Biology subjects

Tanaka, R.

Publications and source records attributed to Tanaka, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Sex peptide-SPR-serotonin signaling regulates mating-induced Sex peptide replenishment in Drosophila, suggesting an ancestral role in males

Seminal fluid not only assists successful fertilization but also induces changes in female post-mating behavior and physiology. Consequently, males with depleted or inadequate seminal fluid produce fewer offspring. Males of iteroparous species therefore increase seminal fluid production to replenish exhausted supplies. However, it remains unclear how mating-induced replenishment of seminal fluid components is regulated at the molecular and cellular levels. Here, we show that replenishment of Sex peptide (SP) in male Drosophila melanogaster requires both SP and its receptor SPR. Mechanistically, mating-induced SP replenishment in the male accessory gland (MAG) is regulated by MAG-projecting serotonergic neurons, and this regulation depends on SPR, which is likely activated by SP as it passes through the reproductive tract during mating. This inter-tissue signaling pathway is not limited to SP but also coordinates the post-mating upregulation of an additional seminal fluid protein gene. While SP-SPR signaling is a well-known regulator of female post-mating responses, we demonstrate that it also modulates male physiology. Furthermore, comparative analyses across 12 drosophilid species are consistent with the hypothesis that SP-SPR signaling acquired a role in males before its role in canonical female post-mating responses evolved. We therefore propose that the role of SP-SPR signaling as a mating signal in males is ancestral to its more familiar but evolutionarily derived role in females.

physiology↗

bioRxiv2026

DiverScan: attention-guided exploratory analysis of animal behaviour across diverse comparative experiments

High-throughput tracking yields rich time-series data of animal behaviour, yet extracting biological insight remains a bottleneck, even for simple control--treatment comparisons. Existing analyses struggle to pinpoint the multiple, nonlinearly intertwined differences hidden in the data and fail to generalise across diverse recording set-ups. Here we present DiverScan, a deep-learning framework for attention-guided exploratory analysis of behavioural time series. DiverScan disentangles condition-dependent behavioural differences by pairing a loss function tailored to data exploration with an attention-branch architecture, and highlights condition-specific behavioural features visually and with explanatory text generated by a large language model. DiverScan is validated on a synthetic benchmark and on datasets from seven laboratories spanning multiple species and both single- and multi-animal behaviour. Beyond recovering known phenotypes, we demonstrate that DiverScan helps researchers generate new hypotheses across these diverse datasets. Available as Jupyter notebooks with a graphical user interface and flexible input formats, DiverScan can integrate into existing behavioural-analysis pipelines.

animal behavior and cognition↗

bioRxiv2026

MpILR1 Hydrolyzes Jasmonate-Amino Acid Conjugates to Activate dn-iso-OPDA Signaling in Marchantia polymorpha.

Jasmonates are essential phytohormones that coordinate defense responses and developmental programs across land plants. In angiosperms, the active jasmonate ligand jasmonoyl-L-isoleucine (JA-Ile), is produced through GH3-mediated conjugation of jasmonic acid to isoleucine and JA-Ile homeostasis is further shaped by ILR1/ILL-family amidohydrolases. In contrast, the primary bioactive jasmonate ligand in bryophytes, dinor-12-oxo-phytodienoic acid (dn-iso-OPDA), is inactivated through conjugation with amino acids, raising the question of whether these conjugates constitute a reversible hormone reservoir or an irreversible catabolic end point. Although the ILR1-like family has been characterized extensively for its role in auxin and jasmonate homeostasis in angiosperms, its function in bryophytes remains basically unexplored. Here we show that MpILR1, the sole Marchantia ortholog of the ILR1/ILL family, hydrolyzes a specific subset of dn-iso-OPDA-amino acid conjugates in vivo. Loss-of-function Mpilr1 mutants exhibit enhanced accumulation of dn-iso-OPDA conjugated to hydrophobic amino acids (Val, Leu and Ile) but not to hydrophilic residues (His, Glu and Gln), demonstrating substrate-selective hydrolysis. MpILR1 hydrolytic activity is required for full dn-iso-OPDA-mediated responses, including transcriptional activation and defense against gastropod herbivory. These findings establish MpILR1 as a key positive regulator of jasmonate signaling in Marchantia polymorpha and suggest that hormone conjugation/deconjugation is an ancient regulatory mechanism evolved during plant terrestrialization.

