Present addresses: Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Science Center, Manhattan, KS 66506, USA. ‡Plant Biotechnology Division, National Institute of Agricultural Biotechnology, RDA, Suwon, 441-100, Korea.
Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae
Article first published online: 22 AUG 2003
Volume 50, Issue 1, pages 145–152, October 2003
How to Cite
Lee, J., Lee, T., Lee, Y.-W., Yun, S.-H. and Turgeon, B. G. (2003), Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae. Molecular Microbiology, 50: 145–152. doi: 10.1046/j.1365-2958.2003.03694.x
- Issue published online: 22 AUG 2003
- Article first published online: 22 AUG 2003
- Accepted 27 June, 2003.
Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild-type haploid MAT1-1; MAT1-2 strain, resulting in MAT1-1; mat1-2, mat1-1; MAT1-2 strains that were self-sterile, yet able to cross to wild-type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self-fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.
Ascomycetous fungi initiate the process of sexual reproduction by requisite interaction with a partner or by selfing. Self-sterile fungi are called heterothallic, whereas self-fertile fungi are homothallic. In ascomycetes, the ability to mate is controlled by the mating type locus (MAT), a single master regulator (Glass et al., 1988; Nelson, 1996; Coppin et al., 1997; Turgeon, 1998). In heterothallic species, this locus carries alternate, dissimilar sequences (idiomorphs) that encode unrelated transcription factors (Nelson, 1996; Coppin et al., 1997; Turgeon, 1998). In most homothallic species, in which it has been examined at the molecular level of resolution, the single MAT locus encodes counterparts of all heterothallic factors (Beatty et al., 1994; Glass and Smith, 1994; Yun et al., 1999).
In previous work with a diverse collection of heterothallic and homothallic isolates of the genus Cochliobolus, we determined that the heterothallic reproductive mode is ancestral to the homothallic mode and that a recombination event within the MAT genes themselves is likely to be the mechanism initiating the change in reproductive mode. Using a closely related heterothallic and homothallic pair of species, we also demonstrated that ‘evolution’ from heterothallic to homothallic reproductive mode could be achieved in vitro, when we expressed MAT from the homothallic isolate in a MAT null strain of the heterothallic species. Conversion to homothallism indicated that the determinants of homothallism resided within the MAT gene (Yun et al., 1999).
To address the issue of whether or not the reverse experiment is possible, i.e. can a homothallic strain be made obligately heterothallic, we turned our attention to Gibberella zeae, a haploid pyrenomycete that is both homothallic and capable of outcrossing. Previous studies have shown that the single G. zeae MAT locus (MAT1) carries closely linked counterparts of the MAT genes found in heterothallic species in this group (Yun et al., 2000). This includes three MAT1-1 genes and one MAT1-2 gene, all required for mating. For clarity, as there is no convention for MAT nomenclature in homothallic ascomycetes (Turgeon and Yoder, 2000), we have used MAT1-1; MAT1-2 as the wild-type designation, reflecting the fact that both types of MAT sequence are present, and we have used MAT1-1; mat1-2 or mat1-1; MAT1-2 for cases in which one has been deleted.
Gibberella zeae is an economically important cereal pathogen of ubiquitous geographical distribution (Cook, 1981; McMullen et al., 1997). It is also a producer of mycotoxins that threaten human and animal health (Marasas et al., 1984). If we could create strains of G. zeae that easily and obligately cross in a heterothallic manner, the cause of analysis of traits of interest, especially those associated with virulence or mycotoxin production, would be advanced. Thus, the objective of this study was to test whether it was possible to convert homothallic G. zeae to an obligately heterothallic fungus. In attempting to do this, we were also testing whether or not both MAT1-1 and MAT1-2 counterparts of heterothallic fungi were required for homothallism.
