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Appl Environ Microbiol. 2007 Nov; 73(22): 7268–7276.
Published online 2007 Sep 21. doi:  10.1128/AEM.00801-07
PMCID: PMC2168228

Aspergillus Volatiles Regulate Aflatoxin Synthesis and Asexual Sporulation in Aspergillus parasiticus

Abstract

Aspergillus parasiticus is one primary source of aflatoxin contamination in economically important crops. To prevent the potential health and economic impacts of aflatoxin contamination, our goal is to develop practical strategies to reduce aflatoxin synthesis on susceptible crops. One focus is to identify biological and environmental factors that regulate aflatoxin synthesis and to manipulate these factors to control aflatoxin biosynthesis in the field or during crop storage. In the current study, we analyzed the effects of aspergillus volatiles on growth, development, aflatoxin biosynthesis, and promoter activity in the filamentous fungus A. parasiticus. When colonies of Aspergillus nidulans and A. parasiticus were incubated in the same growth chamber, we observed a significant reduction in aflatoxin synthesis and asexual sporulation by A. parasiticus. Analysis of the headspace gases demonstrated that A. nidulans produced much larger quantities of 2-buten-1-ol (CA) and 2-ethyl-1-hexanol (EH) than A. parasiticus. In its pure form, EH inhibited growth and increased aflatoxin accumulation in A. parasiticus at all doses tested; EH also stimulated aflatoxin transcript accumulation. In contrast, CA exerted dose-dependent up-regulatory or down-regulatory effects on aflatoxin accumulation, conidiation, and aflatoxin transcript accumulation. Experiments with reporter strains carrying nor-1 promoter deletions and mutations suggested that the differential effects of CA were mediated through separate regulatory regions in the nor-1 promoter. The potential efficacy of CA as a tool for analysis of transcriptional regulation of aflatoxin biosynthesis is discussed. We also identify a novel, rapid, and reliable method to assess norsolorinic acid accumulation in solid culture using a Chroma Meter CR-300 apparatus.

Aflatoxins are environmental carcinogens and mutagens produced by several aspergilli. The entrance of these compounds into the food chain occurs through the contamination of economically important crops (corn, peanuts, tree nuts, dried fruits and vegetables, and medicinal herbs) predominantly by the aflatoxigenic fungi Aspergillus parasiticus and Aspergillus flavus (1, 9, 10).

One goal of our laboratory is to identify biological and environmental factors that control aflatoxin synthesis in aspergillus; these factors could then be manipulated in the field or during crop storage to reduce aflatoxin contamination. Our laboratory recently focused on the identification of fungal volatiles that regulate aflatoxin synthesis; others have studied the correlation between the pattern of specific fungal volatiles and the ability to synthesize ochratoxin (12) or aflatoxin (26) in attempts to use these compounds to identify toxigenic isolates.

We previously analyzed the effects of ethylene and CO2, gases produced naturally by A. parasiticus and Aspergillus nidulans in culture, on development and toxin synthesis (15); these filamentous fungi produce aflatoxin and sterigmatocystin, respectively. Sterigmatocystin is a carcinogenic late pathway precursor of aflatoxin and is generated by a biosynthetic pathway analogous to that used in aflatoxin biosynthesis. Ethylene and CO2, alone or in combination, influenced fungal growth and development and inhibited aflatoxin biosynthesis in laboratory culture and on peanuts. The detailed mechanisms remain to be elucidated; however, we showed that ethylene blocks aflatoxin gene expression, at least in part, at the transcriptional level (15).

In the current study, we sought to identify additional fungal metabolites that control aflatoxin synthesis; we screened A. nidulans and A. parasiticus for volatiles that negatively impact aflatoxin synthesis. Colonies of A. parasiticus were incubated alone or in the same growth chamber as colonies of A. nidulans. The presence of A. nidulans reduced the accumulation of norsolorinic acid by A. parasiticus B62, a nor-1 disruption mutant. nor-1 encodes norsolorinic acid reductase that catalyzes the conversion of the first stable aflatoxin pathway intermediate, norsolorinic acid, to averantin. This observation suggested that A. nidulans could communicate with A. parasiticus by means of gases or volatile compounds. We then identified several volatile compounds produced by both A. parasiticus and A. nidulans in laboratory culture and demonstrated that one of these, 2-buten-1-ol (or crotyl alcohol [CA]), exerts dose-dependent up-regulatory and down-regulatory effects on aflatoxin accumulation, conidiation, nor-1 transcriptional activity, and aflatoxin transcript accumulation in A. parasiticus. The data also suggested that the up-regulatory and down-regulatory effects on nor-1 transcriptional activity are mediated through separate regulatory regions in the nor-1 promoter.

