Signaling Cues Research Articles

A neuro-lymphatic communication guides lymphatic development by CXCL12/CXCR4 signaling.

Lymphatic vessels grow through active sprouting and mature into a vascular complex including lymphatic capillaries and collecting vessels that ensure fluid transport. However, the signaling cues that direct lymphatic sprouting and patterning remains unclear. In this study, we demonstrated the chemokine signaling, specifically through CXCL12/CXCR4 plays critical roles in regulating lymphatic development. We showed that LEC specific CXCR4 deficient embryos and CXCL12 mutant embryos exhibited server defects in lymphatic sprouting, migration, and lymphatic valve formation. We also discovered that CXCL12, originating from peripheral nerves, directs the migration of dermal lymphatic vessels to align with nerves in developing skin. Deletion CXCR4 or blockage of CXCL12/CXCR4 activity results in reduced VEGFR3 levels on the LEC surface. This, in turn, impairs VEGFC mediated VEGFR3 signaling and downstream PI3K/AKT activities. Taken together, these data identify previously unknown chemokine signaling originating from peripheral nerves that guides dermal lymphatic sprouting and patterning. Our work identifies for the first time a neuro-lymphatics communications during mouse development and reveals a novel mechanism by which CXCR4 modulates VEGFC/VEGFR3/AKT signaling.

A common stay-on-goal mechanism in anterior cingulate cortex for information and effort choices.

Humans and non-humans alike often make choices to gain information, even when the information cannot be used to change the outcome. Prior research has shown the anterior cingulate cortex (ACC) is important for evaluating options involving reward-predictive information. Here we studied the role of ACC in information choices using optical inhibition to evaluate the contribution of this region during specific epochs of decision making. Rats could choose between an uninformative option followed by a cue that predicted reward 50% of the time vs. a fully informative option that signaled outcomes with certainty, but was rewarded only 20% of the time. Reward seeking during the informative S+ cue decreased following ACC inhibition, indicating a causal contribution of this region in supporting reward expectation to a cue signaling reward with certainty. Separately in a positive control experiment and in support of a known role for this region in sustaining high-effort behavior for preferred rewards, we observed reduced lever presses and lower breakpoints in effort choices following ACC inhibition. The lack of changes in reward latencies in both types of decisions indicate the motivational value of rewards remained intact, revealing instead a common role for ACC in maintaining persistence toward certain and valuable rewards.

Dopamine release plateau and outcome signals in dorsal striatum contrast with classic reinforcement learning formulations

We recorded dopamine release signals in centromedial and centrolateral sectors of the striatum as mice learned consecutive versions of visual cue-outcome conditioning tasks. Dopamine release responses differed for the centromedial and centrolateral sites. In neither sector could these be accounted for by classic reinforcement learning alone as classically applied to the activity of nigral dopamine-containing neurons. Medially, cue responses ranged from initial sharp peaks to modulated plateau responses; outcome (reward) responses during cue conditioning were minimal or, initially, negative. At centrolateral sites, by contrast, strong, transient dopamine release responses occurred at both cue and outcome. Prolonged, plateau release responses to cues emerged in both regions when discriminative behavioral responses became required. At most sites, we found no evidence for a transition from outcome signaling to cue signaling, a hallmark of temporal difference reinforcement learning as applied to midbrain dopaminergic neuronal activity. These findings delineate a reshaping of striatal dopamine release activity during learning and suggest that current views of reward prediction error encoding need review to accommodate distinct learning-related spatial and temporal patterns of striatal dopamine release in the dorsal striatum.