Deletion of MAT1-1 and MAT1-2
For deletion of MAT1-2, an internal portion of the MAT1-2 gene was replaced with the vector pIGΔMAT2, carrying the marker hygB, which allowed selection on hygromycin B-containing medium; pIGΔMAT2 was linearized to promote integration via double cross-over (Fig. 1A). For deletion of the three MAT1-1 genes, most of the MAT1-1 idiomorph sequence was replaced with vector pΔGzMAT1, carrying chloroamphenicol (chl), which could not be selected for. For selection, the marker gen, which allowed selection on geneticin-containing medium, was introduced at an ectopic site by co-transformation. pΔGzMAT1 was linearized to promote integration via double cross-over (Fig. 1B). These integrations resulted in transgenic strains of strain Z3639 or H-11 that were MAT1-1; mat1-2 or mat1-1; MAT1-2 as determined by gel blot analysis (Fig. 1C and D). DNA corresponding to MAT1-1; mat1-2 transformants T39ΔM2-1, T39ΔM2-3 and T39ΔM2-4 (derived from strain Z3639; Fig. 1C, lanes 2–4) and THΔM2-3, THΔM2-5 and THΔM2-9 (derived from strain H-11; Fig. 1C, lanes 6–8) carried a single 7.8 kb band (Fig. 1C, asterisk) that replaced the wild-type 2.4 kb band (Fig. 1C, arrowhead). This confirmed that a 0.5 kb region of the MAT1-2 gene including the entire 273 bp HMG box sequence (Coppin et al., 1997; Yun et al., 2000) had been deleted and replaced with the linearized fragment of pIGΔMAT2 carrying hygB and GFP; the MAT1-1 genes were left intact (Fig. 1A).
Transformants deleted for the MAT1-1 genes were obtained by co-transformation with linearized pΔGzMAT1 and circular pII99 (Fig. 1B); the expectation was that pΔGzMAT1 would replace the MAT1-1 genes, and pII99, carrying gen, would insert randomly. DNA gel blot analysis revealed one transformant of strain Z3639 (T39ΔM1-3, Fig. 1D, lane 2) that lacked the wild-type 7.5 kb band and had the expected 6.7 kb band. The pattern of hybridization in lane 2 confirmed that, in T39ΔM1-3, a 4.2 kb region of MAT1-1 had been deleted and replaced with pΔGzMAT1 (Fig. 1B).
MAT deletion strains are not self-fertile
Each type of mat deletion strain (MAT1-1; mat1-2 and mat1-1; MAT1-2) was selfed. Wild-type strains (Z3639, H-11 and 88-1) formed abundant perithecia on mating plates by 2 weeks. In contrast, each MAT deletion strain tested formed no perithecia, even after more than 6 weeks, indicating that all were self-sterile.
MAT deletion strains can outcross
Crosses between transgenic mat deletion strains and a wild-type self-fertile tester strain were set up to determine whether the self-sterile transgenic strains (MAT1-1; mat1-2 and mat1-1; MAT1-2) could outcross to a self-fertile strain. For this, conidial suspensions of the wild-type strains were dropped onto 1-week-old mycelial cultures of the self-sterile mat deletion strains to minimize the chance that perithecia would be formed by the self-fertile tester strain, which was thus forced to act as the male.
Most of the outcrosses tested produced normal-looking, mature perithecia (numbers of perithecia were between 30% and 50% of those of wild type). Fertility of the perithecia was about the same as wild type. Mature and healthy-looking perithecia contained asci with complete tetrads of eight ascospores that segregated in a 1:1 ratio for ability to fluoresce or not [in the case of the cross of the MAT1-1; mat1-2 deletion strain THΔM2-5 (hygBR; GFP) to wild type], or for resistance or sensitivity to geneticin [in the case of the mat1-1; MAT1-2 deletion strain T39ΔM1-3 (genR) to wild type]. As with wild-type selfs, some immature or dead asci were also observed, but in no case were partial tetrads or uniparental asci observed. For example, all perithecia examined in the outcross between H-11 MAT1-1; mat1-2 deletion strain THΔM2-5 (hygBR; GFP, and DON type) and wild-type 88-1 (NIV type) yielded tetrads of which four out of eight fluoresced (Fig. 2A and B). Of 159 ascospores picked randomly from five perithecia of this cross, both parental and recombinant phenotypes for two markers (GFP/hygB) and a polymerase chain reaction (PCR) marker that discriminates between trichothecene chemotypes (DON or NIV) were present in equal proportions, as confirmed statistically by χ2 testing (Table 1). The same pattern of segregation was observed in an outcross of Z3639 MAT1-1; mat1-2 deletion strain T39ΔM2-1 to its progenitor strain (Z3639) (data not shown).
