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In JoVE (1)
Other Publications (37)
- Molecular Plant-microbe Interactions : MPMI
- Journal of Food Protection
- Journal of Food Protection
- Mycologia
- Journal of Agricultural and Food Chemistry
- Molecular Plant-microbe Interactions : MPMI
- Analytical and Bioanalytical Chemistry
- Journal of Agricultural and Food Chemistry
- Molecular Plant Pathology
- Molecular Plant Pathology
- Current Genetics
- Molecular Plant-microbe Interactions : MPMI
- Microbiology (Reading, England)
- Journal of Microbiology and Biotechnology
- Journal of Microbiology and Biotechnology
- Journal of Food Protection
- Journal of Microbiology and Biotechnology
- Microbiology (Reading, England)
- Microbiology (Reading, England)
- Journal of Microbiology and Biotechnology
- Journal of Food Protection
- Microbiology (Reading, England)
- Mycologia
- Journal of Agricultural and Food Chemistry
- Journal of Microbiology and Biotechnology
- Journal of Agricultural and Food Chemistry
- Journal of Food Protection
- Current Genetics
- Nature
- Journal of Agricultural and Food Chemistry
- Foodborne Pathogens and Disease
- Fungal Genetics and Biology : FG & B
- Analytical and Bioanalytical Chemistry
- Molecular Plant Pathology
- Molecular Plant Pathology
- Journal of Chromatography. A
- PloS One
Articles by Won-Bo Shim in JoVE
JoVE Immunology and Infection
Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays
Plant Pathology and Microbiology, Texas A&M University
The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.
Other articles by Won-Bo Shim on PubMed
CZK3, a MAP Kinase Kinase Kinase Homolog in Cercospora Zeae-maydis, Regulates Cercosporin Biosynthesis, Fungal Development, and Pathogenesis
The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.
Comparison of Fumonisin B1 Biosynthesis in Maize Germ and Degermed Kernels by Fusarium Verticillioides
Fusarium verticillioides produces a group of mycotoxins known as fumonisins in maize kernels. Fumonisins are associated with a variety of mycotoxicoses in humans and animals; thus, their presence in food is a considerable safety issue. This study addressed fumonisin B1 (FB1) production in two components of the maize kernel, namely the germ tissues and the degermed kernel. Growth of F. verticillioides was similar in colonized germ tissue and degermed kernels, but FB1 production was at least five times higher in degermed maize kernels than in germ tissue. Expression of the fumonisin polyketide synthase gene, FUM1, as measured by beta-glucuronidase (GUS) and Northern blot analysis, followed the same pattern as FB1 production. Also correlated to FB1 was a concomitant drop in pH of the colonized degermed kernels. A time course experiment showed that degermed kernels inoculated with F. verticillioides became acidified over time (from pH 6.4 to 4.7 after 10 days of incubation), whereas colonized germ tissue became alkaline over the same period (from pH 6.5 to 8.5). Because conditions of acidic pH are conducive to FB1 production and alkaline pH is repressive, the observed correlation between the acidification of degermed kernels and the increase in FB1 provides one explanation for the observed differences in FB1 levels.
Detection of Aflatoxin-producing Molds in Korean Fermented Foods and Grains by Multiplex PCR
An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.
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.
Development of Monoclonal Antibodies Against Pirimiphos-methyl and Their Application to IC-ELISA
To detect the organophosphorus (OP) pesticide pirimiphos-methyl in grain samples, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed and optimized. By the active esters method, pirimiphos-methyl hapten A was conjugated to keyhole limpet hemocyanin to be used as the immunogen for the production of monoclonal antibodies, and pirimiphos-methyl hapten B was conjugated to ovalbumin to be used as coating antigen. By using the monoclonal antibody and the coating antigen, an IC-ELISA has been developed. Under the established optimized conditions, the IC-ELISA showed an IC50 of 4.2 ng/mL with a detection limit of 0.07 ng/mL. The IC-ELISA showed negligible cross-reactivity with other OP pesticides except with pirimiphos-ethyl. Recoveries of pirimiphos-methyl from spiked grain samples ranged from 83 to 96%.
