks. BoolDog supports synchronous simulation with events, attractor and steady-state identification, network visualization, and the systematic transformation of logic-based models into continuous ordinary differential equation (ODE) systems -- enabling the seamless integration of discrete and continuous modeling paradigms. Networks can be imported and exported across standard formats, and BoolDog integrates natively with established Python libraries for network analysis and visualisation, including NetworkX, igraph, and py4Cytoscape. Together, these capabilities provide a flexible, accessible, and interoperable platform for logic-based modeling of complex biological systems. Availability and implementationBoolDog is implemented in Python and available at https://github.com/NIB-SI/BoolDog/.

systems biology↗

bioRxiv2026

Reliable quantification of multiplexed genetically encoded biosensors responsiveness in plant tissues

Genetically encoded biosensors are one of the essential tools in biological research. They enable visualization of molecules of interest from the subcellular level to entire organism level in vivo and can be used to monitor presence of small molecules, gene expression, protein activity, and protein degradation. However, multiplexing fluorescent biosensors in plants is notoriously difficult due to signal bleed-through and strong autofluorescence from chlorophyll. In this study, we investigated the potential of multiplexing biosensors based on the selection of reporter fluorescent proteins. We characterized the emission spectra, fluorescence lifetimes, and relative brightness of diverse fluorescent proteins in plant leaves. We show that selected proteins exhibit comparable brightness, supporting their use in co-expression experiments and reliable quantification of individual signals. To separate three overlapping signals, we applied two different linear unmixing approaches and compared them to results obtained without unmixing. We identified channel separation unmixing approach as the most suitable for biosensors. Additionally, we show how unmixing with the selected approach can be applied to separate autofluorescence and five fluorescent proteins. We further validated this approach in virus-infected cells by following organelle dynamics in vivo. Finally, we demonstrate the feasibility of high-throughput segmentation and quantification with a custom MATLAB workflow for nuclei, chloroplasts, and cytoplasm signal analysis. Overall, our work demonstrates that biosensors can be multiplexed, even when their emission spectra overlap. Significance statementMultiplexing genetically encoded biosensors in plants has been limited by overlapping fluorescent signals and strong autofluorescence. This study presents an optimized framework for linear unmixing and provides a MATLAB-based organelle segmentation tool, allowing precise quantification of multiple fluorescent reporters in vivo and advancing real-time visualization of complex cellular processes in plants.

plant biology↗

bioRxiv2026

Potato yield can be predicted by using drone-captured and environmental measurements early in the growing season

Accurate pre-harvest prediction of crop yield informs variety selection, optimizes management, and accelerates breeding. As potato is the worlds leading non-grain staple, here we evaluate a diverse panel of varieties in a three-year field trial across five European locations. Canopy development and environmental parameters are monitored throughout the growing season using drone-based imaging, in-field sensors and gene expression measurements, while tuber yield and quality traits are quantified at harvest. We show that these data enable the identification of climate-resilient, high-yielding genotypes and support the development of machine learning models that explain over 80% of yield variation in independent test sets. Strikingly, measurements collected within the first two months after planting achieve predictive performance comparable to models trained on full-season data. Model interrogation further shows that over 70% of yield variation can already be predicted based on a simple five-parameter linear equation. Our framework thus demonstrates the potential of integrative field phenotyping and data-driven modeling to improve variety selection across heterogeneous environments.

plant biology↗

bioRxiv2026

miR160 controls class III peroxidase via StARF10/17 and couples salicylic acid and ROS signaling networks in potato hypersensitive response to potato virus Y

