IL-6 mediates olfactory dysfunction in a mouse model of allergic rhinitis.

BACKGROUND: Immune-inflammatory response is a key element in the occurrence and development of olfactory dysfunction (OD) in patients with allergic rhinitis (AR). As one of the core factors in immune-inflammatory responses, interleukin (IL)-6 is closely related to the pathogenesis of allergic diseases. It may also play an important role in OD induced by diseases, such as Sjögren's syndrome and coronavirus disease 2019. However, there is no study has reported its role in OD in AR. Thus, this study aimed to investigate the role of IL-6 in AR-related OD, in an attempt to discover a new target for the prevention and treatment of OD in patients with AR. METHODS: Differential expression analysis was performed using the public datasets GSE52804 and GSE140454 for AR, and differentially expressed genes (DEGs) were obtained by obtaining the intersection points between these two datasets. IL-6, a common differential factor, was obtained by intersecting the DEGs with the General Olfactory Sensitivity Database (GOSdb) again. A model of AR mice with OD was developed by sensitizing with ovalbumin (OVA) to verify the reliability of IL-6 as a key factor of OD in AR and explore the potential mechanisms. Furthermore, a supernatant and microglia co-culture model of nasal mucosa epithelial cells stimulated by the allergen house dust mite extract Derp1 was established to identify the cellular and molecular mechanisms of IL-6-mediated OD in AR. RESULTS: The level of IL-6 in the nasal mucosa and olfactory bulb of AR mice with OD significantly increased and showed a positive correlation with the expression of olfactory bulb microglia marker Iba-1 and the severity of OD. In-vitro experiments showed that the level of IL-6 significantly increased in the supernatant after the nasal mucosa epithelial cells were stimulated by Derp1, along with significantly decreased barrier function of the nasal mucosa. The expression levels of neuroinflammatory markers IL-1ß and INOS increased after a conditioned culture of microglia with the supernatant including IL-6. Then knockdown (KD) of IL-6R by small interfering RNA (siRNA), the expression of IL-1ß and INOS significantly diminished. CONCLUSION: IL-6 plays a key role in the occurrence and development of OD in AR, which may be related to its effect on olfactory bulb microglia-mediated neuroinflammation.

Corrigendum: Two small-molecule inhibitors of Toxoplasma gondii proliferation in vitro.

[This corrects the article DOI: 10.3389/fcimb.2023.1145824.].

Brain response in asthma: the role of "lung-brain" axis mediated by neuroimmune crosstalk.

In addition to typical respiratory symptoms, patients with asthma are frequently accompanied by cognitive decline, mood disorders (anxiety and depression), sleep disorders, olfactory disorders, and other brain response manifestations, all of which worsen asthma symptoms, form a vicious cycle, and exacerbate the burden on families and society. Therefore, studying the mechanism of neurological symptoms in patients with asthma is necessary to identify the appropriate preventative and therapeutic measures. In order to provide a comprehensive reference for related research, we compiled the pertinent literature, systematically summarized the latest research progress of asthma and its brain response, and attempted to reveal the possible "lung-brain" crosstalk mechanism and treatment methods at the onset of asthma, which will promote more related research to provide asthmatic patients with neurological symptoms new hope.

P2X7 receptor of microglia in olfactory bulb mediates the pathogenesis of olfactory dysfunction in a mouse model of allergic rhinitis.

The pathogenesis of allergic rhinitis (AR)-related olfactory dysfunction (OD) remains unknown. Inhibiting microglial response in olfactory bulb (OB) can ameliorate AR-related OD, but no precise targets have been available. In this study, we established a mouse model of ovalbumin (OVA)-induced AR and combined with the application of P2X7 receptor (P2X7R)-specific antagonists and cell culture in conditioned medium to investigate the role and mechanism of OB microglial P2X7R in AR-related OD. Serum IgE and IL-5 levels determined via ELISA and federated the number of nose-scratching to affirm the success of OVA-induced AR mouse model. Buried food pellet test was used to evaluate the olfactory function of mice. The changes of IBA1, GFAP, P2X7R, IL-1ß, IL-1Ra, and CASPASE 1 were detected by quantitative polymerase chain reaction and western blotting. The levels of adenosine triphosphate (ATP) were determined by the commercialized kit. The morphological changes of microglia were assessed using immunofluorescence staining and Sholl analysis. Findings showed that AR-related OD was associated with OB microglia-mediated imbalance between IL-1ß and IL-1Ra. Treatment with BBG improved the olfactory function in AR mice with restoring the balance between IL-1ß and IL-1Ra. In vitro, the conditioned medium obtained after HNEpC treatment with Der p1 could activate HMC3 to arise inflammatory reaction basing on "ATP-P2X7R-Caspase 1" axis, while inhibition of its P2X7R suppressed the reaction. In brief, microglial P2X7R in OB is a direct effector molecule in AR-related OD and inhibition of it may be a new strategy for the treatment of AR-related OD.

