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Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77845-2132
Sung Chur Sim
Nancy P. Keller
Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The Aspergillus nidulans sterigmatocystin (ST) gene cluster contains both regulatory (aflR) and biosynthetic genes (stc genes) required for ST production. A total of 26 genes are in the cluster, 13 of which have been assigned a known function in the biosynthetic pathway. This complex secondary pathway represents a physiological cost to the fungus. We tested the amount of asexual spore production using a series of isogenic lines of A. nidulans, differing only in a mutation in aflR (resulting in a strain containing no ST intermediates) or a mutation in three stc genes that produced either no ST intermediates (
stcJ), an early ST intermediate, norsoloroinic acid (stcE) or a late ST intermediate, versicolorin A (stcU). In two independently replicated experiments we compared the numbers of conidia produced by each of these mutant strains and a wild type ST producer in a neutral (growth media) and a host (corn seed) environment. A stepwise increase in asexual spore production was observed with each progressive step in the ST pathway. Thus, the data suggest that recruitment or loss of these secondary metabolite pathway genes has a selective advantage apart from the physiological activity of the metabolite.
Key words: conidia, filamentous fungus, sterigmatocystin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) (Bennett and Ciegler 1983, Cole and Schweikert 2003). AF and ST are among the most potent natural carcinogens known, and they also display toxic, mutagenic and teratogenic properties ( Jelinek et al 1989, Purchase and van der Watt 1971). The ST biosynthetic pathway has been dissected thoroughly in A. nidulans through gene deletions of both ST regulatory and enzymatic genes. The resulting isogenic strains are blocked at different steps along the biosynthetic pathway (FIG. 1; TABLE I). Strains used in this study include wild type and well characterized mutant strains, i.e., aflR, stcJ, stcE and stcU (formerly called verA, Keller et al 1994). Mutants for aflR and stcJ do not produce ST but for different reasons. AflR is the ST/AF pathway transcription factor required for the expression of the ST/AF biosynthetic genes including stcJ, stcE and stcU (Yu et al 1996, Payne and Brown 1998), whereas stcJ encodes the 
-subunit of a fatty acid synthase required for the initial step of the ST pathway (Brown et al 1996). stcE encodes a dehydrogenase required for the conversion of norsolorinic acid to averantin (Butchko et al 1999), and stcU encodes an oxidoreductase required for the conversion of versicolorin A to demethylsterigmatocystin (Keller et al 1994). Mutations in these latter two genes result in strains that accumulate norsolorinic acid and versicolorin A. Versicolorin A is strongly mutagenic and genotoxic, while norsolorinic acid is nonmutagenic and nongenotoxic (Mori et al 1985). Despite the extensive research with these mutants in the context of secondary metabolism, little emphasis has been placed on their morphology and growth. In this study we quantify asexual spore (conidium) production.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. All strains were grown at 37 C on glucose minimal medium (GMM) and stored as glycerol stocks. Appropriate nutritional supplements were added to the medium as required.
Genetic crosses. –
Independent genetic crosses were performed to generate strains for use in each replicate of the two experiments. All the isolates used in crosses (TABLE I) originate from FGSC26 or the progenitor of FGSC26, FGSC4. Thus, our isolates are isogenic. For each replicate conducted, five isogenic and prototrophic lines with or without mutations at different single genes in the ST biosynthetic pathway were generated (TABLE I). The parents bearing mutations in ST biosynthetic pathway genes (RMFV3, TDB2, RJH026, TJK2) and the wild type ST producing parent (FGSC26) have been characterized (Brown et al 1996, Butchko et al 1996, Keller et al 1994, Yu et al 1996). In those studies no morphological or growth differences were observed and the chemotype differences described here were established for each mutation. RAR1 and RSCS1, progeny from independent crosses of FGSC26 and RAMC21.3, were used to generate the isogenic strains. RAR1 and RSCS1 were selected by replica plating the progeny obtained on selective media, choosing an isolate that grew only on minimal media + arginine and not on minimal media with no arginine supplement. RAR2 (ST) is a progeny of a cross between RAR1 and FGSC26. RAR3 (aflR), RAR4 (stcJ), RAR5 (stcE) and RAR6 (stcU) are the progenies of crosses between RAR1 and RMFV3, TDB2, RJH026 and TJK2 respectively. Similarly RSCS2 (ST), RSCS3 (aflR), RSCS4 (stcJ), RSCS5 (stcE) and RSCS6 (stcU) are the progenies of crosses between RSCS1 and FGSC26, RMFV3, TDB2, RJH026 and TJK2 respectively. The genotype of each strain was confirmed by Southern Blotting using cognate ST genes as probes to DNA digested with appropriate enzymes (Brown et al 1996, Butchko et al 1999, Keller et al 1994).