MATERIALS AND METHODS

Strains, media, and growth conditions.

Isogenic A. parasiticus strains used in this study were derived from the parent strain SU-1 (ATCC 56775), a wild-type aflatoxin producer. A. parasiticus D8D3 contains the GUS (uidA) (encodes β-d-glucuronidase) reporter fused to the nor-1 promoter and has been used previously as a nor-1 reporter strain (7). Strain B62 (ATCC 24690) (niaD nor-1 br-1) accumulates the first stable aflatoxin pathway intermediate, norsolorinic acid, due to a mutation in nor-1 (20); because other activities assist Nor-1 in this conversion step, a small quantity of aflatoxin still accumulates in this mutant strain. Accumulation of this brightly colored red pigment in A. parasiticus B62 can be detected visually on the bottom surface of colonies grown on glucose minimal salts (GMS) solid medium (see below) as a red line along the colony margin. A. nidulans FGSC4 (wild-type sterigmatocystin producer) was obtained from the Fungal Genetics Stock Center (Kansas City, KS).

Chemically defined GMS medium supplemented with 5 μM Zn2+ was used as the base growth medium for A. parasiticus (3). Glucose minimal agar medium (GMM) was used for the growth of A. nidulans (5). Yeast extract sucrose agar medium (6) was used as a growth medium for Stachybotrys chartarum, Mucor racemosus, and Penicillium expansum. Media were allowed to solidify in the lids of sterile 60- by 15-mm petri dishes.

Conidiospores (3 × 103 spores/plate) of A. parasiticus D8D3 and B62 and A. nidulans FGSC4 were center inoculated onto appropriate agar media. The small petri dish lids carrying inoculated media were placed inside a larger, 150- by 15-mm petri dish that was then covered. This system allowed free gas or volatile exchange between colonies inside the large dish while preventing direct colony contact. A 150- by 15-mm control dish contained three lids carrying A. parasiticus D8D3 or A. parasiticus B62 only. Treatments contained one lid carrying A. parasiticus D8D3 or B62 placed together with two lids inoculated with A. nidulans (2× treatment) or two lids carrying A. parasiticus D8D3 or B62 placed together with one lid inoculated with A. nidulans (1× treatment). Cultures were incubated at 30°C and in 99.8% relative humidity in the dark for the appropriate time periods (see Results).

To evaluate the effects of 2-ethyl-1-hexanol (EH) and CA on growth, asexual sporulation (conidiation), and aflatoxin synthesis, a designated quantity of the test compound was dispensed into the bottom of a sterile 0.5-ml microcentrifuge tube with a small round opening in its lid made by a sterile 18-gauge needle. This tube was placed in the center of the large petri dish between the small petri dish lids carrying the fungal colonies. Control plates contained an empty tube. The cultures were grown at 30°C in the dark for appropriate periods of time.

Measurements of CO2, O2, and ethylene concentration.

Headspace gases were sampled through a port equipped with a Teflon-lined rubber septum installed in the lid of the 150- by 15-mm petri dish and analyzed for carbon dioxide, oxygen, and ethylene levels by means of gas chromatography (GC) as described previously (15).

Volatile analysis.

Volatiles produced by A. parasiticus and A. nidulans were sampled and analyzed as described previously by Song et al. (17). Briefly, headspace gases in large petri plates containing three colonies of either A. nidulans or A. parasiticus were sampled by means of a solid-phase microextraction (SPME) device (Supelco, Bellefonte, PA) coated with polydimethylsiloxane-divinylbenzene fibers. The SPME was inserted into the large petri dish, the cover was replaced, and the volatiles were absorbed for 4 min. The SPME was then removed from the petri dish and desorbed in the injection port of a gas chromatograph (model 3400; Varian). Detection of volatiles was performed by time-of-flight mass spectrometry using an instrument equipped with an electronic ionization source (FCD-650; LECO Corp., St. Joseph, MI). The volatiles were identified by comparison of mass spectra to a mass spectrum library at the National Institute for Standard Technology (version 1.0). Measurements were performed on two to three replicates. Appropriate controls for medium, plastic ware, and accidental contaminants were conducted.