IL-1ß and Allergy: Focusing on Its Role in Allergic Rhinitis.

Allergic rhinitis (AR) is a chronic upper airway immune-inflammation response mediated by immunoglobulin E (IgE) to allergens and can seriously affect the quality of life and work efficiency. Previous studies have shown that interleukin-1ß (IL-1ß) acts as a key cytokine to participate in and promote the occurrence and development of allergic diseases. It has been proposed that IL-1ß may be a potential biomarker of AR. However, its definitive role and potential mechanism in AR have not been fully elucidated, and the clinical sample collection and detection methods were inconsistent among different studies, which have limited the use of IL-1ß as a clinical diagnosis and treatment marker for AR. This article systematically summarizes the research advances in the roles of IL-1ß in allergic diseases, focusing on the changes of IL-1ß in AR and the possible interventions. In addition, based on the findings by our team, we provided new insights into the use of IL-1ß in AR diagnosis and treatment, in an attempt to further promote the clinical application of IL-1ß in AR and other allergic diseases.

Two small-molecule inhibitors of Toxoplasma gondii proliferation in vitro.

Background: Toxoplasmosis caused by Toxoplasma gondii is a globally distributed zoonosis. Most infections appear asymptomatic in immunocompetent individuals, but toxoplasmosis can be fatal in fetuses and immunocompromised adults. There is an urgent need to research and develop effective and low-toxicity anti-T. gondii drugs because of some defects in current clinical anti-T. gondii drugs, such as limited efficacy, serious side effects and drug resistance. Methods: In this study, 152 autophagy related compounds were evaluated as anti-T. gondii drugs. The activity of ß-galactosidase assay based on luminescence was used to determine the inhibitory effect on parasite growth. At the same time, MTS assay was used to further detect the effects of compounds with over 60% inhibition rate on host cell viability. The invasion, intracellular proliferation, egress and gliding abilities of T. gondii were tested to assess the inhibitory effect of the chosen drugs on the distinct steps of the T. gondii lysis cycle. Results: The results showed that a total of 38 compounds inhibited parasite growth by more than 60%. After excluding the compounds affecting host cell activity, CGI-1746 and JH-II-127 were considered for drug reuse and further characterized. Both CGI-1746 and JH-II-127 inhibited tachyzoite growth by 60%, with IC50 values of 14.58 ± 1.52 and 5.88 ± 0.23 µM, respectively. TD50 values were 154.20 ± 20.15 and 76.39 ± 14.32 µM, respectively. Further research found that these two compounds significantly inhibited the intracellular proliferation of tachyzoites. Summarize the results, we demonstrated that CGI-1746 inhibited the invasion, egress and especially the gliding abilities of parasites, which is essential for the successful invasion of host cells, while JH-II-127 did not affect the invasion and gliding ability, but seriously damaged the morphology of mitochondria which may be related to the damage of mitochondrial electron transport chain. Discussion: Taken together, these findings suggest that both CGI-1746 and JH-II-127 could be potentially repurposed as anti-T. gondii drugs, lays the groundwork for future therapeutic strategies.

Brain response in allergic rhinitis: Profile and proposal.