O_LIHypersensitive response (HR) is an effector-triggered immune response leading to pathogen restriction and local cell death. Small RNAs (sRNAs), mediating RNA silencing, have a well-established role in antiviral immunity, however, their role in HR has not been addressed to date. C_LIO_LIWe applied a spatially resolved sRNAomics approach to uncover changes at the sRNA level and the dynamics of sRNA-gene regulatory networks in potato HR response to potato virus Y (PVY). C_LIO_LIWe show that miR160 is repressed in cells adjacent to HR lesions in the resistant cultivar, but not in PVY-sensitive, salicylic acid (SA)-depleted plants. Beyond its canonical regulation of StARF10 and StARF17, miR160 controls a broader regulatory network spanning auxin signaling, cell-wall remodeling, redox homeostasis, and tuberization. Elevated miR160 levels promote susceptibility-associated traits, reduce thermotolerance, and suppress genes strongly activated in HR. C_LIO_LIMechanistically, we identify a novel miR160-StARF10/StARF17-StPRX28 module that links miR160 repression to the induction of a class III apoplastic peroxidase during HR, with SA acting upstream. Together, these findings establish miR160 as a crucial integrator of SA-auxin-ROS signaling pathways that tunes development, defense, and heat resilience, and highlight miR160 as a promising target for fine-tuning tolerance to multiple stresses with minimal yield loss. C_LI

molecular biology↗

bioRxiv2026

LongPolyASE: An end-to-end framework for allele-specific gene and isoform analysis in polyploids using long-read RNA-seq

MotivationLong-read RNA-seq and phased reference genomes enable haplotype-resolved gene and isoform expression analysis. While methods and tools exist for diploid organisms, analysis tools for polyploids are lacking. ResultsWe developed an end-to-end framework for allele-specific gene and isoform analysis in polyploids with three components: Syntelogfinder identifies syntenic genes in phased assemblies; longrnaseq quantifies transcripts, discovers novel isoforms, and performs quality control of long-read RNA-seq; and PolyASE analyzes differential allelic expression, differential isoform usage between conditions, and structural differences in major isoforms between haplotypes. We demonstrate the use of the framework on diploid rice and autotetraploid potato. Availability and ImplementationSyntelogfinder and longrnaseq are implemented in Nextflow and available on GitHub. PolyASE is a Python package available on PyPI. The framework is fully documented and tutorials are provided. ContactNadja.nolte.franziska@nib.si Supplementary informationSupplementary data are available online and on Zenodo.

bioinformatics↗

bioRxiv2025

Functionality of potato virus Y coat protein in cell-to-cell movement is defined by flexibility of its N terminal region

Potato virus Y (PVY) is one of the top ten economically most important plant viruses and responsible for major yield losses. We previously suggested the involvement of the N terminal region of potato virus Y coat protein (CP) in PVY spread. By constructing different N terminal deletion mutants of PVY N605 strain, we here show that deletions of 40 or more amino acid residues from the N terminal region of the CP resulted in the PVY multiplication limited to primary infected cells in Nicotiana clevelandii plants. Deletion of 26 residues profoundly impaired PVY cell-to-cell movement and prevented systemic PVY spread, while deletions of 19-23 residues allowed delayed systemic PVY spread. Introduced point mutations in the identified region prevent (S21G) or delay (G20P) PVY movement. In summary, this work shows the significance of the CP N-terminus for movement of the PVY.

plant biology↗

bioRxiv2025

An ERF transcription factor StPTI5, a novel regulator of endophyte community maintenance in potato

We have recently identified ERF transcription factor PTI5 as a susceptibility factor, negatively regulating immune response to diverse pathogens. Here we investigated the processes involved in colonisation of potato with beneficial organisms. RNAseq showed that at the time of Bacillus subtilis biofilm establishment, immune responses in interacting roots were attenuated, and complex transcriptional network was triggered, with ethylene signalling being a central module and PTI5 strongly induced. Interestingly, the response is intensified if plants are inoculated by two antagonistic B. subtilis strains. While PTI5 is not involved in the establishment of biofilm on roots, we show that bacterial abundance increases in PTI5-silenced plants. Remarkably, root colonization by the arbuscular mycorrhizal fungus Rhizophagus irregularis was also higher in the PTI5-silenced plants. PTI5 is thus involved both in blocking defence against harmful and blocking colonisation with beneficial microbes. Such mechanistic understanding of plant-microbe interaction paves the way for sustainable crop management.

