Genomic Nucleosome Organization Reconstituted with Pure Proteins.

Chromatin remodelers regulate genes by organizing nucleosomes around promoters, but their individual contributions are obfuscated by the complex in vivo milieu of factor redundancy and indirect effects. Genome-wide reconstitution of promoter nucleosome organization with purified proteins resolves this problem and is therefore a critical goal. Here, we reconstitute four stages of nucleosome architecture using purified components: yeast genomic DNA, histones, sequence-specific Abf1/Reb1, and remodelers RSC, ISW2, INO80, and ISW1a. We identify direct, specific, and sufficient contributions that in vivo observations validate. First, RSC clears promoters by translating poly(dA:dT) into directional nucleosome removal. Second, partial redundancy is recapitulated where INO80 alone, or ISW2 at Abf1/Reb1sites, positions +1 nucleosomes. Third, INO80 and ISW2 each align downstream nucleosomal arrays. Fourth, ISW1a tightens the spacing to canonical repeat lengths. Such a minimal set of rules and proteins establishes core mechanisms by which promoter chromatin architecture arises through a blend of redundancy and specialization.

The interplay between the circadian clock and abiotic stress responses mediated by ABF3 and CCA1/LHY.

Climate change is a global concern for all life on our planet, including humans and plants. Plants' growth and development are significantly affected by abiotic stresses, including adverse temperature, inadequate or excess water availability, nutrient deficiency, and salinity. The circadian clock is a master regulator of numerous developmental and metabolic processes in plants. In an effort to identify new clock-related genes and outputs through bioinformatic analysis, we have revealed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) play a crucial role in regulating a wide range of abiotic stress responses and target ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR3 (ABF3), a key transcription factor in the plant hormone Abscisic acid (ABA)-signaling pathway. Specifically, we found that CCA1 and LHY regulate the expression of ABF3 under diel conditions, as well as seed germination under salinity. Conversely, ABF3 controls the expression of core clock genes and orchestrates the circadian period in a stress-responsive manner. ABF3 delivers the stress signal to the central oscillator by binding to the promoter of CCA1 and LHY. Overall, our study uncovers the reciprocal regulation between ABF3 and CCA1/LHY and molecular mechanisms underlying the interaction between the circadian clock and abiotic stress. This finding may aid in developing molecular and genetic solutions for plants to survive and thrive in the face of climate change.

Systematic Study of Nucleosome-Displacing Factors in Budding Yeast.

Nucleosomes present a barrier for the binding of most transcription factors (TFs). However, special TFs known as nucleosome-displacing factors (NDFs) can access embedded sites and cause the depletion of the local nucleosomes as well as repositioning of the neighboring nucleosomes. Here, we developed a novel high-throughput method in yeast to identify NDFs among 104 TFs and systematically characterized the impact of orientation, affinity, location, and copy number of their binding motifs on the nucleosome occupancy. Using this assay, we identified 29 NDF motifs and divided the nuclear TFs into three groups with strong, weak, and no nucleosome-displacing activities. Further studies revealed that tight DNA binding is the key property that underlies NDF activity, and the NDFs may partially rely on the DNA replication to compete with nucleosome. Overall, our study presents a framework to functionally characterize NDFs and elucidate the mechanism of nucleosome invasion.

General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation.

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

Stacking Structure of Vaterite Revealed by Atomic Imaging and Diffraction Analysis.

Anhydrous calcium carbonate crystals exist as three polymorphs: calcite, aragonite, and vaterite. Although vaterite is a metastable phase rarely found in the geological environment, it is intriguing that various biominerals are composed of vaterite. The processes of stable vaterite formation in biological systems cannot be understood without elucidating the nature of vaterite. The crystal structure of vaterite has been discussed for nearly a century but is still an open question. Here we propose the actual structure of vaterite by combining atomic imaging and diffraction analysis with simulations of disordered stacking sequences. Vaterite basically appears as layers of hexagonal calcium planes and carbonate (CO3 2-)-containing sheets stacked with +60°, -60°, or 180° rotations from the underlying layer. However, equivalent carbonate positions in alternating layers are forbidden, and four-layer stacking in which the fourth layer rotates 180° relative to the first layer are predominant, forming an orthogonal reciprocal lattice in diffraction patterns. These stacking characteristics replicate the intensity distribution in the electron and X-ray diffraction patterns. This study has almost completely elucidated the crystal structure and stacking sequence of vaterite. Our findings provide insights into the thermodynamic stability of vaterite, which facilitates comprehension of the biomineralization processes and growth dynamics of calcium carbonate.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae.

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast.