PmLBD3 links auxin and brassinosteroid signalling pathways on dwarfism in Prunus mume

BackgroundGrafting with dwarf rootstock is an efficient method to control plant height in fruit production. However, the molecular mechanism remains unclear. Our previous study showed that plants with Prunus mume (mume) rootstock exhibited a considerable reduction in plant height, internode length, and number of nodes compared with Prunus persica (peach) rootstock. The present study aimed to investigate the mechanism behind the regulation of plant height by mume rootstocks through transcriptomic and metabolomic analyses with two grafting combinations, ‘Longyan/Mume’ and ‘Longyan/Peach’.ResultsThere was a significant decrease in brassinolide levels in plants that were grafted onto mume rootstocks. Plant hormone signal transduction and brassinolide production metabolism gene expression also changed significantly. Flavonoid levels, amino acid and fatty acid metabolites, and energy metabolism in dwarf plants decreased. There was a notable upregulation of PmLBD3 gene expression in plant specimens that were subjected to grafting onto mume rootstocks. Auxin signalling cues promoted PmARF3 transcription, which directly controlled this upregulation. Through its binding to PmBAS1 and PmSAUR36a gene promoters, PmLBD3 promoted endogenous brassinolide inactivation and inhibited cell proliferation.ConclusionsAuxin signalling and brassinolide levels are linked by PmLBD3. Our findings showed that PmLBD3 is a key transcription factor that regulates the balance of hormones through the auxin and brassinolide signalling pathways and causes dwarf plants in stone fruits.

Motion blur microscopy: in vitro imaging of cell adhesion dynamics in whole blood flow

Imaging and characterizing the dynamics of cellular adhesion in blood samples is of fundamental importance in understanding biological function. In vitro microscopy methods are widely used for this task but typically require diluting the blood with a buffer to allow for transmission of light. However, whole blood provides crucial signaling cues that influence adhesion dynamics, which means that conventional approaches lack the full physiological complexity of living microvasculature. We can reliably image cell interactions in microfluidic channels during whole blood flow by motion blur microscopy (MBM) in vitro and automate image analysis using machine learning. MBM provides a low cost, easy to implement alternative to intravital microscopy, for rapid data generation where understanding cell interactions, adhesion, and motility is crucial. MBM is generalizable to studies of various diseases, including cancer, blood disorders, thrombosis, inflammatory and autoimmune diseases, as well as providing rich datasets for theoretical modeling of adhesion dynamics.

Spatially organized striatal neuromodulator release encodes trajectory errors.

Goal-directed navigation requires animals to continuously evaluate their current direction and speed of travel relative to landmarks to discern whether they are approaching or deviating from their goal. Striatal dopamine and acetylcholine are powerful modulators of goal-directed behavior, but it is unclear whether and how neuromodulator dynamics at landmarks incorporate relative motion for effective behavioral guidance. Using optical measurements in mice, we demonstrate that cue-evoked striatal dopamine release encodes bi-directional 'trajectory errors' reflecting relationships between ongoing speed and direction of locomotion and visual flow relative to optimal goal trajectories. Striatum-wide micro-fiber array recordings resolved an anatomical gradient of trajectory error signaling across the anterior-posterior axis, distinct from trajectory error independent cue signals. Dynamic regression modeling revealed that positive and negative trajectory error encoding emerges early and late respectively during learning and over different time courses in the medial and lateral striatum, enabling region specific contributions to learning. Striatal acetylcholine release also encodes trajectory errors, but encoding is more spatially restricted, opposite polarity, and delayed relative to dopamine, supporting distinct roles in modulating striatal output and behavior. Dopamine trajectory error signaling and task performance were reproduced in a reinforcement learning model incorporating a conjunctive state space representation, suggesting a potential neural substrate for trajectory error generation. Our results establish region specific neuromodulator signals positioned to guide the speed and direction of locomotion to reach goals based on environmental landmarks during navigation.

Structural insights into strigolactone catabolism by carboxylesterases reveal a conserved conformational regulation

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.