plant biology↗

bioRxiv2025

Haplotype-resolved genome assembly of the tetraploid potato cultivar Desiree

Cultivar Desiree is an important model for potato functional genomics studies to assist breeding strategies. Here, we present a haplotype-resolved genome assembly of Desiree, achieved by assembling PacBio HiFi reads and Hi-C scaffolding, resulting in a high-contiguity chromosome-level assembly. We implemented a comprehensive annotation pipeline incorporating gene models and functional annotations from the Solanum tuberosum Phureja DM reference genome alongside RNA-seq reads to provide high-quality gene and transcript annotations. Additionally, we provide a genome-wide DNA methylation profile using Oxford Nanopore reads, enabling insights into potato epigenetics. The assembled genome, annotations, methylation and expression data are visualised in a publicly accessible genome browser (https://desiree.nib.si), providing a valuable resource for the potato research community.

plant biology↗

bioRxiv2024

Evaluating plant growth-defence trade-offs by modelling the interaction between primary and secondary metabolism

Understanding the molecular mechanisms behind plant response to stress can enhance breeding strategies and help us design crop varieties with improved stress tolerance, yield and quality. To investigate resource redistribution from growth-to defence-related processes in an essential tuber crop, potato, here we generate a large-scale compartmentalised genome-scale metabolic model, Potato-GEM. Apart from a large-scale reconstruction of primary metabolism, the model includes the full known potato secondary metabolism, spanning over 600 reactions that facilitate the biosynthesis of 182 distinct potato secondary metabolites. Constraint-based modelling identifies that the activation of the largest amount of secondary (defence) pathways occurs at a decrease of the relative growth rate of potato leaf, due to the costs incurred by defence. We then obtain transcriptomics data from experiments exposing potato leaves to two biotic stress scenarios, a herbivore and a viral pathogen, and apply it as constraints to produce condition-specific models. We show that these models recapitulate experimentally observed decreases in relative growth rates under treatment, enabling us to pinpoint the metabolic rewiring underlying growth-defence trade-offs. Potato-GEM thus presents a useful resource to study and broaden our understanding of potato and general plant defence responses under stress conditions.

systems biology↗

bioRxiv2024

Integration of multi-omics and deep phenotyping provides novel insights into multiple abiotic stress responses in potato

Potato is highly water and space efficient but susceptible to abiotic stresses such as heat, drought, or flooding, which are severely exacerbated by climate change. Understanding of crop acclimation to abiotic stress, however, remains limited. Here, we present a comprehensive molecular and physiological high-throughput profiling of potato (Solanum tuberosum, cv. Desiree) under heat, drought and waterlogging applied as single stresses or in combinations designed to mimic realistic future scenarios. Stress-responses were monitored via daily phenotyping and multi-omics analyses of leaf samples comprising transcriptomics, proteomics, metabolomics and hormonomics at several timepoints during and after stress treatments. Additionally, critical metabolites of tuber samples were analysed at the end of the stress period. Integrative analysis of multi-omics data was performed using a bioinformatic pipeline, which was established here, based on machine learning and knowledge networks. Overall, waterlogging had the most immediate and dramatic effects on potato plants, interestingly activating ABA-responses similar to drought stress. In addition, we observed distinct stress signatures at multiple molecular levels in response to heat or drought and to a combination of both. In response to all treatments, we found a downregulation of photosynthesis at different molecular levels, an accumulation of minor amino acids and diverse stress induced hormones. Our integrative multi-omics analysis provides global insights into plant stress responses, facilitating improved breeding strategies towards climate-adapted potato varieties. One Sentence SummaryIntegrated multi-omics analysis of high-throughput phenotyping in potato reveals distinct molecular signatures of acclimation to single and combined abiotic stresses related to climate change.

plant biology↗

bioRxiv2024

Ethylene signaling is essential for mycorrhiza-induced resistance against chewing herbivores in tomato

Root colonization by certain beneficial microbes can prime plant defenses aboveground, modifying plant responses to potential attackers. Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with most plant species, usually enhancing plant resistance to biotic stresses, leading to Mycorrhiza-Induced Resistance (MIR). Still, our knowledge of the complex molecular regulation leading to MIR is very limited. Here we show that the AM fungus Funneliformis mosseae protects tomato plants against two different chewing herbivores, Spodoptera exigua and Manduca sexta, and we explore the underlying molecular mechanism. We explore the impact of AM symbiosis on the plant response to the herbivores through genome-wide transcriptional profiling, followed by bioinformatics network analyses and functional bioassays. Herbivore-triggered JA-regulated defenses were primed in leaves of mycorrhizal plants, while ET biosynthesis and signaling was also higher both before and after herbivory. We hypothesized that fine-tuned ET signaling is required for the primed defensive response leading to MIR in mycorrhizal plants. We followed analytical, functional, and genetic approaches to test this hypothesis and get mechanistic insights into the ET signaling in MIR. ET is a complex regulator of plant responses to stress, and although ET is generally considered a negative regulator of plant defenses against herbivory, tomato lines deficient in ET synthesis or perception could not develop MIR against either herbivore. Thus, we demonstrate that hormone crosstalk is central to the priming of plant immunity by beneficial microbes, with ET fine-tuning being essential for the primed JA biosynthesis and boosted defenses leading to MIR in tomato.