|Number of progeny of each phenotype|
|GFP; hygBR; DONd||NIVd||DONd||GFP; hygBR; NIVd|
When a Z3639 mat1-1; MAT1-2 deletion strain was outcrossed to strain Z3639, 76 random ascospores examined segregated in equal proportions into the four possible phenotypes for two unlinked markers (hygB and gen) (Table 2).
|Number of progeny of each phenotype|
As a control for these outcross experiments, a G. zeae transformant carrying hygB at an ectopic site (Z3639-1, Table 2) was outcrossed to a transformant containing pII99 (T88-1-G1, from strain 88-1; data not shown). Although abundant perithecia were formed all over the mating plate regardless of mating procedure, identifying perithecia formed by outcrossing was very difficult as both parents were self-fertile. Recombinant progeny (resistant to both hygromycin B and geneticin) could be selected for by plating ascospores from at least 100 perithecia on PDA medium containing both drugs. However, recovery of sexual recombinants (66 putative recombinants from five independent experiments) was very tedious compared with recovery of recombinants from outcrosses using mat deletion strains (J. Lee, unpublished data).
MAT1-1; mat1-2 and mat1-1; MAT1-2 strains can cross in a heterothallic manner
Crosses between transgenic self-sterile mat 1-1 and mat 1-2 deletion strains were set up to test whether these strains could cross with each other (i.e. whether they were heterothallic). Each parent of each cross was tested as both male and female. Crosses between H-11 MAT1-1; mat1-2 strain THΔM2-5 and Z3639 mat1-1; MAT1-2 strain T39ΔM1-3 or Z3639 MAT1-1; mat1-2 strain T39ΔM2-1 and Z3639 mat1-1; MAT1-2 strain T39ΔM1-3 yielded mature perithecia containing fertile asci. Numbers of perithecia formed were 10–20% of those formed by wild-type selfs, and fertility of the perithecia was about 10% of wild type. More than 100 perithecia from the cross of the mat -deleted strains to each other were examined. When tetrads were examined from mature perithecia, all healthy-looking ones contained eight ascospores, only four of which fluoresced (Fig. 2C and D). In addition, when the parental strains were placed at opposite sides of the crossing plate, mature perithecia formed only at the intersection between the two parents (data not shown). Most progeny in these crosses of complementary mating pairs exhibited parental phenotypes (hygBR or genR) only, an unexpected finding, most readily explained by close linkage of the two, different, introduced drug resistance markers in the genomes of the parental strains (Table 3). To prove definitively that strains carrying deletions in opposite MAT genes could cross and that unlinked markers segregated independently in these crosses, we made a nitrate non-utilizing mutant (NitM) (Bowden and Leslie, 1992) of T39ΔM1-3 and crossed this strain to both T39ΔM2-1 and THΔM2-5. In both crosses, drug resistance (hygBR) and nitrate utilization (NitM) segregated in a 1:1 ratio among randomly tested ascospores (Table 4).
|Number of progeny of each phenotype|
|Number of progeny of each phenotype|
This study demonstrated that (i) both MAT1-1 and MAT1-2 homothallic counterparts of heterothallic MAT genes are required for G. zeae matings, i.e. deletion of either MAT1-1 or MAT1-2 genes renders a homothallic isolate self-sterile; (ii) a mat1-1 or a mat1-2 deletion strain can outcross to wild-type homothallic testers; (iii) a mat1-1 deletion strain can cross to a mat1-2 deletion strain; (iv) a mat1-1 deletion strain crossed to a mat1-2 deletion strain is fertile (number of perithecia formed is about 10% of the number formed when wild type selfs); and (v) segregation of unlinked markers in these crosses is as in heterothallic matings.
