Figures
Abstract
We previously characterized members of the Myb protein family, MYT1 and MYT2, in Fusarium graminearum. MYT1 and MYT2 are involved in female fertility and perithecium size, respectively. To expand knowledge of Myb proteins in F. graminearum, in this study, we characterized the functions of the MYT3 gene, which encodes a putative Myb-like transcription factor containing two Myb DNA-binding domains and is conserved in the subphylum Pezizomycotina of Ascomycota. MYT3 proteins were localized in nuclei during most developmental stages, suggesting the role of MYT3 as a transcriptional regulator. Deletion of MYT3 resulted in impairment of conidiation, germination, and vegetative growth compared to the wild type, whereas complementation of MYT3 restored the wild-type phenotype. Additionally, the Δmyt3 strain grew poorly on nitrogen-limited media; however, the mutant grew robustly on minimal media supplemented with ammonium. Moreover, expression level of nitrate reductase gene in the Δmyt3 strain was decreased in comparison to the wild type and complemented strain. On flowering wheat heads, the Δmyt3 strain exhibited reduced pathogenicity, which corresponded with significant reductions in trichothecene production and transcript levels of trichothecene biosynthetic genes. When the mutant was selfed, mated as a female, or mated as a male for sexual development, perithecia were not observed on the cultures, indicating that the Δmyt3 strain lost both male and female fertility. Taken together, these results demonstrate that MYT3 is required for pathogenesis and sexual development in F. graminearum, and will provide a robust foundation to establish the regulatory networks for all Myb-like proteins in F. graminearum.
Citation: Kim Y, Kim H, Son H, Choi GJ, Kim J-C, Lee Y-W (2014) MYT3, A Myb-Like Transcription Factor, Affects Fungal Development and Pathogenicity of Fusarium graminearum. PLoS ONE 9(4): e94359. https://doi.org/10.1371/journal.pone.0094359
Editor: Jae-Hyuk Yu, University of Wisconsin – Madison, United States of America
Received: January 16, 2014; Accepted: March 12, 2014; Published: April 10, 2014
Copyright: © 2014 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT & Future Planning (No. 2008-0061897). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The homothallic ascomycete fungus Fusarium graminearum is a prominent plant pathogen that causes Fusarium head blight (FHB) on cereal crops and ear rot on maize [1], [2], resulting in a severe reduction of crop yield and production of mycotoxins (e.g., trichothecenes and zearalenone) that are dangerous to animals and humans [3]. In particular, sexual spores (ascospores) of F. graminearum are believed to be the primary inocula for FHB epidemics in cereal crops [4]. The spores overwinter within a fruiting body (perithecium) on plant debris, and are forcibly discharged into the air under milder temperatures and moderate moisture [4], [5]. To date, several genes related to sexual development have been studied in F. graminearum, and those genes are known to be closely related to disease development [6], [7], [8], [9], [10].
In the F. graminearum genome, 19 transcription factors encoding Myb-like proteins were identified at a previous work [11]. Of these Myb-like proteins, we found that MYT1 and MYT2 transcription factors are involved in female fertility and perithecium size, respectively [12], [13]. Furthermore, several deletion mutants of putative Myb transcription factors, including FGSG_01915, FGSG_02719, and FGSG_12781, in F. graminearum exhibited abnormality in sexual development [11]. These data suggest that the Myb transcription factors are required for normal sexual development of F. graminearum.
Transcription factors that contain the Myb DNA-binding domain are involved in various cellular processes in eukaryotes, including cell proliferation, apoptosis, differentiation, metabolism, and stress responses [14], [15], [16], [17]. The v-Myb DNA-binding domain was originally identified from avian myeloblastosis virus (AMV) as a truncated version of a cellular progenitor c-Myb that contains three Myb DNA-binding domains, which are referred to as R1, R2, and R3 [18], [19]. The Myb DNA-binding domains identified from vertebrates, plants, and oomycetes are relatively conserved between species, and most Myb proteins contain one to three variants of the Myb DNA-binding domain, such as R1, R2R3, and R1R2R3 [14], [20], [21]. Each Myb DNA-binding domain spans approximately 52 amino acids arranged as tandem repeats of a helix-turn-helix motif [22], [23]. This repeat region is involved in DNA-binding, and the specificity of DNA binding is known to be dependent on the third α-helices of R2 and R3 [24].