In addition to typical nasal symptoms, patients with allergic rhinitis (AR) will further lead to symptoms related to brain function such as hyposmia, anxiety, depression, cognitive impairment, memory loss, etc., which seriously affect the quality of life of patients and bring a heavy burden to the patient's family and society. Some scholars have speculated that there may be potential "nose-brain communication" mechanism in AR that rely on neuro-immunity. This mechanism plays an important role in AR-associated brain response process. However, no study has directly demonstrated which neural circuits will change in the connection between the nose and brain during the onset of AR, and the mechanism which underlines this question is also lack. Focusing on the topic of "nose-brain communication", this paper systematically summarizes the latest research progress between AR and related brain responses and discusses the mechanism of AR-related neurological phenotypes. Hope new diagnostic and therapeutic targets to ameliorate the brain function-related symptoms and improve the quality of life of AR patients will be developed.

Interactivity in learning instructional videos: Sending danmaku improved parasocial interaction but reduced learning performance.

Introduction: The instructional video is considered to be one of the most distinct and effective virtual learning tools. However, one of its biggest drawbacks is the lack of social interaction that occurs. This study tested the impact of participants sending zero danmaku (sending messages on the screen), three danmaku sending, and unlimited danmaku as an instructional video plays on learning performance. Methods: We assessed learners' retention and transfer scores, as well as self-report scores for cognitive load and parasocial interaction. This study sample comprised 104 participants who were randomly assigned to learn from one of three instructional videos on the topic of the heart. Results: The results showed that sending danmaku improved learners' parasocial interaction, while significantly increasing their cognitive load and also hindering learning performance. The observed increase in cognitive load reported by learners was also caused by increased levels of parasocial interaction. Discussion: Our findings suggest that by sending danmaku, learners can promote interactive learning, but that this has a negative impact on learning performance and the process of video learning.

Application of Neurotoxin-Induced Animal Models in the Study of Parkinson's Disease-Related Depression: Profile and Proposal.

Depression can be a non-motor symptom, a risk factor, and even a co-morbidity of Parkinson's disease (PD). In either case, depression seriously affects the quality of life of PD patients. Unfortunately, at present, a large number of clinical and basic studies focused on the pathophysiological mechanism of PD and the prevention and treatment of motor symptoms. Although there has been increasing attention to PD-related depression, it is difficult to achieve early detection and early intervention, because the clinical guidelines mostly refer to depression developed after or accompanied by motor impairments. Why is there such a dilemma? This is because there has been no suitable preclinical animal model for studying the relationship between depression and PD, and the assessment of depressive behavior in PD preclinical models is as well a very challenging task since it is not free from the confounding from the motor impairment. As a common method to simulate PD symptoms, neurotoxin-induced PD models have been widely used. Studies have found that neurotoxin-induced PD model animals could exhibit depression-like behaviors, which sometimes manifested earlier than motor impairments. Therefore, there have been attempts to establish the PD-related depression model by neurotoxin induction. However, due to a lack of unified protocol, the reported results were diverse. For the purpose of further promoting the improvement and optimization of the animal models and the study of PD-related depression, we reviewed the establishment and evaluation strategies of the current animal models of PD-related depression based on both the existing literature and our own research experience, and discussed the possible mechanism and interventions, in order to provide a reference for future research in this area.

Three Small Cysteine-Free Proteins (CFP1-3) Are Required for Insect-Pathogenic Lifestyle of Metarhizium robertsii.

Unique CFP (cysteine-free protein; 120 aa) has been identified as an extraordinary virulence factor in Beauveria bassiana (Cordycipitaceae), a main source of wide-spectrum fungal insecticides. Its homologs exclusively exist in wide-spectrum insect pathogens of Hypocreales, suggesting their importance for a fungal insect-pathogenic lifestyle. In this study, all three CFP homologs (CFP1-3, 128-145 aa) were proven essential virulence factors in Metarhizium robertsii (Clavicipitaceae). Despite limited effects on asexual cycles in vitro, knockout mutants of cfp1,cfp2 and cfp3 were severely compromised in their capability for normal cuticle infection, in which most tested Galleria mellonella larvae survived. The blocked cuticle infection concurred with reduced secretion of extracellular enzymes, including Pr1 proteases required cuticle penetration. Cuticle-bypassing infection by intrahemocoel injection of ~250 conidia per larva resulted in a greater reduction in virulence in the mutant of cfp1 (82%) than of cfp2 (21%) or cfp3 (25%) versus the parental wild-type. Transcriptomic analysis revealed dysregulation of 604 genes (up/down ratio: 251:353) in the Δcfp1 mutant. Many of them were involved in virulence-related cellular processes and events aside from 154 functionally unknown genes (up/down ratio: 56:98). These results reinforce the essential roles of small CFP homologs in hypocrealean fungal adaptation to insect-pathogenic lifestyle and their exploitability for the genetic improvement of fungal insecticidal activity.