Most homothallic ascomycete species examined thus far contain the full complement of MAT genes encoded by the two MAT idiomorphs of their heterothallic relatives (Beatty et al., 1994; Glass and Smith, 1994; Yun et al., 1999; 2000). Whether expression of both MAT1-1 and MAT1-2 counterparts is essential for mating of homothallic isolates has been unclear, as some species, such as certain Neurospora homothallics, appear to carry counterparts of only one of the two mating types (mat A) (Glass et al., 1990; Glass and Smith, 1994). In this study, we have shown that deletion of either MAT1-1 or MAT1-2 genes renders homothallic G. zeae self-sterile, demonstrating that both MAT1-1 and MAT1-2 homothallic counterparts of heterothallic MAT genes are required for homothallic sexual development.
We observed that mat1-1 or mat1-2 deletion strains can outcross to wild-type testers. Approximately 1:1 segregation of single genetic markers (GFP or gen; Tables 1 and 2) in these outcrosses indicated that ascospores recovered resulted from crossing of the two parents and not from selfing of the wild-type strain. Furthermore, the segregation patterns observed when two different pairs of unlinked markers (i.e. GFP and trichothecene chemotype, or gen and hygB) (Jurgenson et al., 2002) were tracked in these outcrosses suggest that each member of the pair of markers sorted independently, as in crosses of unlinked markers in heterothallic fungi. We conclude therefore that mat1-1 or mat1-2 deletion strains of self-fertile G. zeae retain the ability to be sexual but are exclusively heterothallic.
This study also demonstrated that G. zeae could be made strictly heterothallic by manipulation of MAT, i.e. a mat1-1 deletion strain can cross to a mat1-2 deletion strain. Production of 50% fluorescent (GFP) ascospores in crosses between a mat1-1 deletion strain and a GFP-tagged, mat1-2 deletion strain provides direct evidence that fusion of cells of the two, MAT-manipulated, self-sterile, homothallic parents (now expressing only one of two mating types) occurred. Furthermore, the demonstration that perithecia are productive and that pairs of unlinked markers segregate 1:1 is evidence that these strains perform like heterothallic strains and that, like heterothallic haploid species, possess a mechanism that allows unlike nuclei to recognize each other in dikaryotic cells. How is this effected in homothallic isolates when all nuclei have identical MAT loci? Are the MAT genes differentially expressed and only nuclei expressing different MAT genes can fuse with each other (Coppin et al., 1997; Shiu and Glass, 2000)? When a mat1-1 or mat1-2 deletion strain outcrosses to a wild-type strain, ascogenous hyphae would contain both wild-type and mat -deleted nuclei after fertilization (Fig. 2E). The mat -deleted nuclei have the capacity to express only one type of MAT gene, whereas wild-type nuclei have the capacity to express both MAT genes. Wild-type nuclei should have the capacity to fuse with each other or with a mat -deleted nucleus. If mutant and wild-type nuclei fuse, progeny would segregate for markers in the mutant strain, whereas if wild type fuses with wild type, no segregation of markers would occur, i.e. progeny would all be wild type (Fig. 2E). In our outcrosses of mat1-2 deletion strains to a wild-type strain, only asci containing biparental progeny were found, suggesting that wild-type and mat -deleted nuclei fused preferentially. Finding only biparental asci after examination of asci from at least 100 perithecia bolsters the argument that there is a preference for karyogamy between nuclei from the mat -deleted parent and wild type, rather than between like nuclei from the wild-type parent able to self. The mat-deleted strains should prove helpful in resolving how partnering of nuclei is achieved.
MAT gene-mediated nucleus recognition has been studied intensively in the heterothallic ascomycete Podospora anserina. Three MAT genes, FMR1 and SMR2 at the mat– locus and FPR1 at the mat+ locus, orthologues of MAT-1-1-1, MAT-1-1-2 and MAT-1-2-1 of G. zeae, respectively, are required for internuclear recognition; however, the recognition mechanism remains an enigma (Debuchy and Coppin, 1992; Debuchy et al., 1993; Arnaise et al., 1997; 2001). As all three of the MAT genes required for nucleus recognition are transcriptional regulators, characterization of their target genes will be required to unravel the nucleus choice mechanism.