Currently, a few Myb-like proteins have been identified and characterized in fungal species. AgBAS1, a homolog of Saccharomyces cerevisiae BAS1, was identified in Ashbya gossypii, and the deletion mutant of AgBAS1 exhibited auxotrophy for adenine and a delay in spore germination [25]. Furthermore, AgBAS1 controls the adenine-mediated regulation of the purine biosynthetic pathway by regulating the gene expression of ADE4 by binding to the ADE4 promoter [25]. In Aspergillus nidulans, a Myb transcription factor, FlbD, controls asexual and sexual differentiation [26], [27]. The flbD mutants were unable to develop the cleistothecial peridium, a specialized external tissue that differentiates during fruiting body formation, and exhibited severely defective conidiation. In addition, a Myb-like protein PsMYB1 of Phytophthora sojae, which causes soybean root rot, was identified by differential expression between wild type and MAP kinase mutants, and the PsMYB1 is known to be involved in zoospore-mediated pathogenicity [28].
In the present report, as a series of MYT1 and MYT2, we aimed to expand the current knowledge of how Myb transcription factors regulate sexual development of F. graminearum, with an emphasis on the characterization of the gene involved in the formation of perithecia and ascospores. To this end, we chose to investigate MYT3, which encodes a putative Myb-like transcription factor, and further characterized its biological function by molecular approaches. Our results demonstrate that MYT3 is required for conidiation, vegetative growth, sexual development, and pathogenicity.
Materials and Methods
Fungal strains and media
All strains used in this study are listed in Table 1. The wild-type strain Z-3639 and mutants derived from the wild-type strain were maintained on complete media (CM) [29], and stored in 20% glycerol at −80°C. The growth rates of the wild type and transgenic strains were measured in CM and minimal media (MM; 3 g KH2PO4, 0.5 g MgSO4, 0.5 g KCl, 30 g sucrose, and 20 g agar per liter). To observe responses to nitrogen sources, the MM was modified by addition of 2 g NaNO3 or 2 g (NH4)2SO4 per liter. Conidia production was induced in carboxymethyl cellulose (CMC) medium [30]. Minimal liquid medium supplemented with 5 mM agmatine (MMA) was used for trichothecene analysis [31].
Nucleic acid manipulations and protein sequence analysis
Fungal strains grown in CM broth for 5 days at 25°C were harvested and lyophilized. The procedures for isolation of genomic DNA were performed as described [29]. Standard methods were followed for restriction endonuclease digestion, agarose gel electrophoresis, and Southern blotting [32]. To measure expression level of transcripts, total RNA was extracted by using the Easy-Spin Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea), and the first strand of cDNA was synthesized from the RNA by SuperScriptIII reverse transcriptase (Invitrogen, Carlsbad, CA, USA). PCR primers were obtained from Bionics (Seoul, Korea), listed in Table S1. Protein sequence alignment was performed by Clustal Omega [33], and drawn by BOXSHADE 3.31 available at http://www.ch.embnet.org/software/BOX_form.html. InterPro (http://www.ebi.ac.uk/interpro/) and Fungal Transcription Factor Database (FTFD; ftfd.snu.ac.kr) were used to search for the Myb DNA-binding domains. Automated mode of SWISS-MODEL [34] and YASPIN [35] were used for the structural analysis of Myb-like DNA binding domains. Nuclear localization sequences (NLSs) were identified by cNLS Mapper [36].
