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In recent years, significant progress has been made in both three-dimensional (3D) printing technologies and the exploration of silk as an ink to produce biocompatible constructs. Combined with the unlimited design potential of 3D printing, silk can be processed into a broad range of functional materials and devices for various biomedical applications. The ability of silk to be processed into various materials, including solutions, hydrogels, particles, microspheres, and fibers, makes it an excellent candidate for adaptation to different 3D printing techniques. This review presents a didactic overview of the 3D printing of silk-based materials, major categories of printing techniques, and their prototyping mechanisms and structural features. In addition, we provide a roadmap for researchers aiming to incorporate silk printing into their own work by summarizing promising strategies from both technical and material aspects, to relate state-of-the-art silk-based material processing with fast-developing 3D printing technologies. Thus, our focus is on elucidating the techniques and strategies that advance the development of precise assembly strategies for silk-based materials. Precise printing (including high printing resolution, complex structure realization, and printing fidelity) is a prerequisite for the digital design capability of 3D printing technology and would definitely broaden the application era of silk, such as complex biomimetic tissue structures, vasculatures, and transdermal microneedles.

Abstract Organic heterostructures (OHTs) with the desired geometry organization on micro/nanoscale have undergone rapid progress in nanoscience and nanotechnology. However, it is a significant challenge to elucidate the epitaxial-growth process for various OHTs composed of organic units with a lattice mismatching ratio of > 3%, which is unimaginable for inorganic heterostructures. Herein, we have demonstrated a vivid visualization of the morphology evolution of epitaxial-growth based on a doped interfacial-layer, which facilitates the comprehensive understanding of the hierarchical self-assembly of core-shell OHT with precise spatial configuration. Significantly, the barcoded OHT with periodic shells obviously illustrate the shell epitaxial-growth from tips to center parts along the seeded rods for forming the core-shell OHT. Furthermore, the diameter, length, and number of periodic shells were modulated by finely tuning the stoichiometric ratio, crystalline time, and temperature, respectively. This epitaxial-growth process could be generalized to organic systems with facile chemical/structural compatibility for forming the desired OHTs.

The carnivorous fungus Drechslerella dactyloides can form constricting rings through hyphal specialization to capture nematodes. The formation of constricting rings is a prerequisite for capturing nematodes and a characteristic of entering the carnivorous stage. Currently, there is limited research on the molecular mechanism of constricting ring formation. In this study, two D. dactyloides mutants unable to form constricting rings were obtained through UV irradiation mutagenesis, and their growth and development phenotypes were compared with the wild-type strain. Transcriptome comparisons revealed differences between the mutants and the wild-type strain in metabolic pathways related to cell wall structure, peroxisomes, lipid metabolism, and MAPK signal transduction, which we validated through qPCR. We further deleted one differentially expressed gene, DdSTE2, of the MAPK pathway and confirmed its role in conidiogenesis and trap formation in D. dactyloides. Together, our results indicate that the remodeling of cell wall structure, peroxisomes, lipid metabolism, and MAPK signal transduction pathways are involved in the formation and maturation of D. dactyloides constricting rings. We discuss the implications of these results for utilizing these fungi to control animal and plant parasitic nematodes.

Purpureocillium lavendulum is an important biocontrol agent against plant-parasitic nematodes, primarily infecting them with conidia. However, research on the regulatory genes and pathways involved in its conidiation is still limited. In this study, we employed Agrobacterium tumefaciens-mediated genetic transformation to generate 4,870 random T-DNA insertion mutants of P. lavendulum. Among these mutants, 131 strains exhibited abnormal conidiation, and further in-depth investigations were conducted on two strains (designated as #5-197 and #5-119) that showed significantly reduced conidiation. Through whole-genome re-sequencing and genome walking, we identified the T-DNA insertion sites in these strains and determined the corresponding genes affected by the insertions, namely Plhffp and Plpif1. Both genes were knocked out through homologous recombination, and phenotypic analysis revealed a significant difference in conidiation between the knockout strains and the wild-type strain (ku80). Upon complementation of the ΔPlpif1 strain with the corresponding wildtype allele, conidiation was restored to a level comparable to ku80, providing further evidence of the involvement of this gene in conidiation regulation in P. lavendulum. The knockout of Plhffp or Plpif1 reduced the antioxidant capacity of P. lavendulum, and the absence of Plhffp also resulted in decreased resistance to SDS, suggesting that this gene may be involved in the integrity of the cell wall. RT-qPCR showed that knockout of Plhffp or Plpif1 altered expression levels of several known genes associated with conidiation. Additionally, the analysis of nematode infection assays with Caenorhabditis elegans indicated that the knockout of Plhffp and Plpif1 indirectly reduced the pathogenicity of P. lavendulum towards the nematodes. The results demonstrate that Agrobacterium tumefaciens - mediated T-DNA insertion mutagenesis, gene knockout, and complementation can be highly effective for identifying functionally important genes in P. lavendulum.

IntroductionTobacco root-knot nematode (RKN) is a highly destructive soil-borne disease worldwide. However, there is a lack of research on the relationship between RKN and tobacco root microbial community composition under large-scale geographical conditions in China.MethodsIn this study, we collected 65 samples from 28 main tobacco-growing areas across 10 provinces in China and conducted 16S rDNA sequencing to investigate the dynamic microbial changes in tobacco soil infected by RKN compared to healthy tobacco soil. Based on the analysis of rhizosphere soil bacterial communities, changes after RKN infection, and soil environmental factors.ResultsWe found the 28 tobacco-growing areas could be divided into two distinct groups with different microbial compositions and varying responses to RKN infection. In group1 of the provinces of Anhui, Henan, Shanxi, and Heilongjiang, Vicinamibacteria dominated the bacterial community, while Acidobacteriae was present in low abundance. In contrast, group2 of the other six provinces (Yunnan, Guizhou, Chongqing, Guangxi, Hubei, and Shandong) exhibited an opposite pattern. After infected by RKN, the genera Chitinophaga increased significant in group 1, while the genera Rhodococcus in group 2 exhibited a substantial increase. Alpha-diversity analysis revealed that RKN-infected tobacco exhibited a richer and more diverse rhizosphere soil bacterial community compared to healthy tobacco in most growing areas. A total of 12 kinds of soil environmental factors were measured in healthy and RKN-infected tobacco soil, and based on the co-occurrence and correlation analysis between environmental factors and microbial species, the pH level, calcium (Ca), magnesium (Mg), phosphorus (P), iron (Fe), and sodium (Na) were identified as key environmental factors influencing the population composition of rhizosphere microorganisms during RKN infection. We observed that RKN infection further increased the pH in weakly alkaline group 1 soil, while weakly acidic group 2 soil experienced a further decrease in pH. Furthermore, we identified three genera as potential biocontrol or plant growth-promoting bacteria for tobacco.DiscussionThese findings provide valuable reference data for managing RKN disease in different tobacco-growing areas and contribute to the exploration of new and effective biological control methods.

