Biology subjects

Kawano, Y.

Publications and source records attributed to Kawano, Y..

17 recordsLinked to original sources

bioRxiv2026

Rice blast fungus hijacks a rice RALF peptide to initiate infection

Plant-derived peptides regulate immunity and host-microbe interactions, but whether fungal pathogens exploit endogenous plant peptides for infection-site selection remains largely unknown. Rapid alkalinization factors (RALFs) are a family of plant peptide hormones that contribute to plant development, reproduction, stress responses, and cell-wall organization. Here, we show that OsRALF7, which is an immune-activating peptide in rice (Oryza sativa), is co-opted by the rice blast fungus Magnaporthe oryzae as a host-derived cue for appressorium formation, specialized infection structures required for host penetration. While synthetic OsRALF7 peptide induced defense responses in rice suspension cells, the same peptide induced appressorium formation in M. oryzae. OsRALF7-induced appressorium formation was associated with activation of the cAMP/PKA and Pmk1 MAPK pathways and required a hydrophobic surface. M. oryzae inoculated onto Osralf7 knockout plants formed fewer appressoria, and the positioning of these appressoria relative to the boundaries between two adjacent plant epidermal cells was altered. These findings suggest that OsRALF7 provides positional information for infection initiation for M. oryzae. OsRALF7 localized to the plasma membrane and cell wall in rice, and its appressorium-inducing activity required a conserved YISY motif and cysteine residues. This activity was not conserved in Colletotrichum higginsianum, as RALF23 from Arabidopsis thaliana did not induce appressorium formation but instead showed antifungal activity. Together, these findings uncover a dual function of endogenous plant immune-modulating RALF peptides at the plant-pathogen interface, where they can be exploited by M. oryzae as a host-derived cue for infection initiation while also retaining antifungal activity against other fungal species.

plant biology↗

bioRxiv2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

biochemistry↗

bioRxiv2025

Rhizobial infection-specific accumulation ofphosphatidylinositol 4,5-bisphosphate inhibits the excessive infection of rhizobia in Lotus japonicus

O_LIDuring the symbiosis of legume with nitrogen-fixing bacteria, collectively called rhizobia, suppression of excessive rhizobial infection by host plants is important to maximize the benefits of symbiotic nitrogen fixation. However, it remains relatively poorly understood the molecular mechanism involved in the suppression. C_LIO_LIWe performed LC-MS and RNA-seq analysis using rhizobia-infected Lotus japonicus roots and investigated the role of phosphatidylinositol (PI) and phosphatidylinositol phosphates (PIPs) in the symbiosis. Phosphatidylinositol transfer protein (PITP)-like proteins 4 (PLP4), phosphatidylinositol 3-phosphate 5-kinase 4 (PIP5K4) and PIP5K6 mutants, which are involved in the vesicular transport of lipids and phosphorylation of PIPs, were used to show the involvement of the signaling of PI and PIPs. Accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] during rhizobial infection were examined by a fluorescent marker 1xTUBBY-C (TUBBY). C_LIO_LIWe found that PI signaling-related genes were upregulated, and the amount of PIP2 increased in L. japonicus roots during rhizobial infection. In the PLP4, PIP5K4 and PIP5K6 mutants, rhizobial infection increased and PIP2 accumulation was failed. Furthermore, the observation of PI(4,5)P2 in rhizobia-infected roots revealed that the ectopic accumulation was closely related to suppression of rhizobial infection. C_LIO_LIOur findings indicate that the accumulation of PI(4,5)P2, which is mediated by PLP and PIP5Ks, suppresses excessive rhizobial infection in the root epidermis and cortex, leading to the optimal number of nodules. C_LI

plant biology↗

bioRxiv2025

Solubility, Hydration, and Sulphate Coordination in Cubic Insulin Crystals from Esrapid™ Monomers Stabilized in Divalent Anionic Form of Citric Acid