FSR1 is Essential for Virulence and Female Fertility in Fusarium Verticillioides and F. Graminearum
Fusarium verticillioides (teleomorph Gibberella moniliformis) and F. graminearum (teleomorph G. zeae) are well known to cause devastating diseases on cereal crops. Despite their importance, our understanding of the molecular mechanisms involved in these host-pathogen interactions is limited. The FSR1 locus in F. verticillioides was identified by screening REMI mutants for loss of virulence in maize stalk rot inoculation studies. FSR1 encodes an 823-codon open reading frame interrupted by two introns. The Fsr1 protein shares 60% sequence identity with the Sordaria macrospora Pro11, a multimodular protein with four putative protein-protein binding domains (caveolin-binding domain, coiled-coil structure, calmodulin-binding motif, and seven-WD40 repeats), which plays a regulatory role in cell differentiation and ascocarp development. Our data demonstrate that FSR1 is essential for female fertility and virulence in F. verticillioides. Significantly, targeted disruption of the FSR1 ortholog in F. graminearum (FgFSR1) reduced virulence on barley and deterred perithecia formation. Cross-complementation experiments demonstrated that the gene function is conserved in the two Fusarium species. FSR1 is expressed constitutively, and we hypothesize that Fsr1 regulates virulence by acting as a scaffold for a signal transduction pathway. A survey of available genome databases indicates Fsr1 homologs are present in a number of filamentous fungi and animal systems but not in budding yeast or plants. A maximum likelihood analysis of this gene family reveals well-supported monophyletic clades associated with fungi and animals.
Direct Competitive ELISA Based on a Monoclonal Antibody for Detection of Aflatoxin B1. Stabilization of ELISA Kit Components and Application to Grain Samples
A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B1 (AFB1), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB1 concentrations determinable by ELISA ranged from 0.1 to 10 microg L(-1). The IC50 value was 0.62 microg L(-1). Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP-AFB1 conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP-AFB(1) conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L(-1) calcium chloride in 0.05 mol L(-1) Tris-HCl buffer (pH 7.2) maintained the activity of HRP-AFB1 at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4 degrees C, whereas the untreated MAb-coated plate lost its activity within 2 days.
Immunochromatography Using Colloidal Gold-antibody Probe for the Detection of Atrazine in Water Samples
An immunochromatography (ICG) strip test for rapid detection of atrazine in water samples was developed. A monoclonal antibody (MAb) specific to atrazine was produced from the cloned hybridoma cell (AT-1-M3) and used to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and ICG strip. MAb conjugated to colloidal gold, and that was applied to the conjugate pad of the ICG strip. The visual detection limit for the ICG strip was 3 ng/mL. This test required only 10 min to get results and one step of sample to perform the assay. The results of water samples spiked with 5, 10, 20, and 50 ng/mL of atrazine by ICG strip were in good agreement with those obtained by DC-ELISA. The ICG strip was sufficiently sensitive and accurate to be useful for rapid screening of atrazine in various water samples.
The Putative Monomeric G-protein GBP1 is Negatively Associated with Fumonisin B Production in Fusarium Verticillioides
SUMMARY Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides that contaminates maize. FB(1) has been linked to a number of human and animal mycotoxicoses worldwide. Despite its significance, our understanding of the FB(1) biosynthesis regulatory mechanisms is limited. Here, we describe F. verticillioides GBP1, encoding a monomeric G-protein, and its role in FB(1) biosynthesis. GBP1 was discovered as an expressed sequence tag (EST) up-regulated in the F. verticillioides fcc1 mutant that showed reduced conidiation and no FB(1) biosynthesis when grown on maize kernels. Sequence analysis showed that GBP1 encodes a putative 368-amino-acid protein with similarity to DRG and Obg subclasses of G-proteins that are involved in development and stress responses. A GBP1 knockout mutant (Deltagbp1) exhibited normal growth, but increased FB(1) production (> 58%) compared with the wild-type when grown on corn kernels. Complementation of Deltagbp1 with the wild-type GBP1 gene restored FB(1) production levels to that of the wild-type. Our data indicate that GBP1 is negatively associated with FB(1) biosynthesis but not with conidiation in F. verticillioides. The deletion of GBP1 led to up-regulation of key FB(1) biosynthetic genes, FUM1 and FUM8, suggesting that the increased FB(1) production in Deltagbp1 is due to over-expression of FUM genes.