Abstract Background Root-knot nematodes (RKN) are among the most important root-damaging plant-parasitic nematodes, causing severe crop losses worldwide. The plant rhizosphere and root endosphere contain rich and diverse bacterial communities. However, little is known about how RKN and root bacteria interact to impact parasitism and plant health. Determining the keystone microbial taxa and their functional contributions to plant health and RKN development is important for understanding RKN parasitism and developing efficient biological control strategies in agriculture. Results The analyses of rhizosphere and root endosphere microbiota of plants with and without RKN showed that host species, developmental stage, ecological niche, and nematode parasitism, as well as most of their interactions, contributed significantly to variations in root-associated microbiota. Compared with healthy tomato plants at different developmental stages, significant enrichments of bacteria belonging to Rhizobiales, Betaproteobacteriales, and Rhodobacterales were observed in the endophytic microbiota of nematode-parasitized root samples. Functional pathways related to bacterial pathogenesis and biological nitrogen fixation were significantly enriched in nematode-parasitized plants. In addition, we observed significant enrichments of the nifH gene and NifH protein, the key gene/enzyme involved in biological nitrogen fixation, within nematode-parasitized roots, consistent with a potential functional contribution of nitrogen-fixing bacteria to nematode parasitism. Data from a further assay showed that soil nitrogen amendment could reduce both endophytic nitrogen-fixing bacteria and RKN prevalence and galling in tomato plants. Conclusions Results demonstrated that (1) community variation and assembly of root endophytic microbiota were significantly affected by RKN parasitism; (2) a taxonomic and functional association was found for endophytic nitrogen-fixing bacteria and nematode parasitism; and (3) the change of nitrogen-fixing bacterial communities through the addition of nitrogen fertilizers could affect the occurrence of RKN. Our results provide new insights into interactions among endophytic microbiota, RKN, and plants, contributing to the potential development of novel management strategies against RKN. Video Abstract

Peripheral nerve injury (PNI) is a common and complex clinical disease with high morbidity, limited treatment options and poor clinical outcomes. Several million cases of PNI in the world every year have brought a heavy burden to the patients and the social economy. Autologous nerve grafting has long been the “gold standard” in the treatment of PNI repair, but it still has some shortcomings, such as donor area injury, limited graft source and mismatch of nerve thickness after transplantation. In recent years, many artificial nerve guidance conduits (NGCs) have emerged for replacing autologous nerve grafts, and their effectiveness has been proven. Currently, there are already clinical products obtained from the European CE Certification, and approved by the Food and Drug Administration (FDA), China Food and Drug Administration (CFDA), Therapeutic Goods Administration (TGA) in Australia, etc. The preparation of NGCs requires interdisciplinary studies and has received considerable attention from researchers in recent years. At present, among emerging and mature manufacturing technologies, textile methods to prepare NGCs are relatively simple and have wide material sources, which has become a hotspot in textile research. This paper mainly reviewed the current situation and recent technological achievements of NGCs that were prepared by textile methods. Several other common methods were also briefly summarized. Furthermore, current NGCs products and their clinical applications were reported. Finally, the future development direction of textile-based NGCs is discussed in this review.

ABSTRACT Dactylellina haptotyla is a typical nematode-trapping fungus that has garnered the attention of many scholars for its highly effective lethal potential for nematodes. Secondary metabolites play an important role in D. haptotyla-nematode interactions, but which metabolites perform which function remains unclear. We report the metabolic functions based on high-quality, chromosome-level genome assembly of wild D. haptotyla YMF1.03409. The results indicate that a large variety of secondary metabolites and their biosynthetic genes were significantly upregulated during the nematode-trapping stage. In parallel, we identified that 2-furoic acid was specifically produced during nematode trapping by D. haptotyla YMF1.03409 and isolated it from fermentation production. 2-Furoic acid demonstrated strong nematicidal activity with an LD50 value of 55.05 µg/mL against Meloidogyne incognita at 48 h. Furthermore, the pot experiment showed that the number of galls of tomato root was significantly reduced in the experimental group treated with 2-furoic acid. The considerable increase in the 2-furoic acid content during the infection process and its virulent nematicidal activity revealed an essential synergistic effect during the process of nematode-trapping fungal infection. IMPORTANCE Dactylellina haptotyla have significant application potential in nematode biocontrol. In this study, we determined the chromosome-level genome sequence of D. haptotyla YMF1.03409 by long-read sequencing technology. Comparative genomic analysis identified a series of pathogenesis-related genes and revealed significant gene family contraction events during the evolution of D. haptotyla YMF1.03409. Combining transcriptomic and metabolomic data as well as in vitro activity test results, a compound with important application potential in nematode biocontrol, 2-furoic acid, was identified. Our result expanded the genetic resource of D. haptotyla and identified a previously unreported nematicidal small molecule, which provides new options for the development of plant biocontrol agents.

ABSTRACT Accurately recognizing pathogens by the host is vital for initiating appropriate immune response against infecting microorganisms. Caenorhabditis elegans has no known receptor to recognize pathogen-associated molecular pattern. However, recent studies showed that nematodes have a strong specificity for transcriptomes infected by different pathogens, indicating that they can identify different pathogenic microorganisms. However, the mechanism(s) for such specificity remains largely unknown. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum can infect the intestinal tract of the nematode C. elegans and the infection led to the accumulation of reactive oxygen species (ROS) in the infected intestinal tract, which suppressed fungal growth. Co-transcriptional analysis revealed that fungal genes related to anaerobic respiration and ethanol production were up-regulated during infection. Meanwhile, the ethanol dehydrogenase Sodh-1 in C. elegans was also up-regulated. Together, these results suggested that the infecting fungi encounter hypoxia stress in the nematode gut and that ethanol may play a role in the host–pathogen interaction. Ethanol production in vitro during fungal cultivation in hypoxia conditions was confirmed by gas chromatography-mass spectrometry. Direct treatment of C. elegans with ethanol elevated the sodh-1 expression and ROS accumulation while repressing a series of immunity genes that were also repressed during fungal infection. Mutation of sodh-1 in C. elegans blocked ROS accumulation and increased the nematode’s susceptibility to fungal infection. Our study revealed a new recognition and antifungal mechanism in C. elegans. The novel mechanism of ethanol-mediated interaction between the fungus and nematode provides new insights into fungal pathogenesis and for developing alternative biocontrol of pathogenic nematodes by nematophagous fungi. IMPORTANCE Nematodes are among the most abundant animals on our planet. Many of them are parasites in animals and plants and cause human and animal health problems as well as agricultural losses. Studying the interaction of nematodes and their microbial pathogens is of great importance for the biocontrol of animal and plant parasitic nematodes. In this study, we found that the model nematode Caenorhabditis elegans can recognize its fungal pathogen, the nematophagous fungus Purpureocillium lavendulum, through fungal-produced ethanol. Then the nematode elevated the reactive oxygen species production in the gut to inhibit fungal growth in an ethanol dehydrogenase-dependent manner. With this mechanism, novel biocontrol strategies may be developed targeting the ethanol receptor or metabolic pathway of nematodes. Meanwhile, as a volatile organic compound, ethanol should be taken seriously as a vector molecule in the microbial–host interaction in nature.

With the improvement of living standards, people’s awareness of health and environmental protection continues to strengthen. The production of textiles with antibacterial functions is one of the effective ways to protect people from or reduce bacterial invasion. Therefore, textiles with antibacterial functions are increasingly favored by people. At the same time, due to the impact of global warming, people are committed to reducing carbon emissions in all aspects of life. The biodegradable material polypropylidene carbonate (PPC) that can consume carbon dioxide (CO2) in the production process fully conforms to people’s environmental protection concept. However, polypropylidene carbonate itself has poor thermal stability and narrow application range. Polypropylidene carbonate thermoplastic polyurethane (PPC-TPU) is obtained by chain extension modification, which can broaden the application range of polypropylidene carbonate. To develop a yarn with excellent antibacterial and mechanical properties, which can be woven into fabrics for medical applications while taking into account environmental protection and degradability, the antibacterial yarn in this article was prepared by electrospinning using PPC-TPU and polylactic acid (PLA) yarn as raw materials. The preparation process was optimized by adjusting the supply speed, winding collection speed and horn barrel speed, and the optimal concentration of antimicrobial agent was explored by adding different concentrations of benzalkonium bromide antimicrobial. Through the characterization of the properties of the materials, we believe that PPC-PLA yarn with the addition of 5% benzalkonium bromide has broad development prospects in the medical field due to its excellent mechanical and antibacterial properties.

