HapX-mediated H2B deub1 and SreA-mediated H2A.Z deposition coordinate in fungal iron resistance.

Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.

Prophylactic efficacy of Lactobacillus curvatus B67-derived postbiotic and quercetin, separately and combined, against Listeria monocytogenes and Salmonella enterica ser. Typhimurium on processed meat sausage.

This study investigated the antimicrobial and antibiofilm efficacy of separate and combined treatments of Lactobacillus curvatus B67-produced postbiotic and the polyphenolic flavanol quercetin against Listeria monocytogenes and Salmonella enterica ser. Typhimurium. The antimicrobial potentiality of the postbiotic was chiefly associated with organic acids (e.g., lactic and acetic acids). At sub-minimum inhibitory concentration (1/2 MIC), the postbiotic and quercetin effectively reduced the pathogenic biofilm cells on processed pork sausage and meat-processing surfaces (e.g., stainless-steel and rubber). Moreover, the postbiotic exhibited strong residual antimicrobial efficacy over diverse pH and temperature ranges. In addition, the combination of postbiotic with quercetin increased the leakage of pathogenic intracellular metabolites (e.g., nucleic acids and protein) and inhibited pathogenic biofilm formation on both biotic and abiotic surfaces. Therefore, this study confirmed that lactic acid bacteria-derived postbiotic and plant-derived quercetin could be used as potential alternative bioprotective agents in the meat processing industry.

Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production.

Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.

Homogeneous One-Step Immunoassay Based on Switching Peptides for Detection of the Influenza Virus.

In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.

Fusarium verticillioides Induces Maize-Derived Ethylene to Promote Virulence by Engaging Fungal G-Protein Signaling.

Seed maceration and contamination with mycotoxin fumonisin inflicted by Fusarium verticillioides is a major disease concern for maize producers worldwide. Meta-analyses of quantitative trait loci for Fusarium ear rot resistance uncovered several ethylene (ET) biosynthesis and signaling genes within them, implicating ET in maize interactions with F. verticillioides. We tested this hypothesis using maize knockout mutants of the 1-aminocyclopropane-1-carboxylate (ACC) synthases ZmACS2 and ZmACS6. Infected wild-type seed emitted five-fold higher ET levels compared with controls, whereas ET was abolished in the acs2 and acs6 single and double mutants. The mutants supported reduced fungal biomass, conidia, and fumonisin content. Normal susceptibility was restored in the acs6 mutant with exogenous treatment of ET precursor ACC. Subsequently, we showed that fungal G-protein signaling is required for virulence via induction of maize-produced ET. F. verticillioides Gß subunit and two regulators of G-protein signaling mutants displayed reduced seed colonization and decreased ET levels. These defects were rescued by exogenous application of ACC. We concluded that pathogen-induced ET facilitates F. verticillioides colonization of seed, and, in turn, host ET production is manipulated via G-protein signaling of F. verticillioides to facilitate pathogenesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides.

Fusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical Gα, Gß and Gγ subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that Gß protein FvGbb1 directly impacts fumonisin regulation but not other physiological aspects in F. verticillioides. In this study, we identified and characterized a RACK1 (Receptor for Activated C Kinase 1) homolog FvGbb2 as a putative Gß-like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.

FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen.

Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a possibly carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane.

High-sensitivity detection of ATP using a localized surface plasmon resonance (LSPR) sensor and split aptamers.

A highly sensitive localized surface plasmon resonance (LSPR) aptasensor for detection of adenosine triphosphate (ATP) has been developed. The sensor utilizes two split ATP aptamers, one (receptor fragment) being covalently attached to the surface of a gold nanorod (GNR) and the other labeled with a random DNA sequence and TAMRA dye (probe fragment). In the presence of both ATP and the probe fragment, a significant shift takes place in the wavelength of the LSPR band. This phenomenon is a consequence of the fact that the split fragments assemble into an intact folded structure in the presence of ATP, which brings about a decrease in the distance between the GNR surface and TAMRA dye and an associated LSPR wavelength. By using this sensor system, concentrations of ATP in the range of 10 pM-10 µM can be determined. In addition, by taking advantage of its denaturation properties, the LSPR aptasensor can be reused by simply subjecting it to quadruple salt-addition/2M NaCl washing steps. That the new method is applicable to biological systems was demonstrated by its use to measure ATP concentrations in E. coli and, thus to determine cell concentrations as low as 1.0×10(3) CFU.