Assessing inoculum density. – To achieve the appropriate spore densities, spore suspensions were counted using a hemacytometer (Hausser Scientific, Horsham, Pennsylvania) and diluted to generate the desired concentrations. Simultaneously with any given inoculation five replicated mock aliquots were placed in empty tubes, these then were counted to provide for day zero counts of each strain. Thus, it was possible to estimate both the mean and variation associated with the inocula of these experiments.
Conidiation on GMM. –
For each genotype five GMM agar plates (Butchko et al 1999, Shimizu and Keller 2001) (100 x15 mm) were point inoculated with 50 000 spores suspended in 3 µL of water containing 0.01% Tween-20. The plates were incubated at 37 C under two different light conditions (24-h dark and 12-h dark/light) in growth chambers for 7 d. After 7 d, a 1 cm2 plug of agar was removed from the center of each plate and put in a 15 mL conical tube. The plug was homogenized with a pellet pestle (VWR, West Chester, Pennsylvania) using 1 mL of sterile 0.01% Tween-20. The resulting suspension was diluted as needed, vortexed 1 min, and the number of conidia were counted using the hemacytometer.
Conidiation on corn (Zea mays). – Live corn kernels (X516WX, Kaltenberg Seed Farms Inc.) were sterilized in 10% bleach for 2 min and rinsed in sterile water 1 min. Water remaining on surface-sterilized corn seeds was evaporated on sterile filter paper in a laminar flow hood for 2 h. Ten corn kernels were placed in sterile 50 mL conical tubes. Five replicate tubes were used for each genotype, inoculum concentration and light environment combination. Two inoculum concentrations were attempted for each genotype, high concentration (500 000 spores in 250 µL of 0.01% Tween-20 (Sigma-Aldrich, St. Louis, Missouri) and low concentration (50 000 spores in 250 µl of 0.01% Tween-20). The volume for the inoculum was determined experimentally as an appropriate amount of liquid to disperse the spores within the tube without promoting germination of the live corn seed. Screw-top lids were placed loosely on the tubes, with only a quarter turn to ensure they were placed similarly on each tube. The tubes were placed in random positions within Styrofoam racks inside of 32 (L) x20 (W) x15(H) cm clear plastic boxes (Sterilite). To ensure high humidity within the box, an open tube full of water was placed in the center of the rack and the box was covered with clear plastic wrap. The tubes were incubated at 37 C under two different light conditions (24-h dark and 12-h dark/light) in growth chambers for 7 d. Separate incubators were used for the light/dark (Model 2015, low temperature diurnal illumination incubator, VWR, West Chester, Pennsylvania) and the continuous dark (Model 2020, low temperature incubator, VWR, West Chester, Pennsylvania) conditions. At the end of the incubation period, the spores were harvested with an aqueous 0.01% Tween-20 solution. All tubes were vortexed 1 min before conidia were counted using a hemacytometer.