plant biology↗

bioRxiv2026

hTERT Expression, Regulation, and Prognostic Significance in Pediatric Medulloblastoma

BackgroundTelomerase reactivation, a hallmark of many cancers, is associated with expression of its catalytic subunit, hTERT. However, the prognostic significance of telomere maintenance mechanisms in pediatric medulloblastoma remains poorly defined. MethodsIn this multi-institutional retrospective study of telomerase expression and hTERT regulation in newly diagnosed children with medulloblastoma, hTERT and MYC expression were assessed by qRT-PCR, normalized to non-neoplastic brain control samples. hTERT promoter methylation was analyzed using quantitative pyrosequencing and Illumina 450k methylation array. Cox proportional-hazard regression analyses evaluated the association of hTERT expression with progression-free survival (PFS) or overall survival (OS). Spearman correlation and Kruskal-Wallis tests correlated hTERT promoter methylation and expression and assessed variations among medulloblastoma subgroups, respectively. ResultsAmong 74 patients with available hTERT expression and outcome data, higher expression was associated with worse OS (HR=1.22, 95% CI: 1.01-1.47, p=0.036) and PFS (HR=1.17, 95% CI: 1.00-1.37, p=0.051) after adjusting for subgroup. Similar results were obtained when adjusting for metastatic status. Group 3 patients had the highest hTERT expression (p=0.001). Pyrosequencing data were available for 61 patients and 450k methylation array data for 292 patients. hTERT promoter was differentially methylated across subgroups with WNT followed by group 3 demonstrating the highest methylation on 450k (p<0.0001), findings that were confirmed by pyrosequencing. hTERT promoter methylation positively correlated with hTERT expression (Spearman correlation=0.42, p=0.02 by 450k and 0.34, p=0.007 by pyrosequencing). No significant correlation was observed between hTERT and MYC expression. ConclusionElevated hTERT expression is associated with worse PFS and OS in medulloblastoma across subgroups, supporting telomerase inhibition as a potential therapeutic strategy.

cancer biology↗

bioRxiv2026

Adolescent social isolation creates a latent vulnerability in maternal care with intergenerational social consequences, rescued by experienced mothers

Early-life adversity (ELA) can produce lasting alterations on brain and behavior, yet its effects on maternal caregiving and intergenerational social outcomes remain poorly understood. Using a mouse model of mild adolescent social isolation, we demonstrated that females exposed to isolation in late adolescence exhibited progressive deficits in pup-directed maternal behaviors, including nursing, licking, nest-building, and pup retrieval, while self-directed behaviors remain intact. Offspring reared by these dams displayed deficits in adult social behavior, including reduced sociability, impaired social novelty recognition, and diminished social odor discrimination, without affecting non-social memory, anxiety-like behavior, or locomotor activity. Chemogenetic activation of the medial cingulate cortex (mCg) to prelimbic cortex (PrL) pathway in offspring of stressed dams rescued social deficits, whereas inhibition of this pathway in control mice recapitulated the impairments. Notably, co-housing stressed dams with experienced parous females during the early postpartum period restored pup-directed maternal behaviors and normalized offspring social outcomes, demonstrating that social experience can buffer the intergenerational consequences of ELA. Circuit analyses identified the mCg[->]PrL projections as a predominantly excitatory pathway critical for social behavior. Electrophysiological recordings revealed hypofunction of this circuit in offspring of stressed dams, which was ameliorated by postpartum co-housing with parous females. Together, these findings define a neural circuit linking adolescent psychosocial adversity to impaired maternal caregiving and intergenerational social dysfunction, and highlight social support from experienced mothers as a potent modulator of maternal and offspring behavioral outcomes. Significance StatementAdverse social experiences before motherhood can compromise maternal caregiving and transmit social vulnerability across generations, yet the underlying mechanisms remain poorly understood. Using a mouse model of subtle adolescent social isolation, we demonstrate that even mild isolation selectively impairs postpartum caregiving behaviors while sparing other behavioral domains, resulting in social deficits in adult offspring. We identify the midcingulate-prelimbic cortex circuit as a critical neural substrate mediating these effects and show that social support from experienced mothers during the early postpartum period restores both maternal behavior and offspring social outcomes. Together, these findings define a neural and social mechanism for the intergenerational effects of adolescent psychosocial adversity.