Fusarium Verticillioides GBB1, a Gene Encoding Heterotrimeric G Protein Beta Subunit, is Associated with Fumonisin B Biosynthesis and Hyphal Development but Not with Fungal Virulence
SUMMARY: Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a maize pathogen that causes ear rots and stalk rots. The fungus also produces a group of mycotoxins, fumonisins, on infected ears, which cause considerable health and economic concerns for humans and animals worldwide. To date, our understanding of the molecular mechanisms associated with fungal virulence and fumonisin biosynthesis in F. verticillioides is limited. In this study, GBB1, a gene encoding a putative beta subunit of a heterotrimeric G protein, was disrupted and the effects on fumonisin biosynthesis and virulence were evaluated. A GBB1 deletion mutant (Deltagbb1) showed no significant differences in radial growth and mycelial mass but produced significantly less fumonisin B(1 )(FB(1)) than its wild-type progenitor. HPLC analysis showed that Deltagbb1 produced less than 10 p.p.m. FB(1) while the wild-type produced over 140 p.p.m. when strains were grown on cracked corn kernels. Reduced expression of the key FB(1 )biosynthetic genes, FUM1 and FUM8, in Deltagbb1 provides further evidence that GBB1 is involved in FB(1) regulation. Stalk rot virulence, as measured by mean lesion length and by area, was not significantly different in Deltagbb1 compared with the wild-type, suggesting that GBB1 does not regulate virulence in F. verticillioides. Developmentally, hyphae of Deltagbb1 do not deviate from the original axis of polarity established upon germ tube emergence in contrast to wild-type hyphae that meander on and off axis as they grow. Complementation of Deltagbb1 with GBB1 restored FB(1) production and hyphal growth to wild-type. The results of this study demonstrate that heterotrimeric G protein beta subunit plays an important role in regulation of FB(1) biosynthesis and hyphal growth, but not virulence in F. verticillioides.
Functional Characterization of Acetylglutamate Synthase and Phosphoribosylamine-glycine Ligase Genes in Gibberella Zeae
Gibberella zeae (anamorph, Fusarium graminearum) is an important pathogen of cereal crops found in many regions of the world. In this study, we have characterized two auxotrophic strains, designated S4B1279 and S4B3008, which were discovered from a collection of insertional mutants of G. zeae generated by restriction enzyme-mediated integration (REMI). Both mutant strains exhibited pleiotropic phenotypic changes that include reduction of mycelial growth and virulence and abolished sexual reproduction. Molecular analysis of the REMI mutants has shown that the auxotrophy of S4B1279 is due to a mutation of the ARG2 gene encoding an acetylglutamate synthase, and the auxotrophy of S4B3008 is due to a mutation of the ADE5 gene encoding a phosphoribosylamine-glycine ligase. Subsequent gene disruption and complementation studies have confirmed the functions for ARG2 and ADE5, respectively, in G. zeae. Our study has demonstrated the feasibility of using the REMI technique in studying G. zeae virulence mechanisms, in addition to providing two new selectable markers allowing genetic transformation of the fungus.
Disruption of a Maize 9-lipoxygenase Results in Increased Resistance to Fungal Pathogens and Reduced Levels of Contamination with Mycotoxin Fumonisin
Plant oxylipins, produced via the lipoxygenase (LOX) pathway, function as signals in defense and development. In fungi, oxylipins are potent regulators of mycotoxin biosynthesis and sporogenesis. Previous studies showed that plant 9-LOX-derived fatty acid hydroperoxides induce conidiation and mycotoxin production. Here, we tested the hypothesis that oxylipins produced by the maize 9-LOX pathway are required by pathogens to produce spores and mycotoxins and to successfully colonize the host. Maize mutants were generated in which the function of a 9-LOX gene, ZmLOX3, was abolished by an insertion of a Mutator transposon in its coding sequence, which resulted in reduced levels of several 9-LOX-derived hydroperoxides. Supporting our hypothesis, conidiation and production of the mycotoxin fumonisin B1 by Fusarium verticillioides were drastically reduced in kernels of the lox3 mutants compared with near-isogenic wild types. Similarly, conidia production and disease severity of anthracnose leaf blight caused by Colletotrichum graminicola were significantly reduced in the lox3 mutants. Moreover, lox3 mutants displayed increased resistance to southern leaf blight caused by Cochliobolus heterostrophus and stalk rots caused by both F. verticillioides and C. graminicola. These data strongly suggest that oxylipin metabolism mediated by a specific plant 9-LOX isoform is required for fungal pathogenesis, including disease development and production of spores and mycotoxins.