The phytohormone abscisic acid (ABA) regulates plant responses to abiotic stress, such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling poses a major challenge for elucidating individual contributions to the response selectivity and sensitivity of the pathway. Here, we reconstructed single ABA signaling pathways in yeast for combinatorial analysis of ABA receptors and coreceptors, downstream-acting SnRK2 protein kinases, and transcription factors. The analysis shows that some ABA receptors stimulate the pathway even in the absence of ABA and that SnRK2s are major determinants of ABA responsiveness by differing in the ligand-dependent control. Five SnRK2s, including SnRK2.4 known to be active under osmotic stress in plants, activated ABA-responsive transcription factors and were regulated by ABA receptor complexes in yeast. In the plant tissue, SnRK2.4 and ABA receptors competed for coreceptor interaction in an ABA-dependent manner consistent with a tight integration of SnRK2.4 into the ABA signaling pathway. The study establishes the suitability of the yeast system for the dissection of core signaling cascades and opens up future avenues of research on ligand-receptor regulation.

PpyABF3 recruits the COMPASS-like complex to regulate bud dormancy maintenance via integrating ABA signaling and GA catabolism.

Bud dormancy is essential for perennial trees that survive the cold winters and to flower on time in the following spring. Histone modifications have been reported to be involved in the control of the dormancy cycle and DAM/SVPs are considered targets. However, how the histone modification marks are added to the specific gene loci during bud dormancy cycle is still unknown. Using yeast-two hybrid library screening and co-immunoprecipitation assays, we found that PpyABF3, a key protein regulating bud dormancy, recruits Complex of Proteins Associated with Set1-like complex via interacting with PpyWDR5a, which increases the H3K4me3 deposition at DAM4 locus. Chromatin immunoprecipitation-quantitative polymerase chain reaction showed that PpyGA2OX1 was downstream gene of PpyABF3 and it was also activated by H3K4me3 deposition. Silencing of GA2OX1 in pear calli and pear buds resulted in a similar phenotype with silencing of ABF3. Furthermore, overexpression of PpyWDR5a increased H3K4me3 levels at DAM4 and GA2OX1 loci and inhibited the growth of pear calli, whereas silencing of PpyWDR5a in pear buds resulted in a higher bud-break percentage. Our findings provide new insights into how H3K4me3 marks are added to dormancy-related genes in perennial woody plants and reveal a novel mechanism by which ABF3 integrates abscisic acid signaling and gibberellic acid catabolism during bud dormancy maintenance.

ABF1 Positively Regulates Rice Chilling Tolerance via Inducing Trehalose Biosynthesis.

Chilling stress seriously limits grain yield and quality worldwide. However, the genes and the underlying mechanisms that respond to chilling stress remain elusive. This study identified ABF1, a cold-induced transcription factor of the bZIP family. Disruption of ABF1 impaired chilling tolerance with increased ion leakage and reduced proline contents, while ABF1 over-expression lines exhibited the opposite tendency, suggesting that ABF1 positively regulated chilling tolerance in rice. Moreover, SnRK2 protein kinase SAPK10 could phosphorylate ABF1, and strengthen the DNA-binding ability of ABF1 to the G-box cis-element of the promoter of TPS2, a positive regulator of trehalose biosynthesis, consequently elevating the TPS2 transcription and the endogenous trehalose contents. Meanwhile, applying exogenous trehalose enhanced the chilling tolerance of abf1 mutant lines. In summary, this study provides a novel pathway 'SAPK10-ABF1-TPS2' involved in rice chilling tolerance through regulating trehalose homeostasis.

Dehydrin MsDHN1 improves aluminum tolerance of alfalfa (Medicago sativa L.) by affecting oxalate exudation from root tips.

A SK3 -type dehydrin MsDHN1 was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression and suppression of MsDHN1 in alfalfa seedlings or hairy roots. The results showed that MsDHN1 is a typical intrinsically disordered protein that exists in the form of monomers and homodimers in alfalfa. The plant growth rates increased as a result of MsDHN1 overexpression (MsDHN1-OE) and decreased upon MsDHN1 suppression (MsDHN1-RNAi) in seedlings or hairy roots of alfalfa compared with the wild-type or the vector line under Al stress. MsDHN1 interacting with aquaporin (AQP) MsPIP2;1 and MsTIP1;1 positively affected oxalate secretion from root tips and Al accumulation in root tips. MsABF2 was proven to be an upstream transcription factor of MsDHN1 and activated MsDHN1 expression by binding to the ABRE element of the MsDHN1 promoter. The transcriptional regulation of MsABF2 on MsDHN1 was dependent on the abscisic acid signaling pathway. These results indicate that MsDHN1 can increase alfalfa tolerance to Al stress via increasing oxalate secretion from root tips, which may involve in the interaction of MsDHN1 with two AQP.