Fungal insecticidal activity elevated by non-risky markerless overexpression of an endogenous cysteine-free protein gene in Beauveria bassiana.

BACKGROUND: Fungal insecticides are notorious for slow kill action, an intrinsic trait that can be improved by the genetic engineering of an exogenous or endogenous virulence factor. However, transgenic insecticides expressing exogenous toxin and herbicide-resistant marker genes may cause unexpected ecological risks and are hardly permitted for field release due to strict regulatory hurdles. It is necessary to improve biotechnology that can speed up fungal insect-killing action and exclude ecological risk source. RESULTS: A markerless transformation system of Beauveria bassiana, a main source of wide-spectrum fungal insecticides, was reconstructed based on the fungal uridine auxotrophy (Δura3). The system was applied for overexpression of the small cysteine-free protein (120 amino acids) gene cfp previously characterized as a regulator of the fungal virulence and gene expression. Three cfp-overexpressed strains showed much faster kill action to Galleria mellonella larvae than the parental wild-type via normal cuticle infection but no change in vegetative growth and aerial condition. The faster kill action was achieved due to not only significant increases in conidial adherence to insect cuticle and total activity of secreted cuticle-degrading Pr1 proteases and of antioxidant enzymes crucial for collapse of insect immune defense but acceleration of hemocoel localization, proliferation in vivo and host death from mummification. CONCLUSION: The markerless system is free of any foreign DNA fragment as a source of ecologic risk and provides a novel biotechnological approach to enhancing fungal insecticidal activity with non-risky endogenous genes and striding over the regulatory hurdles. © 2022 Society of Chemical Industry.

Electrostatic-Fluid-Structure 3D Numerical Simulation of a MEMS Electrostatic Comb Resonator.

The reliability and stability of MEMS electrostatic comb resonators have become bottlenecks in practical applications. However, there are few studies that comprehensively consider the nonlinear dynamic behavior characteristics of MEMS systems and devices in a coupled field so that the related simulation accuracy is low and cannot meet the needs of design applications. In this paper, to avoid the computational complexity and the uncertainty of the results of three-field direct coupling and take into the damping nonlinearity caused by coupled fields, a novel electrostatic-fluid-structure three-field indirect coupling method is proposed. Taking an actual microcomb resonant electric field sensor as an example, an electrostatic-fluid-structure multiphysics coupling 3D finite element simulation model is established. After considering the influence of nonlinear damping concerning the large displacement of the structure and the microscale effect, multifield coupling dynamics research is carried out using COMSOL software. The multiorder eigenmodes, resonant frequency, vibration amplitude, and the distribution of fluid load of the microresonator are calculated and analyzed. The simulated data of resonance frequency and displacement amplitude are compared with the measured data. The results show that the fluid load distribution of the microelectrostatic comb resonator along the thickness direction is high in the middle and low on both sides. The viscous damping of the sensor under atmospheric pressure is mainly composed of the incompressible flow damping of the comb teeth, which is an order of magnitude larger than those of other parts. Compared with the measured data, it can be concluded that the amplitude and resonance frequency of the microresonator considering the nonlinear damping force and residual thermal stress are close to the experimental values (amplitude error: 15.47%, resonance frequency error: 12.48%). This article provides a reference for studies on the dynamic characteristics of electrostatically driven MEMS devices.

Applying Control-Value Theory and Unified Theory of Acceptance and Use of Technology to Explore Pre-service Teachers' Academic Emotions and Learning Satisfaction.

