Association of aflatoxin biosynthesis and sclerotial development in Aspergillus parasiticus

Mycopathologia 153: 41 48, 2001. 2002 Kluwer Academic Publishers. Printed in the Netherlands. 41 Association of aflatoxin biosynthesis and sclerotial development in Aspergillus parasiticus Perng-Kuang Chang 1, Joan W. Bennett 2 & Peter J. Cotty 1 1 Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124, USA; 2 Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118, USA Received 11 May 2000; accepted 19 September 2001 Abstract Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflr, andaflj, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain, A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflr plus aflj transformants was two to three-fold that produced by the aflr transformants. This increase indicated a synergistic effect of aflr and aflj on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. Key words: Aspergillus parasiticus, aflatoxins, sclerotia Introduction Production of secondary metabolites in fungi is frequently associated with developmental processes, such as sporulation. Years of research have provided many new insights into various physiological, environmental and genetic factors coordinating these cellular processes in the meiotic Aspergillus nidulans [1]. Aflatoxins are toxic and carcinogenic polyketide-derived secondary metabolites that are produced mainly by strictly mitotic Aspergillus parasiticus, Aspergillus flavus and Aspergillus nomius [2]. These compounds are contaminants widely found in preharvest and postharvest agricultural commodities * Published in 2002. [3]. The three Aspergillus species, which do not have a sexual stage, produce specialized structures called sclerotia. Sclerotia are pigmented, compacted aggregates of hyphae, which resist unfavorable environmental conditions and capable of remaining dormant for long periods [4 6]. They are considered by some researchers to be a vestige of the sexual structures, cleistothecia, which also are woven from specialized hyphae but each contains thousands of ascospores [7, 8]. Not all isolates of A. parasiticus and A. flavus which produce aflatoxins produce sclerotia [9 11]. Nonetheless, in contrast to the positive correlation between aflatoxin biosynthesis and conidial production in A. parasiticus [12], an inverse relationship

42 between aflatoxin biosynthesis and sclerotial production has been observed. Some A. flavus strains producing significant levels of aflatoxins have smaller sclerotia [11, 13, 14]. In A. flavus strains that do produce aflatoxins and sclerotia, their formation seems to be inversely related to ph change of the agar medium, i.e., a decrease in ph stimulates aflatoxin production but inhibits sclerotial formation [4]. In A. parasiticus strains inactivation of the production of aflatoxin precursors by disrupting their polyketide synthase gene, pksa, and fatty acid synthase gene, fas-1a, two genes necessary for the formation of anthraquinone during aflatoxin biosynthesis, has been correlated with enhanced levels of sclerotial production by these mutant strains [15, 16]. But, an elevated level of accumulation of versicolorin A in A. parasiticus has been associated with inhibition of sclerotial production [17]. Although it is generally agreed upon that aflatoxin biosynthesis and sclerotial development in A. flavus and A. parasiticus are related [4, 9], the relationship between these processes is yet to be defined. Several molecular approaches have been shown to affect the accumulation levels of aflatoxins and/or their precursors in A. parasiticus. These include disrupting specific steps of the aflatoxin biosynthetic pathway [15, 16], increasing the copy number of the aflatoxin pathwayspecific regulatory gene, aflr [18], altering the promoter region of the introduced aflr gene [19], and overexpressing the carboxyl-terminal coding region of aflr [20]. Some of the approaches also have been used in examining the relationship between aflatoxin biosynthesis and sclerotial development [15, 16, 20]. In this study, we determined the effects of introduction of aflatoxin biosynthetic pathway genes, aflr and aflj [21], on sclerotial development in A. parasiticus SRRC 2043. Elevated levels of accumulation of aflatoxin precursors were found to affect sclerotial development negatively. Discernible changes included decrease in sclerotial size, alteration in sclerotial shape and weakening in sclerotial structure. Materials and methods Fungal strains, media and culture conditions A. parasiticus SRRC 2043 (ATCC 62882) and its isogenic derivatives were used. SRRC 2043 accumulates predominantly OMST due to a defect in the orda gene, which encodes a P450 monooxygenase that converts OMST to aflatoxin B 1 [22]. A. parasiticus RHN1 [18], a NiaD (nitrate reductase) mutant derived from strain 2043, was the recipient strain used in transformation. Production of aflatoxin precursors and sclerotia were carried out on Potato Dextrose Agar (PDA) and Czapek Solution Agar (CZ) plates (Difco, Detroit, Mich.). Five microliters of diluted fungal spore suspensions containing about 1,000 spores were inoculated at the center of the nine-centimeter Petri dish plates containing 25 ml medium. Cultures were incubated in the dark at 30 C. Fungal transformation Vectors containing aflatoxin biosynthetic genes, alfr, aflj and aflr plus aflj were introduced into A. parasiticus RHN1 by a niad-based polyethylene glycol- CaCl 2 mediated fungal transformation protocol [23]. Determination of aflatoxin precursors The semi-quantitative thin layer chromatography (TLC) method [20] was used to examine the precursor profiles of the non-pigmented transformants, Trans J, and the pigmented transformants Trans R and Trans R+J, which had different extents of pigmentation on PDA and CZ plates. For scale-up production of aflatoxin precursors, two-week-old culture plates containing three transformants of each type were extracted twice with acetone and chloroform as described earlier [24]. Extracted aflatoxin precursors were separated by TLC with a toluene-ethyl acetate-acetic acid (50:30:4, vol/vol/vol) solvent system. Norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST were quantified by scanning densitometry with a Shimadzu CS9OOOU dual-wavelength flying-spot scanner using the following conditions: D 2 lamp, no filter and the reflection photo mode were used for AVN and VERA analysis at 290 nm and NOR at 300 nm; xenon lamp, #2 filter and fluorescence mode were used for OMST at 310 nm. Amounts were calculated by comparison with known aflatoxin precursor standards. Sclerotial visualization by scanning electron microscopy Pooled sclerotia, dislodged from PDA plates, were spread onto the 0.5-in Cambridge scanning electron microscopy stubs, coated with 20 to 30 nm of 60:40 gold-palladium in a Technical Hummer II sputter coater, and viewed in Cambridge S-250 scanning electron microscope operating at 6 to 10 KV and a