Assessment of EH and CA concentrations in headspace gases.

Analysis of volatiles was performed essentially as described previously by Song et al. for hexanal (17), with the following modifications. A 0.5-ml plastic microcentrifuge tube containing a designated volume of EH or CA was placed between the lids of three small petri dishes (60 by 15 mm) containing GMS agar medium; the lids were positioned in a large petri dish (150 by 15 mm). The large plate was covered, sealed with Parafilm, and incubated at 30°C for 24 h. Samples of headspace gases were withdrawn by a gas-tight Hamilton syringe through a small, premade, sealed opening in the lid and injected into the gas chromatograph (Carle GC series). Evaporation of 0.1 and 1 μl of EH into a specially made 4.4-liter jar fitted with a Mininert gas-tight sampling valve generated EH standards. The concentration of CA was estimated using a 2.68 nmol/liter CA standard generated by placing 1 μl of the compound into a similar 4.4-liter jar.

Detection of aflatoxins B1, B2, G1, and G2 and norsolorinic acid.

Aflatoxins were extracted from the agar medium and mycelium three times with 5 ml chloroform each. The extracts were combined, dried under a stream of N2, and redissolved in 70% methanol. Aflatoxins were detected by enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) as described previously by Roze et al. (14). ELISA provided a measure of primarily aflatoxin B1 levels, whereas TLC enabled one to measure aflatoxin B1, B2, G1, and G2 levels. Norsolorinic acid was extracted from the agar and mycelium twice with chloroform and once with acetone and then analyzed by TLC (4). The quantity of norsolorinic acid in the agar was also estimated directly by scanning the bottom surface of petri dishes carrying fungal colonies with a Minolta CR-300 Chroma Meter (Konica Minolta, Osaka, Japan); intensity readings from the red-green axis (“a” value) were recorded.

Evaluation of conidiation.

A. parasiticus conidia were harvested and their number per colony was estimated using a hematocytometer as described previously by Roze et al. (14).

Assessment of GUS reporter activity in A. parasiticus D8D3 and in the nor-1 promoter deletion mutants.

A fluorimetric assay, as described previously, was used to determine GUS activity (14).

Total RNA isolation and quantification of transcript levels.

A. parasiticus D8D3 was grown on GMS agar for 3 days at 30°C in the dark. Total RNA was isolated from duplicate colonies by the TRIzol method (TRIzol reagent; Invitrogen, Carlsbad, CA) and treated with RNase-free DNase (QIAGEN, Valencia, CA), and the RNA quality was examined by an Agilent 2100 bioanalyzer (Agilent Technologies). First-strand cDNA was synthesized using 1 μg of total RNA and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Two microliters of cDNA was used as a template in the subsequent PCR using the following thermocycler (Perkin-Elmer GeneAmp PCR system 2400) parameters: initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min, with a final extension step at 72°C for 10 min. Pairs of gene-specific primers for PCR are shown in Fig. Fig.6C.6C. PCR products were separated by electrophoresis on a 1% agarose gel. Band intensities for aflatoxin gene transcripts were compared to the intensity of the transcript of the housekeeping gene (β-tubulin) under the same treatment. Transcript levels as detected by reverse transcription (RT)-PCR for β-tubulin did not change with any treatment, suggesting that the changes in band intensities for aflatoxin gene transcripts reflect real changes in transcript levels in the samples.

FIG. 6.
Aflatoxin transcript accumulation in A. parasiticus D8D3 under volatile exposure. A. parasiticus D8D3 conidiospores were inoculated onto GMS agar medium and incubated for 3 days at 30°C in the dark. Fungal colonies were treated with CA (10 μ1 ...

Analysis of intracellular cAMP levels in A. parasiticus.

