1 Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706, USA
2 Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA
3 Southern Regional Research Center, Agricultural Research Service, US Department of Agriculture, New Orleans, LA 70124, USA
Correspondence
Nancy P. Keller
npk{at}plantpath.wisc.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF528822.
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INTRODUCTION |
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Oleic and linoleic acids are the most common unsaturated fatty acid components of Aspergillus and oil seeds (Bewley & Black, 1985; Evans et al., 1986
; Calvo et al., 2001
). Due to the requirement for oleic acid, linoleic acid and linolenic acid in generating psi factor and LOX products, our investigations have been aimed at understanding the connections between fatty acid metabolism and fungal development and pathogenesis in this hostparasite interaction. In particular, our goals are to generate and characterize Aspergillus mutants that are unable to synthesize polysaturated fatty acids. We previously disrupted the
12-oleic acid desaturase gene, odeA (Calvo et al., 2001
), in the saprophyte Aspergillus nidulans and showed how this mutation leads to almost total loss of polyunsaturated fatty acids and altered conidial and ascospore development. Here, we describe how disrupting an odeA orthologue, ApodeA, in the oilseed pathogen A. parasiticus similarly affects fatty acid biosynthesis and results in delayed spore germination, reduced growth and impaired developmental processes in this agronomically important plant pathogen. In addition, this mutant is also severely compromised in its capacity to colonize host corn and peanut seed, as determined by reduced conidiation on seed.
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METHODS |
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Total RNA was extracted from mycelia by using Trizol (Life Technologies). Aliquots of RNA (10 µg) were separated on a 1·2 % agarose/1·5 % formaldehyde gel. RNA was transferred to a Hybond membrane (Amersham Biosciences) by capillary action. fasA gene expression was analysed by using a 0·5 kbp PCR-amplified fragment of A. nidulans fasA (GenBank accession no. U75347), which was generated with the primers fasAF (5'-GGATTCCACAGCGG-3') and fasAR (5'-GGGAGCACGGAGAG-3') from genomic DNA of strain RDIT9.32.
DNA fragments to be used as probes were radiolabelled with 32P by using the random primer method (Sambrook et al., 1989). Following prehybridization and addition of the probe, membranes were hybridized overnight at 60 °C and washed with increasing stringency up to 0·1x SSC, 0·1 % SDS at 60 °C.
Identification, sequencing and disruption of the 12-oleic acid desaturase-encoding gene, odeA, from A. parasiticus.
The odeA gene was sequenced by using previously described techniques and software (Calvo et al., 2001).
The cosmid pAMC8, containing the A. parasiticus odeA gene, was identified by heterologous hybridization of an A. parasiticus genomic library with a 12-desaturase-encoding gene from Candida albicans, as described by Calvo et al. (2001)
. The sequence of the odeA gene has been deposited in GenBank under accession no. AF528822.
The A. parasiticus odeA disruption vector pAMC37 was created by introducing a pyrG coding-sequence cassette into the ORF of odeA. Firstly, plasmid pBZ5 (kindly provided by Dr J. E. Linz, Department of Food Sciences and Human Nutrition, Michigan State University, East Lansing, MI, USA) was digested to release a 2·8 kbp EcoRIHindIII fragment containing the pyrG gene marker from A. parasiticus. The fragment was ligated into pBluescript KS (Stratagene) that had been previously digested with EcoRI and HindIII, generating the plasmid pAMC34. A 4 kbp fragment corresponding to the 5' flanking region of odeA was amplified from genomic DNA by PCR with proof-reading Pfu polymerase (Stratagene) and the primers ApodeAF1 (5'-GCTGTGAAGCTTCTTCCGCAG-3') and ApodeAR1 (5'-GGTCCGAAGCTTGCTATATCTGG-3'). Thermocycler conditions were 2 min at 94 °C, followed by 30 cycles of 96 °C denaturing (1 min), 48 °C annealing (1 min) and 72 °C extension (4 min 30 s). Each of these two primers incorporates a new HindIII site, which were used to ligate the digested PCR product into the HindIII site of pAMC34. Next, a 6 kbp EcoRI fragment corresponding to the 3' end of the gene was released from pAMC8 and incorporated into the vector at the EcoRI site adjacent to the pyrG cassette, to give pAMC37. Therefore, pAMC37 represents 11 kbp of the gene and flanking regions of odeA, but has 1047 bp of the coding region removed and replaced by pyrG. Transformation of the A. parasiticus pyrG strain CS10 with pAMC37 was performed by using previously described techniques (Skory et al., 1992) and transformants were initially selected based on their ability to grow on GMM lacking the supplements (uracil and uridine) that are required by the pyrG parent.
Physiological studies.