43 magnification range of 20X to 50X as described earlier [25]. For observation of sclerotial structure on agar plates, PDA plugs (1/2 inch in diameter) were cored and immersed in absolute alcohol at room temperature for 30 min with three changes. The agar plugs were then placed onto two sheets of 3MM filter paper, covered with Parafilm and another sheet of 3MM paper, compressed with a light weight to prevent curving due to dehydration, and placed in a vacuum chamber overnight at room temperature. The flattened dehydrated agar plugs were mounted on the stubs and processed as described above. Results and discussion Introduction of aflatoxin biosynthetic pathway genes, aflr, aflj and aflr plus aflj affected the accumulation levels of aflatoxin precursors in A. parasiticus SRRC 2043 differently. The relative amounts of the precursors, NOR, VERA, AVN and OMST accumulated in each type of transformant are shown in Figure 1. The aflr plus aflj transformants appeared to accumulate the highest levels of aflatoxin precursors, followed by the aflr transformants. A detailed analysis indicated that, in contrast to 2043, which produced predominantly OMST on PDA but little OMST on CZ, the aflr transformants and the aflr plus aflj transformants produced substantially higher amounts of NOR, AVN, VERA and OMST on both media (Table 1). The total amount of NOR, AVN, VERA and OMST accumulated in the aflr plus aflj transformants was 2 to 3-fold that in the aflr transformants (Table 1). The role of the aflr gene in activating the transcription of the genes responsible for the synthesis of aflatoxin and sterigmatocystin has been established in A. parasiticus, A. flavus and A. nidulans [26 28]. Although the aflj gene is necessary for aflatoxin biosynthesis [21], its function is not yet clear. Possible roles of the AFLJ protein in the transport of aflatoxin precursors through intracellular compartments and in the localization of aflatoxin biosynthetic enzymes have been proposed [21]. However a specific protein-protein interaction was detected between the A. parasiticus AFLR and AFLJ proteins (unpublished data). This result suggests that AFLJ may be a component of the transcription machinery and function as a co-activator to AFLR in A. parasiticus during aflatoxin biosynthesis. The further elevated accumulation of the aflatoxin precursors in the aflr plus aflj transformants probably results from a synergistic effect of these two genes. There- Figure 1. The representative profile of aflatoxin precursors accumulated in isogenic A. parasiticus strains when grown on PDA medium. 2043: the parental strain; RHN1: the NiaD recipient strain used in transformation; Trans J, aflj transformants; Trans R+J: aflr plus aflj transformants; Trans R: aflr transformants. Numbers 1, 2 and 3 indicate different transformants. Aflatoxin precursors: NOR, norsolorinic acid; VERA, versicolorin A; AVN, averantin; OMST, O-methylsterigmatocystin. Agar plugs from each plate were extracted as previously described [20]. Appropriately diluted amounts of the solvent extracts from each type of the transformant were spotted onto the TLV plate to avoid overloading. fore, aflr and aflj appear to have a direct, positive and coordinated effect on aflatoxin biosynthesis as those activators and co-activators reported for other systems [29, 30]. An increase in the total amounts of aflatoxin precursors (Table 1) seemed to coincide with changes in sclerotial development (Figures 2 and 3). Compare to the sclerotia of 2043, the sclerotia produced by each type of transformant showed some variations in size. Nonetheless, an inverse relationship was found between the levels of accumulation of aflatoxin precursors and the overall sclerotial size of the transformants (Table 1 and Figure 2A D). A comparison of the sclerotial dimension, i.e., length and width, is shown in Figure 4. An increase in the accumulation