ABSTRACT Arthrobotrys flagrans (Duddingtonia flagrans) is a typical nematode-trapping fungus which has been used for nematode biocontrol. The global regulator LaeA is widely distributed in filamentous fungi and plays a crucial role in secondary metabolism and development in addition to pathogenicity in fungal pathogens. In this study, the chromosome-level genome of A. flagrans CBS 565.50 was sequenced and homologous sequences of LaeA were identified in A. flagrans. A. flagrans LaeA (AfLaeA) knockout resulted in slower hyphal growth and a smoother hyphal surface. Importantly, deletion of AfLaeA resulted in the absence of chlamydospores and attenuated glycogen and lipid accumulation in hyphae. Similarly, disruption of the AfLaeA gene led to fewer traps and electron-dense bodies, lower protease activity, and a delay in capturing nematodes. The AfLaeA gene had a large effect on the secondary metabolism of A. flagrans, and both the deletion and overexpression of AfLaeA could yield new compounds, whereas some compounds were lost due to the absence of the AfLaeA. Protein-protein interactions between AfLaeA and another eight proteins were detected. Furthermore, transcriptome data analysis showed that 17.77% and 35.51% of the genes were influenced by the AfLaeA gene on days 3 and 7, respectively. AfLaeA gene deletion resulted in the higher expression level of the artA gene cluster, and multiple differentially expressed genes involved in glycogen and lipid synthesis and metabolism showed opposite expression patterns in wild-type and ΔAfLaeA strains. In summary, our results provide novel insights into the functions of AfLaeA in mycelial growth, chlamydospore production, pathogenicity, secondary metabolism, and energy metabolism in A. flagrans. IMPORTANCE The regulation of biological functions, such as the secondary metabolism, development, and pathogenicity of LaeA, has been reported in multiple fungi. But to date, no study on LaeA in nematode-trapping fungi has been reported. Moreover, it has not been investigated whether or not LaeA is involved in energy metabolism and chlamydospore formation has not been investigated. Especially in the formation mechanism of chlamydospores, several transcription factors and signaling pathways are involved in the production of chlamydospores, but the mechanism of chlamydospore formation from an epigenetic perspective has not been revealed. Concurrently, an understanding of protein-protein interactions will provide a broader perspective on the regulatory mechanism of AfLaeA in A. flagrans. This finding is critical for understanding the regulatory role of AfLaeA in the biocontrol fungus A. flagrans and establishes a foundation for developing high-efficiency nematode biocontrol agents.

Abstract Ischemia-reperfusion (I/R), a leading risk factor of acute kidney injury (AKI), is associated with high mortality and risk of progression to chronic kidney disease. However, the molecular mechanism of I/R-AKI remains not fully understood, which hinders its efficient clinical treatment. In this study, we observed that LIGHT deficiency remarkably attenuated I/R-AKI, as evidenced by rescued renal function, ameliorated tubular cell apoptosis, and alleviated inflammatory responses. Consistently, blocking LIGHT signaling with its soluble receptor fusion proteins (HVEM-IgG-Fc or LTβR-IgG-Fc) improved I/R renal dysfunction. RNA-sequencing and corresponding results indicated that LIGHT promoted oxidative stress and inflammation triggered by ischemic injury. Moreover, LIGHT signaling augmented ischemic stress-induced mitochondrial dysfunction characterized by an imbalance in mitochondrial fission and fusion, decreased mtDNA copies, impaired mitophagy, and increased mitochondrial membrane potential (ΔΨm). Mechanistically, LIGHT promoted mitochondrial fission by enhancing Drp1 phosphorylation (Ser616) and its translocation to the mitochondria. In conclusion, these results suggest that LIGHT-HVEM/LTβR signaling is critical for the I/R-AKI pathogenesis and it is further confirmed to be related to the increase in I/R-induced oxidative stress and mitochondria dysfunction, which may be the underlying mechanism of LIGHT signaling-mediated I/R-AKI.

Compliance mismatch between artificial blood vessel and host vessel can cause abnormal hemodynamics and is the main mechanical trigger of intimal hyperplasia (IH) after transplantation. To explore the specific effects of the degree of compliance mismatch on hemodynamics, the concept of “compliance mismatch degree” was defined in this paper. Numerical results showed that when the compliance mismatch degree was less than −22.21% or more than 3.16%, the minimum WSS in the anastomotic site was less than the safety threshold of 0.5 ​Pa, which is easy to induce IH. In addition, the compliance mismatch caused different radial displacements first, which in turn affected the hemodynamics. Inspired by this, a novel medium convex artificial blood vessel was proposed, and the simulation results theoretically validated its desired effect on reducing the compliance mismatch. An increase of 0.22 ​mm in the diameter of the artificial blood vessel corresponded to a decrease of 0.88% in compliance mismatch degree. The feasibility of preparing real medium convex artificial blood vessel was also investigated. The PLCL medium convex artificial blood vessel with good mechanical properties was prepared by dip-coating and electrospinning composite method. These findings are valuable for the design and preparation of novel artificial blood vessel which can make up for the mismatch of compliance.

Abstract Organic photothermal cocrystals, integrating the advantages of intrinsic organic cocrystals and the fascinating photothermal conversion ability, hold attracted considerable interest in both basic science and practical applications, involving photoacoustic imaging, seawater desalination, and photothermal therapy, and so on. However, these organic photothermal cocrystals currently suffer individual cases discovered step by step, as well as the deep and systemic investigation in the corresponding photothermal conversion mechanisms is rarely carried out, suggesting a huge challenge for their further developments. Therefore, it is urgently necessary to investigate and explore the rational design and synthesis of high‐performance organic photothermal cocrystals for future applications. This review first and systematically summarizes the organic photothermal cocrystal in terms of molecular classification, the photothermal conversion mechanism, and their corresponding applications. The timely interpretation of the cocrystal photothermal effect will provide broad prospects for the purposeful fabrication of excellent organic photothermal cocrystals toward great efficiency, low cost, and multifunctionality.

Wheat gluten (WG) foam was successfully prepared by heating, stirring and freeze-drying. The effects of WG concentration, content of glycerol and glutaraldehyde on the macroscopic appearance, microstructure, swelling property and self-expansion, as well as mechanical compressibility, were studied. Results showed that the density and structure of foam can be changed by adjusting the concentration of WG, glycerol, and glutaraldehyde. The addition of glycerol softens the WG foams, while glutaraldehyde significantly improved their mechanical properties and stability in wet state. The swollen foams were repeatedly pressed and exhibited rapid water-triggered shape recovery performance (less than 3 ​s). Moreover, WG foams displayed excellent biodegradability and hemocompatibility. In vitro studies of drug loading and release were performed using an antibacterial drug, chlorhexidine gluconate (CHG), and showed sustained drug-release activity. The above advantages make the foams hold great potential for application in self-expanding, antibacterial hemostatic medical dressings.

Regulators of G protein signaling (RGSs) are proteins that negatively regulate G protein signal transduction. In this study, seven putative RGSs were characterized in the nematode-trapping (NT) fungus, Arthrobotrys oligospora. Deleting Rgs genes significantly increased intracellular cAMP levels, and caused defects in mycelia growth, stress resistance, conidiation, trap formation, and nematocidal activity. In particular, the ΔAoFlbA mutant was unable to produce conidia and traps. Transcriptomic analysis showed that amino acid metabolic and biosynthetic processes were significantly enriched in the ΔAoFlbA mutant compared to WT. Interestingly, Gas1 family genes are significantly expanded in A. oligospora and other NT fungi that produce adhesive traps, and are differentially expressed during trap formation in A. oligospora. Disruption of two Gas1 genes resulted in defective conidiation, trap formation, and pathogenicity. Our results indicate that RGSs play pleiotropic roles in regulating A. oligospora mycelial growth, development, and pathogenicity. Further, AoFlbA is a prominent member and required for conidiation and trap formation, possibly by regulating amino acid metabolism and biosynthesis. Our results provide a basis for elucidating the signaling mechanism of vegetative growth, lifestyle transition, and pathogenicity in NT fungi.

The family Microthyriaceae is represented by relatively few mycelial cultures and DNA sequences; as a result, the taxonomy and classification of this group of organisms remain poorly understood. During the investigation of the diversity of aquatic hyphomycetes from southern China, several isolates were collected. These isolates were cultured and sequenced and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa are in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the internal transcribed spacers (ITS) and the large subunit (LSU), revealed that these isolates represent eight new taxa in Microthyriaceae, including two new genera, Antidactylaria gen. nov. and Isthmomyces gen. nov. and six new species, Antidactylaria minifimbriata sp. nov., Isthmomyces oxysporus sp. nov., I. dissimilis sp. nov., I. macrosporus sp. nov., Triscelophorus anisopterioideus sp. nov. and T. sinensis sp. nov. These new taxa are described, illustrated for their morphologies and compared with similar taxa. In addition, two new combinations are proposed in this family.