Germination studies were performed by inoculating 106 spores of each strain into 25 ml liquid GMM and shaking at 29 °C. Colony diameters were measured by point-inoculating 5 µl water containing 106 spores of either strain onto the centre of plates containing 25 ml GMM agar, followed by growth at 29 °C for 5 days. Conidial production studies were performed on plates containing 25 ml GMM agar. For each plate, a 5 ml top layer of cool but molten agar that contained 106 spores of the appropriate strain was added. For each strain, there was a minimum of four replicate plates. Strains were grown in continuous light or dark for 72 h (unless otherwise stated) at 29 °C. A core of 15 mm was removed from the plates at the appropriate time interval and homogenized in 2 ml 0·01 % Tween 80 in water to release the spores. Spores were counted on a haemocytometer.
To study the effect of the odeA mutation on sclerotial production in A. parasiticus, strains were grown on coconut agar medium (CAM) (Trail et al., 1995
) and GMM agar. To 30 ml CAM was added a 5 ml top layer of cool but molten agar that contained 106 spores of the appropriate strains. For GMM agar, 5 µl water containing 106 spores of either strain was point-inoculated onto the centre of each plate. Plates were incubated in the dark at 29 °C for 14 days for CAM and 7 days for GMM agar. After this time, plates were sprayed with ethanol to flatten conidia and total numbers of sclerotia per plate were counted by using a dissecting scope. Both CAM and GMM agar experiments were carried out with four replicates.
Colonization studies.
Seeds of the near-isogenic Sunrunner and SunOleic97R peanut lines were kindly provided by Dr C. Holbrook (USDA, GA, USA). Sunrunner has 50 % oleic acid and 30 % linoleic acid content, whilst SunOleic97R is a high oleic acid line that has 80 % oleic acid and approximately 2 % linoleic acid content. Prior to infection by A. parasiticus or A. nidulans strains, the seeds were shelled, the cotyledons separated and the embryo removed. Seeds from both peanut lines were weighed so that all the peanuts used were between 0·4 and 0·6 g in weight. For dead-seed experiments, the cotyledons were autoclaved in a liquid cycle for 30 min. For living-seed experiments, cotyledons were surface-sterilized by immersion in 10 % Clorox bleach for 1 min, followed by immersion in sterile distilled water for 1 min. For A. parasiticus, the seeds were inoculated as follows: for each strain, four peanut cotyledons were placed in a 50 ml Falcon tube and to this was added 500 µl sterile distilled water containing 106 spores of the appropriate strain. Each tube was vortexed for 1 min, the caps were loosened and the samples were placed in the dark at 29 °C for 4 days. These experiments were performed with four replicates. Simultaneously, GMM agar plates were inoculated, using the top-layer method described above, with the same spore suspensions that were used to infect the peanut seed (at a rate of 106 spores per plate, four replicates for each) and placed in the incubator beside the infected seed. After 4 days, spores on the plates were counted as described above. Spores from the infected seed were released by adding 5 ml Tween 80/water to each Falcon tube, vortexing for 1 min and using an aliquot of this for counting using a haemocytometer.
A. nidulans does not readily infect peanut seed and so the inoculation protocol was modified as follows. One cotyledon was immersed in 1 ml sterile distilled water containing 106 spores of the appropriate strain for 30 min, with vigorous vortexing every 10 min. This was repeated six times for each experiment. After 30 min, the infected peanut seeds were placed in a glass Petri dish containing water-saturated filter paper and a water reservoir and left in the dark at 29 °C for 4 days (in the case of dead seed) and 6 days (in the case of live seed). Plates of GMM agar that were inoculated with the same strains were placed in the incubator simultaneously and counted at the same time points as the infected seed. Spores were released from the seed by using Tween 80/water and vortexing for 1 min. Four cotyledons were used for counting per treatment.
Corn seeds were surface-sterilized as described above. Four seeds were placed in one 50 ml Falcon tube and inoculated as described above for A. parasiticus. Tubes were placed at 29 °C under dark conditions for 4 days. Each experiment was performed with four replicates.
In all seed-inoculation experiments, Koch's postulates were determined after spore-counting to verify the identity of the Aspergillus strains infecting the seed.
Fatty acid analysis.
Fatty acid methyl esters were generated from Aspergillus mycelium for GC analysis as follows. After growth in GMM for 72 h under conditions of constant light, mycelia were lyophilized and ground into fine powder and the lipids were extracted three times in chloroform : methanol (2 : 1) following the method of Bligh & Dyer (1959). Heptadecanoic acid (17 : 0; 0·64 ng) was added as an internal standard. The samples were dried down, resuspended in 1 ml 5 % HCl in 90 % methanol and placed at 95 °C for 30 min to generate fatty acid methyl esters (FAMEs). The methylating reaction was quenched with water and the FAMEs were extracted three times in hexane. The samples were concentrated and a 1 µl aliquot of the hexane layer was examined by GC. Identification of peaks was achieved by comparison of sample retention times to those of palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acid standards (FAME-GC mix; Sigma).
