The Ser/Thr protein kinase FonKin4-poly(ADP-ribose) polymerase FonPARP1 phosphorylation cascade is required for the pathogenicity of watermelon fusarium wilt fungus Fusarium oxysporum f. sp. niveum.

Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG), is a kind of post-translational protein modification that is involved in various cellular processes in fungi, plants, and mammals. However, the function of PARPs in plant pathogenic fungi remains unknown. The present study investigated the roles and mechanisms of FonPARP1 in watermelon Fusarium wilt fungus Fusarium oxysporum f. sp. niveum (Fon). Fon has a single PARP FonPARP1 and one PARG FonPARG1. FonPARP1 is an active PARP and contributes to Fon pathogenicity through regulating its invasive growth within watermelon plants, while FonPARG1 is not required for Fon pathogenicity. A serine/threonine protein kinase, FonKin4, was identified as a FonPARP1-interacting partner by LC-MS/MS. FonKin4 is required for vegetative growth, conidiation, macroconidia morphology, abiotic stress response and pathogenicity of Fon. The S_TKc domain is sufficient for both enzyme activity and pathogenicity function of FonKin4 in Fon. FonKin4 phosphorylates FonPARP1 in vitro to enhance its poly(ADP-ribose) polymerase activity; however, FonPARP1 does not PARylate FonKin4. These results establish the FonKin4-FonPARP1 phosphorylation cascade that positively contributes to Fon pathogenicity. The present study highlights the importance of PARP-catalyzed protein PARylation in regulating the pathogenicity of Fon and other plant pathogenic fungi.

Two H3K36 methyltransferases differentially associate with transcriptional activity and enrichment of facultative heterochromatin in rice blast fungus.

Di- and tri-methylation of lysine 36 on histone H3 (H3K36me2/3) is catalysed by histone methyltransferase Set2, which plays an essential role in transcriptional regulation. Although there is a single H3K36 methyltransferase in yeast and higher eukaryotes, two H3K36 methyltransferases, Ash1 and Set2, were present in many filamentous fungi. However, their roles in H3K36 methylation and transcriptional regulation remained unclear. Combined with methods of RNA-seq and ChIP-seq, we revealed that both Ash1 and Set2 are redundantly required for the full H3K36me2/3 activity in Magnaporthe oryzae, which causes the devastating worldwide rice blast disease. Ash1 and Set2 distinguish genomic H3K36me2/3-marked regions and are differentially associated with repressed and activated transcription, respectively. Furthermore, Ash1-catalysed H3K36me2 was co-localized with H3K27me3 at the chromatin, and Ash1 was required for the enrichment and transcriptional silencing of H3K27me3-occupied genes. With the different roles of Ash1 and Set2, in H3K36me2/3 enrichment and transcriptional regulation on the stress-responsive genes, they differentially respond to various stresses in M. oryzae. Overall, we reveal a novel mechanism by which two H3K36 methyltransferases catalyze H3K36me2/3 that differentially associate with transcriptional activities and contribute to enrichment of facultative heterochromatin in eukaryotes. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00127-3.

An immunochromatographic test for serological diagnosis of scrub typhus.

A fluorescent immunochromatographic test (FM-ICT) was developed for rapid detection of anti-Orientia tsutsugamushi antibodies in serum samples. The FM-ICT was constructed based on the dual-antigen sandwich method. Truncated 56 kDa outer membrane protein of O. tsutsugamushi strain SJ, was expressed in E. coli and mixed with those of Ptan and Gillam strains. A thin line of the protein mixture was precisely sprayed across a nitrocellulose membrane making this the "Test" line. Polyclonal antibodies (pAbs) to O.tsutsugamushi were sprayed in another line across the membrane making this the "Control" line. Fluorescent microspheres conjugated 56 kDa proteins reacting with sample serum will be captured on the "Test" line if the sample contains antibodies to O.tsutsugamushi. Several experimental parameters were optimized. After optimizing the reaction procedure, the results are visible, within 6 min, with the naked eye under ultraviolet light. The limit of detection (LOD) was determined to be 7.63 ng/mL with prepared polyclonal antibodies. No cross-reaction was observed with sera samples from other febrile diseases. In clinical evaluations, the strips showed 94.92% sensitivity (106/112) and 93.75% specificity (56/60). The FM-ICT we developed will provide a new tool for on-site diagnosis of scrub typhus.