Statistical analysis. –
Factorial analyses of variance (ANOVA) were performed to discern both differences in inoculum densities (spore number at day zero) and conidia number after 7 d. The parameters tested on media included experimental replicate, light treatment and genotype. For corn, concentration of the initial inoculum was an additional factor. Because genotype represented isogenic variants interrupted along different points of the ST pathway, it was considered to be both a fixed factor and also ordinal data. All other factors also were treated as fixed. JMP-SAS program (SAS Institute, Cary, North Carolina) was used to perform ANOVA, post hoc tests and single degree of freedom contrasts. To determine the effects likely to make the greatest contribution to spore abundance, it was essential to estimate variance components (Winer et al 1991) providing for a percentage of total variance associated with each effect (TABLES II and III).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and III). On both GMM and on corn, the ST-producing isolate made the most conidia and strains blocked at aflR made the fewest conidia. This relationship was true for cultures in continuous darkness and with cycles of light and dark, as well as for both high and inoculum.
Further, in the GMM experiment there was a significant stepwise increase in conidiation associated with each progressive step in the ST pathway (Tukey’s test, all P <1.0 x10–5) (FIG. 2). This effect (i.e. ranking of genotypes) was the same under both light conditions (FIG. 2).
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). In this case, however, the ranking was not linked as precisely to the order of blocked steps in the sterigmatocystin pathway. The clear difference was that the stcE mutant, third in pathway progression represented in these experiments, fewer conidia than the stcJ mutant, second in the progression (F1,80 = 10.62, P = 0.001). The stcE mutant did exhibit significantly greater conidiation, on average, than the aflR mutant, first in the progression (F1,80 = 15.23, P = 0.0001).
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). On GMM all strains produced more spores in the light/dark treatment than in continuous darkness (FIGS. 2 and 4A). A similar light-associated enhancement of sporulation on medium has been observed by Mooney and Yager (1990). On GMM, the degree to which each genotype changed from dark to light varied (FIG. 4A), such that the two mutants arrested earliest in the ST pathway (aflR and stcJ) exhibited the greatest increase in conidiation from dark to light. Furthermore, on media the stcE mutant exhibited no change in conidiation under the two light treatments (F1,80 = 0.001, P = 0.97) and the stcU mutant and wildtype increased from dark to light but not with as great a slope as aflR and stc J mutants (FIG. 4A). All strains on corn produced more spores in continuous darkness than in the light/dark regime (FIG. 4B).
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) and accounts for 22% of the variation in the model (TABLE III). The greatest number of spores were produced in the high inoculum and continuous dark condition (FIG. 3). Accordingly, the light treatment by inoculum density interaction accounted for 15% of the variation in the corn experiment (TABLE III).
Within each replicate of the corn experiment each of the inoculum densities of the genotypes were not significantly different (replicate 1 F4,80 = 1.36, P = 0.25; replicate 2 F4,80 = 0.34, P = 0.85). However, comparison between the two replicates indicates a significantly higher number of spores on average (across all genotypes) for replicate 1 (F1, 160 = 90.16, P <0.0001). The ANOVA results for number of spores in inoculum on day zero of the media experiment revealed, as expected, no significant differences for any factor or interaction. Independent replicates of these experiments accounted for a negligible percentage of the variance each of these models (TABLES II and III). Thus despite some indication of significance differences associated with replication (TABLES II and III) these effects clearly are not paramount relative to those factors associated with substantial variance components.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Shimizu and Keller 2001) however, to the best of our knowledge, no direct relationship between the traits has been observed previously. There was a stepwise increase in conidiation of the strains in these experiments as more of the ST pathway was completed (i.e. the conidiation ranking of the four mutants was identical or quite similar to their ranking in the progression in the ST pathway, e.g., those furthest along make the most conidia on GMM and on corn); these comparisons among mutant strains alone suggest a conidiation advantage for progressing toward ST, in absence of comparisons to the non-mutated ST wild type strain. Two independent replicates of both the experiments (corn and GMM) supported the same overall pattern of relative performance on a given substrate (FIGS. 2 and 3). Thus the independently derived progeny of separate crosses resulted in the same outcome. Again, although replicate was significant in both experiments, it was associated with a negligible percentage of the variation in each model (TABLES II and III). The relative performance of these strains on a given substrate was robust across the treatments (light environment and inoculum concentration [corn only] tested; FIGS. 2 and 3). Once again this trend was reflected well in the variance component analysis, with 82–94% of all the variation in both models associated with the main effects.
