The Auto-Regulation of ATL2 E3 Ubiquitin Ligase Plays an Important Role in the Immune Response against Alternaria brassicicola in Arabidopsis thaliana.

The ubiquitin/26S proteasome system is a crucial regulatory mechanism that governs various cellular processes in plants, including signal transduction, transcriptional regulation, and responses to biotic and abiotic stressors. Our study shows that the RING-H2-type E3 ubiquitin ligase, Arabidopsis Tóxicos en Levadura 2 (ATL2), is involved in response to fungal pathogen infection. Under normal growth conditions, the expression of the ATL2 gene is low, but it is rapidly and significantly induced by exogenous chitin. Additionally, ATL2 protein stability is markedly increased via chitin treatment, and its degradation is prolonged when 26S proteasomal function is inhibited. We found that an atl2 null mutant exhibited higher susceptibility to Alternaria brassicicola, while plants overexpressing ATL2 displayed increased resistance. We also observed that the hyphae of A. brassicicola were strongly stained with trypan blue staining, and the expression of A. brassicicola Cutinase A (AbCutA) was dramatically increased in atl2. In contrast, the hyphae were weakly stained, and AbCutA expression was significantly reduced in ATL2-overexpressing plants. Using bioinformatics, live-cell confocal imaging, and cell fractionation analysis, we revealed that ATL2 is localized to the plasma membrane. Further, it is demonstrated that the ATL2 protein possesses E3 ubiquitin ligase activity and found that cysteine 138 residue is critical for its function. Moreover, ATL2 is necessary to successfully defend against the A. brassicicola fungal pathogen. Altogether, our data suggest that ATL2 is a plasma membrane-integrated protein with RING-H2-type E3 ubiquitin ligase activity and is essential for the defense response against fungal pathogens in Arabidopsis.

Physical impediment to sodium houttuyfonate conversely reinforces ß-glucan exposure stimulated innate immune response to Candida albicans.

Candida albicans is a dimorphic opportunistic pathogen in immunocompromised individuals. We have previously demonstrated that sodium houttuyfonate (SH), a derivative of medicinal herb Houttuynia cordata Thunb, was effective for antifungal purposes. However, the physical impediment of SH by C. albicans ß-glucan may weaken the antifungal activity of SH. In this study, the interactions of SH with cell wall (CW), extracellular matrix (EM), CW ß-glucan, and a commercial ß-glucan zymosan A (ZY) were inspected by XTT assay and total plate count in a standard reference C. albicans SC5314 as well as two clinical fluconazole-resistant strains Z4935 and Z5172. After treatment with SH, the content and exposure of CW ß-glucan, chitin, and mannan were detected, the fungal clearance by phagocytosis of RAW264.7 and THP-1 was examined, and the gene expressions and levels of cytokines TNF-ɑ and IL-10 were also monitored. The results showed that SH could be physically impeded by ß-glucan in CW, EM, and ZY. This impediment subsequently triggered the exposure of CW ß-glucan and chitin with mannan masked in a time-dependent manner. SH-induced ß-glucan exposure could significantly enhance the phagocytosis and inhibit the growth of C. albicans. Meanwhile, the SH-pretreated fungal cells could greatly stimulate the cytokine gene expressions and levels of TNF-ɑ and IL-10 in the macrophages. In sum, the strategy that the instant physical impediment of C. albicans CW to SH, which can induce the exposure of CW ß-glucan may be universal for C. albicans in response to physical deterrent by antifungal drugs.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

Survival in macrophages induces enhanced virulence in Cryptococcus.