The SUMOylation pathway regulates the pathogenicity of Fusarium oxysporum f. sp. niveum in watermelon through stabilizing the pH regulator FonPalC via SUMOylation.

SUMOylation is a key post-translational modification, where small ubiquitin-related modifier (SUMO) proteins regulate crucial biological processes, including pathogenesis, in phytopathogenic fungi. Here, we investigated the function and mechanism of the SUMOylation pathway in the pathogenicity of Fusarium oxysporum f. sp. niveum (Fon), the fungal pathogen that causes watermelon Fusarium wilt. Disruption of key SUMOylation pathway genes, FonSMT3, FonAOS1, FonUBC9, and FonMMS21, significantly reduced pathogenicity, impaired penetration ability, and attenuated invasive growth capacity of Fon. Transcription and proteomic analyses identified a diverse set of SUMOylation-regulated differentially expressed genes and putative FonSMT3-targeted proteins, which are predicted to be involved in infection, DNA damage repair, programmed cell death, reproduction, growth, and development. Among 155 putative FonSMT3-targeted proteins, FonPalC, a Pal/Rim-pH signaling regulator, was confirmed to be SUMOylated. The FonPalC protein accumulation was significantly decreased in SUMOylation-deficient mutant ∆Fonsmt3. Deletion of FonPalC resulted in impaired mycelial growth, decreased pathogenicity, enhanced osmosensitivity, and increased intracellular vacuolation in Fon. Importantly, mutations in conserved SUMOylation sites of FonPalC failed to restore the defects in ∆Fonpalc mutant, indicating the critical function of the SUMOylation in FonPalC stability and Fon pathogenicity. Identifying key SUMOylation-regulated pathogenicity-related proteins provides novel insights into the molecular mechanisms underlying Fon pathogenesis regulated by SUMOylation.

Fish species mislabelling in China: evidence from a survey of frozen, dried, and surimi-based fish products marketed as cod-related species.

'Cod'-related species are among the most appreciated marine fish resources around the world, but are also prone to species mislabelling. In the present study, a total of 76 frozen, dried, and surimi-based fish products, sold as 'Cod' (59 products), 'Atlantic authentic Cod' (11 products), and 'Authentic Cod' (6 products), were collected in China. A species-specific LAMP (loop-mediated isothermal amplification) method was used to screen for the presence of Atlantic cod (Gadus morhua), Pacific cod (G. macrocephalus), Alaska pollock (G. chalcogrammus), Southern hake (Merluccius australis), which was cross-confirmed using real-time PCR and DNA sequencing methods. The results highlighted the greatest species diversity for 'Cod' products, and the identified species were from nine different families. It appears that the practice of assigning a specific type or category of species to the common name 'Cod' has not been widely advocated, and the misuse of this ambiguous common name has been a common practice for species adulteration, negatively impacting consumers' rights and marine conservation. To rebuild consumers' confidence, retail fish suppliers have differentiated their products by adding specific qualifiers in front of the common name 'Cod' on the label, such as 'Authentic cod' and 'Atlantic authentic cod'. The endeavour is highly meaningful, since Gadus morhua was identified as the species for a significant majority of 'Atlantic authentic cod' and 'Authentic cod' products (64.7%, 11/17), with the remaining six products identified as Alaskan pollock (G. chalcogrammus), Pacific cod (G. macrocephalus) and North Pacific hake (Merluccius productus). Despite the positive effort to reverse species mislabelling from retail on-line fish suppliers, a standardized fish nomenclature stipulated by the responsible authorities remains crucial for enhancing transparency and continuing to reduce species mislabelling.