The Arabidopsis IDD14 transcription factor interacts with bZIP-type ABFs/AREBs and cooperatively regulates ABA-mediated drought tolerance.

The INDETERMINATE DOMAIN (IDD) transcription factors mediate various aspects of plant growth and development. We previously reported that an Arabidopsis IDD subfamily regulates spatial auxin accumulation, and thus organ morphogenesis and gravitropic responses. However, its functions in stress responses are not well defined. Here, we use a combination of physiological, biochemical, molecular, and genetic approaches to provide evidence that the IDD14 cooperates with basic leucine zipper-type binding factors/ABA-responsive element (ABRE)-binding proteins (ABRE-binding factors (ABFs)/AREBs) in ABA-mediated drought tolerance. idd14-1D, a gain-of-function mutant of IDD14, exhibits decreased leaf water loss and improved drought tolerance, whereas inactivation of IDD14 in idd14-1 results in increased transpiration and reduced drought tolerance. Altered IDD14 expression affects ABA sensitivity and ABA-mediated stomatal closure. IDD14 can physically interact with ABF1-4 and subsequently promote their transcriptional activities. Moreover, ectopic expression and mutation of ABFs could, respectively, suppress and enhance plant sensitivity to drought stress in the idd14-1 mutant. Our results demonstrate that IDD14 forms a functional complex with ABFs and positively regulates drought-stress responses, thus revealing a previously unidentified role of IDD14 in ABA signaling and drought responses.

Accuracy of Local Polarization Measurements by Scanning Transmission Electron Microscopy.

Accurately determining local polarization at atomic resolution can unveil the mechanisms by which static and dynamical behaviors of the polarization occur, including domain wall motion, defect interaction, and switching mechanisms, advancing us toward the better control of polarized states in materials. In this work, we explore the potential of atomic-resolution scanning transmission electron microscopy to measure the projected local polarization at the unit cell length scale. ZnO and PbMg1/3Nb2/3O3 are selected as case studies, to identify microscope parameters that can significantly affect the accuracy of the measured projected polarization vector. Different STEM imaging modalities are used to determine the location of the atomic columns, which, when combined with the Born effective charges, allows for the calculation of local polarization. Our results indicate that differentiated differential phase contrast (dDPC) imaging enhances the accuracy of measuring local polarization relative to other imaging modalities, such as annular bright-field or integrated-DPC imaging. For instance, under certain experimental conditions, the projected spontaneous polarization for ZnO can be calculated with 1.4% error from the theoretical value. Furthermore, we quantify the influence of sample thickness, probe defocus, and crystal mis-tilt on the relative errors of the calculated polarization.

LoSWEET14, a Sugar Transporter in Lily, Is Regulated by Transcription Factor LoABF2 to Participate in the ABA Signaling Pathway and Enhance Tolerance to Multiple Abiotic Stresses in Tobacco.

Sugar transport and distribution plays an important role in lily bulb development and resistance to abiotic stresses. In this study, a member of the Sugar Will Eventually be Exported Transporters (SWEET) gene family, LoSWEET14, from Oriental hybrid lily 'Sorbonne' was identified. LoSWEET14 encodes a protein of 278 amino acids and is capable of transporting sucrose and some types of hexoses. The transcript level of the LoSWEET14 gene was significantly increased under various stress conditions including drought, cold, salt stresses, and abscisic acid (ABA) treatment. Overexpression of LoSWEET14 in tobacco (Nicotiana tabacum) showed that the transgenic lines had larger leaves, accumulated more soluble sugars, and were more resistant to drought, cold, and salt stresses, while becoming more sensitive to ABA compared with wild-type lines. Promoter analysis revealed that multiple stress-related cis-acting elements were found in the promoter of LoSWEET14. According to the distribution of cis-acting elements, different lengths of 5'-deletion fragments were constructed and the LoSWEET14-pro3(-540 bp) was found to be able to drive GUS gene expression in response to abiotic stresses and ABA treatment. Furthermore, a yeast one hybrid (Y1H) assay proved that the AREB/ABF (ABRE-binding protein/ABRE-binding factor) from lilies (LoABF2) could bind to the promoter of LoSWEET14. These findings indicated that LoSWEET14 is induced by LoABF2 to participate in the ABA signaling pathway to promote soluble sugar accumulation in response to multiple abiotic stresses.

Drought-Responsive NAC Transcription Factor RcNAC72 Is Recognized by RcABF4, Interacts with RcDREB2A to Enhance Drought Tolerance in Arabidopsis.