A. parasiticus was grown in the dark for 2 or 3 days on GMS agar medium overlaid with cellophane, immediately frozen in liquid nitrogen, and stored at −80°C until analysis. The frozen mycelium was ground in liquid nitrogen using a mortar with a pestle, resuspended in 0.1 N HCl, and used for quantitative determination of cyclic AMP (cAMP) by a competitive immunoassay according to instructions provided by the manufacturer (Direct cAMP enzyme immunoassay kit; Assay Designs, Inc., Ann Arbor, MI).

Statistical analysis.

Statistical analyses were performed using SigmaStat one-way analysis of variance scientific statistical software, version 1.0, from the Jandel Corporation.

RESULTS

Effect of A. nidulans on aflatoxin accumulation, conidiation, and colony diameter in A. parasiticus.

Growth of one colony of A. parasiticus B62 in the presence of two colonies of A. nidulans (2× treatment) for 7 days caused a visible reduction of norsolorinic acid accumulation compared with a control containing three colonies of A. parasiticus B62 (Fig. (Fig.1A).1A). We observed a “dose response”; two colonies of A. nidulans produced a larger effect on B62 than one colony of A. nidulans. TLC (Fig. (Fig.1B),1B), ELISA (Fig. (Fig.1C),1C), and a Chroma Meter-based assay (Fig. (Fig.1D)1D) quantified the magnitude of the effect. ELISA demonstrated that 2× treatment caused up to a 20-fold reduction in aflatoxin B1 in strain D8D3, while 1× treatment resulted in only a twofold decrease in this toxin. TLC analysis confirmed these data and demonstrated that the effect extended to aflatoxins B2, G1, and G2. TLC and the Chroma Meter-based assay provided additional evidence that 2× and 1× treatments resulted in similar effects on norsolorinic acid accumulation in strain B62. The Chroma Meter assay proved to be a reliable and rapid screening tool because it does not require the extraction of the compounds from the growth medium.

FIG. 1.
A. nidulans affects toxin accumulation and conidiospore production in A. parasiticus. (A) Growth on solid media. A. parasiticus D8D3 and B62 and A. nidulans FGSC4 were center inoculated onto GMS agar medium and GMM, respectively, and incubated at 30°C ...

The 2× treatment generated up to a twofold reduction in conidiospores by A. parasiticus D8D3 and B62, whereas the 1× treatment did not generate a statistically significant effect (Fig. (Fig.1E).1E). Colony growth (estimated by colony diameter) was not affected by any treatment (Fig. (Fig.1E1E).

Effect on aflatoxin accumulation may be unique to Aspergillus volatiles.

M. racemosus 1216B, S. chartarum, and P. expansum 28830 were tested in place of A. nidulans in experiments similar to those described above. No changes in norsolorinic acid accumulation in A. parasiticus B62 were observed using either 2× or 1× treatments (data not shown) after 7 days. However, 2× treatment with M. racemosus resulted in an approximately threefold reduction in conidiospores produced by A. parasiticus B62 (not shown); none of these treatments affected colony growth.

Volatiles other than CO2, O2, and ethylene are responsible for effects on A. parasiticus.

We hypothesized that A. nidulans produces volatiles that mediate the observed effects on aflatoxin and norsolorinic acid accumulation. CO2 is a potent regulator of fungal morphogenesis (18). We demonstrated previously that CO2 and ethylene are synthesized by A. nidulans and inhibit aflatoxin accumulation (15). Oxygen also affects aflatoxin production (8, 12). To rule out possible effects of CO2, O2, and ethylene on data interpretation in the experiments described above, we measured concentrations of these gases in the headspace of large petri dishes carrying fungal colonies. Gas samples were obtained at day 7 of growth through an F-145 sterile rubber septum plug (Alltech Associates, Inc., Deerfield, IL) installed in the center of the lid of the large petri dish. We observed no significant changes in O2 levels in petri dishes containing any combination of A. parasiticus and/or A. nidulans colonies. We were unable to detect ethylene in the headspace, likely because the concentration was too low under these growth conditions. In contrast, three A. parasiticus colonies produced the highest CO2 levels (0.23%). Three A. nidulans colonies produced the lowest CO2 levels (0.06%), and one A. parasiticus colony with two A. nidulans colonies produced intermediate CO2 levels (0.14%) (Table (Table1);1); these data were not consistent with O2, ethylene, or CO2 being primary contributors to the observed effects of A. nidulans on aflatoxin and conidiospore production in A. parasiticus.