Fusarium Verticillioides GAP1, a Gene Encoding a Putative Glycolipid-anchored Surface Protein, Participates in Conidiation and Cell Wall Structure but Not Virulence
Fusarium verticillioides is an important pathogen of maize that causes ear rot and produces the mycotoxins known as fumonisins. To date, knowledge of pathogenicity and the regulation of fumonisin biosynthesis in F. verticillioides is limited. Here, the molecular characterization of GAP1, a gene encoding a putative 540 aa protein that belongs to a glycolipid-anchored surface (GAS) protein family, is presented. F. verticillioides GAP1 was identified as an expressed sequence tag (EST) upregulated in a culture condition conducive to conidiation and fumonisin B(1) (FB(1)) production. GAP1 null mutants GAM126 (Deltagap1 : : HYG) and GAG8 (Deltagap1 : : GEN) exhibited restricted growth, with more aerial hyphae than their wild-type progenitor on solid media. No defect in mycelial mass or filamentous growth was observed when the GAM126 and GAG8 strains were grown in liquid media under shaking conditions. When grown in suspended conditions, GAM126 and GAG8 strains produced significantly fewer conidia and produced comparatively densely branched hyphae. Concanavalin A staining indicated that the GAP1 deletion altered the cell wall carbohydrate composition/deposition process. Deletion of GAP1 did not affect the production level of FB(1) or F. verticillioides virulence on maize seedlings and stalks. Complementation of GAM126 with the wild-type GAP1 gene restored growth, conidiation and cell wall abnormality phenotypes. The results suggest that GAP1 is associated with growth, development and conidiation in F. verticillioides, but not with pathogenicity or regulation of FB(1).
Production and Characterization of Monoclonal and Recombinant Antibodies Against Antimicrobial Sulfamethazine
A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.
Production of Monoclonal Antibody Against Listeria Monocytogenes and Its Application to Immunochromatography Strip Test
An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.
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.
Development of Immunochromatography Strip-test Using Nanocolloidal Gold-antibody Probe for the Rapid Detection of Aflatoxin B1 in Grain and Feed Samples
An immunochromatography (ICG) strip test using a nanocolloidal gold-antibody probe was developed and optimized for the rapid detection of aflatoxin B1 (AFB1). A monoclonal antibody specific to AFB1 was produced from the cloned hybridoma cell (AF78), coupled with nanocolloidal gold, and distributed on the conjugate pad of the ICG strip test. The visual detection limit of the ICG strip test was 0.5 ng/ml, and this method showed a cross-reaction to aflatoxin B2, G1, and G2. In total, 172 grain and feed samples were collected and analyzed by both the ICG strip test and HPLC. The results of the ICG strip test showed a good agreement with those obtained by HPLC. These results indicated that the ICG strip test has a potential use as a rapid and cost-effective screening tool for the determination of AFB1 in real samples and could be applied to the preliminary screening of mycotoxin in food and agricultural products, generating results within 15 min without complicated steps.
Functional Characterization of Fusarium Verticillioides CPP1, a Gene Encoding a Putative Protein Phosphatase 2A Catalytic Subunit
Fusarium verticillioides produces the mycotoxin fumonisin B(1) (FB(1)) on maize kernels. In this study, we identified a putative protein phosphatase gene CPP1 in F. verticillioides, and investigated its role in FB(1) regulation. Previous work has shown that CPP1 expression is elevated in an FB(1)-suppressing genetic background. Thus, we hypothesized that CPP1 is negatively associated with FB(1) production. To test this hypothesis, we generated a CPP1 knockout mutant, PP179, and studied the effects of gene deletion on FB(1) biosynthesis and fungal development. PP179 showed elevated expression of FUM genes, and in turn produced higher levels of FB(1) than the wild-type progenitor. Other significant mutant phenotypes included reduced radial growth on agar plates, reduced conidia germination rates, significantly increased macroconidia formation, and hyphal swelling. To verify that these phenotypes were directly due to CPP1 deletion, we complemented PP179 with the wild-type CPP1 gene. The complemented strain PPC4 showed FUM1 expression and FB(1) production similar to that of the wild-type, providing evidence that CPP1 is negatively associated with FB(1) biosynthesis. Other PP179 phenotypes, such as macroconidiation and hyphal swelling, were also restored to that of wild-type progenitor. Furthermore, we complemented F. verticillioides PP179 strain with Neurospora crassa wild-type ppe-1 gene, demonstrating that Cpp1 and PPE-1 proteins are functionally conserved. Pleiotropic effects of CPP1 deletion led us to hypothesize that CPP1 is associated with multiple downstream signalling pathways in F. verticillioides. Identification and functional characterization of downstream Cpp1-interacting proteins are necessary to better understand the complex regulatory mechanisms associated with Cpp1.