Great remains great, weak becomes weaker? Examining the influence of platform’s signaling cues on provider performance

In the evolving domain of access-based services characterized by intricate multi-actor interactions, understanding the determinants of provider performance is vital. This study investigates the moderating role of platform-based signaling cues on the relationship between customer experience (CX) and provider performance. We utilize a mixed-methods approach, integrating text mining to quantify CX from customer reviews and panel data fixed-effects regression to assess performance outcomes. Our findings reveal an asymmetry in the impact of CX based on platform signals: non-signaled providers suffer more from negative experiences than their signaled counterparts, while positive experiences do not exhibit a differential impact. Interestingly, customer-reviewed ratings dampen the adverse effects of negative CX, serving as a substitute for platform endorsement. Moreover, high dispersion in customer feedback enhances the benefits of positive CX for signaled providers. These insights offer actionable strategies for access-based companies to optimize customer touchpoints and foster enhanced service delivery.

Poised PABP–RNA hubs implement signal-dependent mRNA decay in development

Signaling pathways drive cell fate transitions largely by changing gene expression. However, the mechanisms for rapid and selective transcriptome rewiring in response to signaling cues remain elusive. Here we use deep learning to deconvolve both the sequence determinants and the trans-acting regulators that trigger extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase kinase (MEK)-induced decay of the naive pluripotency mRNAs. Timing of decay is coupled to embryo implantation through ERK–MEK phosphorylation of LIN28A, which repositions pLIN28A to the highly A+U-rich 3′ untranslated region (3′UTR) termini of naive pluripotency mRNAs. Interestingly, these A+U-rich 3′UTR termini serve as poly(A)-binding protein (PABP)-binding hubs, poised for signal-induced convergence with LIN28A. The multivalency of AUU motifs determines the efficacy of pLIN28A–PABP convergence, which enhances PABP 3′UTR binding, decreases the protection of poly(A) tails and activates mRNA decay to enable progression toward primed pluripotency. Thus, the signal-induced convergence of LIN28A with PABP–RNA hubs drives the rapid selection of naive mRNAs for decay, enabling the transcriptome remodeling that ensures swift developmental progression.

Building better habitats: Spatiotemporal signaling cues in 3D biointerfaces for tailored cellular functionality.

A promising research direction in the field of biological engineering is the design and functional programming of three-dimensional (3D) biointerfaces designed to support living cell functionality and growth in vitro, offering a route to precisely regulate cellular behaviors and phenotypes for addressing therapeutic challenges. While traditional two-dimensional (2D) biointerfaces have provided valuable insights, incorporating specific signaling cues into a 3D biointeractive microenvironment at the right locations and time is now recognized as crucial for accurately programming cellular decision-making and communication processes. This approach aims to engineer cell-centric microenvironments with the potential to recapitulate complex biological functions into a finite set of growing cellular organizations. Additionally, they provide insights into the hierarchical logic governing the relationship between molecular components and higher-order multicellular functionality. The functional live cell-based microenvironment engineered through such innovative biointerfaces has the potential to be used as an in vitro model system for expanding our understanding of cellular behaviors or as a therapeutic habitat where cellular functions can be reprogrammed.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Female cichlids mate with novel androgen receptor mutant males that lack coloration

A key challenge in animal behavior is disentangling the social stimuli that drive conspecific behaviors. For some species, like teleost fish, putative sexual signaling cues are inextricably linked to others, making it difficult to parse the precise roles distinct signals play in driving conspecific behaviors. In the African cichlid Astatotilapia burtoni, males are either dominant or subordinate, wherein bright coloration, territoriality, and courtship behavior inextricably correlate positively with rank. Here, we leveraged androgen receptor (AR) mutant male A. burtoni that lack dominance-typical coloration but not behavior to isolate the role of male coloration in driving female mating behaviors in this species. We found in independent behavioral assays that females behave aggressively towards AR mutant but not WT males, yet still mated with both types of males. Females showed enhanced activation of esr2b + cells in the hypothalamus when housed with either mutant or WT males and this activation scaled with spawning activities. Therefore, there is not a simple relationship between male coloration and female mating behaviors in A. burtoni, suggesting independent sensory mechanisms converge on hypothalamic esr2b cells to coordinate behavioral output.

