Biology subjects

Lukan, T.

Publications and source records attributed to Lukan, T..

7 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↗

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

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↗

bioRxiv2020

Spatial accumulation of salicylic acid is in effector-triggered immunity of potato against viruses regulated by RBOHD

While activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to restriction of pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels to reveal spatiotemporal regulation of immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes, spatiotemporal regulation is completely lost in SA-deficient line, while the others show different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during resistance response. In plants with silenced RBOHD, the functionality of resistance response is perturbed and virus spread is not arrested at the site of infection. RBOHD is required for spatial accumulation of SA, and conversely RBOHD is under transcriptional regulation of SA signaling. Using spatially resolved RNA-Seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

plant biology↗