Academic emotions refer to the emotions related to achievement activities or outcomes. Academic emotions are directly related to learning performance and have been recognized as critical to learners' learning satisfaction and learning effectiveness in the online learning context. This study aimed to explore the relationship between academic emotions and learning satisfaction and their underlying mechanisms in massive open online courses (MOOCs) learning context using mediation models. This study adhered to the theoretical frameworks of the control-value theory (CVT) and the unified theory of acceptance and use of technology (UTAUT). Participants were 283 pre-service teachers who volunteered from a normal university in Southwestern China. Results revealed that: (a) academic emotions did not predict learning satisfaction; (b) learning interest and technology acceptance fully mediated the influence of academic emotions on learning satisfaction; (c) the four dimensions of technology acceptance did not mediate the relationship between academic emotions and learning satisfaction. This study integrated CVT and UTAUT models, and the results emphasized the importance of academic emotions and learning satisfaction in CVT and provision of additional support for UTAUT. Therefore, these findings have significant implications for improving the quality of MOOCs in the post-pandemic era.

Genome-Wide Insight into Profound Effect of Carbon Catabolite Repressor (Cre1) on the Insect-Pathogenic Lifecycle of Beauveriabassiana.

Carbon catabolite repression (CCR) is critical for the preferential utilization of glucose derived from environmental carbon sources and regulated by carbon catabolite repressor A (Cre1/CreA) in filamentous fungi. However, a role of Cre1-mediated CCR in insect-pathogenic fungal utilization of host nutrients during normal cuticle infection (NCI) and hemocoel colonization remains explored insufficiently. Here, we report an indispensability of Cre1 for Beauveriabassiana's utilization of nutrients in insect integument and hemocoel. Deletion of cre1 resulted in severe defects in radial growth on various media, hypersensitivity to oxidative stress, abolished pathogenicity via NCI or intrahemocoel injection (cuticle-bypassing infection) but no change in conidial hydrophobicity and adherence to insect cuticle. Markedly reduced biomass accumulation in the Δcre1 cultures was directly causative of severe defect in aerial conidiation and reduced secretion of various cuticle-degrading enzymes. The majority (1117) of 1881 dysregulated genes identified from the Δcre1 versus wild-type cultures were significantly downregulated, leading to substantial repression of many enriched function terms and pathways, particularly those involved in carbon and nitrogen metabolisms, cuticle degradation, antioxidant response, cellular transport and homeostasis, and direct/indirect gene mediation. These findings offer a novel insight into profound effect of Cre1 on the insect-pathogenic lifestyle of B. bassiana.

Conserved and Noncanonical Activities of Two Histone H3K36 Methyltransferases Required for Insect-Pathogenic Lifestyle of Beauveria bassiana.

Set2 and Ash1 are histone methyltransferases (KMTs) in the KMT3 family normally used to catalyze methylation of histone H3K36 (H3K36me) but remain unexplored in fungal insect pathogens. Here, we report broader/greater roles of Set2 and Ash1 in mono-/di-/trimethylation (me1/me2/me3) of H3K4 than of H3K36 in Beauveria bassiana and function similarly to Set1/KMT2, which has been reported to catalyze H3K4me3 as an epigenetic mark of cre1 (carbon catabolite repressor) to upregulate the classes I and II hydrophobin genes hyd1 and hyd2 required for conidial hydrophobicity and adherence to insect cuticle. H3K4me3 was more attenuated than H3K36me3 in the absence of set2 (72% versus 67%) or ash1 (92% versus 12%), leading to sharply repressed or nearly abolished expression of cre1, hyd1 and hyd2, as well as reduced hydrophobicity. Consequently, the delta-set2 and delta-ash1 mutants were differentially compromised in radial growth on various media or under different stresses, aerial conidiation under normal culture conditions, virulence, and cellular events crucial for normal cuticle infection and hemocoel colonization, accompanied by transcriptional repression of subsets of genes involved in or required for asexual development and multiple stress responses. These findings unravel novel roles of Set2 and Ash1 in the co-catalysis of usually Set1-reliant H3K4me3 required for fungal insect-pathogenic lifestyle.

Student's Learning Strategies and Academic Emotions: Their Influence on Learning Satisfaction During the COVID-19 Pandemic.

