6f348871059c69c2bf85737b8ee8d077 Pla_a2025v14n21p3334.pdf 9d79e38553b5aaf685518eb2344635634c3ca0e6 Pla_a2025v14n21p3334.pdf a41428f3489147bd903d06dcae180bd20ee44181fa16460971e2b45f02d2cc30 Pla_a2025v14n21p3334.pdf Title: Understanding Crosstalk Between Phosphate and Immune-Related Signaling in Rice and ArabidopsisThrough Live Imaging of Phosphate Levels Subject: Phosphate (Pi) is a vital macronutrient for plant growth and development, and precise monitoring of its cellular dynamics is essential to understanding Pi homeostasis and its interaction with stress responses. Genetically encoded FRET-based biosensors such as FLIPPi enable real-time, non-invasive visualization of cytosolic Pi levels in living tissues. In this study, Arabidopsis and rice lines expressing a FLIPPi biosensor were used to monitor cytosolic Pi dynamics in root epidermal cells. Sensor functionality was confirmed by measuring FRET responses to graded Pi supplies, revealing a consistent reduction in FRET ratios with increasing Pi concentrations, reflecting elevated cytosolic Pi levels. Comparisons with a Pi-insensitive FLIPPi variant confirmed the specificity of the observed changes. Furthermore, live imaging demonstrated rapid and dynamic alterations in cytosolic Pi upon treatment with defense-related hormones and elicitors of immune responses supporting a link between Pi signaling and plant immunity. Finally, the application of phosphite, an analog of Pi, altered Pi dynamics in both Arabidopsis and rice, suggesting an interference with Pi accumulation. Collectively, our findings establish FLIPPi as a reliable tool for in vivo monitoring of Pi in Arabidopsis and rice plants, the model systems for studies in dicotyledonous and monocotyledonous species, respectively. Keywords: FRET biosensor; phosphate; cytosolic Pi; FLIPPi; Arabidopsis; rice; phosphite; nutrient sensing; plant immunity; live imaging; nutrient-immunity crosstalk Author: Mani Deepika Mallavarapu, María Ribaya, Beatriz Val-Torregrosa and Blanca San Segundo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Fri Oct 31 09:49:53 2025 CET ModDate: Fri Oct 31 10:01:08 2025 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3314695 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YHPIKO+URWPalladioL-Bold Type 1 Custom yes yes yes 138 0 BFCLLF+URWPalladioL-BoldItal Type 1 Custom yes yes yes 139 0 NLEVXE+URWPalladioL-Roma Type 1 Custom yes yes yes 140 0 BOSQPW+URWPalladioL-Ital Type 1 Custom yes yes yes 141 0 XEYJZU+CMSY10 Type 1 Builtin yes yes yes 194 0 OVNEXW+EURM10 Type 1 Builtin yes yes yes 257 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 236 0 AJKIAC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 249 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 250 0 AJKIAC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 292 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 293 0 AJKIAC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 309 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 310 0 AJKIAC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 345 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 346 0 AJKIAC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 370 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 371 0 AJKIAC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 395 0 AJKHEF+PalatinoLinotype TrueType WinAnsi yes yes no 396 0 MPUDFL+PazoMath Type 1 Builtin yes yes yes 449 0 AXGJOC+VnURWPalladioL Type 1 Custom yes yes yes 517 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-07-29 04:19:29 CEST RepresentationInformation: Pla_a2025v14n21p3334.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-07-28 18:45:16 CEST Size: 3314695 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 554 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: Generation and Validation of FLIPPi Sensor Lines in Arabidopsis and Rice for Live Imaging of Cytosolic Pi Item: Title: Pi Treatment Increases Cytosolic Pi Levels in Arabidopsis Roots Corresponding to Decreasing FRET Emission Item: Title: Pi Treatment Increases Cytosolic Pi Levels in Rice Roots Reflected by Decreased FRET Emission Item: Title: Cytosolic Pi Levels Increase in Response to Treatment with Defense-Related Hormones and Elicitors of Immune Responses in Arabidopsis and Rice Roots Item: Title: Phosphite Application Alters Cytosolic Pi Dynamics in Roots of Arabidopsis and Rice Plants Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Plant Material and Growth Conditions Item: Title: Generation of Transgenic Arabidopsis and Rice Plants Expressing FLIPPi Sensors Item: Title: RT-qPCR Item: Title: Pi Treatments Item: Title: Phosphate Quantification Item: Title: Hormone and Elicitor Treatments Item: Title: Phosphite Treatments Item: Title: Live Imaging of Pi and FRET Analysis Item: Title: Statistical Analysis Item: Title: References Info: Title: Understanding Crosstalk Between Phosphate and Immune-Related Signaling in Rice and ArabidopsisThrough Live Imaging of Phosphate Levels Author: Mani Deepika Mallavarapu, María Ribaya, Beatriz Val-Torregrosa and Blanca San Segundo Subject: Phosphate (Pi) is a vital macronutrient for plant growth and development, and precise monitoring of its cellular dynamics is essential to understanding Pi homeostasis and its interaction with stress responses. Genetically encoded FRET-based biosensors such as FLIPPi enable real-time, non-invasive visualization of cytosolic Pi levels in living tissues. In this study, Arabidopsis and rice lines expressing a FLIPPi biosensor were used to monitor cytosolic Pi dynamics in root epidermal cells. Sensor functionality was confirmed by measuring FRET responses to graded Pi supplies, revealing a consistent reduction in FRET ratios with increasing Pi concentrations, reflecting elevated cytosolic Pi levels. Comparisons with a Pi-insensitive FLIPPi variant confirmed the specificity of the observed changes. Furthermore, live imaging demonstrated rapid and dynamic alterations in cytosolic Pi upon treatment with defense-related hormones and elicitors of immune responses supporting a link between Pi signaling and plant immunity. Finally, the application of phosphite, an analog of Pi, altered Pi dynamics in both Arabidopsis and rice, suggesting an interference with Pi accumulation. Collectively, our findings establish FLIPPi as a reliable tool for in vivo monitoring of Pi in Arabidopsis and rice plants, the model systems for studies in dicotyledonous and monocotyledonous species, respectively. Keywords: FRET biosensor; phosphate; cytosolic Pi; FLIPPi; Arabidopsis; rice; phosphite; nutrient sensing; plant immunity; live imaging; nutrient-immunity crosstalk Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Fri Oct 31 09:49:53 CET 2025 ModDate: Fri Oct 31 10:01:08 CET 2025 ID: 0x847d68eb0efc2c84586e34c084f97bcd, 0x72ec1a3b153d2d9ff2a3d9202d80174b Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true ToUnicode: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true ToUnicode: true Font: BaseFont: XEYJZU+CMSY10 FontSubset: true FirstChar: 0 LastChar: 20 FontDescriptor: FontName: XEYJZU+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 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