Statistical analysis.
Spore data and colony diameters were evaluated by analysis of variance (ANOVA) using the Statistical Analysis system (SAS Institute, Cary, NC, USA).
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RESULTS |
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Disruption of the A. parasiticus odeA gene
The 12-oleic acid desaturase gene of A. parasiticus was disrupted by homologous recombination as depicted in Fig. 1
a. A rapid screen of 96 pyrG+ transformants for integration of pyrG at the odeA locus was employed. Undigested genomic DNA from these transformants was run out on an agarose gel and probed with the 0·7 kbp BamHIEcoRI internal fragment of odeA, shown hatched in Fig. 1a
. One transformant, TAMC37.41, did not hybridize to the odeA probe (Fig. 1b
, lane 2), indicating removal of the odeA coding region from the genome. Single-copy integration of pyrG at the odeA locus in TAMC37.41 was subsequently confirmed by Southern blot analysis following PstI restriction digestion, using the full-length odeA coding region as a probe, in addition to DNA sequencing and PCR analysis (data not shown).
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12-desaturase mutation reduced A. parasiticus conidial production and inhibited sclerotial development
Conidial production of A. parasiticus odeA and wild-type strains after 3 days growth at 29 °C in conditions of continuous light and 4 days growth in conditions of continuous darkness is shown in Fig. 3
. The A. parasiticus
odeA mutant was significantly reduced in conidial development, compared to the wild-type strain, under both conditions.
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odeA mutant strains were impaired in the colonization of peanut seed
We assayed for the ability of A. parasiticus and A. nidulans odeA strains, termed
ApodeA and
AnodeA, respectively, and the corresponding wild-type controls, to colonize two isogenic lines of peanut seed that differed only in oleic acid : linoleic acid ratios. Colonization was assessed in terms of conidial production on seed. Sunrunner has typical peanut seed oil content, containing 50 % oleic acid and 30 % linoleic acid [see Norden et al. (1985)
and references therein]. SunOleic97R has increased oleic acid (80 %) and reduced linoleic acid (25 %) contents (Gorbet & Knauft, 2000
; Andersen & Gorbet, 2002
). These differences are due to recessive mutations of odeA orthologues in SunOleic97R (Knauft et al., 1993
; Jung et al., 2000
). Both live and dead seeds were used to assess the interactions between host seed and the fungus.
Wild-type A. parasiticus produced significantly more conidia (P0·05) on all peanut seeds than did
ApodeA. Wild-type A. parasiticus showed no significant differences (P
0·05) in conidiation on any peanut seed, live or dead (Fig. 4
), after 96 h growth. In control-plate growth tests, wild-type produced significantly more conidia than did
ApodeA (Fig. 3b
, P
0·05) on GMM under dark conditions at 29 °C for 96 h.
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DISCUSSION |
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The odeA gene, which encodes a 12-oleic acid desaturase that is involved in converting oleic acid to linoleic acid, was identified and disrupted in A. parasiticus. Mycelia of the resulting
odeA mutant (
ApodeA) had a similar fatty acid content to the A. nidulans
odeA mutant (
AnodeA; Calvo et al., 2001
), with dramatically increased oleic acid content and reduced contents of linoleic and linolenic acids (Table 2
). In addition, total fatty acid content accrued in
ApodeA to a level five times greater than that seen for wild-type. Calvo et al. (2001)
reported a similar increase in the total fatty acid content of
AnodeA and attributed it to a lack of polyunsaturated fatty acid inhibition of fasA gene expression, leading to upregulation of fatty acid biosynthesis. The increase in fatty acid content in
ApodeA (Table 2
), coupled with increased fasA gene expression (Fig. 2
), suggests that the feedback inhibition mechanism is conserved in A. parasiticus.