Synergistic induction of blood–brain barrier properties

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/β-catenin signaling and inhibition of the TGF-β pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/β-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Signaling cues and focused prompts for professional vision support: The interplay of instructional design and situational interest in preservice teachers’ video analysis

In teacher education, video representations of practice offer a motivating means for applying conceptual teaching knowledge toward real-world settings. With video analysis, preservice teachers can begin cultivating professional vision skills through noticing and reasoning about presented core teaching practices. However, with novices’ limited prior knowledge and experience, processing transient information from video can be challenging. Multimedia learning research suggests instructional design techniques for support, such as signaling keyword cues during video viewing, or presenting focused self-explanation prompts which target theoretical knowledge application during video analysis. This study investigates the professional vision skills of noticing and reasoning (operationalized as descriptions and interpretations of relevant noticed events) from 130 preservice teachers participating in a video-analysis training on the core practice of small-group instruction. By means of experimental comparisons, we examine the effects of signaling cues and focused self-explanation prompts on professional vision performance. Further, we explore the impact of these techniques, considering preservice teachers’ situational interest. Overall, results demonstrated that preservice teachers’ professional vision skills improved from pretest to posttest, but the instructional design techniques did not generally offer additional support. However, moderation analysis indicated that training with cues fostered professional vision skills for preservice teachers with low situational interest. This suggests that for uninterested novices, signaling cues may compensate for the generative processing boost typically associated with situational interest. Research and practice implications involve the consideration of situational interest as a powerful component of instructional design, and that keyword cueing can offer an alternative when interest is difficult to elicit.

Skeletal stem and progenitor cells in bone development and repair.

Bone development, growth, and repair are complex processes involving various cell types and interactions, with central roles played by skeletal stem and progenitor cells. Recent research brought new insights into the skeletal precursor populations that mediate intramembranous and endochondral bone development. Later in life, many of the cellular and molecular mechanisms determining development are reactivated upon fracture, with powerful trauma-induced signaling cues triggering a variety of postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect. Interestingly, in this injury context, the current evidence suggests that the fates of both SSPCs and differentiated skeletal cells can be considerably flexible and dynamic, and that multiple cell sources can be activated to operate as functional progenitors generating chondrocytes and/or osteoblasts. The combined implementation of in vivo lineage tracing, cell surface marker-based cell selection, single-cell molecular analyses, and high-resolution in situ imaging has strongly improved our insights into the diversity and roles of developmental and reparative stem/progenitor subsets, while also unveiling the complexity of their dynamics, hierarchies, and relationships. Albeit incompletely understood at present, findings supporting lineage flexibility and possibly plasticity among sources of osteogenic cells challenge the classical dogma of a single primitive, self-renewing, multipotent stem cell driving bone tissue formation and regeneration from the apex of a hierarchical and strictly unidirectional differentiation tree. We here review the state of the field and the newest discoveries in the origin, identity, and fates of skeletal progenitor cells during bone development and growth, discuss the contributions of adult SSPC populations to fracture repair, and reflect on the dynamism and relationships among skeletal precursors and differentiated cell lineages. Further research directed at unraveling the heterogeneity and capacities of SSPCs, as well as the regulatory cues determining their fate and functioning, will offer vital new options for clinical translation toward compromised fracture healing and bone regenerative medicine.

Engineering a robust and anisotropic cardiac-specific extracellular matrix scaffold for cardiac patch tissue engineering