Background: Based on the control-value theory (CVT), learning strategies and academic emotions are closely related to learning achievement, and have been considered as important factors influencing student's learning satisfaction and learning performance in the online learning context. However, only a few studies have focused on the influence of learning strategies on academic emotions and the interaction of learning strategies with behavioral engagement and social interaction on learning satisfaction. Methods: The participants were 363 pre-service teachers in China, and we used structural equation modeling (SEM) to analyze the mediating and moderating effects of the data. Results: The main findings of the current study showed that learning strategies influence students' online learning satisfaction through academic emotions. The interaction between learning strategies and behavioral engagement was also an important factor influencing online learning satisfaction. Conclusions: We explored the internal mechanism and boundary conditions of how learning strategies influenced learning satisfaction to provide intellectual guarantee and theoretical support for the online teaching design and online learning platform. This study provides theoretical contributions to the CVT and practical value for massive open online courses (MOOCs), flipped classrooms and blended learning in the future.

Essential Role of COP9 Signalosome Subunit 5 (Csn5) in Insect Pathogenicity and Asexual Development of Beauveria bassiana.

Csn5 is a subunit ofthe COP9/signalosome complex in model fungi. Here, we report heavier accumulation of orthologous Csn5 in the nucleus than in the cytoplasm and its indispensability to insect pathogenicity and virulence-related cellular events of Beauveria bassiana. Deletion of csn5 led to a 68% increase in intracellular ubiquitin accumulation and the dysregulation of 18 genes encoding ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) enzymes and ubiquitin-specific proteases, suggesting the role of Csn5 in balanced ubiquitination/deubiquitination. Consequently, the deletion mutant displayed abolished insect pathogenicity, marked reductions in conidial hydrophobicity and adherence to the insect cuticle, the abolished secretion of cuticle penetration-required enzymes, blocked haemocoel colonisation, and reduced conidiation capacity despite unaffected biomass accumulation. These phenotypes correlated well with sharply repressed or abolished expressions of key hydrophobin genes required for hydrophobin biosynthesis/assembly and of developmental activator genes essential for aerial conidiation and submerged blastospore production. In the mutant, increased sensitivities to heat shock and oxidative stress also correlated with reduced expression levels of several heat-responsive genes and decreased activities of antioxidant enzymes. Altogether, Csn5-reliant ubiquitination/deubiquitination balance coordinates the expression of those crucial genes and the quality control of functionally important enzymes, which are collectively essential for fungal pathogenicity, virulence-related cellular events, and asexual development.

SET1/KMT2-governed histone H3K4 methylation coordinates the lifecycle in vivo and in vitro of the fungal insect pathogen Beauveria bassiana.

Biological control potential of insect-pathogenic fungi against pests is an overall output of various cellular processes regulated by signalling and epigenetic networks. In Beauveria bassiana, mono/di/trimethylation of histone H3 Lys 4 (H3K4me1/me2/m3) was abolished by inactivation of the histone lysine methyltransferase SET1/KMT2, leading to marked virulence loss, reductions in conidial hydrophobicity and adherence to insect cuticle, impeded proliferation in vivo, severe defects in growth and conidiation, and increased sensitivities to cell wall perturbation, H2 O2 and heat shock. Such compromised phenotypes correlated well with transcriptional abolishment or repression of carbon catabolite-repressing transcription factor Cre1, classes I and II hydrophobins Hyd1 and Hyd2 required for cell hydrophobicity, key developmental regulators, and stress-responsive enzymes/proteins. Particularly, expression of cre1, which upregulates hyd4 upon activation by KMT2-mediated H3K4me3 in Metarhizium robertsii, was nearly abolished in the Δset1 mutant, leading to abolished expression of hyd1 and hyd2 as homologues of hyd4. These data suggest that the SET1-Cre1-Hyd1/2 pathway function in B. bassiana like the KMT2-Cre1-Hyd4 pathway elucidated to mediate pathogenicity in M. robertsii. Our findings unveil not only a regulatory role for the SET1-cored pathway in fungal virulence but also its novel role in mediating asexual cycle in vitro and stress responses in B. bassiana.

DIM5/KMT1 controls fungal insect pathogenicity and genome stability by methylation of histone H3K4, H3K9 and H3K36.