Linoleic acid is a major constituent of fungal lipid and commonly comprises 3050 % of the total fatty acid content in mycelia and conidia of aspergilli (Table 2; Singh & Sood, 1973
; Sood & Singh, 1973
; Rambo & Bean, 1974
; Budinska et al., 1981
; Evans et al., 1986
; Chattopadhyay et al., 1987
; Calvo et al., 2001
). Several studies have shown that various unsaturated fatty acids affect Aspergillus developmental processes (Mazur et al., 1990
, 1991
; Calvo et al., 1999
), whilst ratios of unsaturated : saturated fatty acids are important for Aspergillus and Mucor development (Calvo et al., 1999
; Khunyoshyeng et al., 2002
). In A. nidulans, specific individual fatty acids are also required at different stages of development. For example, Evans et al. (1986)
reported that mycelial linolenic acid concentration was prominent in 1-day-old cultures, but declined to trace levels in cultures older than 3 days. The
ApodeA mutant was altered in all these parameters of fatty acid metabolism, displaying reduced production of linoleic and linolenic acids and altered ratios of unsaturated : saturated fatty acids. It was consequently defective in rates of spore germination, colony growth and development. In a parallel study, a
odeA mutation that was generated in an O-methylsterigmatocystin-accumulating strain of A. parasiticus, SRRC 2043, also resulted in altered conidiation and sclerotial development (Chang et al., 2004
). This supports our conclusion that the reduction in conidiation and abolishment of sclerotial production seen for
ApodeA compared to the wild-type is due solely to aberrant polyunsaturated fatty acid metabolism and serves to emphasize the genetic connection between fatty acid metabolism and fungal development.
The role of fatty acid metabolism in the plantfungal interaction was studied by using two peanut lines and two corn lines. Because of an increasing interest in developing peanut seed with higher oleic and lower linoleic acid content, due to the value of monounsaturated fatty acids in the diet (Massaro et al., 1999), and considering the importance of fatty acids in Aspergillus development, we investigated whether Aspergillus species differentially colonized two near-isogenic commercial peanut lines with altered fatty acid content. One line is SunOleic97R, which contains high amounts of oleic acid (80 %), but reduced amounts (25 %) of linoleic acid and undetectable amounts of linolenic acid. Another is Sunrunner, which contains 50 % oleic acid and 30 % linoleic acid (Holbrook et al., 2000
). For the corn lines, we chose Asgrow 404 and Tex6, lines that exhibit some susceptibility and resistance to aflatoxin contamination, respectively, in the field (Hamblin & White, 2000
). Conidiation of wild-type A. parasiticus and A. nidulans on live peanut seed did not differ between these two lines, indicating that the reduced amount of linoleic acid in SunOleic97R is still sufficient to induce and support conidiation. In contrast, the
odeA mutants of A. parasiticus and A. nidulans exhibited a significant reduction in conidiation on live peanut seed (Figs 4 and 5
), indicating a direct or indirect role for the odeA gene in fungal development on peanut seed. On corn seed, the A. parasiticus
odeA strain also produced fewer conidia on both corn lines, compared to the wild-type (Fig. 6
). Wild-type A. parasiticus did not produce significantly different amounts of conidia on dead Asgrow 404, live Tex6 and dead Tex6 seed (data not shown). However, conidial production of wild-type A. parasiticus was reduced significantly on live Asgrow 404 (P
0·05), suggesting an inhibitory effect of this line that is not seen for Tex6. This observation is interesting as it contrasts with the situation observed in the field, where the Asgrow 404 line is more susceptible to aflatoxin contamination, whereas Tex6 has some resistance (Hamblin & White, 2000
). Nonetheless, reducing fungal colonization and sporulation would be a prerequisite for curbing aflatoxin contamination of crops. Taken together, these results suggest that whilst altering the fatty acid content of oil seeds might not reduce Aspergillus conidiation and probable spread of the pathogen, inactivation of the fungal odeA gene may do so. Therefore, a promising strategy for controlling contamination could be the selective targeting and inactivation of fungal
12-desaturases by fungicides.
Wild-type A. parasiticus did not produce different amounts of conidia on either live or dead peanut lines, suggesting that the plant defensive response of live seed is not effective to reduce the development of this fungus. In contrast, all A. nidulans strains produced more conidia on dead seed than on live seed, even when growth on live seed was prolonged. This indicates the effectiveness of the plant defensive response against A. nidulans, which is not a pathogen of peanut in nature.
This study shows that pathogenic differences exist between A. nidulans and A. parasiticus and that loss-of-function desaturase mutants of both species are impaired in colonization abilities. One future course of our study will be an in-depth analysis of the interactions that are involved in the colonization of oilseed crops by A. nidulans and A. parasiticus and the closely related A. flavus. By using available genomic and expressed sequence tag resources of A. nidulans and A. flavus, coupled with microarray technology, subtractive libraries and proteomics, one could further dissect the Aspergillusseed interaction to identify the molecular bases for differences in colonization by saprophytic, pathogenic and mutant aspergilli as new targets for reducing the infestation of oilseed crops by these fungi.
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ACKNOWLEDGEMENTS |
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Received 1 April 2004;
revised 16 June 2004;
accepted 24 June 2004.
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