Functional Analyses of Heterotrimeric G Protein G Alpha and G Beta Subunits in Gibberella Zeae
The homothallic ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is a major toxigenic plant pathogen that causes head blight disease on small-grain cereals. The fungus produces the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) in infected hosts, posing a threat to human and animal health. Despite its agricultural and toxicological importance, the molecular mechanisms underlying its growth, development and virulence remain largely unknown. To better understand such mechanisms, we studied the heterotrimeric G proteins of G. zeae, which are known to control crucial signalling pathways that regulate various cellular and developmental responses in fungi. Three putative Galpha subunits, GzGPA1, GzGPA2 and GzGPA3, and one Gbeta subunit, GzGPB1, were identified in the F. graminearum genome. Deletion of GzGPA1, a homologue of the Aspergillus nidulans Galpha gene fadA, resulted in female sterility and enhanced DON and ZEA production, suggesting that GzGPA1 is required for normal sexual reproduction and repression of toxin biosynthesis. The production of DON and ZEA was also enhanced in the GzGPB1 mutant, suggesting that both Galpha GzGPA1 and Gbeta GzGPB1 negatively control mycotoxin production. Deletion of GzGPA2, which encodes a Galpha protein similar to A. nidulans GanB, caused reduced pathogenicity and increased chitin accumulation in the cell wall, implying that GzGPA2 has multiple functions. Our study shows that G. zeae heterotrimeric G protein subunits can regulate vegetative growth, sexual development, toxin production and pathogenicity.
Identification of Genes Associated with Fumonisin Biosynthesis in Fusarium Verticillioides Via Proteomics and Quantitative Real-time PCR
In this study, we used functional genomic strategies, proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production in the fungus Fusarium verticillioides. Earlier studies have demonstrated that deletion of the FCC1 gene, which encodes a C-type cyclin, leads to a drastic reduction in fumonisin production and conidiation in the mutant strain (FT536). The premise of our research was that comparative analysis of F. verticillioides wild-type and FT536 proteomes will reveal putative proteins, and ultimately corresponding genes, that are important for fumonisin biosynthesis. We isolated proteins that were significantly upregulated in either the wild type or FT536 via two-dimensional polyacrylamide gel electrophoresis, and subsequently obtained sequences via mass spectrometry. Homologs of identified proteins, e.g., carboxypeptidase, laccase, and nitrogen metabolite repression protein, are known to have functions involved in fungal secondary metabolism and development. We also identified gene sequences corresponding to the selected proteins and investigated their transcriptional profiles via quantitative real-time (qRT)-PCR in order to identify genes that show concomitant expression patterns during fumonisin biosynthesis. These genes can be selected as targets for functional analysis to further verify their roles in FB1 biosynthesis.
Enhanced Rapidity for Qualitative Detection of Listeria Monocytogenes Using an Enzyme-linked Immunosorbent Assay and Immunochromatography Strip Test Combined with Immunomagnetic Bead Separation
An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.
The Coiled-coil Protein-binding Motif in Fusarium Verticillioides Fsr1 is Essential for Maize Stalk Rot Virulence
Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is one of the key pathogens of maize stalk rot disease. However, a clear understanding of stalk rot pathogenesis is still lacking. Previously, we identified the F. verticillioides FSR1 gene, which plays a key role in fungal virulence and sexual mating. The predicted Fsr1 protein contains multiple protein-binding domains, namely a caveolin-binding domain, a coiled-coil structure, and a calmodulin-binding motif at the N terminus and a WD40 repeat domain at the C terminus. Fsr1 shares significant similarity to a family of striatin proteins that play a critical role in cellular mechanisms that regulate a variety of developmental processes. Significantly, FSR1 function is conserved in Fusarium graminearum, where it also plays a direct role in pathogenesis. In this study, our goal was to determine the motif(s) in Fsr1 that are directly associated with fungal virulence. We complemented the FSR1 knockout (Deltafsr1) strain with mutated versions of the FSR1 gene, and determined that the Fsr1 C-terminal WD40 repeat domain is dispensable for vegetative growth and maize stalk rot virulence. We also examined the potential link between FSR1-mediated virulence and cell wall-degrading enzyme (alpha-amylase, pectinase and cellulase) activities. Further characterization of the N-terminal region revealed that the coiled-coil structure is essential for virulence in F. verticillioides. The coiled-coil domain is involved in a variety of protein-protein interactions in eukaryotic systems, and thus we hypothesize that the interaction between Fsr1 and the putative Fsr1-binding protein triggers downstream gene signalling that is associated with F. verticillioides virulence.