animal behavior and cognition↗

bioRxiv2026

The REFLEX system enables in vivo identification of perivascular angiogenic macrophages in the heart

Direct identification of physically interacting cells in vivo remains challenging because conventional interactome analyses infer signaling partners from transcriptomes and cannot reveal which cells are in direct contact. In pressure-overload induced cardiac remodeling, VEGF-A plays a central role in the maintenance of vascular integrity and cardiac function. However, the cell type which produces VEGF-A and how the VEGF-A peptide is delivered to vascular endothelial cells remains unclear. Here, we developed a genetically encoded platform that combines REFLEX mice with HUNTERuni-seq, enabling unbiased detection and transcriptional profiling of the cells that physically interact with vascular endothelial cells. The REFLEX and HUNTERuni-seq approach identified subpopulations of Vegfa positive macrophages which we named perivascular angiogenic macrophages (PVAMs). Although the amount of VEGF-A in PVAMs is small, loss of VEGF-A in PVAMs impaired angiogenesis and systolic function during pressure overload. We additionally show that direct contact between PVAMs and endothelial cells is critical for the delivery of VEGF-A to endothelial cells. Conventional interactome analysis predicted that cardiomyocytes as dominant sources of VEGF-A in the heart. However, cardiomyocyte Vegfa deletion had no effect on capillary density nor systolic function in a model of heart failure. These results suggest that VEGF-A signaling does not rely on free diffusion through the interstitium and that cellular proximity and physical contact between PVAMs and endothelial cells are the key determinants of effective signal delivery. Together, these findings establish REFLEX and HUNTERuni-seq as a versatile platform for uncovering biologically critical cell-to-cell interactions and provide new insight into intercellular communication in pathological tissue contexts.

cell biology↗

bioRxiv2025

Conservation and diversification of mating behavior patterns among three sibling species in the Drosophila subobscura species subgroup

Behavioral traits are known to evolve rapidly, often even preceding morphological or physiological changes. However, the genomic and neural bases for such rapid behavioral changes remain to be clarified. Drosophila subobscura is a rare example of a species that performs nuptial gift giving, while the mating behaviors of the other two members of the D. subobscura species subgroup, D. madeirensis and D. guanche, remain largely unstudied. In the present study, we characterize and compare mating behaviors of three sibling species of the D. subobscura species subgroup, with the aim of providing a starting point for investigating the neural mechanisms underlying reproductive behavioral divergence in the D. subobscura subgroup. We find that D. madeirensis males exhibit a rich repertoire of courtship behaviors--very similar to that of D. suboscura--including tapping, midleg swinging, proboscis extension and nuptial gift giving. In contrast, D. guanche males perform only tapping and lack the other premating displays, yet they still copulate successfully. We postulate that female promiscuity has promoted the loss of multiple components of the male courtship repertoire in D. guanche. The relatively recent divergence among these species ([~]1.72 Myr between D. guanche and other two species) suggests that only a few genomic and neural changes underpin the striking differences in mating behavior within the subobscura species subgroup. This system offers a promising platform for uncovering the mechanistic basis of rapid behavioral evolution.

evolutionary biology↗

bioRxiv2025

Distinct effects of nonselective Rho-kinase inhibitor fasudil and selective Rho-kinase 2 inhibitor KD025 on serotonin and dopamine release in the nucleus accumbens of mice

Recent studies have indicated that the Rho GTPase family and Rho-kinases are associated with psychiatric diseases, such as schizophrenia. Rho-kinases have two subtypes, Rho-kinases 1 and 2 that regulate actin dynamics and mediate neurite outgrowth, spine morphology in neurons, and neurotransmitter release in vitro and ex vivo. However, the precise role of Rho-kinases in neurotransmitter release in vivo remains unclear. To clarify the role of Rho-kinases 1 and 2 in serotonin and dopamine release in the nucleus accumbens (NAc) of mice in vivo, we investigated the effect of a nonselective Rho-kinase inhibitor, fasudil, and a selective Rho-kinase 2 inhibitor, KD025, using an in vivo microdialysis technique. Fasudil perfusion (1-20 M) into the NAc increased the basal extracellular serotonin level but did not affect dopamine levels, whereas KD025 (10-20 M) had little effect on basal serotonin and dopamine levels. Notably, fasudil perfusion into the NAc suppressed depolarization-induced serotonin and dopamine release in a dose-dependent manner, whereas KD025 selectively suppressed depolarization-induced serotonin release. Our results suggested that Rho-kinases 1 and 2 are associated with dopamine and serotonin release, respectively, and that both may have significant but distinct roles in the regulation of serotonin and dopamine release in the NAc.