TABLE 1.
CO2 and O2 concentrations in headspace gasesa

Volatile analysis of headspace gases.

Headspace gases obtained from large petri dishes carrying three colonies of A. parasiticus or A. nidulans were subjected to GC/mass spectrum analyses to identify compounds uniquely produced or produced in different quantities by A. nidulans and A. parasiticus in culture.

In initial experiments, gas samples were obtained directly from large petri dishes carrying fungal colonies incubated for 6 days. Using this procedure, we identified two volatile alcohols that were produced in significantly larger quantities by A. nidulans than by A. parasiticus (not shown); these included EH (up to 10-fold-greater quantities) and CA (up to 1,000-fold-greater quantities). Ethanol was produced in significantly higher quantities by A. parasiticus than by A. nidulans (trace) under these growth conditions (not shown).

In follow-up experiments, large petri dishes were incubated for 6 days and placed into 1-liter Teflon containers. The containers were sealed and incubated at 30°C for an additional 20 h, and headspace gases were obtained. Under these growth conditions, A. nidulans produced one additional alcohol (1-penten-3-ol) in higher quantities than A. parasiticus; two additional compounds were detected at low levels that we suspect were contaminants derived from the Teflon container (not shown).

Effects of CA and EH on aflatoxin biosynthesis in A. parasiticus.

We focused attention on CA and EH because of their structural similarity to ethylene and hexanal, biologically active compounds with strong effects on fungi (15, 17, 22). To generate different concentrations of these volatiles in the headspace of a petri dish, microcentrifuge tubes containing different volumes (1 to 100 μl) of these compounds were placed into large petri dishes as described in Materials and Methods. To determine the initial concentration of each volatile in the headspace, large petri dishes (no fungal colonies) were sealed with Parafilm, samples of headspace gases were taken after 6 h, and GC analysis was performed (Table (Table2).2). We observed a roughly linear correlation between the initial volume of the compound in the microcentrifuge tube and the concentration of the volatilized compound detected in the headspace of the sealed plates.

TABLE 2.
Initial concentrations of EH and CA in headspace gasesa

A. parasiticus B62 and D8D3 were then grown on GMS agar medium in the presence of different concentrations of CA or EH for 6 days at 30°C. We measured norsolorinic acid, aflatoxins B1 and G1, and colony diameter as a function of dose. EH inhibited growth by approximately 10% (not shown) and increased the production of norsolorinic acid, aflatoxin B1, and aflatoxin G1 at doses ranging from 10 μl to 100 μl (Fig. (Fig.2).2). Treatment with EH also resulted in the accumulation of norsolorinic acid on the entire bottom surface of the colony compared to untreated controls in which NA accumulated along the margin of the colony. The effect of CA on visible norsolorinic acid accumulation depended on dose (Fig. (Fig.3).3). One hundred microliters inhibited norsolorinic acid accumulation; in contrast, lower doses (1 μl to 25 μl) strongly increased norsolorinic acid accumulation but did not produce an effect on growth (Fig. (Fig.3A).3A). By day 6 of growth on a control plate, we observed norsolorinic acid accumulation along the margin of a colony. Low doses (1 μl and 10 μl) of CA resulted in norsolorinic acid accumulation over almost the entire bottom surface of a colony (Fig. (Fig.3A),3A), similar to treatment with EH (not shown). TLC data confirmed data from visual analysis (Fig. (Fig.3B):3B): low doses of CA (1 μl and 10 μl) stimulated toxin accumulation, while a high dose greatly reduced toxin accumulation (Fig. (Fig.3B3B).

FIG. 2.
TLC analysis of toxin accumulation under EH exposure. A. parasiticus B62 conidiospores were center inoculated onto GMS agar medium and incubated for 6 days at 30°C in the dark. Fungal colonies were treated with EH (see Materials and Methods); ...
FIG. 3.
Dose-response effect of CA on toxin accumulation in A. parasiticus B62. A. parasiticus B62 conidiospores were center inoculated onto GMS agar and incubated for 6 days at 30°C in the dark (see Materials and Methods). Fungal colonies were treated ...