Artificial oil bodies: A review on composition, properties, biotechnological applications, and improvement methods.

In order to simulate the structure of natural oil body, artificial oil bodies (AOBs) are fabricated by the integration of oleosins, triacylglycerols (TAGs) and phospholipids (PLs) in vitro. Recently, AOBs have gained great research interest both in the food and biological fields due to its ability to act as a novel delivery system for bioactive compounds and as a carrier for target proteins. This review aims to summarize the composition and the preparation methods of AOBs, examine the factors influencing their stability. Moreover, this contribution focusses on exploring the application of AOBs to encapsulate functional ingredients that are prone to oxidation as well as improve efficiency involved in protein purification, renaturation and immobilization by reducing the complex steps. In addition, the improvement measures to further enhance the stability and efficacy of AOBs are also discussed. The application of AOBs is expected to be a big step towards replacing existing bioreactors and delivery systems.

Near-Infrared Light-Driven Nanoparticles for Cancer Photoimmunotherapy by Synergizing Immune Cell Death and Epigenetic Regulation.

Histone deacetylases (HDACs) are a class of epigenetic enzymes that are closely related to tumorigenesis and suppress the expression of tumor suppressor genes. Whereas the HDACs inhibitors can release DNA into the cytoplasm and trigger innate immunity. However, the high density of chromatin limits DNA damage and release. In this study, suitable nanosized CycNHOH NPs (150 nm) and CypNHOH NPs (85 nm) efficiently accumulate at the tumor site due to the enhanced permeability and retention (EPR) effect. In addition, robust single-linear oxygen generation and good photothermal conversion efficiency under NIR laser irradiation accelerated the DNA damage process. By effectively initiating immune cell death, CypNHOH NPs activated both innate and adaptive immunity by maturing dendritic cells, infiltrating tumors with natural killer cells, and activating cytotoxic T lymphocytes, which offer a fresh perspective for the development of photo-immunotherapy.

A rapid colloidal gold immunochromatographic assay based on polyclonal antibodies against HtpsC protein for the detection of Streptococcus suis.

An efficient and rapid immunochromatographic assay (ICA) has been engineered for the detection of Streptococcus suis (S. suis). The underpinning principle of this ICA test lies in the use of polyclonal antibodies (pAbs) decorated with colloidal gold, which are specific to S. suis. These pAbs were derived from rabbits immunized with type II histidine triad protein (HtpsC) and HtpsC-N of S. suis. The sensitivity of the ICA was noteworthy, identifying S. suis at bacterial concentrations as diminutive as 1.0 × 103 CFU/mL. Moreover, the assay demonstrated respectable specificity and did not indicate false positives for other bacterial species (Escherichia coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus lactis, or Enterococcus faecalis). The assay was also capable of detecting multiple S. suis serotypes containing the htpsC gene, including serotypes 1-9, 12, 14, 16 and 23. Nonetheless, the detection of S. suis that lacks the htpsC gene remained beyond the capabilities of this assay. A simultaneous analysis of 16 samples utilizing PCR substantiated the reliability of the ICA test. The assay's results can be procured within a 15-min window, making it a suitable option for field application. Broadly, this study underscores the potential of the HtpsC protein as a target antigen for the detection of S. suis, and proposes that the HtpsC protein be evaluated further in other detection assays specific for S. suis.

First Report of Anthracnose Caused by Collectotrichum higginsianum on Rumex crispus in China.