plant biology↗

bioRxiv2023

Evidence based Unification of poTato gene models with UniTato collaborative genome browser

Potato (Solanum tuberosum) is the most popular tuber crop and model organism. Though its gene models are frequently updated, they are not unified, leading to missing or wrongly annotated genes. Here, we thus unify the recent potato double monoploid v4 and v6 gene models by automatic merging. We established an Apollo web server that enables access to the Unified poTato genome annotation database (UniTato) as well as further community-based manual curation at unitato.nib.si. We demonstrate how the web server interface can help to resolve problems with missing or misplaced genes and can be used to further update them or consolidate a wider set of gene models or genome information. Genome annotation files and a comprehensive translation table are provided at github.com/NIB-SI/unitato.

plant biology↗

bioRxiv2023

Stress Knowledge Map: A knowledge graph resource for systems biology analysis of plant stress responses

Stress Knowledge Map (SKM, https://skm.nib.si) is a publicly available resource containing two complementary knowledge graphs describing current knowledge of biochemical, signalling, and regulatory molecular interactions in plants: a highly curated model of plant stress signalling (PSS, 543 reactions) and a large comprehensive knowledge network (CKN, 488,390 interactions). Both were constructed by domain experts through systematic curation of diverse literature and database resources. SKM provides a single entrypoint for plant stress response investigations and the related growth tradeoffs. SKM provides interactive exploration of current knowledge. PSS is also formulated as qualitative and quantitative models for systems biology, and thus represents a starting point of a plant digital twin. Here, we describe the features of SKM and show, through two case studies, how it can be used for complex analyses, including systematic hypothesis generation, design of validation experiments, or to gain new insights into experimental observations in plant biology.

plant biology↗

bioRxiv2023

Identification and characterisation of Planococcus citri cis- and trans-isoprenyl diphosphate synthase genes, supported by short- and long-read transcriptome data

Many insect species rely on diverse terpenoids for their development and interorganismal interactions. However, little is known about terpenoid biosynthesis in insects. The monoterpenoid sex pheromones of mealybugs and scale insects (Coccoidea) are particularly enigmatic, with several species producing unique structures presumed to result from the irregular coupling activity of unidentified isopentenyl diphosphate synthases (IDSs). Enzymes capable of similar transformations have previously only been described from a few plant, bacterial and archaeal species. To investigate if insect irregular monoterpenes can be biosynthesised by similar enzymes, we performed a comprehensive search for IDS coding sequences in the genome of Planococcus citri, a widespread agricultural pest. We complemented the available P. citri genome data with newly generated short- and long-read transcriptome data. The identified candidate genes had homology to both short- and long-chain IDSs and some appeared to be paralogous, indicating gene duplications and consequent IDS gene family expansion in P. citri. We tested the activity of eleven candidate gene products, confirming in vitro regular activity for five enzymes, one of which (transIDS5) also produced the irregular prenyl diphosphates, maconelliyl and lavandulyl diphosphate. Targeted mutagenesis of selected aspartates and a lysine in the active site of transIDS5 uncovered their importance for chain-length preference and irregular coupling. This work provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, as well as a potential source of enzymes for the biotechnological production of sustainable insect pest management products.

biochemistry↗

bioRxiv2022

A mini-TGA protein, lacking a functional DNA-binding domain, modulates gene expression through heterogeneous association with transcription factors

