Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of Aspergillus parasiticus

Lee, Li Wei; Chiou, Ching Hsun; Klomparens, Karen L.; Cary, Jeffrey W.; Linz, John E.

doi:10.1007/s00203-003-0643-3

Cited by 42 publications

(46 citation statements)

References 35 publications

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“…Our current analysis indicates a linear correlation (R 2 = 0.855) between the function of the gpd promoter driving expression of the gfp gene and aflatoxin production. We are currently in the process of evaluating other strains of A. flavus transformed with gfp driven by aflatoxin gene promoters such as omtA or ver-1 [30,31] so that aflatoxin levels can be directly correlated to GFP values. …”

Section: Discussionmentioning

confidence: 99%

Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

et al. 2008

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Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination.

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Section: Discussionmentioning

confidence: 99%

Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

et al. 2008

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“…The strains used in this study are listed in Table 1 : the model proposes that Nor-1 (early), Ver-1 (middle), and OmtA (late) are synthesized on free ribosomes in the cytoplasm, packaged into transport vesicles (6 -8), and transported to vacuoles via the cytoplasm-to-vacuole targeting (Cvt) pathway (6,8). In contrast Vbs localizes in the cytoplasm and in structures thought to be Golgi (29), suggesting it is transported to vacuoles via the classical secretory pathway; VBS is glycolsylated in support of this notion.…”

Section: Methodsmentioning

confidence: 99%

“…We previously observed that early (Nor-1), middle (Ver-1), and late (OmtA) aflatoxin enzymes localize in vesicles and vacuoles in A. parasiticus (6)(7)(8). To determine the functional role of these organelles in aflatoxin synthesis, a vesicle-vacuole fraction (V) was first isolated from A. parasiticus grown for 36 h in aflatoxin-inducing medium (YES) using a high-density sucrose cushion method.…”

Section: Vesicles and Vacuoles Are One Primary Site For The Late Stepmentioning

confidence: 99%

“…In A. parasiticus, the delivery of OmtA to vacuoles appears to occur through fusion of vesicles containing OmtA to vacuoles (8); this fusion event represents an important late step in vacuole biogenesis (18). To clarify which compartments (vesicles or vacuoles) associate functionally with aflatoxin synthesis, we targeted the class C Vps tethering complex (Fig.…”

Section: Blocking Vesicle-vacuole Fusion Increases Aflatoxin Synthesimentioning

confidence: 99%

“…1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the ␤-lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6)(7)(8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of the end product or biosynthetic enzymes.…”

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A key role for vesicles in fungal secondary metabolism

Chanda

Roze

Kang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B 1 ; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.S econdary metabolites, natural products generated by filamentous fungi, plants, bacteria, algae, and animals, have an enormous impact on humans due to their application in health, medicine, and agriculture. Many secondary metabolites are beneficial (antibiotics, statins, morphine, etc.), though phytotoxins (e.g., ricin, crotin, amygdalin) and fungal poisons called mycotoxins (e.g., aflatoxin, sterigmatocystin, fumonisin) are detrimental to humans and animals. To control or customize biosynthesis of these natural products we must understand how and where secondary metabolism is orchestrated within the cell.Vacuoles and vesicles are known to sequester secondary metabolites to protect host cells from self-toxicity (1). Enzymes involved in secondary metabolism are often found in vesicles and vacuoles, including those for biosynthesis of alkaloids (e.g., berberine, sanguinarine, camptotecin, and morphine; reviewed in refs. 1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the ␤-lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6-8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of...

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