neuroscience↗

bioRxiv2025

Twinfilin-1 phosphorylation in reelin signaling regulates actin dynamics and spine development

Reelin is an extracellular glycoprotein essential for neuronal migration, spine development, and synaptic plasticity. Impaired reelin signaling is linked to neurological disorders, including schizophrenia and autism. While reelin mutant (reeler) mice exhibit behavioral deficits associated with impaired spine formation, the underlying molecular mechanisms remain unclear. We identified Twinfilin-1 (Twf1) as a downstream effector of reelin signaling via phosphoproteomic analysis, based on its reduced tyrosine phosphorylation in reeler mice. We found that Src regulated Twf1 phosphorylation at tyrosine 309, and reelin stimulation increased Twf1 phosphorylation in neurons, an effect blocked by the Src inhibitor PP2. A phospho-resistant Twf1 mutant (Twf1 Y309F) showed reduced capping protein binding and a lower F/G-actin ratio. Twf1Y309F mice exhibited cognitive deficits, reduced spine density, smaller spine head size, and a decreased F/G-actin ratio in synaptosomes. These findings highlight Twf1 phosphorylation as a key component of reelin signaling involved in actin remodeling and spine development.

neuroscience↗

bioRxiv2025

PAH-former: Transfer Learning for Efficient Discovery of Pulmonary Arterial Hypertension-Associated Genes

Single-cell RNA sequencing (scRNA-seq) of patient samples holds promise for understanding disease mechanisms, but faces the challenge of excessive cost and effort in acquisition, processing, and data analysis, making it essential to leverage existing data. Pulmonary artery hypertension (PAH) is a refractory disease characterized by pulmonary vascular remodeling, and access to patient specimens is limited due to difficulties in tissue collection. In this study, we employed transfer learning with Geneformer, a deep learning algorithm pre-trained with scRNA-seq datasets and fine-tuned it with public PAH lung tissue data to identify the disease-relevant genes. The resulting algorithm, which we named PAH- former, demonstrated that its prediction accuracy varied significantly depending on the dataset used for fine-tuning. PAH-former enabled us to perform in silico perturbation analysis and identified PAH related genes. Loss-of-function PAH related genes in human pulmonary artery endothelial cells increased the expression of SOX18, a signature gene of PAH. This integration of artificial intelligence and biological experiments can significantly advance our understanding of molecular mechanisms of PAH.

bioinformatics↗

bioRxiv2025

Mating status-dependent dopaminergic modulation of auditory sensory neurons in Drosophila

Mating status often modulates responses to courtship sounds in animals. The neural mechanisms underlying this modulation, however, have not been well clarified. Here, we show that dopaminergic signals are involved in modulating the responses of auditory sensory neurons in Drosophila melanogaster females depending on their mating status. These neurons abundantly express three types of dopamine receptors, with some having direct synaptic connections with dopaminergic neurons. Of these receptors, suppressing the expression of Dop1R2 reduces sound responses of auditory sensory neurons in unmated but not mated females. Moreover, expression of Dop1R2 in auditory sensory neurons enhances the song response behavior of unmated females, manifested by copulation receptivity when exposed to songs. Our research suggests that dopaminergic modulation via Dop1R2 is involved in mating state-dependent regulation of auditory sensory processing.