Rumex crispus L. is a perennial herb with medicinal properties derived from its roots and whole plant (Bhandari et al. 2022). In December 2022, symptoms of anthracnose were observed in approximately 40% of naturally occurring R. crispus plants in Longquan Reservior, Nanchang city (115°53' N, 28°43' E), Jiangxi Province, China. Initially, red lesions appeared randomly on various parts of the leaf blade, which gradually became dry and brown at the center, eventually leading to leaf death. To isolate the fungal pathogen responsible, ten symptomatic leaves were randomly collected and their lesions were cut into small pieces (4 × 4 mm). The leaf fragments were surface-sterilized in 70% ethanol for 45 s and then in 1% NaClO for 45 s. The leaf pieces were rinsed three times with sterile distilled water. The surface-sterilized leaf pieces were then placed onto potato dextrose agar (PDA) and incubated at 28 ℃, dark condition for 3 days. Twelve isolates were obtained, characterized by a milky white and uneven growth pattern with a white root-like structure branching out at the edge, along with scattered black deposits on the bottom of the plate. Conidiogenous cells cylindrical, smooth-walled, hyaline, 9.3-23.2 × 3.6-4.2 µm. Conidia elliptical, aseptate, smooth-walled, with one end blunt and the other truncate, ranging in size from 10.4 to 22.3 (mean 16.7) µm in length and 3.2 to 5.0 (mean 4.1) µm in width (n = 50), which are consistent with the characteristics of the members of Colletotrichum destructivum species complex (Damm et al. 2014). To accurately identify the strain, three representative isolates, namely JFRL 03-930, JFRL 03-931, and JFRL 03-935, were selected for further identification. The internal transcribed spacer (ITS) region, actin (ACT), chitin synthase (CHS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GADPH), histone3 (HIS3), and beta-tubulin (TUB2) genes were amplified and sequenced using specific primer pairs, including ITS5/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, GDF1/GDR1, GYLH3F/CYLH3R, and T1/Bt2b (Damm et al. 2014). All sequences were deposited in GenBank with accession numbers OQ560476-OQ560478 (ITS), OQ576154-OQ576156 (ACT), OQ576157-OQ576159 (CHS), OQ576160-OQ576162 (HIS3), OQ576163-OQ576165 (GADPH), and OQ576166-OQ576168 (TUB2). A maximum likelihood phylogenetic tree was constructed using IQtree v1.5.6 based on the combined ITS, ACT, CHS, GAPDH, HIS3 and TUB2 data set (Nguyen et al. 2015), The phylogenetic tree showed that the three isolates clustered with C. higginsianum in a clade with 91% bootstrap support. Based on morphology and molecular characters, the isolates were identified as C. higginsianum of the C. destructivum species complex. To confirm the pathogenicity, One-year-old R. crispus were collected from the wild and potted in an climate chamber. Six healthy leaves of R. crispus were surface sterilized with 70% ethanol and wounded by sterile needle, and a 20-µl conidial suspension (3×105 conidia/ml) of the isolate JFRL 03-931 was inoculated on the wound. Another set of six leaves of R. crispus was inoculated with distilled water as controls. The potted plants were incubated under conditions of 25 ℃ and 80% humidity. After 10 days, reddish brown spots were observed on all inoculated leaves, while the control leaves remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated leaves and confirmed as C. higginsianum by morphological and molecular analysis. It has been reported that C. higginsianum caused anthracnose disease on several cruciferous vegetables, Boehmeria nivea and Rumex acetosa in China (Damm et al. 2014; Wang et al. 2011; Patel et al. 2014; Zhang et al. 2018). But to our knowledge, this is the first report of C. higginsianum casued anthracnose on Rumex crispus in China. Therefore, we should pay more attention to this pathogen and develop appropriate control strategies.

Rapid screening of electrochemically active bacteria based on a biocathode-functional bipolar electrode-electrochemiluminescence platform.

A functional bipolar electrode-electrochemiluminescence (BPE-ECL) platform based on biocathode reducing oxygen was constructed for detecting electrochemically active bacteria (EAB) in this paper. Firstly, thiolated trimethylated chitosan quaternary ammonium salt (TMC-SH) layer was assembled on the gold-plated cathode of BPE. TMC-SH contains quaternary ammonium salt branch chain, which can inhibit the growth of microorganisms on the surface or in the surrounding environment while absorbing bacteria. Then, the peristaltic pump was used to flow all of the samples through the cathode, and the EAB was electrostatically adsorbed on the electrode surface. Finally, applying a constant potential to the BPE, bacteria can catalyze electrochemical reduction of O2, and decrease the overpotential of O2 reduction at the cathode, which in turn generates an ECL reporting intensity change at the anode. In this way, live and dead bacteria can be distinguished, and the influence of complex food substrates on detection can be greatly reduced.