Whatever the nucleus recognition mechanism, it is clear that the G. zeae mat-deleted strains are fertile when outcrossed to wild type or to each other. The feasibility of MAT manipulation for changing the sexual reproductive mode from heterothallic to homothallic was demonstrated earlier with Cochliobolus heterostrophus (Yun et al., 1999). Each type of manipulation demonstrates that determinants of reproductive strategy reside at MAT. In both cases, fertility is less than in wild-type selfs or wild-type crosses. We do not have an explanation for this at this point. Note, however, that haploid C. heterostrophus (Turgeon et al., 1993) and P. anserina (Picard et al., 1991) transgenic strains, carrying both MAT genes (alternative idiomorph introduced via transformation), are self-maters, although they are essentially barren. Obviously, the mere association of both MAT idiomorphs from heterothallic species in the same haploid heterothallic genome does not lead to an optimal homothallic reproductive mode.
Conversion of homothallic G. zeae to obligate heterothallism is beneficial on a practical level. Differential deletion of MAT genes eliminated self-fertility without impairing its ability to cross. It is a simple matter to produce sexual recombinants using mat deletion strains retaining opposite MAT genes. Once a pair of tester strains is available, they can be used as testers for any cross. Other types of markers used for forcing heterothallism, such as Nit markers, must be present in both partners. Furthermore, Nit mutant crosses allow recovery of recombinant progeny only, whereas all ascospores can be recovered from crosses involving mat-deleted strains. The mat-deleted tester strains will facilitate genetic analyses of important traits, such as pathogenicity and ability to produce mycotoxins. This may speed up analysis of mutants generated by high-throughput gene deletion. Deletions carried out in a wild-type strain could be used as the male parent in a cross to a mat-deleted tester. Alternatively, if deletions are done in a mat1-1 or mat1-2 strain, these strains could be crossed to deletion strains of opposite mating type and segregation of any character of interest determined.
Strains and culture conditions
Gibberella zeae wild-type strain Z3639 (Bowden and Leslie, 1992) and two Korean strains, H-11 and 88-1 isolated from corn and barley, respectively (Lee et al., 2001; 2002), as well as appropriate media, growth conditions, storage and transformation protocols have been described previously (Bowden and Leslie, 1992; 1999; Lee et al., 2001; 2002). Transformants were purified by single conidium isolation and stored in 25% glycerol at −80°C. NitM mutants of mat1-1 deletion strain T39ΔM1-3 were generated on minimal medium (MM) with 2.5% chlorate as described previously (Bowden and Leslie, 1992). All strains were recovered from storage for each experiment. Escherichia coli strains DH5α and Top10 (Invitrogen) were used for propagation of plasmids. E. coli transformants were grown on Luria–Bertani (LB) agar or liquid medium, supplemented with an appropriate antibiotic (75 µg ml−1 ampicillin or 25 µg ml−1 chloramphenicol).
Isolation of fungal DNA and PCR amplifications were performed as described previously (Lee et al., 2001; 2002). E. coli colonies carrying recombinant plasmids were screened by a single-tube mini-prep method (Liu and Mishra, 1995). For fungal transformation, plasmids were purified from 5 ml E. coli cultures using a Qiagen mini-prep kit. Standard procedures were used for restriction endonuclease digestions, ligations, agarose gel electrophoresis and gel blot hybridizations (Sambrook et al., 1989).
The G. zeae MAT genes manipulated in this study have been described previously (Yun et al., 2000). Plasmids pBCSK– (Stratagene) and pIGPAPA (Horwitz et al., 1999) carrying the gene for resistance to hygromycin B (hygB) and the gene for expression of green fluorescent protein (GFP) were modified for use in this study.