GAC1, a Gene Encoding a Putative GTPase-activating Protein, Regulates Bikaverin Biosynthesis in Fusarium Verticillioides
Fusarium verticillioides (teleomorph Gibberella moniliformis) is an ascomycete known to produce a variety of secondary metabolites, including fumonisins, fusaric acid and bikaverin. These metabolites are synthesized when the fungus is under stress, notably nutrient limitations. To date we have limited understanding of the complex regulatory process associated with fungal secondary metabolism. In this study we generated a collection of F. verticillioides mutants by using REMI (restriction enzyme mediated integration) mutagenesis and in the process identified a strain, R647, that carries a mutation in a gene designated GAC1. Mutation in the GACI locus, which encodes a putative GTPase activating protein, resulted in the increased production of bikaverin, suggesting that GAC1 is negatively associated with bikaverin biosynthesis. Complementation of R647 with the wildtype GAC1 gene restored the bikaverin production level to that of the wild-type progenitor, demonstrating that gac1 mutation was directly responsible for the overproduction of bikaverin. We also demonstrated that AREA, encoding global nitrogen regulator, and PKS4, encoding polyketide synthase, are downstream genes that respectively are regulated positively and negatively by GAC1. Our results suggest that GAC1 plays an important role in signal transduction regulating bikaverin production in F. verticillioides.
Development of Fluorescence Polarization Immunoassay for the Rapid Detection of 6-chloronicotinic Acid: Main Metabolite of Neonicotinoid Insecticides
A fluorescence polarization immunoassay (FPIA) for the quantitative determination of 6-chloronicotinic acid (6-CNA) using polyclonal antibody was developed. The 6-CNA-protein (bovine serum albumin and soybean trypsin inhibitor) conjugates and fluorescein-labeled 6-CNA derivative (tracer) were prepared and used as the immunogens and tracer, respectively. The synthesized tracer was purified by thin layer chromatography (TLC) and showed a good binding to antiserum (73/5) which was obtained from the immunized rabbit (No. 73) with 6-CNA-BSA conjugate. The detection limit (10% inhibition) of FPIA was 4 microg/mL, and IC(50) value was 32 microg/mL. The FPIA showed a cross-reaction for 5-amino-2-chloropyridine (60%), but no cross-reaction for other pesticides was observed. Recoveries for spiked apple, urine, soil, and water samples (5, 50, and 500 ppm) averaging between 78.6 +/- 8.8 and 114 +/- 18% were reasonable and in good agreement with the amounts spiked. Although the developed FPIA possesses low sensitivity, this assay is more simple and quick than other analytical methods, such as high performance liquid chromatography and gas chromatography. Thus, the developed FPIA method could be a useful tool for express screening 6-CNA in agricultural, environmental, and biological samples.
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.
Development and Validation of a Gold Nanoparticle Immunochromatographic Assay (ICG) for the Detection of Zearalenone
A monoclonal antibody (mAb)-based gold nanoparticle immunochromatographic assay (ICG) for zearalenone detection was developed, optimized, and validated. The detection limits of ICG optimized with appropriate amounts of zearalenone-bovine serum albumin and gold nanoparticle-mAb to zearalenone were 2.5 ng/mL and 30 μg/kg for the standard solution and spike sample, respectively, and a weak cross-reaction for α-zearalenol and β-zearalenol was observed. The assay required only 15 min to obtain results and one step to perform the assay. In validation, the results obtained from spiked corn (10, 20, 30, 50, and 100 μg/kg) and naturally contaminated corn samples by the ICG were in good agreement with those obtained by direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and high-performance liquid chromatography (HPLC). Therefore, the results obtained in this study could be used as basic research for the development of zearalenone-ICG, and the ICG developed could be a useful on-site screening tool for the rapid detection of zearalenone in corn without special instrumentation.
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.