neuroscience↗

bioRxiv2025

Algorithmic dissection of optic flow memory in larval zebrafish

The visual stabilization behavior in the larval zebrafish reflects the history of optic flow experienced in the recent past. This integrative process has gained traction in recent years as a simplified, tractable model of working memory and decision-making. Yet, its algorithmic bases are poorly understood. In the present study, we first demonstrate that fish integrate externally generated optic flow over time, disregarding self-generated optic flow. This observation suggests that the fish integrates optic flow to better estimate the state of the environment, rather than to keep track of their position. Second, through reverse correlation and delay-based paradigms, we reveal multiple timescales involved in the optic flow integration. With the help of quantitative modeling, we show that the fish becomes more forgetful about the past optic flow in a more dynamic visual environment. Next, with whole-brain, light-sheet calcium imaging, we find that optic flow selective neurons throughout the brain exhibit neural signatures of motor efference copies, mirroring the behavioral findings. Lastly, with two photon calcium imaging, we show that inferior olive neurons integrate forward and backward flow separately, giving clues to how the multiple timescales of the optic flow integration is implemented. Overall, the results here refine our algorithmic and functional understanding of the history dependence of the visual stabilization behaviors in the larval zebrafish, paving the way for deciphering its circuit implementations.

neuroscience↗

bioRxiv2024

Visual circuitry for distance estimation in Drosophila

Animals must infer the three-dimensional structure of their environment from two-dimensional images on their retinas. In particular, visual cues like motion parallax and binocular disparity can be used to judge distances to objects. Studies across several animal models have found neural signals that correlate with visual distance, but the causal role of these neurons in distance estimation as well as the range of possible neural properties that can inform distance estimation have remained poorly understood. Here, we developed a novel high-throughput behavioral assay to identify neurons in the Drosophila visual system that are involved in distance estimation during free locomotion. We found that silencing the primary motion detectors in the fly visual system eliminated their ability to perceive distance, consistent with a reliance on motion parallax to judge distance. Through a targeted silencing screen of visual neurons during behavior and through in vivo two-photon microscopy, we identified a visual projection neuron that encodes the parallax signal in the relative motion of foreground and background. Interestingly, it differs from previously identified parallax-tuned neurons in its lack of direction selectivity both to moving bars and to moving backgrounds. This non-canonical tuning is interpretable in the context of parallax signals that the fly would likely encounter during naturalistic walking behavior. Our results demonstrate how both direction selective and non-direction selective feature-detecting neurons can contribute to distance estimation using parallax cues, providing a framework for considering broader classes of parallax-encoding neurons in distance estimation across visual systems.

neuroscience↗

bioRxiv2024

Mechanisms for plastic landmark anchoring in zebrafish compass neurons

Vision is a sensory modality particularly important for navigation, as it can inform animals of their current heading (i.e. visual landmarks) as well as its changes (i.e. optic flow). It has been shown that head direction (HD) neurons in various species incorporate the visual cues into their heading estimates. However, circuit mechanisms underlying this process remain still elusive in vertebrates. Here, we asked if and how the recently identified HD cells in the larval zebrafish - one of the smallest vertebrate models - incorporate visual information. By combining two-photon microscopy with a panoramic virtual reality setup, we demonstrate that the zebrafish HD cells can reliably track the orientation of multiple visual scenes, exploiting both landmark and optic flow cues. The mapping between landmark cues and the heading estimates is idiosyncratic across fish, and experience-dependent. Furthermore, we show that the landmark tracking requires the lateralized projection from the habenula to the interpeduncular nucleus (IPN), where the HD neuron processes innervate. The physiological and morphological parallels suggest that a Hebbian mechanism similar to the fly ring neuron is at work in the habenula axons. Overall, the observations that the hindbrain HD cells of the larval zebrafish lacking an elaborate visual telencephalon shed new light on the evolution of the navigation circuitry in vertebrates.

neuroscience↗

bioRxiv2024

YORU: social behavior detection based on user-defined animal appearance using deep learning

The creation of tools using deep learning methodologies for animal behavior analysis has revolutionized neuroethology. They allow researchers to analyze animal behaviors and reveal causal relationships between specific neural circuits and behaviors. However, the application of such annotation/manipulation systems to social behaviors, in which multiple individuals interact dynamically, remains challenging. Here, we applied an object detection algorithm to classify animal social behaviors. Our system, packaged as "YORU" (Your Optimal Recognition Utility), classifies animal behaviors, including social behaviors, based on the shape of the animal as a "behavior object". It successfully classified several types of social behaviors ranging from vertebrates to insects. We also integrated a closed-loop control system for operating optogenetic devices into the YORU package. YORU enables real-time delivery of photostimulation feedback to specific individuals during specific behaviors, even when multiple individuals are close together. We hope that the YORU system will accelerate the understanding of the neural basis of social behaviors.