Complementation and overexpression of MYT3
For the complementation of the Δmyt3 strain, intact copies of MYT3 (FGSG_00324; Fusarium Comparative Sequencing Project at the Broad Institute of MIT and Harvard, http://www.broadinstitute.org) were amplified from F. graminearum wild-type Z-3639 with primer pair MYT3-5N/MYT3-3N. The selection marker, hygromycin phosphotransferase (HPH), was amplified from the HK33 strain with primer pair HPH-F/HPH-R [37]. These two fragments were co-transformed into the protoplast of the Δmyt3 strain. For fungal transformations, the methods for the preparation of protoplasts and regeneration of transformants were performed as described previously [38]. Transformants were confirmed by Southern blot analysis. To generate the MYT3 overexpression strains, the 5′ flanking region and ORF region of MYT3 were amplified with primer pairs UP-5F/5R-GEN and MYT3-F/MYT3-R, respectively. A PCR fragment with the geneticin resistant gene (GEN) and the elongation factor 1α promoter (PEF1α) was amplified from pSKGEN [37] using primer pair Neo-F/EF-R. These three amplicons were combined as previously described [39]. The resulting product was amplified with nested primers MYT3-5N and MYT3-RN to create a 4.3-kb product containing GEN and PEF1α fused to the MYT3 ORF. The final construct was transformed into the wild-type strain.
Cellular localization of MYT3
To investigate cellular localization of MYT3, we generated a strain carrying green fluorescent protein (GFP) fused to C-terminus of MYT3. A PCR fragment including GFP and HPH was amplified from the pIGPAPA plasmid [40] with primers GFP-F and HYG-F1. The 5′ flanking region, which is the MYT3 C-terminus without its own terminator sequence, was amplified from the wild-type strain with primers MYT3-5F and MYT3-5R. The 3′ flanking region of MYT3 was amplified by primers MYT3-3F and MYT3-3R. These three resulting PCR products were fused as described [39]. The resulting PCR product was used as a template with primers MYT3-gfpF and MYT3-gfpR for a final construct. Subsequently, the final PCR products were transformed into the wild-type strain.
To observe co-localization of MYT3 with a nuclear protein, a resulting MYT3-gfp transformant was outcrossed with a mat1r strain that contains red fluorescent protein (RFP) fused to histone H1 as generated previously [41]. Strains containing both MYT3::GFP and hH1::RFP were selected by antibiotic resistance and confirmed by PCR. Microscopic observation was performed by a DE/Axio Imager A1 microscope (Carl Zeiss, Oberkochen, Germany) with the filter set 38HE (excitation 470/40; emission 525/50) for GFP and the filter set 15 (excitation 546/12; emission 590) for RFP.
Western blot analysis
Mycelia and perithecia were harvested from carrot agar cultures, and ground by liquid nitrogen. The ground tissues (approximately 100 to 150 mg) were used for the extraction of total protein with 300 μl of extraction buffer [50 mM Tris-Cl, pH 8.4; 192 mM glycine; 0.1% sodium dodecyl sulfate (SDS); 1 mM protease inhibitor phenylmethanesulfonylfluoride]. After centrifugation at 16000 g for 10 min, supernatant was used for the protein analysis. The samples containing 1.5 to 2.0 μg of total protein were separated on 5% sodium dodecyl sulfate polyacrylamide gels. Preparing gels and electrophoresis were performed as previously described [42]. Western blot analysis was performed using chemiluminescence (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. Primary anti-GFP (Abcam, Cambridge, UK) and anti-rabbit secondary antibody conjugated to horseradish peroxidase (GE Healthcare) were used at 1 4000 and 1 5000 dilution, respectively.
Conidiation, germination, and pathogenicity assay
To measure conidiation, the same sized agar blocks of the wild type, Δmyt3, Δmyt3-com, and MYT3-oe strains grown on CM were used to inoculate 5 ml of CMC liquid medium. The conidia were counted after 5 days of incubation. To measure germination rate, conidial suspensions (106/ml) of each strain were inoculated into 20 ml of CM and MM including NaNO3. The germinated conidia per 100 conidia were counted 0, 2, 4, 8, and 16 h after inoculation. For the pathogenicity assay, 10 μl of conidial suspension (106/ml) obtained from each strain were point-inoculated into a spikelet of the wheat head at early anthesis. Infected plants were incubated in a humidity chamber for 3 days, and subsequently transferred to a green house. After 2 weeks, spikelets showing disease symptoms were counted.