44 Table 1. Aflatoxin precursors produced by A. parasiticus strains grown on PDA and CZ media Strains Medium Aflatoxin precursor (mg) a NOR AVN VERA OMST Total est. b 2043 PDA Tr. c Tr. Tr. 0.98 0.98 CZ Tr. Tr. Tr. 0.06 0.06 Trans J PDA Tr. Tr. Tr. 0.94 0.94 CZ Tr. Tr. Tr. 0.09 0.09 Trans R PDA 2.15 1.15 0.53 4.15 7.98 CZ 4.26 1.27 2.68 2.39 10.6 Trans R+J PDA 7.48 4.39 1.12 8.14 21.13 CZ 14.84 5.07 2.61 4.93 27.45 a The averages of Trans J, Trans R and Trans R+J were obtained from three independent transformants grown on Petri dish plates; standard deviations are within 15%. b The total estimated amount was calculated as the sum of NOR, AVN, VERA and OMST. c Trace (<0.02). of aflatoxin precursors by the aflr transformants and the aflr plus aflj transformants was associated with a progressive decrease in their sclerotial size. Likewise, A. flavus strains producing significant levels of aflatoxins tend to produce smaller sclerotia [11, 13, 14]. In this study. the decrease in the sclerotial size was also accompanied by a change in the sclerotial shape from being oval to elongated (Figure 2) as evidenced by reduction of the width to length ratio from 0.73 to 0.54 (Figure 4). A change of the sclerotia from round shape to a pointed, somewhat bullet-like shape was observed on CZ plates when substantial amounts of the aflatoxin precursors were accumulated in the aflr plus aflj transformants (Table 1 and Figure 3). This morphological change was also reproducible when the aflr and aflj genes were introduced into an aflatoxigenic A. parasiticus strain, SU-1, which produced much lower numbers of sclerotia than SRRC 2043 (unpublished data). Termination of sclerotial maturation has been proposed to be associated with a signal that modulates aflatoxin biosynthesis [31]. The elongation of sclerotia on both media thus may indicate delayed termination/completion of a pre-melaninization phase in sclerotial development (see below). An increase in the levels of accumulation of aflatoxin precursors generally was associated with an increased production of sclerotia on PDA plates. But, such a correlation was not observed for transformants on CZ plates (unpublished data). Morphological stages associated with sclerotial formation consist of (i) the appearance of white tufts of thick mycelial initials, (ii) the enlargement and hardening of the mycelial tufts and the appearance of exudate on the surface, (iii) the continued hardening due to surface delimitation, and (iv) melanin deposition in the peripheral rind cells, leaving a smooth, hard, dark surface [32, 33]. The sclerotial surface of 2043, which does not produce high levels of aflatoxin precursors, was relatively smooth (Figure 2E). In contrast, a substantial increase in the accumulation of aflatoxin precursors as that seen in the aflr and aflj transformants not only decreased the sclerotial size, but also altered the sclerotial surface to a sieve-like appearance (Figure 2F). Sclerotia having the sievelike structure were fragile and easily crushed open by an inoculation needle. This sieve-like structure suggests that the sclerotia produced by the aflr plus aflj transformants may not have reached full maturation. The weakening in sclerotial structure might be, in part, due to fewer layers of interwoven hyphae (rind cells) or less development of the extracellular matrix and melaninization, which normally obscure the rind hyphae on the sclerotial surface in the maturation phase. Overexpression of pathway-specific developmental regulatory genes has been used to assess the relationship between vegetative growth and asexual development in A. nidulans [34]. New insights into the mechanisms associated with sexual/asexual development, secondary metabolism and mycelial proliferation are emerging. Hicks et al. (1997) demonstrated that a signal transduction pathway is critical