A small molecule species specifically inhibits Fusarium myosin I.

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease of cereal crops worldwide. Recently, a novel fungicide JS399-19 has been launched into the marketplace to manage FHB. It is compelling that JS399-19 shows highly inhibitory activity towards some Fusarium species, but not to other fungi, indicating that it is an environmentally compatible fungicide. To explore the mode of action of this species-specific compound, we conducted a whole-genome transcript profiling together with genetic and biochemical assays, and discovered that JS399-19 targets the myosin I of F. graminearum (FgMyo1). FgMyo1 is essential for F. graminearum growth. A point mutation S217L or E420K in FgMyo1 is responsible for F. graminearum resistance to JS399-19. In addition, transformation of F. graminearum with the myosin I gene of Magnaporthe grisea, the causal agent of rice blast, also led to JS399-19 resistance. JS399-19 strongly inhibits the ATPase activity of the wild-type FgMyo1, but not the mutated FgMyo1(S217L/E420K) . These results provide us a new insight into the design of species-specific antifungal compounds. Furthermore, our strategy can be applied to identify novel drug targets in various pathogenic organisms.

Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers.

We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner.

Proteomic comparison of Gibberella moniliformis in limited-nitrogen (fumonisin-inducing) and excess-nitrogen (fumonisin-repressing) conditions.

The maize pathogen Gibberella moniliformis produces fumonisins, a group of mycotoxins associated with several disorders in animals and humans, including cancer. The current focus of our research is to understand the regulatory mechanisms involved in fumonisin biosynthesis. In this study, we employed a proteomics approach to identify novel genes involved in the fumonisin biosynthesis under nitrogen stress. The combination of genome sequence, mutant strains, EST database, microarrays, and proteomics offers an opportunity to advance our understanding of this process. We investigated the response of the G. moniliformis proteome in limited nitrogen (N0, fumonisininducing) and excess nitrogen (N+, fumonisin-repressing) conditions by one- and two-dimensional electrophoresis. We selected 11 differentially expressed proteins, six from limited nitrogen conditions and five from excess nitrogen conditions, and determined the sequences by peptide mass fingerprinting and MS/MS spectrophotometry. Subsequently, we identified the EST sequences corresponding to the proteins and studied their expression profiles in different culture conditions. Through the comparative analysis of gene and protein expression data, we identified three candidate genes for functional analysis and our results provided valuable clues regarding the regulatory mechanisms of fumonisin biosynthesis.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Fumonisins B1 and B2 in agricultural products consumed in South Korea: an exposure assessment.

To survey fumonisins B1 (FB1) and B2 (FB2) in agricultural products consumed in South Korea and provide an exposure assessment, ground samples were extracted (80% MeOH), filtered (0.2 microm), and cleaned up. After evaporation, dry residues were reconstituted in 50% MeOH, and a 50-micro1 aliquot of this sample was mixed with 200 micro1 of o-phthaldialdehyde for derivatization. The derivatives were analyzed with a high-performance liquid chromatography system equipped with a fluorescence detector. For validation of the detection procedure, linearity, accuracy, precision, detection limit, and quantification limit were determined. The validated detection method was then used to survey fumonisins in white rice, brown rice, barley, barley tea, beer, wheat flour, millet, dried corn, corn flour, corn tea, canned corn, popcorn, and breakfast cereal. Retention times for FB1 and FB2 standards were 7 and 18 min, respectively. Linearity (R2 = 0.99995 to 0.99998), accuracy (81.47 to 108.83%), precision (2.35 to 5.77), detection limit (25 ng/g or ng/ml), and quantification limit (37 ng/g or ng/ml) indicated that this procedure is capable of quantifying fumonisins in agricultural products. Only FB1-positive samples (5.12%, three dried corn samples and five corn flour samples) were found at 90.89 to 439.67 ng/g. According the survey results, an estimated daily intake of FB1 and FB2 in Korea was 0.087 ng/kg of body weight per day. These results indicate that continuous monitoring of these mycotoxins is necessary to establish appropriate risk assessment, and the maximum tolerable daily intake of fumonisins in Korea is lower than the 2 microg/kg set by the Joint Food and Agriculture Organization-World Health Organization Expert Committee.