Quantitative real-time (qRT)-PCR of gene expression
The qRT-PCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and a 7500 real-time PCR system (Applied Biosystems, Foster city, CA, USA). Each reaction contained 10 μl of SYBR green Supermix, 500 nM of forward and reverse primers, cDNA template, and nuclease-free water to a final volume of 20 μl. PCR cycling conditions were 2 min at 50°C (one cycle); 10 min at 95 °C (one cycle); and 15 s at 95°C followed by 1 min at 60°C (40 cycles). Experiments were repeated two times with three replicates. Expression levels were calculated using the comparative Ct method (Applied Biosystems). CYP1 gene (FGSG_07439) served as the endogenous reference for normalization.
Trichothecene analysis
To analyze total trichothecene production, conidial suspensions (104/ml) of each strain were inoculated in MMA, and the cultures were incubated for 7 days at 25°C. The culture media was filtered with Miracloth. Trichothecenes were extracted from 150 μl of culture filtrates by mixing with 250 μl of ethyl acetate/methanol solution (4 1, v/v) [43]. Extracts were dried and derivatized with Sylon BTZ (BSA + TMCS + TMS1, 3 2 3; Supelco, Bellefonte, PA, USA). Sequentially, 200 μl of n-hexane and 200 μl of distilled water were added to the reaction products. This reaction was left standing until two layers separated. Trichothecenes in the upper layer were analyzed with a Shimadzu QP-5000 gas chromatograph-mass spectrometer (GC-MS; Shimadzu, Kyoto, Japan) [44]. The total trichothecene concentration was quantified based on the biomass of each strain in MMA.
Sexual crosses
From strains grown on carrot agar media [29] for 5 days, aerial mycelia were removed with 700 μl of 2.5% Tween 60 solution to induce fertilization. The plates were incubated under a near-UV light for 7 to 10 days. For outcrosses, mycelia of a female strain grown on carrot agar media were fertilized with 1 ml of conidial suspension (106/ml) obtained from male strain, which was induced in CMC. The heterothallic Δmat2 mutant, a deletion strain of MAT1-2, was used as a tester mutant for outcrosses [45]. Perithecia and ascospores were observed 9 days after sexual crosses.
Results
Identification of Myb-like transcription factor MYT3
MYT3 has an open reading frame (ORF) of 7,340 base pairs (bp) with three introns, and is predicted to encode a 2,304-amino acid protein that contains two Myb-like DNA-binding domains (residues 1,263 to 1,314 and 1,534 to 1,586; Figure 1A). BLAST and phylogenetic analysis of homologous proteins in other fungi demonstrated that MYT3 is more highly conserved within species of the subphylum Pezizomycotina of the Ascomycota than in the phyla Oomycota, Basidiomycota and the subphylum Saccharomycotina (Figure S1A). Additionally, the Myb-like DNA binding domains of MYT3 showed high sequence similarities to F. oxysporum (FOXG_00743, 93%), Magnaporthe oryzae (MGG_14558, 74%), Neurospora crassa (NCU_10346, 69%), and Botrytis cinerea (BC1G_02829, 54%) (Figure 1A and S1B) although the other regions of MYT3 homolog proteins were variable. Currently, none of the MYT3 homologs has been functionally characterized. Each Myb-like DNA binding domain of MYT3 consists of 52 amino acids, forming a helix-turn-helix structure with hydrophobic residues (Figure 1A and B). The third helix of the second domain contains residues (KN) that are implicated to be in direct contact with DNA (Figure 1A) [46], suggesting that MYT3 possibly functions in transcriptional regulation.
(A) MYT3 homologs in Fusarium oxysporum (FOXG_00743), Magnaporthe oryzae (MGG_14558), Neurospora crassa (NCU_10346), Botrytis cinerea (BC1G_02829) were used for this analysis. Each Myb domain contains three helices, indicated with an underline, and residues that form their hydrophobic cores, indicated by black dots above the residue. The second domain contains residues that are implicated to have direct contact with the DNA, indicated by asterisks [21], [46]. (B) Ribbon diagram of Myb-like DNA binding domains in MYT3.