Genomic DNA from strain Z3639 was digested with XhoI, self-ligated and used as template for inverse PCR with primers GzMAT2/delp1 (5′-CGGGATCCGGTTCCCGC CGCCCAGCCTACTC-3′) and GzMAT2/delp2 (5′-CGGGATCCCACATGAAGAGGTTGGCGAGAGC-3′) corresponding to nucleotide positions 8861–8839 and 9414–9434 of the G. zeae MAT sequence (accession number AF318048) (Fig. 1A). Each included a BamHI recognition sequence (underlined). The PCR amplified a 1.9 kb fragment corresponding to 0.7 kb of the 5′ and 1.2 kb of the 3′ sequences flanking the MAT1-2-1 open reading frame (ORF) (Fig. 1A). The amplified product was phenol extracted, digested with BamHI and ligated into BamHI-digested pIGPAPA that had been modified to remove a XhoI site. The resulting 7.8 kb plasmid was designated pIGΔMAT2. To delete the MAT1-2-1 gene, pIGΔMAT2 was linearized with XhoI and transformed into G. zeae protoplasts.
Primers GzMAT1/delp1 (5′-TCTACAAAGGGA GGACGCGATAACTA-3′) and GzMAT1/delp2 (5′-CAAGCCC TATTCGGTCCTGATTACG-3′) corresponding to nucleotide positions 3628–3603 and 7858–7882 of the G. zeae MAT sequence (accession number AF318048), respectively (Fig. 1B), were used to amplify a 3.3 kb fragment from NarI-digested and self-ligated G. zeae genomic DNA. This inverse PCR product carrying 1.6 kb of sequence flanking the MAT1-1-3 and 1.7 kb sequence flanking the MAT1-1-1 ORFs was cloned into pCR2.1-TOPO (Invitrogen). The insert was then removed from the cloning vector by digestion with EcoRI and subcloned into the EcoRI site of pBCSK– that had been modified to remove a NarI site. The resulting 6.7 kb plasmid was designated pΔGzMAT1. For deletion of MAT1-1-1, MAT1-1-2 and MAT1-1-3 from strain Z3639, pΔGzMAT1 was linearized with NarI and added to fungal protoplasts together with 5.3 kb of circular pII99 (kindly provided by T. Tsuge, Nagoya University, Japan), which carries the gene for resistance to geneticin (gen) as a selectable marker.
Mycelial plugs (3 mm) of transformants sustaining a deletion of MAT1-1 or MAT1-2 were placed on carrot agar plates (Klittich and Leslie, 1988; Bowden and Leslie, 1999) and incubated at 25°C under a mixture of fluorescent cool white and black lights with a 12 h photoperiod. After 7 days, conidial suspensions (105 ml−1) of the wild-type strain were dropped onto the mycelia of the mat deletion transformants that had grown up, and plates were incubated for an additional 10–14 days. Alternatively, mycelial plugs of two transgenic strains were placed at opposite sides of a plate of carrot medium and incubated for 3 weeks under the conditions described above. All asci from more than 100 perithecia from each cross were examined.
Confocal laser microscopy
Microscopic observation of progeny from crosses of transgenic strains carrying GFP was done at the National Instrumentation Center for Environmental Management (NICEM, Seoul National University, Suwon, Korea) using confocal laser microscopy (Radiance 2000 MP; Bio-Rad) with excitation wavelength at 488 nm and emission wavelength at 515/30 nm.
PCR assay for determination of trichothecene chemotype
PCRs were performed using primer pairs developed previously to correspond to the Tri7 or Tri13 genes at the locus involved in trichothecene biosynthesis (Lee et al., 2001; 2002) and genomic DNAs of progeny from an outcross of a DON-producing transgenic strain to a NIV-producing wild-type strain 88-1 as template. This procedure identified deoxynivalenol (DON) versus nivalenol (NIV) chemotype-specific fragments.
This study was supported by a grant (M1-01-KG-01-0001-01-K07-01-028-1-0) from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Korean Ministry of Science and Technology and by a grant (2000-2-22100-004-3) from the Korean Science and Engineering Foundation. J.L. and T.L. were supported by graduate and postdoctoral fellowships, respectively, from the Korean Ministry of Education through the Brain Korea 21 project. We thank Robert L. Bowden, USDA ARS Plant Science and Entomology Research Unit, Manhattan, KS, USA, for providing G. zeae strain Z3639 for this study.
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