Gibberella Zeae Chitin Synthase Genes, GzCHS5 and GzCHS7, Are Required for Hyphal Growth, Perithecia Formation, and Pathogenicity
Gibberella zeae causes Fusarium head blight of cereal crops, and sexual spores of the fungus play an important role as primary inocula. We isolated a restriction enzyme-mediated integration (REMI) transformant, ZH431, of G. zeae with defects in perithecia formation and virulence. Integration of the REMI vector resulted in disruption of GzCHS7 gene, which encodes a putative class VII chitin synthase with high similarity to Fusarium oxysporum ChsVb. A second chitin synthase, GzCHS5, is adjacently located in a head-to-head configuration with GzCHS7, and its deduced protein sequence showed similarity with a class V chitin synthase in F. oxysporum. Neither DeltaGzChs5 nor DeltaGzChs7 mutants produced perithecia or caused disease on barley heads. Microscopic observation revealed that both mutants formed balloon-shaped hyphae and intrahyphal hyphae and that cell wall rigidity of the mutants was weaker than that of the wild-type strain. Transcription profiles of GzCHS5 and GzCHS7 were not altered in DeltaGzChs7 and DeltaGzChs5, respectively, suggesting that transcription regulations of the genes are independent of each other. Our results demonstrate that GzCHS5 and GzCHS7 are indispensable for perithecia formation and pathogenicity as well as normal septa formation and hyphal growth in G. zeae.
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.
Rapid Detection of Enterotoxigenic Clostridium Perfringens in Meat Samples Using Immunomagnetic Separation Polymerase Chain Reaction (IMS-PCR)
Rapid detection of enterotoxigenic Clostridium perfringens in meat samples was accomplished with an immunomagnetic separation polymerase chain reaction (IMS-PCR). First, a monoclonal antibody (mAb) specific to C. perfringens was generated. The antibody showed strong binding to C. perfringens and no binding to non- Clostridia bacteria, except a weak cross-reaction to Staphylococcus aureus based on the enzyme-linked immunosorbent assay (ELISA). Then, magnetic beads were coated with the mAb, and the IMS-PCR system was developed. With the optimized conditions, the IMS-PCR assay was capable of detecting as few as 10 colony forming units (CFU)/g of C. perfringens cells in the meat sample within 10 h. Of the 116 collected samples (26 chicken samples, 20 beef samples, 30 pork samples, 20 fish samples, and 20 processed meat samples) examined with IMS-PCR, 36 (31%) were C. perfringens -positive samples and 2 (1.7%) were enterotoxigenic C. perfringens -positive samples. The IMS-PCR results gave a good agreement with the results obtained by conventional culture methods. In comparison to conventional culture methods, the IMS-PCR is a rapid and specific method and has potential use as a screening tool for enterotoxigenic C. perfringens in food samples.
Microbiological Assessment in Strawberry Production and Recommendations to Establish a Good Agricultural Practice System
This study conducted microbiological assessment in tunnel style strawberry greenhouses and packaging centers and suggested recommendations to establish a good agricultural practice for strawberry production. The samples from irrigation water, workers' gloves, harvest bins, soil, strawberry leaves and strawberries in greenhouses, packers' gloves, conveyor belts, packaging tables, and door knobs of entrances in packaging centers were collected. Bacterial cell counts of aerobic plate counts, coliforms, Escherichia coli, E. coli O157:H7, Salmonella, Staphylococcus aureus, and Bacillus cereus were then enumerated on appropriate selective media. In general, bacterial populations were similar (p ≥ 0.05) among strawberry greenhouses but not among packaging houses. E. coli and E. coli O157:H7 were negative in all samples, and low levels of Salmonella and B. cereus were detected. However, high bacterial cell counts of aerobic plate counts, coliforms, and S. aureus were found in most samples. These results suggest that food safety practice in strawberry greenhouses and packaging centers should be improved, and the results may be useful in the establishment of a good agricultural practice system for strawberry production.
Aspergillus Nidulans Striatin (StrA) Mediates Sexual Development and Localizes to the Endoplasmic Reticulum
Striatin family proteins have been identified in animals and fungi and are considered to be scaffolding proteins. In fungi striatin orthologs have been associated with sexual development and virulence to plants. In this study, we characterized the functions and localization of the striatin ortholog, StrA, in Aspergillus nidulans. deltastrA strains showed multiple defects in conidium germination, mycelial radial growth, production of diffusible red pigment, and reduced conidiation. The most striking phenotype is the production of abnormally small cleistothecia that are defective in ascosporogenesis. Over-expression of strA enhanced cleistothecium development and increased the production of Hülle cells in shaking liquid cultures. In addition, we generated strains expressing StrA::eGFP under the endogenous promoter. By co-labeling with FM4-64 and co-localization with nuclear localized StuA(NLS)::DsRed or CxnA (an endoplasmic reticulum marker), we determined that StrA mainly localizes to endoplasmic reticulum and the nuclear envelope.