CA was also added to 100 ml of liquid GMS medium, and B62 was inoculated and incubated under standard shake culture conditions (30°C, shaking at 150 rpm, and in the dark) (Fig. 4A and B). Similar to effects on solid media, low doses (1 μl or 10 μl) strongly stimulated the accumulation of norsolorinic acid and aflatoxins B1, B2, G1, and G2 (Fig. (Fig.4B).4B). The addition of 100 μl of CA in liquid culture completely blocked detectable norsolorinic acid (Fig. (Fig.4A)4A) and aflatoxin (Fig. (Fig.4B)4B) accumulation; these effects were accompanied by a clearly observable reduction in mycelial growth.

FIG. 4.
Effect of CA on toxin accumulation of A. parasiticus B62 in liquid medium. A. parasiticus B62 conidiospores were inoculated into 250-ml flasks containing 100 ml liquid GMS medium and incubated for 4 days under standard conditions (see Materials and Methods); ...

Ethanol is one volatile produced by A. parasiticus in significantly higher quantities than by A. nidulans. The addition of 1% ethanol to liquid GMS medium produced a strong positive effect on toxin accumulation (Fig. (Fig.4B4B).

CA affects transcriptional activity of the nor-1 promoter.

To determine whether CA exerts an influence on aflatoxin biosynthesis at the level of gene transcription, A. parasiticus D8D3, a nor-1::GUS reporter strain carrying a 3,000-bp nor-1 promoter fragment integrated at 3′ end of nor-1 in the A. parasiticus genome, was center inoculated onto GMS agar and grown in the presence of 0 μl, 10 μl, or 100 μl of CA for 72 h at 30°C. This time point was empirically determined; analysis at 48 h did not generate detectable GUS activity, while at 72 h, we did detect reproducible levels of GUS activity. GUS activity, which provides a useful indicator of nor-1 promoter activity (13), increased up to fourfold with 10 μl of CA but decreased over 30-fold with 100 μl of this compound (Fig. (Fig.5).5). These are representative data from two independent experiments.

FIG. 5.
Separate regulatory elements mediate activation and inhibition of nor-1 promoter activity by CA. (A) Schematic of the intergenic region between nor-1 and pksA in the A. parasiticus aflatoxin gene cluster. Positions of the AflR, CRE1, and NorL transcription ...

CA and EH affect transcript accumulation of aflatoxin genes.

To determine if CA and EH affect the expression of aflatoxin genes in addition to nor-1, we analyzed transcript accumulation for ver-1, aflR (a transcription factor which activates aflatoxin gene expression), and β-tubulin (a housekeeping gene) by semiquantitative RT-PCR (Fig. (Fig.6).6). A. parasiticus D8D3 was incubated for 72 h on GMS agar medium and grown in the presence of 10 or 100 μl CA, 100 μl EH, or no volatile (control). Purified RNA from duplicate colonies was analyzed by RT-PCR (see Materials and Methods). Volatile treatment had little detectable effect on aflR and β-tubulin transcript accumulation; CA (10 μl) and EH increased ver-1 transcript accumulation, while CA (100 μl) reduced ver-1 transcript accumulation.

Differential effects of CA on nor-1 promoter activity are mediated through different regulatory regions.

We previously identified three transcription factors (AflR, NorL, and CRE1bp) required for maximum levels of nor-1 transcription and characterized their DNA binding sites (13, 16). Each of these cis-acting sites was located within a 332-bp nor-1 promoter fragment situated between the nor-1 transcriptional start site and the stop codon of the upstream gene (orf3) (Fig. (Fig.5A).5A). Reporter strains carrying a 3,000-bp genomic region including the 332-bp promoter, orf3, and a small portion of the coding region of pksA (Fig. (Fig.5A)5A) had 31-fold-higher levels of GUS activity than reporter strains carrying the 332-bp nor-1 promoter fragment. These data suggested that additional regulatory sites lie upstream from the 332-bp promoter fragment and influence nor-1 transcriptional activity.

We therefore analyzed nor-1::GUS reporter strains carrying 3,000-bp, 1,250-bp, 1,200-bp, and 332-bp promoter fragments upstream of the nor-1 transcriptional start site (Fig. (Fig.5B)5B) to identify regions that mediate the CA regulatory response. In addition, we analyzed nor-1::GUS reporter strains carrying the 332-bp fragment in which the consensus AflR cis-acting site was replaced by a restriction endonuclease site (Fig. (Fig.5B)5B) to determine if this site was necessary for the observed regulatory response. All reporter strains contained the reporter constructs integrated at the 3′ end of nor-1 in the A. parasiticus genome; we demonstrated previously that this integration site generated normal patterns of promoter activity (13).

