Biology subjects

Levak, V.

Publications and source records attributed to Levak, V..

4 recordsLinked to original sources

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

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↗

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↗