REPOSITORY > RESULTS

Abstract

Genetically encoded biosensors are powerful tools for studying plant immune response, as they enable real-time, in vivo monitoring of defence-related signals with high spatial and temporal resolution. By shifting the focus from static endpoint measurements to dynamic analyses of signal timing, intensity, and localization, biosensors reveal important features that are critical for effective immune responses.
This work provides a critical review of biosensor applications in plant immunity and crops, together with original experimental research. We investigated jasmonic acid (JA) and salicylic acid (SA) signalling in potato (Solanum tuberosum L.) response to herbivory by the Colorado potato beetle (CPB, Leptinotarsa decemlineata [Say]) and to mechanical wounding. Three complementary biosensors were employed: a transcriptional reporter of JA-dependent gene expression (MC::EYFP), a direct reporter of JA-Ile signalling based on JAZ degron degradation (AtJas9-Venus-N7), and a bacterial bioluminescent reporter for SA (Acinetobacter ADPWH_lux). Imaging intact plants was essential, as tissue excision or leaf detachment abolished detectable JA-dependent responses, highlighting the importance of in vivo approaches. Together, these sensors enabled visualization of distinct stages of the JA signalling cascade and their spatial relationship with SA accumulation.
Our results revealed a previously undescribed spatial organization of wound-induced hormone signalling. JA-dependent gene expression was consistently suppressed in cells immediately adjacent to wound sites, despite active JA-Ile signalling in the same region. In contrast, JA responses propagated into some millimetres of surrounding tissue, while SA accumulated preferentially near the wound edge. Depletion of SA and treatments with SA analogue INA and JA demonstrated that local SA signalling suppresses JA-dependent transcription in wound-proximal cells. This spatial antagonism between SA and JA was observed following both mechanical damage and herbivory, although the suppressed zone was broader after insect feeding. Cells at the wound margin showing persistent JA suppression later exhibited physiological features consistent with secondary cell wall formation, suggesting a role for localized hormone crosstalk in wound healing and barrier formation.
To enable following more analytes simultaneously, we systematically assessed strategies for multiplexing fluorescent biosensors in living plant tissues. Multiple fluorescent proteins were characterized for brightness and spectral properties, and methods for separating overlapping signals were evaluated. Channel dye separation using standard multichannel imaging provided reliable signal discrimination while minimizing acquisition time, photobleaching, and motion artifacts, and an objective segmentation pipeline enabled quantitative analysis of compartment-specific signals.
Overall, this work demonstrates how genetically encoded biosensors can resolve the spatiotemporal dynamics of plant defence signalling. Particularly, it identifies spatially confined SA–JA antagonism as a key regulatory feature of wound responses. These approaches complement omics-based analyses and advance mechanistic understanding of plant immunity directly in crop species.