45 Figure 2. Comparison of size, shape and surface structure of the sclerotia produced by isogenic A. parasiticus strains grown on PDA medium. Representative scanning electron micrographs of the pooled, dislodged sclerotia from (A) SRRC 2043 (B) aflj transformants (C) aflr transformants (D) aflr plus aflj transformants; magnification: 20. Sclerotia produced by (E) SRRC 2043 (F) the aflr plus aflj transformants on PDA plates before being dislodged; magnification: 50. for the regulation of conidiation and sterigmatocystin biosynthesis in A. nidulans [35]. Zhou et al. (2000) reported that disruption of the A. parasiticus flup gene, which encodes a polyketide synthase, resulted in substantial reduction or elimination of asexual spores, the appearance of fluffy, cotton-like hyphae, and two-fold reduction in aflatoxin accumulation [361. Guzman-de- Pena et al. (1998) found a positive correlation between cleistothecial formation and sterigmatocystin production in wild type and mutant strains of Emericella nidulans (Aspergillus nidulans); conditions which favored sporulation stimulated sterigmatocystin formation [37]. Although Calvo et al. (1999) recently showed that the development of conidial spores and production of sclerotia in A. parasiticus and A. flavus was affected by linoleic acid, whose degraded product might mimic the action of psi (precocious sexual inducer) factors [38], little is known about the mechanisms that regulate aflatoxin biosynthesis and sclerotial development. Sclerotial morphogenesis is a complex process. A variety of environmental and nutritional factors are

46 Figure 3. Representative sclerotial morphology of aflr plus aflj transformants (A) and niad vector RHN1 transformants (B) grown on CZ medium. Twenty-five visually largest sclerotia from each type of transformant were selected and adhered to a Petri dish with two sided tape [4]. Images of sclerotia were captured on a transilluminator using a video camera with macro lens. The niad vector transformants produced sclerotia having the morphology similar to that of SRRC 2043. RNH1, a NiaD mutant, is unable to utilize nitrate; it does not grow on CZ medium, which contains nitrate as the sole nitrogen source. Figure 4. Comparison of the length and the width of the sclerotia produced by isogenic A. parasiticus strains grown on PDA medium. Approximately 50 to 100 of the pooled sclerotia of each type in scanning electron micrographs were manually measured. The curved line shows the change of the width to length ratio, which is used as an indicator for the change in the sclerotial shape. known to affect its development, including light, temperature, substrate, ph, organic acids, stale products, phenolics, polyphenoloxidase activities and endogenous camp levels, [6, 9, 32, 33, 38]. An examination of our data and those from the above studies [35 38] suggests that the observed changes are not caused by the introduced aflr and aflr plus aflj on a upper level of the regulatory cascade involved in sclerotial development. Rather, it implies that a competition for the same factor(s) could affect aflatoxin biosynthesis and sclerotial development in A. parasiticus [11, 13 16]. Larroche [39] proposed that competition for nutrients and environmental factors governs growth and development in fungi. Acetate, the building block

47 for polyketides such as aflatoxins and their precursors (nosolorinic acid, averatin, versicolorin A, O- methylsterigmatocystin, etc.) [40], is also the basic component for polyphenolic compounds, such as sclerin, melanin, conidial pigments, and cleistin, which are important for the development of sclerotia, conidia and cleistothecia, respectively [8, 32, 33, 41, 42]. An elevation of the activities of aflatoxin biosynthetic enzymes, which results from the introduction of additional aflr and aflr plus aflj genes into A. parasiticus, would divert the metabolic flux (the utilization of acetate) toward aflatoxin biosynthesis. This and other related physiological changes could result in a competition that favors aflatoxin biosynthesis (compared to the state in the wild-type). Consequently, it affects sclerotial development, as manifested in the altered sclerotial size, shape, surface structure and numbers. Acknowledgments We thank B. Ingber, H. Holen and B. 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