Cryptococcus is a ubiquitous environmental fungus and frequent colonizer of human lungs. Colonization can lead to diverse outcomes, from clearance to long-term colonization to life-threatening meningoencephalitis. Regardless of the outcome, the process starts with an encounter with phagocytes. Using the zebrafish model of this infection, we have noted that cryptococcal cells first spend time inside macrophages before they become capable of pathogenic replication and dissemination. What "licensing" process takes place during this initial encounter, and how are licensed cryptococcal cells different? To address this, we isolated cryptococcal cells after phagocytosis by cultured macrophages and found these macrophage-experienced cells to be markedly more virulent in both zebrafish and mouse models. Despite producing a thick polysaccharide capsule, they were still subject to phagocytosis by macrophages in the zebrafish. Analysis of antigenic cell wall components in these licensed cells demonstrated that components of mannose and chitin are more available for staining than they are in culture-grown cells or cells with capsule production induced in vitro. Cryptococcus is capable of exiting or transferring between macrophages in vitro, raising the likelihood that this fungus alternates between intracellular and extracellular life during growth in the lungs. Our results raise the possibility that intracellular life has its advantages over time, and phagocytosis-induced alteration in mannose and chitin exposure is one way that makes subsequent rounds of phagocytosis more beneficial to the fungus.IMPORTANCECryptococcosis begins in the lungs and can ultimately travel through the bloodstream to cause devastating infection in the central nervous system. In the zebrafish model, small amounts of cryptococcus inoculated into the bloodstream are initially phagocytosed and become far more capable of dissemination after they exit macrophages. Similarly, survival in the mouse lung produces cryptococcal cell types with enhanced dissemination. In this study, we have evaluated how phagocytosis changes the properties of Cryptococcus during pathogenesis. Macrophage-experienced cells (MECs) become "licensed" for enhanced virulence. They out-disseminate culture-grown cells in the fish and out-compete non-MECs in the mouse lung. Analysis of their cell surface demonstrates that MECs have increased availability of cell wall components mannose and chitin substances involved in provoking phagocytosis. These findings suggest how Cryptococcus might tune its cell surface to induce but survive repeated phagocytosis during early pathogenesis in the lung.

An overview of viral chitinases: General properties and biotechnological potential.

Chitin is a biopolymer profusely present in nature and of pivotal importance as a structural component in cells. It is degraded by chitinases, enzymes naturally produced by different organisms. Chitinases are proteins enrolled in many cellular mechanisms, including the remodeling process of the fungal cell wall, the cell growth process, the autolysis of filamentous fungi, and cell separation of yeasts, among others. These enzymes also have properties with different biotechnological applications. They are used to produce polymers, for biological control, biofilm formation, and as antitumor and anti-inflammatory target molecules. Chitinases are classified into different glycoside hydrolase (GH) families and are widespread in microorganisms, including viruses. Among them, the GH18 family is highly predominant in the viral genomes, being present and active enzymes in baculoviruses and nucleocytoplasmic large DNA viruses (NCLDV), especially chloroviruses from the Phycodnaviridae family. These viral enzymes contain one or more GH domains and seem to be involved during the viral replication cycle. Curiously, only a few DNA viruses have these enzymes, and studying their properties could be a key feature for biological and biotechnological novelties. Here, we provide an overview of viral chitinases and their probable function in viral infection, showing evidence of at least two distinct origins for these enzymes. Finally, we discuss how these enzymes can be applied as biotechnological tools and what one can expect for the coming years on these GHs.

Soy protein/ß-chitin sponge-like scaffolds laden with human mesenchymal stromal cells from hair follicle or adipose tissue promote diabetic chronic wound healing.

Chronic wounds are a worldwide problem that affect >40 million people every year. The constant inflammatory status accompanied by prolonged bacterial infections reduce patient's quality of life and life expectancy drastically. An important cell type involved in the wound healing process are mesenchymal stromal cells (MSCs) due to their long-term demonstrated immunomodulatory and pro-regenerative capacity. Thus, in this work, we leveraged and compared the therapeutic properties of MSCs derived from both adipose tissue and hair follicle, which we combined with sponge-like scaffolds (SLS) made of valorized soy protein and ß-chitin. In this regard, the combination of these cells with biomaterials permitted us to obtain a multifunctional therapy that allowed high cell retention and growing rates while maintaining adequate cell-viability for several days. Furthermore, this combined therapy demonstrated to increase fibroblasts and keratinocytes migration, promote human umbilical vein endothelial cells angiogenesis and protect fibroblasts from highly proteolytic environments. Finally, this combined therapy demonstrated to be highly effective in reducing wound healing time in vivo with only one treatment change during all the experimental procedure, also promoting a more functional and native-like healed skin.

Multi-enzyme Machinery for Chitin Degradation in the Chitinolytic Bacterium Chitiniphilus shinanonensis SAY3T.

The chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T was examined to characterize its chitin-degrading enzymes in view of its potential to convert biomass chitin into useful saccharides. A survey of the whole-genome sequence revealed 49 putative genes encoding polypeptides that are thought to be related to chitin degradation. Based on an analysis of the relative quantity of each transcript and an assay for chitin-degrading activity of recombinant proteins, a chitin degradation system driven by 19 chitinolytic enzymes was proposed. These include sixteen endo-type chitinases, two N-acetylglucosaminidases, and one lipopolysaccharide monooxygenase that catalyzes the oxidative cleavage of glycosidic bonds. Among the 16 chitinases, ChiL was characterized by its remarkable transglycosylation activity. Of the two N-acetylglucosaminidases (ChiI and ChiT), ChiI was the major enzyme, corresponding to > 98% of the total cellular activity. Surprisingly, a chiI-disrupted mutant was still able to grow on medium with powdered chitin or GlcNAc dimer. However, its growth rate was slightly lower compared to that of the wild-type SAY3. This multi-enzyme machinery composed of various types of chitinolytic enzymes may support SAY3 to efficiently utilize native chitin as a carbon and energy source and may play a role in developing an enzymatic process to decompose and utilize abundant chitin at the industrial scale.

Chitooligosaccharide boosts the immunity of immunosuppressed blunt snout bream against bacterial infections.

The immunosuppression hazard of fish brought by intensive aquaculture needs to be addressed urgently, while chitooligosaccharide (COS) shows the potential application in the prevention the immunosuppression of fish due to its superior biological properties. In this study, COS reversed the cortisol-induced immunosuppression of macrophages and improved the immune activity of macrophages in vitro, promoting the expression of inflammatory genes (TNF-α, IL-1ß, iNOS) and NO production, and increasing the phagocytic activity of macrophages. In vivo, the oral COS was absorbed directly through the intestine, significantly ameliorating the innate immunity of cortisol-induced immunosuppression of blunt snout bream (Megalobrama amblycephala). Such as facilitated the gene expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6) and pattern recognition receptors (TLR4, MR) and potentiated bacterial clearance, resulting in an effective improvement in survival and tissue damage. Altogether, this study demonstrates that COS offers potential strategies in the application of immunosuppression prevention and control in fish.

Chitooligosaccharide accelerated wound healing in potato tubers by promoting the deposition of suberin polyphenols and lignin at wounds.

Chitooligosaccharide (COS) is a low molecular weight product of chitosan degradation. Although COS induces plant resistance by activating phenylpropanoid metabolism, there are few reports on whether COS accelerates wound healing in potato tubers by promoting the deposition of phenolic acids and lignin monomers at wounds. The results showed that COS activated phenylalanine ammonialyase and cinnamate 4-hydroxylase and promoted the synthesis of cinnamic, caffeic, p-coumaric, ferulic acids, total phenolics and flavonoids. COS activated 4-coumaric acid coenzyme A ligase and cinnamyl alcohol dehydrogenase and promoted the synthesis of sinapyl, coniferyl and cinnamyl alcohols. COS also increased H2O2 levels and peroxidase activity and accelerated the deposition of suberin polyphenols and lignin on wounds. In addition, COS reduced weight loss and inhibited lesion expansion in tubers inoculated with Fusarium sulfureum. Taken together, COS accelerated wound healing in potato tubers by inducing phenylpropanoid metabolism and accelerating the deposition of suberin polyphenols and lignin at wounds.

The role of FIBCD1 in response to Aspergillus fumigatus in lung epithelial cells.

Chitin, a polysaccharide, is ubiquitously found in nature and has been known to be an active immunogen in mammals, and interacts with Toll-like, mannose and glucan receptors, to induce cytokine and chemokine secretions. FIBCD1 is a tetrameric type II transmembrane endocytic vertebrate receptor that binds chitin, is found in human lung epithelium and modulates lung epithelial inflammatory responses to A. fumigatus cell wall polysaccharides. We previously reported the detrimental role of FIBCD1 in a murine model of pulmonary invasive aspergillosis. However, the effect that chitin and chitin-containing A. fumigatus conidia exerts on lung epithelium following exposure through FIBCD1 is not yet fully explored. Using both in vitro and in vivo strategies, we examined how lung and lung epithelial gene expression are modified after exposure to fungal conidia or chitin fragments in the presence or absence of FIBCD1. FIBCD1 expression was associated with a decrease in inflammatory cytokines with increasing size of chitin (dimer-oligomer). Thus, our results demonstrate that FIBCD1 expression modulates cytokine and chemokine expression in response to A. fumigatus conidia that is modified by the presence of chitin particles.