One-step simultaneous immunochromatographic strip test for multianalysis of ochratoxin a and zearalenone.

Individual immunochromatographic assays (ICG) for ochratoxin A (OTA) and zearalenone (ZEA) were optimized and used in the development of a one-step simultaneous immunochromatographic assay (OS-ICG) for the rapid multianalysis of two mycotoxins in corn samples. The nitrocellulose membrane of the OS-ICG was treated with OTA-bovine serum albumin (BSA), ZEA-ovalbumin (OVA), and anti-mouse IgG in the OTA test, ZEA test, and control zones, respectively. Monoclonal antibody-gold conjugates (OTA3 MAb-gold and ZEA2C5 MAb-gold) were sprayed onto the conjugate pad. The visual detection limits were 2.5 and 5 ng/ml for OTA and ZEA, resepectively, and the results were obtained within 15 min after starting the analysis. An efficient, simple, and rapid extraction method using 30% MeOH/PBS was established and validated by analyzing the corn samples spiked with OTA/ZEA mixtures (0/0, 5/10, 10/20, and 20/30 microg/kg). The cut-off values of the OS-ICG for the spiked corn were 5 and 10 microg/kg for OTA and ZEA, respectively. Natural corn samples were analyzed by OS-ICG, direct competitive enzyme-linked immunosorbent assay (DC-ELISA), and HPLC. Results of the OS-ICG were in good agreement with those obtained by DC-ELISA and HPLC. The developed OS-ICG offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site simultaneous determination of OTA and ZEA in cereals, food, and agricultural products in one analytical cycle.

Natural occurrence of aflatoxin B1 in marketed foods and risk estimates of dietary exposure in Koreans.

Aflatoxin B1 (AFB1) is an unavoidable food contaminant. To evaluate the potential health risk of AFB1 to Koreans posed by food consumption, we determined the natural occurrence of AFB1 in food and estimated the excess risk for liver cancer through dietary exposure to AFB1. A total of 694 food samples collected from six different regions of South Korea were analyzed for their AFB, content. One hundred four of the 694 samples were found to give positive enzyme-linked immunosorbent assay (ELISA) readings for AFB1 and were further investigated with high-performance liquid chromatography. Thirty-two samples, including 2 maize samples, 3 soybean products, 20 peanut samples, nut samples, and their products, and 7 spices, were found to be contaminated with AFB1 (4.6% incidence), up to 48.6 microg kg(-1). The level of AFB1 contamination in 28 of the 32 food products was below 10 microg kg(-1), which is the legal tolerance limit in Korea. From data on daily food consumption, the exposure dose of AFB1 was estimated to be 6.42 x 10(-7) mg kg(-1) body weight (bw) day(-1). The major contributors to the dietary intake of AFB1 were soybean paste and soy sauce, which composed 91% of the total exposure to AFB1. The excess risk of liver cancer for those exposed to AFB1 through food intake was estimated to be 5.78 x 10(-6) for hepatitis B-negative individuals and 1.48 x 10(-4) for hepatitis B-positive individuals. These results suggest that special consideration is required to reduce the intake of AFB1 in hepatitis B-positive individuals.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis.

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylogenetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.