Degradation of α-Subunits, Doa1 and Doa4, are Critical for Growth, Development, Programmed Cell Death Events, Stress Responses, and Pathogenicity in the Watermelon Fusarium Wilt Fungus Fusarium oxysporum f. sp. niveum.

The ubiquitin-proteasome system (UPS) regulates protein quality or control and plays essential roles in several biological and biochemical processes in fungi. Here, we present the characterization of two UPS components, FonDoa1 and FonDoa4, in watermelon Fusarium wilt fungus, Fusarium oxysporum f. sp. niveum (Fon), and their biological functions. FonDoa1 localizes in both the nucleus and cytoplasm, while FonDoa4 is predominantly present in the cytoplasm. Both genes show higher expression in germinating macroconidia at 12 h. Deletion of FonDoa1 or FonDoa4 affects vegetative growth, conidiation, conidial germination/morphology, apoptosis, and responses to environmental stressors. FonDoa1, but not FonDoa4, positively regulates autophagy. The targeted disruption mutants exhibit significantly attenuated pathogenicity on watermelon due to defects in the infection process and invasive fungal growth. Further results indicate that the WD40, PFU, and PUL domains are essential for the function of FonDoa1 in Fon pathogenicity and environmental stress responses. These findings demonstrate the previously uncharacterized biological functions of FonDoa1 and FonDoa4 in phytopathogenic fungi, providing potential targets for developing strategies to control watermelon Fusarium wilt.

Mpro-targeted anti-SARS-CoV-2 inhibitor-based drugs.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 is a global health emergency. The main protease is an important drug target in coronaviruses. It plays an important role in the processing of viral RNA-translated polyproteins and is highly conserved in the amino acid sequence and three-dimensional structure, making it a good drug target for which several small molecule inhibitors are available. This paper describes the various anti-severe acute respiratory syndrome coronavirus 2 inhibitor drugs targeting Mpro discovered since the severe acute respiratory syndrome coronavirus 2 outbreak at the end of 2019, with all these compounds inhibiting severe acute respiratory syndrome coronavirus 2 Mpro activity in vitro. This provides a reference for the development of severe acute respiratory syndrome coronavirus 2 Mpro-targeted inhibitors and the design of therapeutic approaches to address newly emerged severe acute respiratory syndrome coronavirus 2 mutant strains with immune evasion capabilities.

Double bipolar electrode electrochemiluminescence color switch for food-borne pathogens detection.

Color-switch electrochemiluminescence (ECL) sensing platform based on a dual-bipolar electrode (D-BPE) is reported in this work. The D-BPE was composed of a cathode filled with buffer and two anodes filled with [Ru(bpy)3]2+-TPrA and luminol-H2O2 solutions, respectively. Both anodes were modified with capture DNA and served as ECL reporting platforms. After introducing ferrocene-labeled aptamer (Fc-aptamer) on both anodes, the ECL emission signal of the [Ru(bpy)3]2+ was difficult to be observed (anode 1), while luminol emitted a strong and visible ECL signal (anode 2). Ferrocene (Fc) did not only prevent the oxidation of [Ru(bpy)3]2+ due to its lower oxidation potential, its oxidation product Fc+ also quenched the [Ru(bpy)3]2+ ECL through efficient energy transfer. For luminol, Fc+ catalyzes the accelerated formation of the excited-state of the luminol anion radical, which leads to the enhancement of the luminol ECL. In the presence of food-borne pathogens, the aptamer was assembled with them, leading to the leaving of Fc from the surface of the D-BPE anodes. The ECL intensity of [Ru(bpy)3]2+ was enlarged, meanwhile, the blue emission signal of luminol became weakened. By self-calibrating the ratio of the two signals, 1-106 CFU mL-1 food-borne pathogenic bacteria can be sensitively detected with a detection limit of 1 CFU mL-1. Ingeniously, the color-switch biosensor can be used to detect S. aureus, E. coli and S. typhimurium by assembling the corresponding aptamers onto the D-BPE anodes.