RcNAC72, a key transcription factor that may respond to drought stress in Rosa chinensis 'Old Blush', was selected in our previous study. In the present study, we found that RcNAC72 is localized in the nucleus and is a transcriptional activator. RcNAC72 expression could be significantly induced by drought, low temperature, salt as well as abscisic acid (ABA) treatment. Analysis of the promoter revealed that multiple abiotic stress and hormone response elements were located in the promoter region. The promoter could respond to drought, low temperature, salt and ABA treatments to activate GUS gene expression. Overexpressing RcNAC72 in Arabidopsis thaliana enhanced sensitivity to ABA and tolerance to drought stress. Silencing of RcNAC72 by virus-induced gene silencing (VIGS) in rose leaves significantly reduced leaf water loss tolerance and leaf extension capacity. Physical interaction of RcNAC72 with RcDREB2A was shown by means of the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. RcABF4 was demonstrated to be able to bind to the promoter of RcNAC72 by means of the yeast one-hybrid (Y1H) assay. These results provide new insights into the regulatory network of RcNAC72 response to drought stress in roses.

Lighting programme as a management tool for broilers raised without antibiotics - impact on productivity and welfare.

1. This study investigated the impact of photoperiod on the productivity and welfare of broilers reared without antibiotics (RWA).2. A total of 8,064 mixed sex Ross 308 broilers were allocated to two trials. Lighting treatments were 14L:10D, 17L:7D, 20L:4D or 23L:1D.3. Significance defined when P ≤ 0.05 and trends noted when P ≤ 0.10. Highest body weights and feed consumption were found in the 4D treatment. Longer dark periods resulted in improved feed conversion. Uniformity was improved with 1D.4. Heterophil/lymphocyte ratios were highest in birds reared on 1D. Longer photoperiods negatively impacted gait and footpad scores. Birds spent more time performing feeding behaviours under longer photoperiods. Birds reared under 1 and 10D spent more time standing while those under 4 and 7D spent more time preening. Birds raised on 4D spent more time environmental pecking, while object pecking occurred more under 10D. Condemnations were lowest in birds reared under 7D.5. In conclusion, rearing RWA broilers on longer photoperiods negatively impacted welfare, as indicated by poorer mobility, higher stress, more severe footpad lesions, and altered behaviour.

Analysis of the Function of the Alfalfa Mslea-D34 Gene in Abiotic Stress Responses and Flowering Time.

A novel late embryogenesis abundant (LEA) gene, MsLEA-D34, was cloned from alfalfa (Medicago sativa L.). Its function and gene regulatory pathways were studied via overexpression (OE) and RNA interference (RNAi) of the gene in Arabidopsis and in hairy roots of alfalfa, as well as via analyzing key genes related to MsLEA-D34 during developmental phases in alfalfa. The results showed that MsLEA-D34 was a typical intrinsically disordered protein with a high capability for protein protection. Overexpression of MsLEA-D34 increased plant tolerance to osmotic and salt stresses, and caused Arabidopsis early flowering under drought and well-watered conditions. Overexpressing MsLEA-D34 induced up-regulation of FLOWERING LOCUS T (FT) and GIGANTEA (GI) at the flowering phase of Arabidopsis and hairy roots of alfalfa, but only FT was down-regulated in MsLEA-D34-RNAi lines. A positive effect of MsLEA-D34 on FT accumulation was demonstrated in alfalfa hairy roots. An ABA-responsive element (ABRE)-binding transcription factor (MsABF2), a novel transcription factor cloned from alfalfa, directly bound to the RY element in the MsLEA-D34 promoter and activated MsLEA-D34 expression. The above results indicate that MsLEA-D34 can regulate abiotic stress response in plants and influence flowering time of Arabidopsis.

Deep learning supported disease detection with multi-modality image fusion.

Multi-modal image fusion techniques aid the medical experts in better disease diagnosis by providing adequate complementary information from multi-modal medical images. These techniques enhance the effectiveness of medical disorder analysis and classification of results. This study aims at proposing a novel technique using deep learning for the fusion of multi-modal medical images. The modified 2D Adaptive Bilateral Filters (M-2D-ABF) algorithm is used in the image pre-processing for filtering various types of noises. The contrast and brightness are improved by applying the proposed Energy-based CLAHE algorithm in order to preserve the high energy regions of the multimodal images. Images from two different modalities are first registered using mutual information and then registered images are fused to form a single image. In the proposed fusion scheme, images are fused using Siamese Neural Network and Entropy (SNNE)-based image fusion algorithm. Particularly, the medical images are fused by using Siamese convolutional neural network structure and the entropy of the images. Fusion is done on the basis of score of the SoftMax layer and the entropy of the image. The fused image is segmented using Fast Fuzzy C Means Clustering Algorithm (FFCMC) and Otsu Thresholding. Finally, various features are extracted from the segmented regions. Using the extracted features, classification is done using Logistic Regression classifier. Evaluation is performed using publicly available benchmark dataset. Experimental results using various pairs of multi-modal medical images reveal that the proposed multi-modal image fusion and classification techniques compete the existing state-of-the-art techniques reported in the literature.