Two single-spore isolates of each reporter strain were grown on GMS agar medium in the presence of 0 μl, 10 μl, or 100 μl of CA for 72 h, and GUS activity was measured. Reporter strains carrying the 332-bp promoter fragment showed approximately 15-fold-lower GUS activity, and strains carrying the 1,200-bp and 1,250-bp promoter fragments showed approximately 30-fold-lower overall GUS activity than strain D8D3 carrying the 3,000-bp promoter fragment. Nevertheless, strains carrying the 3,000-bp, 1,250-bp, and 1,200-bp promoter fragments showed clear up-regulatory and down-regulatory responses to 10 μl and 100 μl of CA, respectively (only one of two strains of carrying the 1,250-bp promoter fragment showed consistent down-regulatory effects). However, only the up-regulatory (and not the down-regulatory) effect was detected in reporter strains carrying the 332-bp promoter fragment. Mutation in the AflR cis-acting site in the background of the 332-bp promoter resulted in low GUS activity with or without CA treatments. Neither up-regulatory nor down-regulatory effects could be observed in the AflR mutant (Fig. (Fig.5C5C).

Steady-state cAMP levels in A. parasiticus under treatment with CA.

A cAMP/PKA-signaling pathway is involved in the regulation of aflatoxin biosynthesis and conidiation in A. parasiticus (15). We previously demonstrated that ethylene blocked the cAMP-mediated stimulation of aflatoxin biosynthesis and decreased the accumulation of aflatoxin in A. parasiticus (16), suggesting that ethylene could influence the cAMP/PKA signaling pathway downstream from PKA. To evaluate the possible influence of CA on cAMP/PKA signaling, three independent experiments were performed to estimate cAMP levels in A. parasiticus D8D3 mycelium treated with 0 μl, 10 μl, or 100 μl CA for 48 h and 72 h at 30°C. We consistently observed an approximately twofold decrease in steady-state cAMP levels with the 100-μl CA treatment for 72 h. No other treatment provided consistent results.

DISCUSSION

Living organisms have evolved sensitive and efficient mechanisms to utilize gases as signals to communicate and to adapt to the changing environment. Carbon dioxide, oxygen, and ethylene are common gases that serve as regulators of gene expression in a diverse group of organisms including bacteria, fungi, and plants (18). Signaling by gases is conserved in higher vertebrates (21). Directly relevant to our line of research, several cotton leaf volatiles, neem leaf volatiles, and volatile aldehydes were previously shown to produce effects on fungal growth (22) and aflatoxin biosynthesis (11, 22, 24, 25, 27).

Our data clearly demonstrate that the A. nidulans volatiles EH, CA, and ethanol have strong regulatory influences on asexual development and aflatoxin synthesis in A. parasiticus. Because these volatiles are also synthesized by A. parasiticus in culture, albeit at much different levels, we speculate that they represent part of the normal control circuitry, together with CO2 and ethylene, that the mold utilizes to control the timing and level of aflatoxin synthesis in the soil and on plant material. The fact that Mucor, Stachybotrys, and Penicillium do not mimic this biological effect suggests that the regulatory effect is specific to aspergilli.

In the current study, we observed that CA and EH produce differential effects on A. parasiticus in culture when added in pure form. In nature, the regulatory effects of these volatiles might require modification to the active derivative by cell metabolism, may result from combined actions of several biologically active molecules, and may depend on dose. For example, the biological effects of EH may be based on its structural similarity to hexanal; this compound inhibits growth (17) and aflatoxin production in aspergilli (22). Alternatively, hexanal could be metabolized to hexanol (17) and, possibly, to EH by these filamentous fungi. The down-regulatory effects of high doses of CA (in contrast to low doses) on aflatoxin synthesis may be due to an adaptive response to nonphysiologically high levels of CA. In support of this idea, we demonstrated previously that A. parasiticus adapts to nonphysiologically high exogenous cAMP levels by down-regulating PKA activity (14). Follow-up studies are required to clarify these possibilities.