Fusarium oxysporum f. sp. niveum Pumilio 1 Regulates Virulence on Watermelon through Interacting with the ARP2/3 Complex and Binding to an A-Rich Motif in the 3' UTR of Diverse Transcripts.

Fusarium oxysporum f. sp. niveum (Fon), a soilborne phytopathogenic fungus, causes watermelon Fusarium wilt, resulting in serious yield losses worldwide. However, the underlying molecular mechanism of Fon virulence is largely unknown. The present study investigated the biological functions of six FonPUFs, encoding RNA binding Pumilio proteins, and especially explored the molecular mechanism of FonPUF1 in Fon virulence. A series of phenotypic analyses indicated that FonPUFs have distinct but diverse functions in vegetative growth, asexual reproduction, macroconidia morphology, spore germination, cell wall, or abiotic stress response of Fon. Notably, the deletion of FonPUF1 attenuates Fon virulence by impairing the invasive growth and colonization ability inside the watermelon plants. FonPUF1 possesses RNA binding activity, and its biochemical activity and virulence function depend on the RNA recognition motif or Pumilio domains. FonPUF1 associates with the actin-related protein 2/3 (ARP2/3) complex by interacting with FonARC18, which is also required for Fon virulence and plays an important role in regulating mitochondrial functions, such as ATP generation and reactive oxygen species production. Transcriptomic profiling of ΔFonPUF1 identified a set of putative FonPUF1-dependent virulence-related genes in Fon, possessing a novel A-rich binding motif in the 3' untranslated region (UTR), indicating that FonPUF1 participates in additional mechanisms critical for Fon virulence. These findings highlight the functions and molecular mechanism of FonPUFs in Fon virulence. IMPORTANCE Fusarium oxysporum is a devastating plant-pathogenic fungus that causes vascular wilt disease in many economically important crops, including watermelon, worldwide. F. oxysporum f. sp. nievum (Fon) causes serious yield loss in watermelon production. However, the molecular mechanism of Fusarium wilt development by Fon remains largely unknown. Here, we demonstrate that six putative Pumilio proteins-encoding genes (FonPUFs) differentially operate diverse basic biological processes, including stress response, and that FonPUF1 is required for Fon virulence. Notably, FonPUF1 possesses RNA binding activity and associates with the actin-related protein 2/3 complex to control mitochondrial functions. Furthermore, FonPUF1 coordinates the expression of a set of putative virulence-related genes in Fon by binding to a novel A-rich motif present in the 3' UTR of a diverse set of target mRNAs. Our study disentangles the previously unexplored molecular mechanism involved in regulating Fon virulence, providing a possibility for the development of novel strategies for disease management.

HPMC films functionalized by zein/carboxymethyl tamarind gum stabilized Pickering emulsions: Influence of carboxymethylation degree.

Pickering emulsions are promising systems to act as carriers of active hydrophobic components, and to improve compatibility and the water vapor barrier properties of bio-based films. This study aimed to investigated the effects of cinnamon essential oil Pickering emulsions (CEOEs) using zein/carboxymethyl tamarind gum as stabilizers on the mechanical, barrier, antibacterial and antioxidant properties of Hydroxypropyl methyl cellulose (HPMC) films, and assessed the influence of carboxymethylation degree. In addition, the effect of the packaging was studied on the shelf life of cherry tomatoes. Results showed that the droplet size reduced approximately from 93.03 to 10.59 µm with the increasing degree of substitution (DS), greatly facilitating the droplet uniform distribution in film matrix. Moreover, with the addition of CEOEs, significant increase was observed with the tensile strength from 8.46 to 25.41 MPa, and the water vapor permeability decreased from 6.18 × 10-10 to 4.24 × 10-10 g·m-1·s-1·Pa-1. The films exhibited good UV barrier properties without sacrificing the transparency after adding CEO. Furthermore, the antibacterial and antioxidant activities of the prepared films have also been greatly improved. Consequently, the CEOEs was an ideal alternative for incorporation with HPMC based films for increasing the shelf life of cherry tomatoes.