Insulins structural adaptations have been extensively studied at neutral and basic pH; however, the effects of water and anion coordination on allosteric regions under various acidic conditions remain unexplored. Given the critical role of polar interactions in allosteric modulation, investigating structured water movements across extended pH intervals is essential for understanding solvent-mediated stabilization mechanisms in the monomer form of insulin. Structures of acid-stable cubic insulin crystals were determined in the divalent anionic form of citric acid solutions over a pH range of 2 to 6 to investigate the effects of water and anion coordination, along with charge distribution, on protein conformation. Synchrotron X-ray diffraction data were collected at resolutions ranging from 1.4 to 1.76 [A], with refined models exhibiting R-factors between 0.19 and 0.21. While the spatial arrangement of most proteins is highly conserved, [~]90% of the water coordination network and polar interactions alter local residue motion and intrinsic dynamics of the structures as the pH is changed. This is in line with previously determined structures at pH 7-11, allowing for a comprehensive structural analysis in the pH range of pH 2-11. Three key observations emerged: (i) water coordinations undergo a prominent shift toward the isoelectric point of insulin (pH 5-7), (ii) water molecules and anions function as allosteric modulators to stabilize the T-state of insulin at the pH range 2 to 6, and (iii) at extreme pH values 2 and 11, increased solubility correlates with the structural adoption of insulins most active form, wherein hydration within the allosteric pocket supports monomer stabilization in the T-state. Combined with the computational analyses, pH-dependent electrostatic redistributions primarily affect side-chain dynamics and local protein motion. This solvent-coupled allosteric regulation provides a mechanistic framework for solvent-mediated protein stabilization, offering a novel insight into the rational design of insulin formulations through controlled protonation and hydration strategies.

biophysics↗

bioRxiv2025

Local delivery of SBRT and IL-12 to Murine PDAC Tumors Modulates Hematopoiesis

BackgroundStandard of care therapies such as radiotherapy and chemotherapy have shown little efficacy against pancreatic ductal adenocarcinoma (PDAC). Immunotherapy is a newly emerging form of treatment that has shown promise; however, toxic systemic effects resulted in limited use in the clinic. Shifting from systemic to local delivery of cancer therapeutics reduces adverse systemic effects and increases response rates in multiple malignancies. Importantly, the effects of tumor-targeted therapies on distal tissues, such as the bone marrow, have not been thoroughly investigated. MethodsUsing a murine model of PDAC, we treated tumors with targeted stereotactic body radiation therapy (SBRT) and intratumoral interleukin-12 (IL-12). 13 days-13 months after tumor injection, the cells in the tumor, blood, and bone marrow were analyzed for therapy-induced changes. Hematopoietic cell numbers and lymphocytes were quantified by flow cytometry, and cytokine levels were quantified by enzyme-linked immunosorbent assays (ELISAs). ResultsWe demonstrated that although SBRT/IL-12 delivered locally to PDAC tumors successfully eradicated primary disease, it also induced significant acute and long-term effects in the bone marrow. Within days of intratumoral SBRT/IL-12 treatment, we observed acute lymphopenia in the blood, accompanied by an immunostimulatory response in the bone marrow characterized by an increase in hematopoiesis. Long-term effects included a decrease in hematopoietic stem cells (HSCs) and skewing toward a myeloid lineage bias, which could indicate premature aging of the HSC population. ConclusionsThese findings demonstrate that despite being locally delivered to the tumor, SBRT/IL-12 therapy exerts significant effects on the distal bone marrow, reinforcing the need for further investigations into the long-term systemic immunological outcomes of localized cancer treatments. Key MessagesWhat is already known on this topic: Systemic cancer therapies used to combat pancreatic ductal adenocarcinoma (PDAC) often induce toxic systemic effects. Local delivery of radiation and immunotherapy reduces adverse effects; however, the systemic spread of these therapies and the resulting effects on distal tissues such as the bone marrow have yet to be elucidated. What this study adds: Intratumoral delivery of stereotactic body radiation therapy (SBRT) and interleukin-12 (IL-12) augment hematopoiesis in the bone marrow soon after treatment and induce long-term alterations in the hematopoietic stem cells (HSCs). These effects are mainly a result of IL-12 that is transiently increased in the bone marrow after treatment. How this study might affect research, practice, or policy: Targeted SBRT/IL-12 therapy induces long-term systemic effects on the bone marrow, indicating the need for further investigation of the systemic spread of locally delivered therapeutics.

immunology↗

bioRxiv2024

Modeling suggests SARS-CoV-2 rebound after nirmatrelvir-ritonavir treatment is driven by target cell preservation coupled with incomplete viral clearance