To begin to understand the mechanistic basis for the regulatory effects of CA and EH, we analyzed aflR, ver-1, and β-tubulin transcript accumulation under volatile treatment. Effects of volatile treatment on transcript accumulation of the aflatoxin structural gene paralleled effects on aflatoxin accumulation; β-tubulin transcript accumulation was unaffected. These data suggest that EH and CA affect aflatoxin synthesis in a relatively specific manner. Negligible effects on aflR transcript levels were observed, strongly suggesting that CA and EH regulatory effects were mediated via changes in AflR activity or by an independent mechanism.

The specific effects of CA on nor-1 promoter activity were analyzed using a set of promoter deletions in a GUS reporter construct. The data suggested that the region from positions −1200 to −332 mediated nor-1 activation, whereas the region from positions −332 to −1 mediated nor-1 inhibition. As a confounding factor, we recognize that the highest dose of CA used (100 μl) in these studies may influence nor-1 promoter activity, at least in part, due to growth inhibition.

We compared the nucleotide sequence of the 868-bp activation region with that of the 332-bp inhibition region using ClustalW (version 1.83); the analysis revealed 42% identity between these regions. The analysis also identified a GC box, CCGCCC, in both regions. It is of interest that the GC box within the 332-bp region is adjacent to the 5′ end of the AflR consensus DNA binding site. These two elements together with the TATA box and cAMP response element (CRE1) are located within 110 bp of the transcriptional start site of nor-1 (16). Others previously demonstrated that a group of transcription factors (including Sp factors) selectively bind GC-rich consensus sequences (including the GC box) in many promoters and are capable of either activating or repressing transcription (2, 19). In addition, Sp1 can form complexes with the cAMP response element binding protein (23). Based on these observations, we hypothesize that CA affects nor-1 transcription via these specific GC-rich response elements (GC boxes) located in the activation and inhibition regulatory regions of the nor-1 promoter.

We previously presented a model in which parallel signal transduction pathways control aflatoxin gene transcription and biosynthesis in response to a preferred carbon source (glucose or sucrose) (16). Recent work in our laboratory (L. V. Roze, A. E. Arthur, S. Y. Hong, A. Chanda, and J. E. Linz, unpublished data) allowed us to provide new insights into the sequence of events that govern transcriptional activation of the entire gene aflatoxin cluster. In the expanded model, activation of CRE1bp results in the recruitment of histone acetyltransferase, generating a bidirectional wave of histone H4 acetylation and, subsequently, in gene activation within the aflatoxin cluster in the order and at the specific times required by the biochemical pathway. The proposed role of the GC box in the regulation of the nor-1 transcription introduces an additional player to the set of cis-regulatory elements (CRE1 and AflR), which together may mediate the initiation of histone H4 acetylation followed by the activation of gene transcription. However, due to inconclusive cAMP data, the role of the cAMP/PKA pathway in mediating CA activity is unclear; future experiments are required to identify the signal transduction pathway(s) that mediates the CA response and its relationship to the regulatory model presented above.

Studying natural gaseous compounds and the mechanisms of their action on aflatoxin biosynthesis has practical applications; these metabolites offer great promise in the development of safe, practical, and inexpensive compounds that block aflatoxin synthesis in the field or during storage of plant materials without adverse effects on food quality. To this end, we clearly demonstrated that treatment of peanuts with an ethylene-CO2 gas mixture could provide an effective antiaflatoxin postharvest measure (15). Although the chemical and biological properties of CA (an irritant harmful to the respiratory system, eyes, and skin) restrict its use as an antiaflatoxin agent, further analysis of the effects of CA on aflatoxin gene transcription may have broader implications for understanding the role of signaling pathways and transcriptional mechanisms on aflatoxin gene activation; this in turn may facilitate the identification of novel and effective means to control aflatoxin contamination.

Acknowledgments

We thank James Pestka and Zahidul Islam, who kindly provided Stachybotrys chartarum, and Kerri Harris for Penicillium expansum 28830 (Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI). Mucor racemosus strain 1216B was obtained from Paul Sypherd, University of California, Irvine.

Footnotes

Published ahead of print on 21 September 2007.

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