ABSTRACT DNA methylation is an important factor in the regulation of gene expression. In analyzing genomic data of Stereum hirsutum FP-91666, we found a hypothetical bifunctional transcription regulator/O6Meguanine-DNA methyltransferase (named SaraC), which is widely present in both bacteria and fungi, and confirmed that its function in bacteria is mainly for DNA reparation. In this paper, we confirmed that SaraC has the function of DNA binding and demethylation through surface plasma resonance and reaction experiments in vitro. Then, we achieved the overexpression of SaraC (OES) in S. hirsutum, sequenced the methylation and transcription levels of the whole-genome, and further conducted untargeted metabolomics analyses of the OES transformants and the wild type (WT). The results confirmed that the overall-methylation levels of the transformants were significantly downregulated, and various genes related to secondary metabolism were upregulated. Through comparative untargeted metabolomic analyses, it showed that OES SA6 transformant produced a greater number of hybrid polyketides, and we identified 2 novel hybrid polyketides from the fermentation products of SA6. Our results show that overexpression SaraC can effectively stimulate the expression of secondary-metabolism-related genes, which could be a broad-spectrum tool for discovery of metabolites due to its cross-species conservation. IMPORTANCE Fungi are one of the important sources of active compounds. However, in fungi, most of the secondary metabolic biosynthetic gene clusters are weakly expressed or silenced under conventional culture conditions. How to efficiently excavate potential new compounds contained in fungi is becoming a research hot spot in the world. In this study, we found a DNA demethylation protein (SaraC) and confirmed that it is a global secondary metabolism regulator in Stereum hirsutum FP-91666. In the past, SaraC-like proteins were mainly regarded as DNA repair proteins, but our findings proved that it will be a powerful tool for mining secondary metabolites for overexpression of SaraC, which can effectively stimulate the expression of genes related to secondary metabolism.

Small-diameter artificial blood vessels are prone to cause intimal hyperplasia (IH) after transplantation, which leads to restenosis and low long-term patency rates. The main biomechanical factor for IH formation is the compliance mismatch between the artificial and host blood vessels which can cause abnormal hemodynamics. Although there have been many studies on vascular compliance mismatches, however, little attention has been paid to the effect of the degree of compliance mismatch between graft and the host vessel on hemodynamics. At present, the research on compliance mismatch between the artificial and host blood vessels is still very limited, especially with regard to the specific impact of the compliance mismatch degree on hemodynamics. Therefore, three end-to-end anastomosis models (the compliance of the artificial blood vessel is lower than, similar to, and higher than that of the host blood vessel, called model 1, model 2, model 3, respectively) were constructed and simulated in this study. Simulation results showed that the radial displacement difference between the artificial and host blood vessels were 0.281 ​mm, 0.183 ​mm and 0.485 ​mm in model 1, model 2 and model 3, respectively. A low-velocity recirculation zone near the distal anastomosis was formed in model 1 which resulted in excessively low TAWSS (9.261 ​E-5Pa) and high OSI (0.497). Similarly, a low-velocity recirculation zone near the proximal anastomosis was formed in model 3 and lead to low TAWSS (6.007 ​E-4Pa) and high OSI (0.480). However, there was no low-velocity recirculation zone near the anastomosis stoma in model 2. The results are instructive for the design and preparation of artificial blood vessels.

ABSTRACT AMP-activated protein kinase (AMPK), a heterotrimeric complex, can sense energy and nutritional status in eukaryotic cells, thereby participating in the regulation of multiple cellular processes. In this study, we characterized the function of the catalytic α-subunit (SNF1) and the two regulatory β- and γ-subunits (GAL83 and SNF4) of AMPK in a representative nematode-trapping fungus, Arthrobotrys oligospora, by gene knockout, phenotypic analysis, and RNA sequencing. The ability of the AMPK complex mutants (including ΔAosnf1, ΔAogal83, and ΔAosnf4) to utilize a nonfermentable carbon source (glycerol) was reduced, and the spore yields and trap formation were remarkably decreased. Moreover, AMPK plays an important role in regulating stress response and nematode predation efficiency. Transcriptomic profiling between the wild-type strain and ΔAosnf1 showed that differentially expressed genes were enriched for peroxisome, endocytosis, fatty acid degradation, and multilipid metabolism (sphingolipid, ether lipid, glycerolipid, and glycerophospholipid). Meanwhile, a reduced lipid droplet accumulation in ΔAosnf1, ΔAogal83, and ΔAosnf4 mutants was observed, and more vacuoles appeared in the mycelia of the ΔAosnf1 mutant. These results highlight the important regulatory role of AMPK in the utilization of carbon sources and lipid metabolism, as well as providing novel insights into the regulatory mechanisms of the mycelia development, conidiation, and trap formation of nematode-trapping (NT) fungi. IMPORTANCE NT fungi are widely distributed in various ecosystems and are important factors in the control of nematode populations in nature; their trophic mycelia can form unique infectious devices (traps) for capturing nematodes. Arthrobotrys oligospora is a representative NT fungi which can develop complex three-dimensional networks (adhesive networks) for nematode predation. Here, we demonstrated that AMPK plays an important role in the glycerol utilization, conidiation, trap formation, and nematode predation of A. oligospora, which was further confirmed by transcriptomic analysis of the wild-type and mutant strains. In particular, our analysis indicated that AMPK is required for lipid metabolism, which is primarily associated with energy regulation and is essential for trap formation. Therefore, this study extends the functional study of AMPK in NT fungi and helps to elucidate the molecular mechanism of the regulation of trap development, as well as laying the foundation for the development of efficient nematode biocontrol agents.

Abstract Objective Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS) are the two most common hyperglycemic emergencies (HEs) associated with diabetes mellitus. Individuals with HEs can present with combined features of DKA and HHS. The objective of this study is to assess the clinical characteristics, therapeutic outcomes, and associated predisposing factors of type 2 diabetic patients with isolated or combined HEs in China. Methods We performed a retrospective analysis of 158 patients with type 2 diabetes (T2DM), complicated with DKA, HHS, or DKA combined with HHS (DKA-HHS) in Shanghai Tongji Hospital, China from 2010 to 2015. Admission clinical features, therapeutic approaches and treatment outcomes of those patients were extracted and analyzed. Results Of the 158 patients with T2DM, 65 (41.1%) patients were DKA, 74 (46.8%) were HHS, and 19 (12.0%) were DKA-HHS. The most common precipitants were infections (111, 70.3%), newly diagnosed diabetes (28,17.7%) and non-compliance to medications (9, 5.7%). DKA patients were divided into mild, moderate and severe group, based on arterial blood gas. Spearman correlation analysis revealed that C-reaction protein (CRP) was positively correlated with severity of DKA, whereas age and fasting C peptide were inversely correlated with severity of DKA (P

ABSTRACT Arthrobotrys oligospora (A. oligospora) is a typical nematode-trapping (NT) fungus that can capture nematodes by producing adhesive networks. Peroxisomes are single membrane-bound organelles that perform multiple physiological functions in filamentous fungi. Peroxisome biogenesis proteins are encoded by PEX genes, and the functions of PEX genes in A. oligospora and other NT fungi remain largely unknown. Here, our results demonstrated that two PEX genes (AoPEX1 and AoPEX6) are essential for mycelial growth, conidiation, fatty acid utilization, stress tolerance, and pathogenicity in A. oligospora. AoPEX1 and AoPEX6 knockout resulted in a failure to produce traps, conidia, peroxisomes, and Woronin bodies and damaged cell walls, reduced autophagosome levels, and increased lipid droplet size. Transcriptome data analysis showed that AoPEX1 and AoPEX6 deletion resulted in the upregulation of the proteasome, membranes, ribosomes, DNA replication, and cell cycle functions, and the downregulation of MAPK signaling and nitrogen metabolism. In summary, our results provide novel insights into the functions of PEX genes in the growth, development, and pathogenicity of A. oligospora and contribute to the elucidation of the regulatory mechanism of peroxisomes in trap formation and lifestyle switching in NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are important resources for the biological control of plant-parasitic nematodes. They are widely distributed in various ecological environments and capture nematodes by producing unique predatory organs (traps). However, the molecular mechanisms of trap formation and lifestyle switching in NT fungi are still unclear. Here, we provided experimental evidence that the AoPEX1 and AoPEX6 genes could regulate mycelial growth and development, trap formation, and nematode predation of A. oligospora. We further analyzed the global transcription level changes of wild-type and mutant strains using RNA-seq. This study highlights the important role of peroxisome biogenesis genes in vegetative growth, conidiation, trap formation, and pathogenicity, which contribute to probing the mechanism of organelle development and trap formation of NT fungi and lays a foundation for developing high-efficiency nematode biocontrol agents.

