Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis.

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis . FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT -producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1 's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 ( SEP3 ), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

Deep imaging reveals dynamics and signaling in one-to-one pollen tube guidance.

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, we develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument-dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI-independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

Novel inhibitors of microtubule organization and phragmoplast formation in diverse plant species.

Cell division is essential for development and involves spindle assembly, chromosome separation, and cytokinesis. In plants, the genetic tools for controlling the events in cell division at the desired time are limited and ineffective owing to high redundancy and lethality. Therefore, we screened cell division-affecting compounds in Arabidopsis thaliana zygotes, whose cell division is traceable without time-lapse observations. We then determined the target events of the identified compounds using live-cell imaging of tobacco BY-2 cells. Subsequently, we isolated two compounds, PD-180970 and PP2, neither of which caused lethal damage. PD-180970 disrupted microtubule (MT) organization and, thus, nuclear separation, and PP2 blocked phragmoplast formation and impaired cytokinesis. Phosphoproteomic analysis showed that these compounds reduced the phosphorylation of diverse proteins, including MT-associated proteins (MAP70) and class II Kinesin-12. Moreover, these compounds were effective in multiple plant species, such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes conserved across diverse plant species.

Cellular dynamics of coenocytic endosperm development in Arabidopsis thaliana.

After double fertilization, the endosperm in the seeds of many flowering plants undergoes repeated mitotic nuclear divisions without cytokinesis, resulting in a large coenocytic endosperm that then cellularizes. Growth during the coenocytic phase is strongly associated with the final seed size; however, a detailed description of the cellular dynamics controlling the unique coenocytic development in flowering plants has remained elusive. By integrating confocal microscopy live-cell imaging and genetics, we have characterized the entire development of the coenocytic endosperm of Arabidopsis thaliana including nuclear divisions, their timing intervals, nuclear movement and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive actin filament (F-actin) organization. Microtubules promote nuclear movement after division, while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and the mature seed. The characterization of cytoskeleton dynamics in real time throughout the entire coenocyte endosperm period provides foundational knowledge of plant coenocytic development, insights into the coordination of F-actin and microtubules in nuclear dynamics, and new opportunities to increase seed size and our food security.

Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips.

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

Live imaging-based assay for visualising species-specific interactions in gamete adhesion molecules.

Successful gamete fusion requires species-specific membrane adhesion. However, the interaction of adhesion molecules in gametes is difficult to study in real time through low-throughput microscopic observation. Therefore, we developed a live imaging-based adhesion molecule (LIAM) assay to study gamete adhesion molecule interactions in cultured cells. First, we modified a fusion assay previously established for fusogens introduced into cultured cells, and confirmed that our live imaging technique could visualise cell-cell fusion in the modified fusion assay. Next, instead of fusogen, we introduced adhesion molecules including a mammalian gamete adhesion molecule pair, IZUMO1 and JUNO, and detected their temporal accumulation at the contact interfaces of adjacent cells. Accumulated IZUMO1 or JUNO was partly translocated to the opposite cells as discrete spots; the mutation in amino acids required for their interaction impaired accumulation and translocation. By using the LIAM assay, we investigated the species specificity of IZUMO1 and JUNO of mouse, human, hamster, and pig in all combinations. IZUMO1 and JUNO accumulation and translocation were observed in conspecific, and some interspecific, combinations, suggesting potentially interchangeable combinations of IZUMO1 and JUNO from different species.

Development of microfluidic chip for entrapping tobacco BY-2 cells.

The tobacco BY-2 cell line has been used widely as a model system in plant cell biology. BY-2 cells are nearly transparent, which facilitates cell imaging using fluorescent markers. As cultured cells are drifted in the medium, therefore, it was difficult to observe them for a long period. Hence, we developed a microfluidic device that traps BY-2 cells and fixes their positions to allow monitoring the physiological activity of cells. The device contains 112 trap zones, with parallel slots connected in series at three levels in the flow channel. BY-2 cells were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell filaments consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell filaments at 25 out of 112 trap zones (22.3%). The cell numbers increased through cell division from 1 to 4 days after trapping with a peak of mitotic index on day 2. Recovery experiments of fluorescent proteins after photobleaching confirmed cell survival and permeability of plasmodesmata. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe cell activity in real time under controllable conditions.

Optical Clearing of Plant Tissues for Fluorescence Imaging.

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis.