Physicochemical and Structural Properties of Gluten-Konjac glucomannan Conjugates Prepared by Maillard Reaction.

Gluten (Glu) is important to wheat products by forming a three-dimensional matrix. This study aimed to investigate the physicochemical and structural properties of gluten after conjugation with konjac glucomannan (KGM) through the Maillard reaction. The study revealed that the degree of graft increased with the prolonged reaction time. The Glu-KGM conjugates were possessed of increased ß-sheet but decreased α-helix and ß-turn, as well as unfolding and loose tertiary structures as the reaction proceeded. Among three different proportions, the Glu-KGM 1:1 conjugate was proved to have the most excellent foaming and emulsifying properties, and could form more rigid and firm gelation structures, which could be related to the decreased particle size and increased zeta potential of the conjugate. Overall, the physicochemical and structural properties of gluten were significantly related to the KGM ratios as well as the reaction period.

The SUMOylation Pathway Components Are Required for Vegetative Growth, Asexual Development, Cytotoxic Responses, and Programmed Cell Death Events in Fusarium oxysporum f. sp. niveum.

SUMOylation is an essential protein modification process that regulates numerous crucial cellular and biochemical processes in phytopathogenic fungi, and thus plays important roles in multiple biological functions. The present study characterizes the SUMOylation pathway components, including SMT3 (SUMO), AOS1 (an E1 enzyme), UBC9 (an E2 enzyme), and MMS21 (an E3 ligase), in Fusarium oxysporum f. sp. niveum (Fon), the causative agent of watermelon Fusarium wilt, in terms of the phylogenetic relationship, gene/protein structures, and basic biological functions. The SUMOylation components FonSMT3, FonAOS1, FonUBC9, and FonMMS21 are predominantly located in the nucleus. FonSMT3, FonAOS1, FonUBC9, and FonMMS21 are highly expressed in the germinating macroconidia, but their expression is downregulated gradually in infected watermelon roots with the disease progression. The disruption of FonUBA2 and FonSIZ1 seems to be lethal in Fon. The deletion mutant strains for FonSMT3, FonAOS1, FonUBC9, and FonMMS21 are viable, but exhibit significant defects in vegetative growth, asexual reproduction, conidial morphology, spore germination, responses to metal ions and DNA-damaging agents, and apoptosis. The disruption of FonSMT3, FonAOS1, FonUBC9, and FonMMS21 enhances sensitivity to cell wall-perturbing agents, but confers tolerance to digestion by cell wall-degrading enzymes. Furthermore, the disruption of FonSMT3, FonAOS1, and FonUBC9 negatively regulates autophagy in Fon. Overall, these results demonstrate that the SUMOylation pathway plays vital roles in regulating multiple basic biological processes in Fon, and, thus, can serve as a potential target for developing a disease management approach to control Fusarium wilt in watermelon.

Palmitoyl Transferase FonPAT2-Catalyzed Palmitoylation of the FonAP-2 Complex Is Essential for Growth, Development, Stress Response, and Virulence in Fusarium oxysporum f. sp. niveum.