ABSTRACT Nematode-trapping (NT) fungi can form unique infection structures (traps) to capture and kill free-living nematodes and, thus, can play a potential role in the biocontrol of nematodes. Arthrobotrys oligospora is a representative species of NT fungi. Here, we performed a time course transcriptome sequencing (RNA-seq) analysis of transcriptomes to understand the global gene expression levels of A. oligospora during trap formation and predation. We identified 5,752 unique differentially expressed genes, among which the rac gene was significantly upregulated. Alternative splicing events occurred in 2,012 genes, including the rac and rho2 gene. Furthermore, we characterized three Rho GTPases (Rho2, Rac, and Cdc42) in A. oligospora using gene disruption and multiphenotypic analysis. Our analyses showed that AoRac and AoCdc42 play an important role in mycelium growth, lipid accumulation, DNA damage, sporulation, trap formation, pathogenicity, and stress response in A. oligospora. AoCdc42 and AoRac specifically interacted with components of the Nox complex, thus regulating the production of reactive oxygen species. Moreover, the transcript levels of several genes associated with protein kinase A, mitogen-activated protein kinase, and p21-activated kinase were also altered in the mutants, suggesting that Rho GTPases might function upstream from these kinases. This study highlights the important role of Rho GTPases in A. oligospora and provides insights into the regulatory mechanisms of signaling pathways in the trap morphogenesis and lifestyle transition of NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are widely distributed in terrestrial and aquatic ecosystems. Their broad adaptability and flexible lifestyles make them ideal agents for controlling pathogenic nematodes. Arthrobotrys oligospora is a model species employed for understanding the interaction between fungi and nematodes. Here, we revealed that alternative splicing events play a crucial role in the trap development and lifestyle transition in A. oligospora. Furthermore, Rho GTPases exert differential effects on the growth, development, and pathogenicity of A. oligospora. In particular, AoRac is required for sporulation and trap morphogenesis. In addition, our analysis showed that Rho GTPases regulate the production of reactive oxygen species and function upstream from several kinases. Collectively, these results expand our understanding of gene expression and alternative splicing events in A. oligospora and the important roles of Rho GTPases in NT fungi, thereby providing a foundation for exploring their potential application in the biocontrol of pathogenic nematodes.

Plant-parasitic nematodes cause severe economic losses to agriculture. As important biocontrol agents, nematophagous fungi evolved the ability to obtain nitrogen sources from nematodes. However, the impact of nitrogen sources on the growth and development of these fungi is largely unknown. In this study, we aimed to better understand how nitrogen sources could influence vegetative growth and conidiation through epigenetic regulation in the nematophagous fungus, Purpureocillium lavendulum. Through nutrition screening, we found a phenomenon of the fungus, limited colony extension with a large amount of conidia production when cultured on PDA media, can be altered by adding ammonia nitrate. Characterized by site-directed mutagenesis, the histone H3K14 acetylation was found to be involved in the alternation. Furthermore, the acetyltransferase PlGCN5 was responsible for H3K14 acetylation. Knockout of Plgcn5 severely diminished conidiation in P. lavendulum. Chip-seq showed that H3K14ac distributed in conidiation regulating genes, and genes in the MAPK pathway which may be the downstream targets in the regulation. These findings suggest that histone modification and nitrogen sources coordinated lifestyle regulation in P. lavendulum, providing new insight into the mechanism of growth regulation by nutritional signals for the carnivorous fungus.

Autophagy is a conserved cellular recycling and trafficking pathway in eukaryotes that plays an important role in cell growth, development, and pathogenicity. Atg1 and Atg13 form the Atg1–Atg13 complex, which is essential for autophagy in yeast. Here, we characterized the roles of the Aolatg1 and Aolatg13 genes encoding these autophagy-related proteins in the nematode-trapping fungus Arthrobotrys oligospora. Investigation of the autophagy process by using the AoAtg8-GFP fusion protein showed that autophagosomes accumulated inside vacuoles in the wild-type (WT) A. oligospora strain, whereas in the two mutant strains with deletions of Aolatg1 or Aolatg13, GFP signals were observed outside vacuoles. Similar results were observed by using transmission electron microscopy. Furthermore, deletion of Aolatg1 caused severe defects in mycelial growth, conidiation, conidial germination, trap formation, and nematode predation. In addition, transcripts of several sporulation-related genes were significantly downregulated in the ΔAolatg1 mutant. In contrast, except for the altered resistance to several chemical stressors, no obvious differences were observed in phenotypic traits between the WT and ΔAolatg13 mutant strains. The gene ontology analysis of the transcription profiles of the WT and ΔAolatg1 mutant strains showed that the set of differentially expressed genes was highly enriched in genes relevant to membrane and cellular components. The Kyoto Encyclopedia of Genes and Genomes analysis indicated that differentially expressed genes were highly enriched in those related to metabolic pathways, autophagy and autophagy-related processes, including ubiquitin-mediated proteolysis and SNARE interaction in vesicular transport, which were enriched during trap formation. These results indicate that Aolatg1 and Aolatg13 play crucial roles in the autophagy process in A. oligospora. Aolatg1 is also involved in the regulation of asexual growth, trap formation, and pathogenicity. Our results highlight the importance of Aolatg1 in the growth and development of A. oligospora, and provide a basis for elucidating the role of autophagy in the trap formation and pathogenicity of nematode-trapping fungi.

The predominant nematode-trapping fungus Arthrobotrys oligospora harbors a unique polyketide synthase-prenyltransferase (PKS-PTS) gene cluster AOL_s00215g responsible for the biosynthesis of sesquiterpenyl epoxy-cyclohexenoids (SECs) that are involved in the regulation of fungal growth, adhesive trap formation, antibacterial activity, and soil colonization. However, the function of one rare gene (AOL_s00215g275 (275)) embedded in the cluster has remained cryptic. Here, we constructed two mutants with the disruption of 275 and the overexpression of 275, respectively, and compared their fungal growth, morphology, resistance to chemical stress, nematicidal activity, transcriptomic and metabolic profiles, and infrastructures, together with binding affinity analysis. Both mutants displayed distinct differences in their TCA cycles, SEC biosynthesis, and endocytosis, combined with abnormal mitochondria, vacuoles, septa formation, and decreased nematicidal activity. Our results suggest that gene 275 might function as a separator and as an integrated gene with multiple potential functions related to three distinct genes encoding the retinoic acid induced-1, cortactin, and vacuolar iron transporter 1 proteins in this nematode-trapping fungus. Our unexpected findings provide insight into the intriguing organization and functions of a rare non-biosynthetic gene in a biosynthetic gene cluster.