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation and final cellularization, it remains unknown when and how the cell fate is determined during development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of individual cell types to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the 1-nucleate stage to the 7-cell stage after cellularization using an in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal (distal-proximal) axis. During cellularization, the polar nuclei migrated while associating with the forming edge of the cell plate, and then, migrated toward each other to fuse linearly. We also tracked the gene expression dynamics and identified that the expression of MYB98pro::GFP-MYB98, a synergid-specific marker, was initiated just after cellularization in the synergid, egg, and central cells and was then restricted to the synergid cells. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild-type and myb98 mutant revealed that the myb98 synergid cells had egg cell-like gene expression profiles. Although in myb98, egg cell-specific gene expression was properly initiated in the egg cells only after cellularization, but subsequently expressed ectopically in one of the 2 synergid cells. These results, together with the various initiation timings of the egg cell-specific genes, suggest complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

ClearSeeAlpha: Advanced Optical Clearing for Whole-Plant Imaging.

To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits-tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.

Arabidopsis GEX1 Is a Nuclear Membrane Protein of Gametes Required for Nuclear Fusion During Reproduction.

During the life cycle of flowering plants, nuclear fusion, or karyogamy, occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. In Arabidopsis thaliana, nuclear fusion events during sexual reproduction proceed without the breakdown of the nuclear envelope, indicating that nuclear membrane fusion is essential for the completion of this process. Arabidopsis gamete expressed 1 (GEX1) is a membrane protein that is conserved among plant species. GEX1 shares homology with the yeast karyogamy protein Kar5, which is primarily expressed in the nuclear membrane. The GEX1 family represents a putative karyogamy factor. Herein, we show that GEX1 is required for the nuclear fusion events in Arabidopsis reproduction. GEX1-deficient mature female gametophytes were found to contain two unfused polar nuclei in close proximity within the central cell. Electron microscopy showed that the outer membrane of the polar nuclei was connected via the endoplasmic reticulum, whereas the inner membrane remained unfused. These results indicate that GEX1 is involved in polar nuclear membrane fusion following the fusion of the outer nuclear membrane. Furthermore, sperm nuclear fusion events were defective in the fertilized egg and central cell following plasmogamy in the fertilization of gex1-1 female gametophytes by gex1-1 pollen. An analysis of GEX1 localization in the female gametophyte using a transgenic line expressing GFP-tagged GEX1 driven by the GEX1 promoter showed that GEX1 is a nuclear membrane protein in the egg and central cell. Time-lapse live-cell imaging showed that in developing female gametophytes, the nuclear GFP-GEX1 signal was first detectable in the central cell shortly before the polar nuclei came in close contact, and then in the egg cell. Thus, we suggest that the GEX1-family proteins are nuclear membrane proteins involved in karyogamy in the reproduction of eukaryotes including flowering plants.

A Peptide Pair Coordinates Regular Ovule Initiation Patterns with Seed Number and Fruit Size.

Ovule development in Arabidopsis thaliana involves pattern formation, which ensures that ovules are regularly arranged in the pistils to reduce competition for nutrients and space. Mechanisms underlying pattern formation in plants, such as phyllotaxis, flower morphogenesis, or lateral root initiation, have been extensively studied, and genes controlling the initiation of ovules have been identified. However, the fundamental patterning mechanism that determines the spacing of ovule anlagen within the placenta remained unexplored. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing gynoecium and fruits is controlled by two secreted peptides, EPFL2 and EPFL9 (also known as Stomagen), and their receptors from the ERECTA (ER) family that act from the carpel wall and the placental tissue. We found that a signaling pathway controlled by EPFL9 acting from the carpel wall through the LRR-receptor kinases ER, ERL1, and ERL2 promotes fruit growth. Regular spacing of ovules depends on EPFL2 expression in the carpel wall and in the inter-ovule spaces, where it acts through ERL1 and ERL2. Loss of EPFL2 signaling results in shorter gynoecia and fruits and irregular spacing of ovules or even ovule twinning. We propose that the EPFL2 signaling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which may serve to minimize competition between seeds or facilitate equal resource allocation. Together, EPFL2 and EPFL9 help to coordinate ovule patterning and thereby seed number with gynoecium and fruit growth through a set of shared receptors.

Live-Cell Imaging of Zygotic Intracellular Structures and Early Embryo Pattern Formation in Arabidopsis thaliana.