ABA-responsive ABRE-BINDING FACTOR3 activates DAM3 expression to promote bud dormancy in Asian pear.

Bud dormancy is indispensable for the survival of perennial plants in cold winters. Abscisic acid (ABA) has essential functions influencing the endo-dormancy status. Dormancy-associated MADS-box/SHORT VEGETATIVE PHASE-like genes function downstream of the ABA signalling pathway to regulate bud dormancy. However, the regulation of DAM/SVP expression remains largely uncharacterized. In this study, we confirmed that endo-dormancy maintenance and PpyDAM3 expression are controlled by the ABA content in pear (Pyrus pyrifolia) buds. The expression of pear ABRE-BINDING FACTOR3 (PpyABF3) was positively correlated with PpyDAM3 expression. Furthermore, PpyABF3 directly bound to the second ABRE in the PpyDAM3 promoter to activate its expression. Interestingly, both PpyABF3 and PpyDAM3 repressed the cell division and growth of transgenic pear calli. Another ABA-induced ABF protein, PpyABF2, physically interacted with PpyABF3 and disrupted the activation of the PpyDAM3 promoter by PpyABF3, indicating DAM expression was precisely controlled. Additionally, our results suggested that the differences in the PpyDAM3 promoter in two pear cultivars might be responsible for the diversity in the chilling requirements. In summary, our data clarify the finely tuned regulatory mechanism underlying the effect of ABA on DAM gene expression and provide new insights into ABA-related bud dormancy regulation.

Transcription factor TCP20 regulates peach bud endodormancy by inhibiting DAM5/DAM6 and interacting with ABF2.

The dormancy-associated MADS-box (DAM) genes PpDAM5 and PpDAM6 have been shown to play important roles in bud endodormancy; however, their molecular regulatory mechanism in peach is unclear. In this study, by use of yeast one-hybrid screening, we isolated a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR transcription factor, PpTCP20, in the peach cultivar 'Zhongyou 4' (Prunus persica var. nectarina). The protein was localized in the nucleus and was capable of forming a homodimer. Electrophoretic mobility shift assays demonstrated that PpTCP20 binds to a GCCCR element in the promoters of PpDAM5 and PpDAM6, and transient dual luciferase experiments showed that PpTCP20 inhibited the expression of PpDAM5 and PpDAM6 as the period of the release of flower bud endodormancy approached. In addition, PpTCP20 interacted with PpABF2 to form heterodimers to regulate bud endodormancy, and the content of abscisic acid decreased with the release of endodormancy. PpTCP20 also inhibited expression of PpABF2 to regulate endodormancy. Taken together, our results suggest that PpTCP20 regulates peach flower bud endodormancy by negatively regulating the expression of PpDAM5 and PpDAM6, and by interacting with PpABF2, thus revealing a novel regulatory mechanism in a perennial deciduous tree.

Simultaneous visualization of callose deposition and plasma membrane for live-cell imaging in plants.

KEY MESSAGE: The appropriate combination of fluorescent probes enabled the simultaneous visualization of callose deposition and plasma membrane in living Arabidopsis and can be useful for the cell biological study of papilla formation in plants. Localized callose deposition at the site of fungal infection is a central part of papilla formation, which creates a barrier between the host plasma membrane and the cell wall and plays an important role in preventing the penetration of fungal hyphae into the host cells. Using chitin-induced callose deposition as a model system, we examined suitable conditions for the simultaneous visualization of callose deposition and plasma membrane dynamics in living Arabidopsis cotyledons. We found that aniline blue fluorochrome (ABF) for callose staining selectively interferes with FM dyes for membrane visualization depending on the structure of the latter compounds and the proper combination of these fluorescent dyes and staining conditions is a key for successful live-cell imaging. The established conditions enabled the live-cell imaging of chitin-induced callose deposition and host membrane systems. The established system/conditions would also be useful for the cell biological studies on the localized callose deposition in other stress/development-associated processes. The finding that the slight difference in the structure of FM dyes affects the interaction with another fluorescent dye, ABF, would also give useful suggestions for the studies where multiple fluorescent dyes are utilized for live-cell imaging.