neuroscience↗

bioRxiv2024

Arabidopsis SUFB and CLP protease regulate SUFBC2D to manipulate iron-sulfur cluster biosynthesis in chloroplast

Iron-sulfur (Fe-S) clusters are essential cofactors for Fe-S proteins. SUFBC2D complex is the scaffold responsible for Fe-S cluster assembly in chloroplasts. However, the regulatory mechanism on SUFBC2D remains elusive. In this study, we report that the transcription of SUFB responds rapidly to leaf senescence, whereas the transcription of SUFC and SUFD does not. Intriguingly, their protein contents remain stable during leaf senescence. We further found that leaf death was occurred only when SUFB RNAi was induced, and SUFB and SUFC contents decreased much faster in the SUFB-RNAi lines than in the SUFC-RNAi lines, indicating that SUFB had a faster turnover rate than SUFC. Moreover, overexpressing SUFB increased the contents of SUFC and SUFD, and SUFBC2D, whereas overexpressing SUFC did not increase SUFB and SUFD. Our findings reveal that SUFB stabilizes SUFC and SUFD via forming SUFBC2D, whereas SUFC lacks this function. Furthermore, SUFB expression was sharply downregulated when the plants were subjected to iron deficiency, whereas SUFC and SUFD expression was not. Interestingly, the contents of all three SUF members decreased, indicating that plants degrade SUFBC2D in response to iron deficiency by downregulating SUFB transcription. We subsequently studied the degradation mechanism of SUFBC2D. Our results indicated that all SUFs are substrates of the caseinolytic protease (CLP) because they all accumulated in the CLP impaired mutant, and they physically interact with CLPS1, the substrate recognition adaptor of CLP. Collectively, our findings provide novel insights into how plants regulate SUFBC2D complex via SUFB to adapt to leaf senescence and iron deficiency.

plant biology↗

bioRxiv2024

Antipsychotic-like effects of the selective Rho-kinase 2 inhibitor KD025 in genetic and pharmacological mouse models of schizophrenia

1Copy number variations in the ARHGAP10 gene encoding Rho GTPase-activating protein 10 are significantly associated with schizophrenia. ARHGAP10 negatively regulates RhoA/Rho-kinase (ROCK) signaling. We previously demonstrated that fasudil, a non-selective ROCK inhibitor, exhibited antipsychotic-like effects in several mouse models of schizophrenia. ROCK has two subtypes, ROCK1 and ROCK2. ROCK1 is mainly expressed in the thymus and blood, while ROCK2 is predominantly expressed in the brain. Therefore, it is expected that like fasudil, selective ROCK2 inhibitors will exhibit antipsychotic-like effects, accompanied by a lower incidence of adverse effects due to ROCK1 inhibition. Here, we used genetic and pharmacological models of schizophrenia to investigate whether the selective ROCK2 inhibitor KD025 would show antipsychotic-like effects with a favorable adverse effect profile. Oral administration of KD025 suppressed the abnormal increase in the phosphorylation level of myosin phosphatase-targeting subunit 1, a substrate of ROCK, and ameliorated the decreased spine density of layer 2/3 pyramidal neurons in the medial prefrontal cortex of Arhgap10 S490P/NHEJ mice. Furthermore, KD025 mitigated the methamphetamine-induced impairment of visual discrimination (VD) in Arhgap10 S490P/NHEJ and wild-type mice. KD025 also reduced MK-801-induced impairments of VD, novel object recognition, and hyperlocomotion. Regarding side effects that are commonly seen with typical antipsychotics, KD025 did not affect systolic blood pressure and did not induce extrapyramidal symptoms, hyperprolactinemia, or hyperglycemia at the effective dosage in naive wild-type mice. Taken together, KD025 shows antipsychotic-like effects with a favorable adverse effect profile in genetic and pharmacological mouse models of schizophrenia.

pharmacology and toxicology↗

bioRxiv2024

In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E. coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E. coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E. coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method is expected to potentially advance further understanding of influenza viruses and apply to other RNA viruses.

synthetic biology↗