Mono-, di- and tri-methylation of histone H3 Lys 9, Lys 4, and Lys 36 (H3K_me1/me2/me3) required for mediation of DNA-based cellular events in eukaryotes usually rely upon the activities of histone lysine methyltransferases (KMTs) classified to the KMT1, KMT2, and KMT3 families, respectively. Here, an H3K9-specific DIM5/KMT1 orthologue, which lacks a C-terminal post-SET domain and localizes mainly in nucleus, is reported to have both conserved and noncanonical roles in methylating the H3 core lysines in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of wide-spectrum fungal insecticides. Disruption of dim5 led to abolishment of H3K9me3 and marked attenuation of H3K4me1/me2, H3K9me1/me2 and H3K36me2. Consequently, the Δdim5 mutant lost the whole insect pathogenicity through normal cuticle infection, and was compromised severely in virulence through cuticle-bypassing infection (hemocoel injection) and also in a series of cellular events critical for the fungal virulence and lifecycle in vivo and in vitro, including reduced hyphal growth, blocked conidiation, impeded proliferation in vivo, altered carbohydrate epitopes, disturbed cell cycle, reduced biosynthesis and secretion of cuticle-degrading enzymes, and increased sensitivities to various stresses. Among 1,201 dysregulated genes (up/down ratio: 712:489) associated with those phenotypic changes, 92 (up/down ratio: 59:33) encode transcription factors and proteins or enzymes involved in posttranslational modifications, implying that the DIM5-methylated H3 core lysines could act as preferential marks of those transcription-active genes crucial for global gene regulation. These findings uncover a novel scenario of DIM5 and its indispensability for insect-pathogenic lifestyle and genome stability of B. bassiana.

A Small Cysteine-Free Protein Acts as a Novel Regulator of Fungal Insect-Pathogenic Lifecycle and Genomic Expression.

Small secreted proteins (SSPs), particularly cysteine-rich proteins secreted during fungal infection, comprise virulence effectors in plant-pathogenic fungi but remain unknown in insect-pathogenic fungi. We report here that only a small cysteine-free protein (CFP) is indispensable for insect pathogenicity of Beauveria bassiana among 10 studied SSPs (99 to 274 amino acids [aa]), including seven hypothetical proteins containing 0 to 12 Cys residues. CFP (120 aa) features an N-terminal signal peptide (residues 1 to 17), a nuclear localization signal motif (residues 24 to 57), and no predictable domain. Its homologs exist exclusively in insect-pathogenic Cordycipitaceae and Clavicipitaceae. Fluorescence-tagged CFP fusion protein was localized in the nucleus but extracellularly undetectable, suggesting an inability for CFP to be secreted out. Disruption of cfp resulted in abolished pathogenicity via normal cuticle infection, attenuated virulence via hemocoel injection, compromised conidiation capacity versus little growth defect, impaired conidial coat, blocked secretion of cuticle-degrading enzymes, impeded proliferation in vivo, disturbed cell cycle, reduced stress tolerance, and 1,818 dysregulated genes (genomic 17.54%). Hundreds of those genes correlated with phenotypic changes observed in the disruption mutant. Intriguingly, nearly 40% of those dysregulated genes encode hypothetical or unknown proteins, and another 13% encode transcription factors and enzymes or proteins collectively involved in genome-wide gene regulation. However, purified CFP showed no DNA-binding activity in an electrophoretic mobility shift assay. These findings unveil that CFP is a novel regulator of fungal insect-pathogenic life cycle and genomic expression and that cysteine richness is dispensable for distinguishing virulence effectors from putative SSPs in B. bassiana IMPORTANCE Small cysteine-rich proteins secreted during plant-pathogenic fungal infection comprise virulence effectors. Our study confirms that only a cysteine-free protein (CFP) is determinant to insect-pathogenic fungal virulence among 10 small putatively secreted proteins containing 0 to 12 Cys residues. Disruption of cfp abolished insect pathogenicity and caused not only a series of compromised cellular events associated with host infection and disease development but also dysregulation of 1,818 genes, although no DNA-binding activity was detected in purified CFP samples. Nearly 13% of those genes encode transcription factors and enzymes or proteins collectively involved in transcriptional regulation. Altogether, CFP serves as a novel regulator of the fungal insect-pathogenic life cycle and genomic expression. Cysteine richness is dispensable for distinguishing virulence effectors from the fungal SSPs.