Plant embryogenesis begins with fertilization and ends with the generation of the basic body plan of the future plant. Despite its importance, the dynamics of flowering plant ontogeny have long been a mystery, because the embryo develops deep in the maternal tissue. Recently, an embryonic live-cell imaging system was established in Arabidopsis thaliana by developing an in vitro ovule cultivation method and utilizing two-photon excitation microscopy (2PEM), which is suitable for deep imaging. This system enabled us to visualize intracellular dynamics during zygote polarization and monitor the cell division pattern during embryogenesis from the zygote until organ formation. In this chapter, we describe a method that allows for high-resolution imaging of cytoskeletal rearrangements in the zygote and long-term tracing of embryo patterning.

Mitochondrial dynamics and segregation during the asymmetric division of Arabidopsis zygotes.

The zygote is the first cell of a multicellular organism. In most angiosperms, the zygote divides asymmetrically to produce an embryo-precursor apical cell and a supporting basal cell. Zygotic division should properly segregate symbiotic organelles, because they cannot be synthesized de novo. In this study, we revealed the real-time dynamics of the principle source of ATP biogenesis, mitochondria, in Arabidopsis thaliana zygotes using live-cell observations and image quantifications. In the zygote, the mitochondria formed the extended structure associated with the longitudinal array of actin filaments (F-actins) and were polarly distributed along the apical-basal axis. The mitochondria were then temporally fragmented during zygotic division, and the resulting apical cells inherited mitochondria at higher concentration compared to the basal cells. Further observation of postembryonic organs showed that these mitochondrial behaviours are characteristic of the zygote. Overall, our results showed that the zygote has spatiotemporal regulation that unequally distributes the mitochondria.

The formation of perinucleolar bodies is important for normal leaf development and requires the zinc-finger DNA-binding motif in Arabidopsis ASYMMETRIC LEAVES2.

In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant-specific nuclear protein that contains the AS2/LOB domain, which includes a zinc-finger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co-localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2-1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes.

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana.

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

Successful endoscopic submucosal dissection of colon cancer with severe fibrosis after tattooing.

Endoscopic tattooing is often used to facilitate the identification of colorectal lesions before endoscopic treatments. However, tattooing under the lesion can result in technical difficulties because of the dark endoscopic field and submucosal fibrosis. A 65-year-old man with a non-granular-type laterally spreading tumor was referred to our hospital after tattooing with India ink for surgery. However, endoscopic submucosal dissection (ESD) was selected for the resection of this lesion because the findings of magnifying endoscopy suggested an intramucosal cancer. Dissection around a dense section was difficult because of the dark endoscopic field and non-lifting as a result of severe fibrosis. We performed ESD using the following strategy: (1) injection with a smaller amount of indigo carmine and (2) cut and dissection from the side of the thinly tattooed area. The lesion was curatively resected en bloc without any complications. This finding suggests that endoscopic tattooing before endoscopic treatment should be performed one or two folds away from the lesion.

Safety and efficacy of simultaneous colorectal ESD for large synchronous colorectal lesions.

BACKGROUND AND STUDY AIMS: Multiple large colorectal lesions are sometimes diagnosed during colonoscopy. However, there have been no investigations of the feasibility of simultaneous endoscopic submucosal dissection (ESD) for multiple lesions. This study aims to reveal the strategy of simultaneous ESD for multiple large colorectal lesions. PATIENTS AND METHODS: 246 patients who underwent ESD for 274 colorectal lesions were retrospectively evaluated in this study. Fifty-one large colorectal lesions among 23 patients were treated by ESD simultaneously (simultaneous group), and 223 patients were treated with ESD for a single lesion (single group). RESULTS: En-bloc resection and curative resection rates did not differ. Compared with the single group, each procedure time was faster (31.8 ±â€Š23.6 min vs. 45.8 ±â€Š44.8, P  = 0.002), but total procedure time was significantly longer in the simultaneous group (70.6 ±â€Š33.4 vs. 45.8 ±â€Š44.8 min, P  = 0.01). Rates of adverse events including bleeding and perforation were not higher in the simultaneous group but the mean blood pressure, incidence of bradycardia and the amount of sedative drug used during ESD were significantly higher in the simultaneous group. Multiple logistic regression analysis identified non-experienced physician, lesion size ≥ 40 mm and submucosal fibrosis as an independent risk factor for procedure duration (≥ 90 min) (Odds ratio 11.852, 18.280, and 3.672; P  < 0.05, respectively). CONCLUSIONS: Simultaneous ESD for multiple synchronous colorectal lesions is safe and feasible compared with single ESD and can reduce the burden to patients, length of hospital stay and medical expense. These results need to be elucidated by further studies.

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode Heterodera glycines.

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled "Parthenon pillars." In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.