TGA transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigate the role of StTGA2.1, a potato TGA lacking the full bZIP, which we name a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirm that StTGA2.1 overexpression compensates for SA-deficiency. To understand the underlying mechanisms, we show that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigate the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirm that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases, which are known to play role in immune response. Finally, via structural modelling and molecular dynamics simulations, we hypothesise that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to novel functions and that such events should be studied carefully in other protein families.

plant biology↗

bioRxiv2021

pISA-tree - a data management framework for life science research projects using a standardised directory tree

We developed pISA-tree, a straightforward and flexible data management solution for organisation of life science project-associated research data and metadata. pISA-tree was initiated by end-user requirements thus its strong points are practicality and low maintenance cost. It enables on-the-fly creation of enriched directory tree structure (project/Investigation/Study/Assay) based on the ISA model, in a standardised manner via consecutive batch files. Templates-based metadata is generated in parallel at each level enabling guided submission of experiment metadata. pISA-tree is complemented by two R packages, pisar and seekr. pisar facilitates integration of pISA-tree datasets into bioinformatic pipelines and generation of ISA-Tab exports. seekr enables synchronisation with the FAIRDOMHub repository. Applicability of pISA-tree was demonstrated in several national and international multi-partner projects. The system thus supports findable, accessible, interoperable and reusable (FAIR) research and is in accordance with the Open Science initiative. Source code and documentation of pISA-tree are available at https://github.com/NIB-SI/pISA-tree.

bioinformatics↗

bioRxiv2021

Chloroplast redox state changes indicate cell-to-cell signalling during the hypersensitive response

Hypersensitive response (HR)-conferred resistance is associated with an induction of programmed cell death (PCD) and pathogen spread restriction in its proximity. While the pivotal role of salicylic acid (SA) in the restriction of pathogen spread during HR has been confirmed, the exact role of chloroplastic reactive oxygen species and the link between SA signalling and chloroplast redox state during HR remain unexplained. To unravel these relationships, we performed detailed spatiotemporal analysis of chloroplast redox response to potato virus Y (PVY) infection in resistant Ny-1-gene-bearing potato and its transgenic counterpart with impaired SA accumulation and compromised resistance. We found that the chloroplasts are highly oxidized in the cells adjacent to the cell death zone at different stages after virus inoculation in both genotypes. Moreover, we detected individual cells with moderately oxidized chloroplasts, which we call "signalling cells", in close proximity as well as farther from the cell death zone. These are relatively rare in SA-deficient plants, suggesting their role in signalling for HR-conferred resistance. This hypothesis is further supported by highly induced formation of stroma filled tubules that extend from chloroplasts (stromules) in the cells adjacent to signalling cells. Unexpectedly, the cells with the highest occurrence of stromules have chloroplasts in more reduced state than the adjacent ones. In addition, we show that stromules are induced also at the edge of PVY multiplication zone. We conclude that chloroplast redox state and stromule formation are tightly spatiotemporally regulated by SA-signalling which leads to effective HR response.

plant biology↗

bioRxiv2021

Orchestration of the stilbene synthase gene family and their regulators by subgroup 2 MYB genes

The stilbenoid pathway is responsible for the production of resveratrol and its derivatives in grapevine. A few transcription factors (TFs) have been previously identified as regulators of this pathway but the extent of this control is yet to be fully understood. Here we demonstrate how DNA affinity purification sequencing (DAP-Seq) allows for genome-wide TF binding site interrogation in a non-model species. We obtained 5,190 and 4,443 binding events assigned to 4,041 and 3,626 genes for MYB14 and MYB15, respectively (around 40% of peaks being located within -10kb of transcription start sites). DAP-Seq of MYB14 and MYB15 was combined with aggregate gene centred co-expression networks built from more than 1,400 transcriptomic datasets from leaves, fruits and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15 and MYB13, a third uncharacterised member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS ) targets, these three regulators bind to 30 out of 47 STS family genes. Moreover all three MYBs bind to several PAL, C4H and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and novel resveratrol-modifying gene candidates amongst which ROMT2 -3 were validated enzymatically. A high proportion of DAP-Seq bound genes was induced in the activated transcriptomes of transient MYB15 -overexpressing stilbenoid-producing grapevine leaves, validating our methodological approach for identifying gene regulatory networks of specialised metabolism. Overall, MYB genes from Subgroup 2 appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production.

plant biology↗