In a subset of SARS-CoV-2 infected individuals treated with the oral antiviral nirmatrelvir-ritonavir, the virus rebounds following treatment. The mechanisms driving this rebound are not well understood. We used a mathematical model to describe the longitudinal viral load dynamics of 51 individuals treated with nirmatrelvir-ritonavir, 20 of whom rebounded. Target cell preservation, either by a robust innate immune response or initiation of nirmatrelvir-ritonavir near the time of symptom onset, coupled with incomplete viral clearance, appear to be the main factors leading to viral rebound. Moreover, the occurrence of viral rebound is likely influenced by time of treatment initiation relative to the progression of the infection, with earlier treatments leading to a higher chance of rebound. Finally, our model demonstrates that extending the course of nirmatrelvir-ritonavir treatment, in particular to a 10-day regimen, may greatly diminish the risk for rebound in people with mild-to-moderate COVID-19 and who are at high risk of progression to severe disease. Altogether, our results suggest that in some individuals, a standard 5-day course of nirmatrelvir-ritonavir starting around the time of symptom onset may not completely eliminate the virus. Thus, after treatment ends, the virus can rebound if an effective adaptive immune response has not fully developed. These findings on the role of target cell preservation and incomplete viral clearance also offer a possible explanation for viral rebounds following other antiviral treatments for SARS-CoV-2. ImportanceNirmatrelvir-ritonavir is an effective treatment for SARS-CoV-2. In a subset of individuals treated with nirmatrelvir-ritonavir, the initial reduction in viral load is followed by viral rebound once treatment is stopped. We show the timing of treatment initiation with nirmatrelvir-ritonavir may influence the risk of viral rebound. Nirmatrelvir-ritonavir stops viral growth and preserves target cells but may not lead to full clearance of the virus. Thus, once treatment ends, if an effective adaptive immune response has not adequately developed, the remaining virus can lead to rebound. Our results provide insights into the mechanisms of rebound and can help develop better treatment strategies to minimize this possibility.

immunology↗

bioRxiv2024

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF-Trap

The constant fragment (Fc) of the Immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half-life. A prime example is VEGF-Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)-1 and VEGFR-2. The Fc regions role in N-glycosylation is particularly important, as it significantly influences protein stability. In this study, we present the first near-physiological temperature structures of the N-glycan-bound Fc fragment of IgG1 subtype from a biosimilar VEGF-Trap, determined using the SPring-8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including our own data, reveals alternate conformations within the glycan-binding pocket. Furthermore, molecular dynamics (MD) simulations highlight an unexpected degree of structural plasticity. These findings offer new insights into the molecular basis of Fc-mediated functions and provide valuable information for the design of next-generation therapeutics.

molecular biology↗

bioRxiv2023

Study on Vibrational Behavior of Cytoskeletons Modeled by Cylindrical Tensegrity Structure

The dynamic mechanism of a cellular cytoskeleton is essential for the role of the cell, and its accurate characterization has been a long-standing problem for cell scientists. A cytoskeletons vibrations are highly influenced by interactions of filamentous proteins mediated by axial vibration of the stiff microtubules (compressive member) and lateral vibration of F-actin (tensile member). Among various structures in a cell, the cytoplasmic contractile bundles, lamellipodia, and filipodia cells can be modeled by a symmetrical cylinder-shaped self-equilibrium tensegrity structure with different radii at the top and bottom of the cylinder. The truncated conelike cylinder model is made to be small in height compared to both radii. This study investigates the tensegrity self-vibrational behavior of the cellular cytoskeleton to calculate its natural frequencies, composed of the individual vibration of microtubules and F-actins from measured data. The spectral element method is adopted based on the Wittrick-Williams procedure to solve the vibrational behaviors of the cellular cytoskeleton. Various n-polygon cylindrical truncated cone-shaped tensegrity structures to mimic the cellular cytoskeletons are presented to demonstrate the robustness of the present study.

cell biology↗

bioRxiv2023

MicroRNA-221/222-expression in HSC and MPP safeguards their quiescence and multipotency by downregulating stress-independent and dependent expression of IEG and of several myelo/granulopoiesis-enhancing target genes.