Maize Antifungal Protein AFP1 Elevates Fungal Chitin Levels by Targeting Chitin Deacetylases and Other Glycoproteins.

Pathogenic fungi convert chitin to chitosan to evade plant perception and disarm chitin-triggered immune responses. Whether plants have evolved factors to counteract this evasion mechanism remains obscure. Here, we decipher the mechanism underlying the antifungal activity of maize secretory mannose-binding cysteine-rich receptor-like secreted protein (CRRSP), antifungal protein 1 (AFP1). AFP1 binds to multiple sites on the surface of sporidial cells, filaments, and germinated spores of the biotrophic fungus Ustilago maydis. It inhibits cell growth and budding, as well as spore germination. AFP1 promiscuously interacts with most chitin deacetylases (CDAs) by recognizing the conserved NodB domain to interfere with the enzyme activity. Deletion of O-mannosyltransferase 4 decreases protein mannosylation, which correlates with reduced AFP1 binding and antifungal activity, suggesting that AFP1 interacts with mannosylated proteins to exhibit an inhibitory effect. AFP1 also has extended inhibitory activity against Saccharomyces cerevisiae; however, AFP1 did not reduce binding to the double ΔΔcda1,2 mutant, suggesting the targets of AFP1 have expanded to other cell surface glycoproteins, probably facilitated by its mannose-binding property. Increasing chitin levels by modulating the activity of cell surface glycoproteins is a universal feature of AFP1 interacting with a broad spectrum of fungi to inhibit their growth. IMPORTANCE Plants alert immune systems by recognizing the fungal pathogen cell wall component chitin via pattern recognition cell surface receptors. Successful fungal pathogens escape the perception by deacetylating chitin to chitosan, which is also necessary for fungal cell development and virulence. Targeting glycoproteins that are associated with regulating chitin metabolism and maintaining cell wall morphogenesis presents an effective strategy to combat fungal pathogens by simultaneously altering cell wall plasticity, activating chitin-triggered immunity, and impairing fungal viability. Our study provides molecular insights into a plant DUF26 domain-containing secretory protein in warding off a broad range of fungal pathogens by acting on more than one glycoprotein target.

LysM receptor-like kinases involved in immunity perceive lipo-chitooligosaccharides in mycotrophic plants.

Symbiotic microorganisms such as arbuscular mycorrhizal fungi (AMF) produce both conserved microbial molecules that activate plant defense and lipo-chitooligosaccharides (LCOs) that modulate plant defense. Beside a well-established role of LCOs in the activation of a signaling pathway required for AMF penetration in roots, LCO perception and defense modulation during arbuscular mycorrhiza is not well understood. Here we show that members of the LYRIIIA phylogenetic group from the multigenic Lysin Motif Receptor-Like Kinase family have a conserved role in dicotyledons as modulators of plant defense and regulate AMF colonization in the Solanaceae species Nicotiana benthamiana. Interestingly, these proteins have a high-affinity for LCOs in plant species able to form a symbiosis with AMF but have lost this property in species that have lost this ability. Our data support the hypothesis that LYRIIIA proteins modulate plant defense upon LCO perception to facilitate AMF colonization in mycotrophic plant species and that only their role in plant defense, but not their ability to be regulated by LCOs, has been conserved in non-mycotrophic plants.

The secreted immune response peptide 1 functions as a phytocytokine in rice immunity.

Small signalling peptides play important roles in various plant processes, but information regarding their involvement in plant immunity is limited. We previously identified a novel small secreted protein in rice, called immune response peptide 1 (IRP1). Here, we studied the function of IRP1 in rice immunity. Rice plants overexpressing IRP1 enhanced resistance to the virulent rice blast fungus. Application of synthetic IRP1 to rice suspension cells triggered the expression of IRP1 itself and the defence gene phenylalanine ammonia-lyase 1 (PAL1). RNA-seq results revealed that 84% of genes up-regulated by IRP1, including 13 OsWRKY transcription factors, were also induced by a microbe-associated molecular pattern (MAMP), chitin, indicating that IRP1 and chitin share a similar signalling pathway. Co-treatment with chitin and IRP1 elevated the expression level of PAL1 and OsWRKYs in an additive manner. The increased chitin concentration arrested the induction of IRP1 and PAL1 expression by IRP1, but did not affect IRP1-triggered mitogen-activated protein kinases (MAPKs) activation. Collectively, our findings indicate that IRP1 functions as a phytocytokine in rice immunity regulating MAPKs and OsWRKYs that can amplify chitin and other signalling pathways, and provide new insights into how MAMPs and phytocytokines cooperatively regulate rice immunity.