Protein palmitoylation, one of posttranslational modifications, is catalyzed by a group of palmitoyl transferases (PATs) and plays critical roles in the regulation of protein functions. However, little is known about the function and mechanism of PATs in plant pathogenic fungi. The present study reports the function and molecular mechanism of FonPATs in Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt. The Fon genome contains six FonPAT genes with distinct functions in vegetative growth, conidiation and conidial morphology, and stress response. FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for Fon virulence on watermelon mainly through regulating in planta fungal growth within host plants. Comparative proteomics analysis identified a set of proteins that were palmitoylated by FonPAT2, and some of them are previously reported pathogenicity-related proteins in fungi. The FonAP-2 complex core subunits FonAP-2α, FonAP-2ß, and FonAP-2µ were palmitoylated by FonPAT2 in vivo. FonPAT2-catalyzed palmitoylation contributed to the stability and interaction ability of the core subunits to ensure the formation of the FonAP-2 complex, which is essential for vegetative growth, asexual reproduction, cell wall integrity, and virulence in Fon. These findings demonstrate that FonPAT1, FonPAT2, and FonPAT4 play important roles in Fon virulence and that FonPAT2-catalyzed palmitoylation of the FonAP-2 complex is critical to Fon virulence, providing novel insights into the importance of protein palmitoylation in the virulence of plant fungal pathogens. IMPORTANCE Fusarium oxysporum f. sp. niveum (Fon), the causal agent of watermelon Fusarium wilt, is one of the most serious threats for the sustainable development of the watermelon industry worldwide. However, little is known about the underlying molecular mechanism of pathogenicity in Fon. Here, we found that the palmitoyl transferase (FonPAT) genes play distinct and diverse roles in basic biological processes and stress response and demonstrated that FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for virulence in Fon. We also found that FonPAT2 palmitoylates the core subunits of the FonAP-2 complex to maintain the stability and the formation of the FonAP-2 complex, which is essential for basic biological processes, stress response, and virulence in Fon. Our study provides new insights into the understanding of the molecular mechanism involved in Fon virulence and will be helpful in the development of novel strategies for disease management.

A rapid and closed-tube method based on the dual-color fluorescence loop-mediated isothermal amplification for visual detection of Atlantic salmon (Salmo salar).

Loop-mediated isothermal amplification (LAMP) visual detection based on hydroxyl naphthol blue (HNB) and SYTO 9 is often confounded by the narrow color variation window and the requirement of empirical preset of cutoff intensity value. To improve the suitability for naked-eye inspection, the present work proposed a strategy based on the fluorescence property of SYTO 9 and HNB to enlarge the contrast and a novel dual-color fluorescence LAMP (dfLAMP) assay was developed for visual detection of Atlantic salmon. Specifically, HNB of 26.25 µM, blended with SYTO 9 of 0.75-1.5 µM, was added in the mixture before amplification, producing light green fluorescence for both positive and negative samples. After amplification, green or yellow-green fluorescence was observed for positive samples, while only orange-red fluorescence emitted for negative ones, enabling an easy and rapid distinguish. The optimized dfLAMP assay has proved its specificity and can detect as little as 1 fg Atlantic salmon DNA.

Biocathodes reducing oxygen in BPE-ECL system for rapid screening of E. coli O157:H7.

After discovery of electron transfer from bacteria, most bacteria known to be electrochemically active are utilized as a self-regenerable catalyst at the anode of microbial fuel cells (MFCs). However, the reverse phenomenon, cathodic catalysts is not so widely researched. This present study demonstrated that E. coli O157:H7 was electrochemically active, and it was able to catalyze oxygen reduction at the cathode of bipolar electrode (BPE). Applying a constant potential to the BPE, E. coli O157:H7 can catalyze electrochemical reduction of O2, decrease the overpotential of O2 reduction at the cathode, which in turn generates an electrochemiluminescence (ECL) reporting intensity change at the anode. Significantly, a majority of food matrix does not exhibit catalytic activity for electrochemical reduction of O2. Meanwhile, due to the physically separation of two poles of closed BPE, complex food matrix at the cathode does not interfere with the ECL reaction at the anode. Therefore, the effect of food matrix is negligible when measuring E. coli O157:H7 levels in food. A low detection limit of 10 CFU mL-1 E. coli O157:H7 could be identified within 1 h. Thus, biocathodes reducing oxygen in BPE-ECL system has shown excellent characteristics in the field of rapid detection of electroactive bacteria in food.