Extracellular matrix (ECM) fabricated using human induced pluripotent stem cells (hiPSCs)-derived cardiac fibroblasts (hiPSC-CFs) could serve as a completely biological scaffold for an engineered cardiac patch, leveraging the unlimited source and outstanding reproducibility of hiPSC-CFs. Additionally, hiPSC-CF-derived ECM (hiPSC-CF-ECM) holds the potential to enhance maturation of exogenous cardiomyocytes, such as hiPSC-derived cardiomyocytes (hiPSC-CMs), by providing a microenvironment rich in cardiac-specific biochemical and signaling cues. However, achieving sufficient robustness of hiPSC-CF-ECM is challenging. This study aims to achieve appropriate ECM deposition, scaffold thickness, and mechanical strength of an aligned hiPSC-CF-ECM by optimizing the culture period, ranging from 2 to 10 weeks, of hiPSC-CFs grown on micro-grated substrates, which can direct the alignment of both hiPSC-CFs and their secreted ECM. The hiPSC-CFs demonstrated a production rate of 13.5 µg ECM per day per 20,000 cells seeded. An anisotropic nanofibrous hiPSC-CF-ECM scaffold with a thickness of 20.0 ± 2.1 µm was achieved after 6 weeks of culture, followed by decellularization. Compositional analysis through liquid chromatography-mass spectrometry (LC-MS) revealed the presence of cardiac-specific fibrillar collagens, non-fibrillar collagens, and matricellular proteins. Uniaxial tensile stretching of the hiPSC-CF-ECM scaffold indicated robust tensile resilience. Finally, hiPSCs-CMs cultured on the hiPSC-CF-ECM exhibited alignment following the guidance of ECM nanofibers and demonstrated mature organization of key structural proteins. The culture duration of the anisotropic hiPSC-CF-ECM was successfully refined to achieve a robust scaffold containing structural proteins that resembles cardiac microenvironment. This completely biological, anisotropic, and cardiac-specific ECM holds great potential for cardiac patch engineering.

Genetic Tools for Cell Lineage Tracing and Profiling Developmental Trajectories in the Skin

The epidermis is the body's first line of protection against dehydration and pathogens, continually regenerating the outermost protective skin layers throughout life. During both embryonic development and wound healing, epidermal stem and progenitor cells must respond to external stimuli and insults to build, maintain, and repair the cutaneous barrier. Recent advances in CRISPR-based methods for cell lineage tracing have remarkably expanded the potential for experiments that track stem and progenitor cell proliferation and differentiation over the course of tissue and even organismal development. Additional tools for DNA-based recording of cellular signaling cues promise to deepen our understanding of the mechanisms driving normal skin morphogenesis and response to stressors as well as the dysregulation of cell proliferation and differentiation in skin diseases and cancer. In this review, we highlight cutting-edge methods for cell lineage tracing, including in organoids and model organisms, and explore how cutaneous biology researchers might leverage these techniques to elucidate the developmental programs that support the regenerative capacity and plasticity of the skin.

Mutation of the ALS-/FTD-Associated RNA-Binding Protein FUS Affects Axonal Development.

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.

Betaglycan sustains HGF/Met signaling in lung cancer and endothelial cells promoting cell migration and tumor growth

Persistent HGF/Met signaling drives tumor growth and dissemination. Proteoglycans within the tumor microenvironment might control HGF availability and signaling by affecting its accessibility to Met (HGF receptor), likely defining whether acute or sustained HGF/Met signaling cues take place. Given that betaglycan (BG, also known as type III TGFβ receptor or TGFBR3), a multi-faceted proteoglycan TGFβ co-receptor, can be found within the tumor microenvironment, we addressed its hypothetical role in oncogenic HGF signaling. We found that HGF/Met promotes lung cancer and endothelial cells migration via PI3K and mTOR. This effect was enhanced by recombinant soluble betaglycan (solBG) via a mechanism attributable to its glycosaminoglycan chains, as a mutant without them did not modulate HGF effects. Moreover, soluble betaglycan extended the effect of HGF-induced phosphorylation of Met, Akt, and Erk, and membrane recruitment of the RhoGEF P-Rex1. Data-mining analysis of lung cancer patient datasets revealed a significant correlation between high MET receptor, HGF, and PREX1 expression and reduced patient survival. Soluble betaglycan showed biochemical interaction with HGF and, together, they increased tumor growth in immunocompetent mice. In conclusion, the oncogenic properties of the HGF/Met pathway are enhanced and sustained by GAG-containing soluble betaglycan.

Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii.

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.