Chitin contributes to the formation of a feeding structure in a predatory nematode.

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P. pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicates a key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

Antifungal activity, mycotoxin inhibitory efficacy, and mode of action of hop essential oil nanoemulsion against Fusarium graminearum.

This work aims to investigate antifungal, mycotoxin inhibitory efficacy of the hop essential oil (HEO) nanoemulsion and their mode of action (MOA) against Fusarium graminearum isolate, a fungal pathogen causing Fusarium Head Blight (FHB) in cereal crops. The HEO, primarily consisting of terpenes and terpenoids, was encapsulated in nanoemulsion droplets. Physically stable HEO-in-water nanoemulsion was fabricated using 0.5 wt% of tween 80 and 5 wt% oil phase comprising 30 % of Ostwald ripening inhibitor and 70 % of HEO. In terms of antifungal effect, HEO nanoemulsion could not only effectively inhibit mycelial growth and spore germination of F. graminearum isolates, but also remarkably suppress the production of deoxynivalenol (DON) and its derivatives in rice culture by applying 750 µg of HEO/g rice. Our studies on the MOA showed that HEO nanoemulsion could alter the contents of total lipid and chitin in outer cell membrane as well as damaging cytoplasmic membrane.

Chitin powder enhances growth factor production and therapeutic effects of mesenchymal stem cells in a chronic kidney disease rat model.

Previously, we fabricated a device with polylactic acid nonwoven filters and mesenchymal stem cells (MSCs), which effectively reduced urinary protein levels in a rat model of chronic kidney disease (CKD) but could not suppress CKD progression. Therefore, to improve the therapeutic effects of MSCs, in this study, we analyzed the ability of rat adipose tissue-derived MSCs (ADSCs) in contact with chitin nonwoven filters or chitin powder to produce growth factors and examined their therapeutic effect in an adriamycin (ADR)-induced CKD rat model. Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) production was significantly enhanced by ADSCs cultured in a medium containing chitin powder (C-ADSCs) compared with that by ADSCs cultured in a standard medium without chitin (N-ADSCs). However, the production of HGF and VEGF by ADSCs on chitin nonwoven filters was not significantly enhanced compared with that by the control. Intravenous C-ADSC injection significantly increased podocin expression and improved proteinuria compared with those in saline-treated CKD rats; however, no such improvements were observed in the N-ADSC-treated group. These results showed that ADSCs cultured in a medium supplemented with chitin powder suppressed proteinuria via enhanced HGF and VEGF production in ADR-induced CKD rats to mitigate podocyte damage, offering a new strategy to reduce the dose of MSC therapy for safe and effective treatment of kidney disease.

Cloning and Characterization of Two Novel PR4 Genes from Picea asperata.

Pathogenesis-related (PR) proteins are important in plant pathogenic resistance and comprise 17 families, including the PR4 family, with antifungal and anti-pathogenic functions. PR4 proteins contain a C-terminal Barwin domain and are divided into Classes I and II based on the presence of an N-terminal chitin-binding domain (CBD). This study is the first to isolate two PR4 genes, PaPR4-a and PaPR4-b, from Picea asperata, encoding PaPR4-a and PaPR4-b, respectively. Sequence analyses suggested that they were Class II proteins, owing to the presence of an N-terminal signal peptide and a C-terminal Barwin domain, but no CBD. Tertiary structure analyses using the Barwin-like protein of papaya as a template revealed structural similarity, and therefore, functional similarity between the proteins. Predictive results revealed an N-terminal transmembrane domain, and subcellular localization studies confirmed its location on cell membrane and nuclei. Real-time quantitative PCR (RT-qPCR) demonstrated that PaPR4-a and PaPR4-b expression levels were upregulated following infection with Lophodermium piceae. Additionally, PaPR4-a and PaPR4-b were induced in Escherichia coli, where the recombinant proteins existed in inclusion bodies. The renatured purified proteins showed antifungal activity. Furthermore, transgenic tobacco overexpressing PaPR4-a and PaPR4-b exhibited improved resistance to fungal infection. The study can provide a basis for further molecular mechanistic insights into PR4-induced defense responses.