ABSTRACT Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soils. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi is little studied. In this study, it was found that ammonia (NH3) induced global protein misfolding marked by increased ubiquitination levels of proteins (ubiquitylome data and Western blot verification). The misfolded proteins should trigger the endoplasmic reticulum (ER) stress, which was indicated by electron microscope image and proteome data. Results from the mutants of BiP and proteasome subunit alpha 7 suggested that ER stress played a mechanistic role in inhibiting conidial germination. Results from proteome data indicated that, to survive ammonia fungistasis, conidia first activated the unfolded protein response (UPR) to decrease ER stress and restore ER protein homeostasis, and the function of UPR in surviving ammonia was confirmed by using mutant strains. Second, ammonia toxicity could be reduced by upregulating carbon metabolism-related proteins, which benefited ammonia fixation. The results that metabolites (especially glutamate) could relieve the ammonia fungistasis confirmed this indirectly. Finally, results from gene knockout mutants also suggested that the fungistatic mechanism of ammonia is common for soil fungistasis. This study increased our knowledge regarding the mechanism of soil fungistasis and provided potential new strategies for manipulating soil fungistasis. IMPORTANCE Soil fungistasis is a phenomenon in which the germination and growth of fungal propagules is widely inhibited in soil. Although fungistatic compounds are known to play important roles in the formation of soil fungistasis, how such compounds act on soil fungi remains little studied. This study revealed an endoplasmic reticulum stress-related fungistatic mechanism with which ammonia acts on Arthrobotrys oligospora and a survival strategy of conidia under ammonia inhibition. Our study provides the first mechanistic explanation of how ammonia impacts fungal spore germination, and the mechanism may be common for soil fungistasis. This study increases our knowledge regarding the mechanism of soil fungistasis in fungal spores and provides potential new strategies for manipulating soil fungistasis.

Summary: The nematode-trapping fungi are ideal agents for controlling pathogenic nematodes. Arthrobotrys oligospora is a representative species of the same, producing traps for nematode predation. Here, three orthologous Ras GTPases (Ras2, Ras3, and Rheb) were characterized in A. oligospora. Our results indicate that they play pleiotropic roles in regulating the mycelial growth, conidiation, stress resistance, and pathogenicity of A. oligospora. Furthermore, deletion of Aoras2 and Aorheb significantly affected the mitochondrial activity, reactive oxygen species levels, lipid storage, and autophagy. Transcriptome analyses of ΔAoras2 mutant revealed that many repressed genes were associated with signal transduction, energy production, and carbohydrate transport and metabolism. Moreover, metabolic profile analyses showed that AoRas2 and AoRheb affect the biosynthesis of secondary metabolites in A. oligospora. Collectively, these findings provide an in-depth insight into the essential roles of Ras GTPases in vegetative growth, development, and pathogenicity and highlight their importance in the lifestyle switch of the nematode-trapping fungi.

The complete mitochondrial genome of Purpureocillium lavendulum was characterized in this study. This mitogenome is a closed circular molecule of 23,567 bp in length with a GC content of 28.46%, including 15 protein-coding genes, 25 transfer RNA genes, 2 ribosomal RNA genes. Phylogenetic analyses based on sequences at the 14 concatenated mitochondrial protein-coding genes showed that P. lavendulum was closely related to Hirsutella minnesotensis.

Autophagy and apoptosis have been regarded as important processes in the development of diabetic erectile dysfunction (DMED). Probucol is considered to have anti-apoptotic effects, but its relationship with autophagy has not been reported. The aim of this study was to investigate the effects and mechanisms of probucol on erectile function. Thirty Sprague–Dawley (SD) male rats (12 weeks old) were fasted for 12 h. Twenty SD rats were injected with a single intraperitoneal injection of 60 mg kg−1 streptozotocin (STZ). Ten rats were given vehicle only and used as a sham group. After 72 h, 20 STZ-treated rats with random blood glucose concentrations consistently greater than 16.7 mmol l−1 were used as successfully established diabetic rats. The diabetic rats were divided randomly into two groups and treated with a daily gavage of probucol at a dose of 0 or 500 mg kg−1 for 12 weeks. After treatment, the intracavernous pressure (ICP) was used to measure erectile function upon electrical stimulation of the cavernous nerve. After euthanasia, penile tissue was examined using immunohistochemistry and Western blot to assess the protein levels of B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), microtubule-associated protein light chain 3-II (LC3-II), mammalian target of rapamycin (mTOR), and sequestosome 1 (P62). Caspase-3 activity was measured to determine apoptosis using a caspase-3 assay kit. After 12 weeks of treatment, the erectile function of the probucol group was significantly better than that of the DM group (P < 0.05). Bax and LC3-II protein expression and caspase-3 activity were significantly lower in the probucol group than those in the DM group (all P < 0.05), while Bcl-2, mTOR, and P62 protein expression levels were significantly higher than those in the DM group (all P < 0.05). We demonstrated that probucol inhibited apoptosis and autophagy in STZ-induced diabetic rats.

Decreased temperatures lengthen C. elegans lifespan by mechanisms that are not fully understood. Here the authors show that autophagy activation contributes to lifespan extension at low temperatures and that this involves adiponectin receptor PAQR-2 signaling.

The cell wall integrity (CWI) pathway is composed of three mitogen-activated protein kinases (MAPKs), Bck1, Mkk1/2, and Slt2, and is one of the main signaling pathways for fungal pathogenesis, cell wall synthesis, and integrity maintenance. In this study, we characterized orthologs of Saccharomyces cerevisiae Bck1 and Mkk1 in the nematode-trapping (NT) fungus Arthrobotrys oligospora by multiple phenotypic comparison, and the regulation of conidiation and cell wall synthesis was analyzed using real-time PCR (RT-PCR). Both ΔAoBck1 and ΔAoMkk1 mutants showed severe defects in vegetative growth, cell nucleus number, and stress resistance. Both the mutants were unable to produce spores, and the transcription of several genes associated with sporulation and cell wall biosynthesis was markedly downregulated during the conidiation stage. Further, cell walls of the ΔAoBck1 and ΔAoMkk1 mutants were severely damaged, and the Woronin body failed to respond to cellular damage. In particular, the mutants lost the ability to produce mycelial traps for nematode predation. Taken together, AoBck1 and AoMkk1 play a conserved role in mycelial growth and development, CWI, conidiation, multi-stress tolerance, trap formation, and pathogenicity. We highlighted the role of AoBck1 and AoMkk1 in regulating the Woronin body response to cellular damage and cell nucleus development in A. oligospora.

For decades, more and more long non-coding RNAs (lncRNAs) have been confirmed to play important functions in key biological processes of different organisms. At present, most identified lncRNAs and those with known functional roles are from mammalian systems. However, lncRNAs have also been found in primitive eukaryotic fungi, and they have different functions in fungal development, metabolism, and pathogenicity. In this review, we highlight some recent researches on lncRNAs in the primitive eukaryotic fungi, particularly focusing on the identification of lncRNAs and their regulatory roles in diverse biological processes.

As both host and pathogen require iron for survival, iron is an important regulator of host-pathogen interactions. However, the molecular mechanism by which how the availability of iron modulates host innate immunity against bacterial infections remains largely unknown. Using the metazoan Caenorhabditis elegans as a model, we demonstrate that infection with a pathogenic bacterium Salmonella enterica serovar Typhimurium induces autophagy by inactivating the target of rapamycin (TOR). Although the transcripts of ftn-1 and ftn-2 encoding two H-ferritin subunits are upregulated upon S. Typhimurium infection, the ferritin protein is kept at a low level due to its degradation mediated by autophagy. Autophagy, but not ferritin, is required for defense against S. Typhimurium infection under normal circumstances. Increased abundance of iron suppresses autophagy by activating TOR, leading to an increase in the ferritin protein level. Iron sequestration, but not autophagy, becomes pivotal to protect the host from S. Typhimurium infection in the presence of exogenous iron. Our results show that TOR acts as a regulator linking iron availability with host defense against bacterial infection.