The microRNA cluster-221/222 is expressed in hematopoietic stem cells (HSC) and multipotent progenitors (MPP). To study its function in hematopoiesis, we generated mice, in which this cluster is selectively deleted by Vav-cre in HSC and, thus, in all hematopoietic cells. Fluorescence-activated cell sorting analyses of the lineage-negative HSC and MPP compartments in bone marrow at unperturbed, steady state hematopoiesis detect strong activation of HSC to MPP, as well as increased granulocytes in the periphery, induced by miR-221/222-deficiency. Short-term social stress on mice also activates HSC to MPP, but the time of stress is too short to detect further increases in granulocyte numbers. Single cell deep mRNA sequencing identifies Fos as direct, and Jun as well as six other immediate early genes (IEG) as indirect targets of miR-221/222 at unperturbed hematopoiesis. Three of these IEG - Klf6, Nr4a1 and Zfp36 - have previously been found to influence myelo/granulopoiesis. Short stress induces higher levels of the same, and an even larger number of IEGs, now also in MPP, indicating, that stress and miR-221/222 both activate HSC to MPP by IEG upregulation in perturbed hematopoiesis. Furthermore, combined stress and miR-221/222-deficiency rapidly increase numbers of myelo/granulocyte progenitors (MEP, GMP) in bone marrow. Additional indirect miR-221/222-targets become detectable in MPP, of which H3f3b has previously been found to influence myelopoiesis. In serial transplantations, miR-221/222-deficient HSC retain their capacity to home to, and become resident in bone marrow, but they loose their lymphopoietic capacities, thus their multipotency. Our results suggest, that miR-221/222-expression in HSC and MPP safeguards their quiescence and multipotency by downregulating the expression of IEG and of myelo/granulopoiesis-enhancing target genes. Since miR-221/222 is also expressed in human HSC and MPP, its expression should improve clinical settings of human bone marrow transplantations.

immunology↗

bioRxiv2022

Elucidating polymorphs of crystal structures with intensity-based hierarchical clustering analysis on multiple diffraction datasets

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference for defining a threshold used for grouping the isomorphous datasets ( isomorphic threshold) poses a challenge. Here, we have applied unit cell-based and intensity-based HCAs to the datasets of apo-trypsin and inhibitor-bound trypsin that were mixed post-data acquisition to investigate how effective HCA is in classifying polymorphous datasets. Single-step intensity-based HCA successfully classified polymorphs with a certain isomorphic threshold. In datasets of several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested isomorphic threshold. Polymorphs were also detected in single crystals using the data collected by the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis. SynopsisSingle-step intensity-based hierarchical clustering is demonstrated to allow the detection of structural polymorphs in the diffraction datasets obtained from multiple crystals. By splitting the datasets collected by continuous helical scheme into several chunks, both inter and intra-crystal polymorphs can be successfully analyzed.

molecular biology↗

bioRxiv2022

Germinal center-derived broadly neutralizing antibodies adapt to SARS-CoV-2 antigenic drift

The outbreak of SARS-CoV-2 variant Omicron which harbors a striking number of mutations in the spike protein has been raising concerns about the effectiveness of vaccines and antibody treatment1. Here, we confirmed a substantial reduction in neutralizing potency against Omicron in all convalescent and vaccinated sera. However, we found that some people infected by the early strain show relatively higher neutralization to Omicron. From those B cells, we developed neutralizing antibodies inhibiting broad variants including Delta and Omicron. Unlike reported antibodies, one had an extremely large interface and widely covered receptor binding motif of spike, thereby interfering with diversified variants. Somatic mutations introduced by long-term germinal center reaction contributed to the key structure of antibodies and the universal interaction with spike variants. Recalling such rare B cells may confer sustainable protection against SARS-CoV-2 variants emerging one after another.

immunology↗

bioRxiv2021

Water droplet-in-oil digestion method for single-cell proteomics

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

systems biology↗

bioRxiv2021

Three conserved hydrophobic residues in the CC domain of Pit contribute to its plasma membrane localization and immune induction