Shell Disease Syndrome Is Associated with Reduced and Shifted Epibacterial Diversity on the Carapace of the Crustacean Cancer pagurus.

Cancer pagurus is highly susceptible to shell disease syndrome. However, little is known about concomitant changes in the epibacterial community. We compared the bacterial communities of black spot affected and nonaffected areas of the carapace by amplicon sequencing of 16S rRNA genes and 16S rRNA. Within each spot, bacterial communities of affected areas were less diverse compared to communities from nonaffected areas. Communities of different affected spots were, however, more divergent from each other, compared to those of different nonaffected areas. This indicates a reduced and shifted microbial community composition caused by the black spot disease. Different communities found in black spots likely indicate different stages of the disease. In affected areas, Flavobacteriaceae rose to one of the most abundant and active families due to the increase of Aquimarina spp., suggesting a significant role in shell disease syndrome. We isolated 75 bacterial strains from diseased and healthy areas, which are primarily affiliated with Proteobacteria and Bacteroidetes, reflecting the dominant phyla detected by amplicon sequencing. The ability to degrade chitin was mainly found for Gammaproteobacteria and Aquimarina spp. within the Flavobacteriia, while the ability to use N-acetylglucosamine, the monomer of the polysaccharide chitin, was observed for most isolates, including many Alphaproteobacteria. One-third of the isolates, including most Aquimarina spp., showed antagonistic properties, indicating a high potential for interactions between the bacterial populations. The combination of bacterial community analysis and the physiological properties of the isolates provided insights into a functional complex epibacterial community on the carapace of C. pagurus. IMPORTANCE In recent years, shell disease syndrome has been detected for several ecologically and economically important crustacean species. Large proportions of populations are affected, e.g., >60% of the widely distributed species Cancer pagurus in different North Sea areas. Bacteria play a significant role in the development of different forms of shell disease, all characterized by microbial chitinolytic degradation of the outer shell. By comparing the bacterial communities of healthy and diseased areas of the shell of C. pagurus, we demonstrated that the disease causes a reduced bacterial diversity within affected areas, a phenomenon co-occurring also with many other diseases. Furthermore, the community composition dramatically changed with some taxa rising to high relative abundances and showing increased activity, indicating strong participation in shell disease. Characterization of bacterial isolates obtained from affected and nonaffected spots provided deeper insights into their physiological properties and thus the possible role within the microbiome.

Diflubenzuron Induces Cardiotoxicity in Zebrafish Embryos.

Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.

NAC Transcription Factor GmNAC12 Improved Drought Stress Tolerance in Soybean.

NAC transcription factors (TFs) could regulate drought stresses in plants; however, the function of NAC TFs in soybeans remains unclear. To unravel NAC TF function, we established that GmNAC12, a NAC TF from soybean (Glycine max), was involved in the manipulation of stress tolerance. The expression of GmNAC12 was significantly upregulated more than 10-fold under drought stress and more than threefold under abscisic acid (ABA) and ethylene (ETH) treatment. In order to determine the function of GmNAC12 under drought stress conditions, we generated GmNAC12 overexpression and knockout lines. The present findings showed that under drought stress, the survival rate of GmNAC12 overexpression lines increased by more than 57% compared with wild-type plants, while the survival rate of GmNAC12 knockout lines decreased by at least 46%. Furthermore, a subcellular localisation analysis showed that the GmNAC12 protein is concentrated in the nucleus of the tobacco cell. In addition, we used a yeast two-hybrid assay to identify 185 proteins that interact with GmNAC12. Gene ontology (GO) and KEGG analysis showed that GmNAC12 interaction proteins are related to chitin, chlorophyll, ubiquitin-protein transferase, and peroxidase activity. Hence, we have inferred that GmNAC12, as a key gene, could positively regulate soybean tolerance to drought stress.