The origin and prevalence of azole-resistant Aspergillus fumigatus

Purpureocillium lavendulum is a fungus with promising biocontrol applications. Here, transcriptome data acquired during the infection of Caenorhabditis elegans by Purpureocillium lavendulum showed that the transcription of metabolite synthesis genes was significantly up-regulated after 24 and 48 h of the fungus-nematode interaction. Then, the up-regulated transcription level of lipoxygenase was confirmed by RT-qPCR. The ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of differential metabolites revealed that this interaction resulted in the emergence of new metabolites or enhanced the production of metabolites. The results of the UPLC-MS analysis and the nematicidal assay were used to establish optimal culturing conditions under which 12 metabolites, including 3 hydroxylated C18 fatty acids and 9 steroids, were isolated and identified. Among them, hydroxylated fatty acids showed pronounced nematicidal activity against Meloidogyne incognita, and two degradative sterols showed chemotaxis activity to M. incognita. This study lays a foundation for the function of lipoxygenase and its products during the infection of Purpureocillium lavendulum.

Autophagy plays an important role in cell growth and development. The autophagy-related gene atg4 encodes a cysteine protease, which can cleave the carboxyl terminus of Atg8, thus plays a role in autophagosome formation in yeast and filamentous fungi. Arthrobotrys oligospora is well known for producing special trapping-devices (traps) and capturing nematodes. In this study, two ΔAolatg4 mutants were generated using targeted gene replacement and were used to investigate the biological functions of autophagy in A. oligospora. Autophagic process was observed using the AoAtg8-GFP fusion protein. The mutants showed a defective in hyphal growth and sporulation and were sensitive to chemical stressors, including menadione and Congo red. The spore yield of the ΔAolatg4 mutants was decreased by 88.5% compared to the wild type (WT), and the transcript levels of six sporulation-related genes, such as abaA, fluG, brlA, and wetA, were significantly downregulated during the conidiation stage. Deletion of Aolatg4 also affected the cell nuclei and mycelial septal development in A. oligospora. Importantly, autophagosome formation and the autophagic process were impaired in the ΔAolatg4 mutant. Moreover, the ΔAolatg4 mutant lost its ability to form mature traps. Our results provide novel insights into the roles of autophagy in A. oligospora.

ABSTRACT The nematophagous fungus Purpureocillium lavendulum is a natural enemy of plant-parasitic nematodes, which cause severe economic losses in agriculture worldwide. The production of asexual spores (conidia) in P. lavendulum is crucial for its biocontrol activity against nematodes. In this study, we characterized the core regulatory genes involved in conidiation of P. lavendulum at the molecular level. The central regulatory pathway is composed of three genes, P. lavendulum brlA (PlbrlA), PlabaA, and PlwetA, which regulate the early, middle, and late stages of asexual development, respectively. The deletion of PlbrlA completely inhibited conidiation, with only conidiophore stalks produced. PlAbaA determines the differentiation of conidia from phialides. The deletion of PlwetA affected many phenotypes related to conidial maturation, including abscission of conidia from conidium strings, thickening of the cell wall layers, vacuole generation inside the cytoplasm, production of trehalose, tolerance to heat shock, etc. Comparative analyses showed that the upstream regulators of the core regulatory pathway of conidiation, especially the “fluffy” genes, were different from those in Aspergillus. Besides their roles in conidiation, the central regulators also influence the production of secondary metabolites, such as the leucinostatins, in P. lavendulum. Our study revealed a set of essential genes controlling conidiation in P. lavendulum and provided a framework for further molecular genetic studies on fungus-nematode interactions and for the biocontrol of plant-parasitic nematodes. IMPORTANCE Plant-parasitic nematodes cause serious damage to crops throughout the world. Purpureocillium lavendulum is a nematophagous fungus which is a natural enemy of nematodes and a potential biocontrol agent against plant-parasitic nematodes. The conidia play an important role during infection of nematodes. In this study, we identified and characterized genes involved in regulating asexual development of P. lavendulum. We found that these genes not only regulate conidiation but also influence secondary-metabolite production. This work provides a basis for future studies of fungus-nematode interactions and nematode biocontrol.

Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that "innate immune response" and "defense response to Gram-negative bacterium" were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.

Summary: Understanding how fungi interact with other organisms has significant medical, environmental, and agricultural implications. Nematode-trapping fungi (NTF) can switch to pathogens by producing various trapping devices to capture nematodes. Here we perform comparative genomic analysis of the NTF with four representative trapping devices. Phylogenomic reconstruction of these NTF suggested an evolutionary trend of trapping device simplification in morphology. Interestingly, trapping device simplification was accompanied by expansion of gene families encoding adhesion proteins and their increasing adhesiveness on trap surfaces. Gene expression analysis revealed a consistent up-regulation of the adhesion genes during their lifestyle transition from saprophytic to nematophagous stages. Our results suggest that the expansion of adhesion genes in NTF genomes and consequential increase in trap surface adhesiveness are likely the key drivers of fungal adaptation in trapping nematodes, providing new insights into understanding mechanisms underlying infection and adaptation of pathogenic fungi.

Predatory fungi in Orbiliaceae (Ascomycota) have evolved a diversity of trapping devices that enable them to trap and kill nematodes, other small animals, and protozoans. These trapping devices include adhesive hyphae, adhesive knobs, adhesive networks, constricting rings, and non-constricting rings. Their diversity and practical importance have attracted significant attention from biologists, making them excellent model organisms for studying adaptative evolution and as biological control agents against parasitic nematodes. The putative origins and evolutionary relationships among these carnivorous fungi have been investigated using nuclear protein-encoding genes, but their patterns of mitogenome relationships and divergences remain unknown. Here we analyze and compare the mitogenomes of 12 fungal strains belonging to eight species, including six species representing all four types of nematode trapping devices and two from related but non-predatory fungi. All 12 analyzed mitogenomes were of circular DNA molecules, with lengths ranging from 146,101 bp to 280,699 bp. Gene synteny analysis revealed several gene rearrangements and intron transfers among the mitogenomes. In addition, the number of protein coding genes (PCGs), GC content, AT skew, and GC skew varied among these mitogenomes. The increased number and total size of introns were the main contributors to the length differences among the mitogenomes. Phylogenetic analyses of the protein-coding genes indicated that mitochondrial and nuclear genomes evolved at different rates, and signals of positive selection were found in several genes involved in energy metabolism. Our study provides novel insights into the evolution of nematode-trapping fungi and shall facilitate further investigations of this ecologically and agriculturally important group of fungi.

Long non-coding RNAs (lncRNAs), including linear lncRNAs and circular RNAs (circRNAs), exhibit a surprising range of structures. Linear lncRNAs and circRNAs are generated by different pathways. Linear lncRNAs perform functions that depend on their specific sequences, transcription, and DNA elements of their gene loci. In some cases, linear lncRNAs contain a short open reading frame encoding a peptide. circRNAs are covalently closed RNAs with tissue-specific and cell-specific expression patterns that have recently been extensively investigated. Pioneering work focusing on their biogenesis and functional characterization indicates that circRNAs regulate cell development via multiple mechanisms and play critical roles in the immune system. Furthermore, circRNAs in exosomes function on target cells. As with linear lncRNAs, specific circRNAs can also be translated. In this review, we summarize current understanding and highlight the diverse structure, regulation, and function of linear lncRNAs and circRNAs.

Nematode-trapping (NT) fungi play a significant role in the biological control of plant- parasitic nematodes. NT fungi, as a predator, can differentiate into specialized structures called “traps” to capture, kill, and consume nematodes at a nutrient-deprived condition. Therefore, trap formation is also an important indicator that NT fungi transition from a saprophytic to a predacious lifestyle. With the development of gene knockout and multiple omics such as genomics, transcriptomics, and metabolomics, increasing studies have tried to investigate the regulation mechanism of trap formation in NT fungi. This review summarizes the potential regulatory mechanism of trap formation in NT fungi based on the latest findings in this field. Signaling pathways have been confirmed to play an especially vital role in trap formation based on phenotypes of various mutants and multi-omics analysis, and the involvement of small molecule compounds, woronin body, peroxisome, autophagy, and pH-sensing receptors in the formation of traps are also discussed. In addition, we also highlight the research focus for elucidating the mechanism underlying trap formation of NT fungi in the future.