Nucleotide-binding leucine-rich repeat (NLR) proteins work as crucial intracellular immune receptors. N-terminal domains of NLRs fall into two groups, namely coiled-coil (CC) and Toll-interleukin 1 receptor (TIR) domains, which play critical roles in signal transduction and disease resistance. However, the activation mechanisms of NLRs, and how their N-termini are involved in immune induction, remain largely unknown. Here, we revealed that the rice NLR Pit self-associates through its CC domain. The CC domain of Pit possesses three conserved hydrophobic residues that are known to be involved in homodimer formation in two NLRs, barley MLA10 and Arabidopsis RPM1. Interestingly, the function of these residues in Pit is different from that in MLA10 and RPM1. Although the three hydrophobic residues are important for Pit-induced disease resistance against rice blast fungus, they do not participate in self-association or in binding to downstream signaling molecules. Based on homology modeling of Pit using the structure of the Arabidopsis NLR ZAR1, we tried to clarify the role of the three conserved hydrophobic residues and found that they are involved in the plasma membrane localization. Our findings provide novel insights for understanding the mechanisms of NLR activation as well as the relationship between subcellular localization and immune induction.

plant biology↗

bioRxiv2021

The secreted peptide IRP functions as a DAMP in rice immunity

Small signaling peptides play important roles in various plant processes, but information regarding their involvement in plant immunity is limited. We previously identified a novel small secreted protein in rice, named immune response peptide (IRP) by the integrated multi-omics analyses. Here, we studied IRP functions in rice immunity. Rice plants overexpressing IRP enhanced resistance to the blast fungus. Application of the IRP peptide to rice suspension cells triggered the expression of IRP itself and defense gene PAL1. RNA-seq results revealed that 84% of genes upregulated by IRP peptide were also induced by chitin, including 13 OsWRKY transcription factors, indicating that IRP and chitin share the similar signaling pathway. Co-treatment with chitin and IRP elevated the expression level of PAL1 and OsWRKYs in an additive manner. When results of IRP and PAL1 expression and MAPK activation by IRP were compared with those by chitin, IRP had a stronger effect on MAPK activation rather than IRP and PAL1 expression. Collectively, our findings indicate that IRP functions as a damage-associated molecular pattern (DAMP) in rice immunity, regulating MAPKs and OsWRKYs to amplify chitin signaling, and provide new insights into how PAMPs and DAMPs cooperatively regulate rice immunity.

plant biology↗

bioRxiv2020

The Small GTPase OsRac1 forms two distinct immune receptor complexes containing the PRR OsCERK1 and the NLR Pit

Plants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and Leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we reveal that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane. Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the plasma membrane and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Ergothioneine, a metabolite of the gut bacterium Lactobacillus reuteri, protects against stress-induced sleep disturbances

The relationships between depression and gut microbiota, particularly those involving the immune system, have become a major focus of recent research. Here, we analyzed changes in gut microbiota and their sulfur metabolites in the feces of a depression rat model using the modified 14-day social defeat stress (SDS) paradigm. Our results showed that SDS increased fecal Lactobacillus reuteri in correlation with ergothioneine levels at around day 11, which continued for at least one month following SDS administration. In vitro study further revealed that L. reuteri is capable of producing ergothioneine. Although the known anti-inflammatory and anti-oxidative actions of ergothioneine suggested that the increased fecal ergothioneine levels may be related to intestinal anti-inflammatory defense mechanisms, no change was observed in the plasma ergothioneine levels during the same observation period, indicating that the defense mechanisms may not be sufficiently reflected in the body. As ergothioneine is a natural ingredient that is absorbed mainly from the upper gastrointestinal tract, we hypothesized that oral ergothioneine may exert antidepressant effects. As expected, oral administration of ergothioneine prior to and during the SDS paradigm had a preventative effect on SDS-induced depressive behaviors, such as social avoidance and depression-like sleep abnormalities, particularly those of rapid eye movement sleep. These findings indicate that ergothioneine, a metabolite of L. reuteri, may be a common substance in the microbiota-gut-brain axis that prevents stress-induced sleep disturbances, especially those associated with depression.

neuroscience↗

bioRxiv2018

In vivo monitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor

Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. We have established a plant version of a Forster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in rice cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images. Our protocol should be widely adaptable for monitoring activation for other plant small GTPases and for other FRET sensors in various plant cells.

plant biology↗