Determination of Ochratoxin A in Grains by Immuno-ultrafiltration and HPLC-fluorescence Detection After Postcolumn Derivatisation in an Electrochemical Cell
The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 μg kg(-1) and 1 μg kg(-1). The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.
Involvement of a Putative Response Regulator FgRrg-1 in Osmotic Stress Response, Fungicide Resistance and Virulence in Fusarium Graminearum
Response regulator (RR) proteins are core elements of the high-osmolarity glycerol (HOG) pathway, which plays an important role in the adaptation of fungi to a variety of environmental stresses. In this study, we constructed deletion mutants of two putative RR genes, FgRRG-1 and FgRRG-2, which are orthologues of Neurospora crassa RRG-1 and RRG-2, respectively. The FgRRG-1 deletion mutant (ΔFgRrg1-6) showed increased sensitivity to osmotic stress mediated by NaCl, KCl, sorbitol or glucose, and to metal cations Li(+) , Ca(2+) and Mg(2+) . The mutant, however, was more resistant than the parent isolate to dicarboximide and phenylpyrrole fungicides. Inoculation tests showed that the mutant exhibited decreased virulence on wheat heads. Quantitative real-time polymerase chain reaction assays indicated that the expression of FgOS-2, the putative downstream gene of FgRRG-1, was decreased significantly in ΔFgRrg1-6. All of the defects were restored by genetic complementation of ΔFgRrg1-6 with the wild-type FgRRG-1 gene. Different from the FgRRG-1 deletion mutant, FgRRG-2 deletion mutants were morphologically indistinguishable from the wild-type progenitor in virulence and in sensitivity to the dicarboximide fungicide iprodione and osmotic stresses. These results indicate that the RR FgRrg-1 of F. graminearum is involved in the osmotic stress response, pathogenicity and sensitivity to dicarboximide and phenylpyrrole fungicides and metal cations.
Regulators of G-protein Signalling in Fusarium Verticillioides Mediate Differential Host-pathogen Responses on Nonviable Versus Viable Maize Kernels
GBB1, a heterotrimeric G-protein β-subunit gene, was shown to be a key regulator of fumonisin B(1) (FB(1) ) biosynthesis in the maize pathogen Fusarium verticillioides. In this study, we performed functional analyses of genes that encode putative RGS (regulators of G-protein signalling) proteins and PhLPs (phosducin-like proteins) in F. verticillioides. These proteins are known to regulate heterotrimeric G-protein activity by altering the intrinsic guanosine triphosphatase (GTPase) activity, which, in turn, influences the signalling mechanisms that control fungal growth, virulence and secondary metabolism. Our aim was to isolate and characterize gene(s) that are under the transcriptional control of GBB1, and to test the hypothesis that these genes are directly associated with FB(1) regulation and fungal development in F. verticillioides on maize kernels. We first identified eight genes (two PhLPs and six RGSs) in the F. verticillioides genome, and a subsequent transcriptional expression study revealed that three RGS genes were up-regulated in the gbb1 deletion (Δgbb1) mutant and one RGS gene was up-regulated in the wild-type. To characterize their function, we generated knockout mutants using a homologous recombination strategy. When grown on autoclaved nonviable kernels, two mutants (ΔflbA2 and ΔrgsB) produced significantly higher levels of FB(1) compared with the wild-type progenitor, suggesting that the two mutated genes are negative regulators of FB(1) biosynthesis. ΔflbA2 also showed a severe curly conidia germination pattern, which was contradictory to that observed in the Δgbb1 strain. Strikingly, when these mutants were grown on live maize kernels, we observed contrasting FB(1) and conidiation phenotypes in fungal mutants, which strongly suggests that these G-protein regulators have an impact on how F. verticillioides responds to host/environmental factors. Our data also provide evidence that fungal G-protein signalling is important for modulating the ethylene biosynthetic pathway in maize kernels.
Sol-gel Immunoaffinity Chromatography for the Clean Up of Ochratoxin A Contaminated Grains
This paper describes the application of sol-gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol-gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82±5%, 90±6% and 91±3%, were obtained for wheat, barley and rye, respectively, with sol-gel columns containing 1mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 μg/kg. Sol-gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.
A Type 2C Protein Phosphatase FgPtc3 is Involved in Cell Wall Integrity, Lipid Metabolism, and Virulence in Fusarium Graminearum
Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.