Ssk1, a response regulator of the two-component signaling system, plays an important role in the cellular response to hyperosmotic stress in fungi. Herein, an ortholog of ssk1 (Aossk1) was characterized in the nematode-trapping fungus Arthrobotrys oligospora using gene disruption and multi-phenotypic comparison. The deletion of Aossk1 resulted in defective growth, deformed and swollen hyphal cells, an increased hyphal septum, and a shrunken nucleus. Compared to the wild-type (WT) strain, the number of autophagosomes and lipid droplets in the hyphal cells of the ΔAossk1 mutant decreased, whereas their volumes considerably increased. Aossk1 disruption caused a 95% reduction in conidial yield and remarkable defects in tolerance to osmotic and oxidative stress. Meanwhile, the transcript levels of several sporulation-related genes were significantly decreased in the ΔAossk1 mutant compared to the WT strain, including abaA, brlA, flbC, fluG, and rodA. Moreover, the loss of Aossk1 resulted in a remarkable increase in trap formation and predation efficiency. In addition, many metabolites were markedly downregulated in the ΔAossk1 mutant compared to the WT strain. Our results highlight that AoSsk1 is a crucial regulator of asexual development, stress responses, the secondary metabolism, and pathogenicity, and can be useful in probing the regulatory mechanism underlying the trap formation and lifestyle switching of nematode-trapping fungi.

Phosphodiesterases are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Two phosphodiesterases, PdeH and PdeL, have been identified from yeast and filamentous fungi. Here, the orthologs of PdeH and PdeL were characterized in a typical nematode-trapping fungus Arthrobotrys oligospora by gene disruption and phenotypic comparison. Deletion of AopdeH caused serious defects in mycelial growth, conidiation, stress response, trap formation, and nematicidal efficiency compared to the wild-type strain. In contrast, these phenotypes have no significant difference in the absence of AopdeL. In addition, deletion of AopdeH and AopdeL resulted in a remarkable increase in cAMP level during vegetative growth and trap formation, and the number of autophagosomes was decreased in ΔAopdeH and ΔAopdeL mutants, whereas their volumes considerably increased. Moreover, metabolomic analyses revealed that many metabolites were downregulated in ΔAopdeH mutant compared to their expression in the wild-type strain. Our results indicate that AoPdeH plays a crucial role in mycelial growth, conidiation, stress response, secondary metabolism, and trap formation. In contrast, AoPdeL only plays a minor role in hyphal and conidial morphology, autophagy, and trap formation in A. oligospora. This work expands the roles of phosphodiesterases and deepens the understanding of the regulation of trap formation in nematode-trapping fungi.

Maintaining the effects of nematode-trapping fungi (NTF) agents in order to control plant-parasitic nematodes (PPNs) in different ecological environments has been a major challenge in biological control applications. To achieve such an objective, it is important to understand how populations of the biocontrol agent NTF are geographically and ecologically structured. A previous study reported evidence for ecological adaptation in the model NTF species Arthrobotrys oligospora. However, their large-scale geographic structure, patterns of gene flow, their potential phenotypic diversification, and host specialization remain largely unknown. In this study, we developed a new panel of 20 polymorphic short tandem repeat (STR) markers and analyzed 239 isolates of A. oligospora from 19 geographic populations in China. In addition, DNA sequences at six nuclear gene loci and strain mating types (MAT) were obtained for these strains. Our analyses suggest historical divergence within the A. oligospora population in China. The genetically differentiated populations also showed phenotypic differences that may be related to their ecological adaptations. Interestingly, our analyses identified evidence for recent dispersion and hybridization among the historically subdivided geographic populations in nature. Together, our results indicate a changing population structure of A. oligospora in China and that care must be taken in selecting the appropriate strains as biocontrol agents that can effectively reproduce in agriculture soil while maintaining their nematode-trapping ability.

The efficiency of hybrid rice seed production can be improved by increasing the percentage of exserted stigmas. To identify quantitative trait loci (QTLs) for this trait, we conducted QTL mapping using 75 chromosome segment substitution lines (CSSLs) developed from a cross between the donor parent, Xieqingzao B (XQZB), a maintainer line which has high stigma exsertion and the recurrent parent, Zhonghui 9308 (ZH9308), a restorer line which has low stigma exsertion. A total of nine QTLs (qSSE5, qSSE10, qSSE11, qDSE10, qDSE11, qTSE5, qTSE6, qTSE10, and qTSE11) for single stigma exsertion (SSE), dual stigma exsertion (DSE) and total stigma exsertion (TSE) were assessed in two environments (Hainan and Zhejiang). Six of these QTLs (qSSE10, qSSE11, qDSE10, qDSE11, qTSE10, and qTSE11) were found in both environments, while one QTL (qTSE6) was found in only Hainan, and two QTLs (qSSE5 and qTSE5) were found in only Zhejiang. The qSSE10, qSSE11, qDSE10, qDSE11, qTSE6, qTSE10, and qTSE11 alleles, which are derived from the parent XQZB, exhibited a positive additive effect. In contrast, the qSSE5 and qTSE5 alleles, which are derived from the parent ZH9308, exhibited a negative additive effect. The SSE, DSE and TSE traits were significantly correlated with each other in an environmentally dependent manner. These results indicated that the lines showing higher values for SSE were more likely to exhibit increased values for DSE, which would ultimately increase TSE. To evaluate the advantage of exserted stigmas for cross-pollination, single, dual and total stigma exsertion should be considered separately in future attempts at genetic improvement to achieve increased production of rice hybrid seeds. This study also provides information for fine mapping, gene cloning and particularly marker-assisted selection (MAS), on the latter and with an emphasis the phenotypic effects and implications of the QTLs for practical use in hybrid rice breeding.

Abstract Arthrobotrys oligospora is a typical nematode-trapping fungus. In this study, 37 transformants of A. oligospora were obtained by REMI (restriction enzyme mediated integration) method and phenotypic properties of nine transformants were analyzed. The nine transformants showed differences in growth, conidiation, trap formation, stress tolerance, and/or pathogenicity among each other and with those of the parental wild-type strain (WT). The insertional sites of the hph cassette were identified in transformants X5 and X13. In X5, the cassette was inserted in the non-coding region between AOL_s00076g273 (76g273) and AOL_s00076g274 (76g274) and the transcription of 76g274, but not 76g273, was enhanced in X5. 76g274p had two conserved domains and was predicted as a nucleoprotein, which we confirmed by its nuclear localization in Saccharomyces cerevisiae using the green fluorescent protein-fused 76g274p. The transcription of 76g274 was stimulated or inhibited by several environmental factors. The sporulation yields of 76g274-deficient mutants were decreased by 70%, and transcription of several sporulation-related genes was severely diminished compared to the WT during the conidiation. In summary, a method for screening mutants was established in A. oligospora and using the method, we identified a novel C2H2-type transcription factor that positively regulates the conidiation of A. oligospora.

Abstract In the majority of sexual eukaryotes, the mitochondrial genomes are inherited uniparentally. As a result, individual organisms are homoplasmic, containing mitochondrial DNA (mtDNA) from a single parent. Here we analyzed the mitochondrial genotypes in Clade I of the gourmet mushroom Thelephora ganbajun from its broad geographic distribution range. A total of 299 isolates from 28 geographic locations were sequenced at three mitochondrial loci: the mitochondrial small ribosomal RNA gene, and the cytochrome c oxidase subunits I (COX1) and III (COX3) genes. Quantitative PCR analyses showed that the strains had about 60–160 copies of mitochondrial genomes per cell. Interestingly, while no evidence of heteroplasmy was found at the 12S rRNA gene, 262 of the 299 isolates had clear evidence of heterogeneity at either the COX1 (261 isolates) or COX3 (12 isolates) gene fragments. The COX1 heteroplasmy was characterized by two types of introns residing at different sites of the same region and at different frequencies among the isolates. Allelic association analyses of the observed mitochondrial polymorphic nucleotide sites suggest that mtDNA recombination is common in natural populations of this fungus. Our results contrast the prevailing view that heteroplasmy, if exists, is only transient in basidiomycete fungi.