The most striking difference between the GMM and corn experiments was the opposite response to light (FIG. 4). Strains grown on GMM conidiated more in the light (Mooney and Yager 1990). On corn, conidiation was greater in the dark. Corn is a substrate frequently colonized by Aspergillus spp. in agroecosystems (Adisa 1994). Moreover, on corn, the stepwise increase in conidiation response observed on GMM was not duplicated as faithfully. On corn the stcE strains showed less conidiation than the stcJ strain. The stcJ might have been able to use some of the naturally occurring fatty acids present in corn seed partially to remediate the fatty acid defect and allow low level production of ST, thus increasing the conidiation of this strain in this environment. Studies by Brown et al (1996) have demonstrated this possibility. It is noteworthy, that the stcE strains showed the least plastic response to light on media (FIG. 4A). These results might imply that accumulation of norsolorinic acid constrains conidiation more than accumulation of the other intermediates.
Fitness is defined as an individual’s contribution to the gene pool of the next generation (Roughgarden 1979). We think the use of isogenic strains and the highly clonal nature of most Aspergillus species (i.e., highly dependent on asexual sporulation) justifies speculation that the individual differences in conidiation can serve as a surrogate for fungal fitness, a parameter advocated by Pringle and Taylor (2002). Conidia represent the major form of reproduction for A. nidulans and many other cosmopolitan fungi. An obvious extension of this study will be to confirm that these individual differences in conidiation levels associated with mutations along the ST pathway actually translate into greater contribution to the next generation in mixed populations. However, individual fitness differences do not necessarily predict the behavior of strains in mixed populations. For example, presence of a fungus that completes the ST pathway might partially or fully compensate the fitness of a mutant, especially if ST itself is sporogenic. Conversely, under competition the cost of executing the ST pathway might become more pronounced, especially under nutrient-limiting conditions (Siemens et al 2002, Strauss et al 2002), resulting in reducing the conidiation of strains that complete more of the pathway. In evolutionary ecology a distinction has been made between direct effects of a trait on fitness and indirect (or ecological) effects of a trait (Strauss et al 2002). To our knowledge this study is the first demonstration of a possible fitness advantage associated with progression in a secondary metabolism pathway. While the presence of a direct effect of AF/ST does not preclude an ecological role for these secondary metabolites, it does bring to light broader questions to be tested beyond defining the ecological role, if any, of the products of these complex pathways. The discovery of direct benefits to the fungus under neutral conditions, like those in the defined media experiments, is fascinating because it sets up a mechanism by which the incremental addition of pathway components might have been favored. Recruitment or loss of pathway genes might be a target of natural selection. Finally, this study demonstrates the advantages of using a genetic model fungus for studying the ecological implications of secondary metabolism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: h-wilkinson{at}tamu.edu.
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LITERATURE CITED |
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Roughgarden J. 1979. Theory of population genetics and evolutionary ecology: an introduction. New York: Macmillan Publishing Co.
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Siemens DH, Garner SH, Mitchell-Olds T, Callaway RM. 2002. Costs of defense in the context of plant competition: Brassica rapa may grow and defend. Ecology 83: 505–517.
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Winer BJ, Donald RB, Michels KM. 1991. Statistical principles in experimental design. New York: McGraw-Hill.
Yu JH, Butchko AE, Fernandes M, Keller NP, Leonard TJ, Adams TH. 1996. Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Curr Genet 29:549–555.[Medline]
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