MYT3 is consistently expressed in nuclei
In addition to the Myb DNA-binding domains, we identified several nuclear localization sequences (NLSs) in MYT3, including mono- and bi-partite sequences (data not shown). Therefore, we next investigated whether MYT3 localizes in the nucleus. To do this, we generated a strain that has GFP fused to C-terminus of MYT3 in the wild-type strain, named by MYT3-gfp (). Thirteen transformants containing single MYT3::GFP copy were obtained, and nuclear GFP signals were observed in these strains. For further confirmation of nuclear localization of MYT3, the MYT3-gfp strain was outcrossed with a mat1r strain that contains RFP fused to histone H1 protein in MAT1-1 deletion background, with expectation to observe co-localization of MYT3::GFP and hH1::RFP in nuclei. From perithecia by this outcrossing, we isolated 20 progeny resistant to both hygromycin and geneticin, and observed that all progeny expressed GFP and RFP in nucleus of the most developmental stages such as hyphae, conidia, and ascospores (Figure 2), indicating that MYT3 is continuously expressed in nuclei.
Representative examples show co-localization of MYT3 fused with green fluorescent protein (GFP) and histone H1 fused with red fluorescent protein (RFP). A strain containing MYT3::GFP and hH1::RFP was grown on CM (hypha), YMA (conidia), and carrot agar media (ascospores) for the microscopic observation, respectively. DIC, differential interference contrast; scale bar = 20 μm.
Genetic complementation and expression of MYT3
To confirm the roles of MYT3, we generated a complementation strain, Δmyt3-com, by co-transforming the MYT3 gene and hygromycin resistance gene into the protoplast of the Δmyt3 strain (Figure S3A). Southern blot analysis indicated that the resulting mutant, designated strain Δmyt3-com, obtains a 2.3-kb band of hybridization present in the wild-type strain (Figure S3A). To further dissect the molecular mechanism of the function of MYT3, overexpression of MYT3 was achieved with a construct containing the EF1α promoter fused to the MYT3 ORF (Figure S3B). Strain MYT3-oe, obtained by insertion of the constitutive expression construct into the wild type, was confirmed by Southern blot analysis (Figure S3B), and the expression level of MYT3 in the MYT3-oe strain was measured by qRT-PCR (Figure 3A).
(A) Transcript levels of MYT3 in each strain were analyzed by qRT-PCR. Total RNA of each strain was extracted from vegetative cultures grown on carrot agar media, and from sexual cultures 3, 5, and 7 days after induction of sexual development. WT, wild-type strain Z-3639; Δmyt3, MYT3 deletion mutant; MYT3-oe, over expression strain where MYT3 expression is driven by the EF1α promoter. d, day; n.d., not detected. (B) Western blot analysis of MYT3-gfp stain. Lane 1, whole cell extracts were prepared from vegetative cultures grown on carrot agar media for 5 days; Lane 2–4, whole cell extracts were from sexual cultures 3, 5, and 7 days after induction of sexual development, respectively. Top, western blotting data; bottom, coomassie blue staining used as a loading control.
Expression of MYT3 in the MYT3-oe strain was consistent at all stages of development, with approximately two- to three-fold higher expression than that of the wild-type strain (Figure 3A). The expression level of MYT3 in the wild-type strain was slightly increased at 3 days after sexual induction, and decreased over time to the similar level of expression in vegetative growth (Figure 3A). At translational level, the expected 283-kDa protein (MYT3::GFP) in a MYT3-gfp strain was detected by anti-GFP (Figure 3B). However, expression of MYT3 was high during sexual development with the highest level at 7 days after sexual induction (Figure 3B). Taken together, these results suggest that MYT3 play an important role during sexual development.
Deletion of MYT3 affects fungal development in response to nitrogen
The deletion mutant of MYT3 exhibited a growth defect on CM in comparison to the wild type, Δmyt3-com, and MYT3-oe strains (Figure 4A and Table 2). Also, the Δmyt3 strain poorly grew on MM lacking nitrogen in comparison to the growth on CM (Table 2). With these observations, increasing expression of ESTs, corresponding to FGSG_00324 (MYT3), in nitrogen starvation condition (MIPS, http://www.plexdb.org/) [47] gave rise to a question whether nutrient such as nitrogen sources affects fungal growth of Δmyt3 strain. On MM containing nitrate, the mutant grew poorly compared to the wild type, whereas fungal growth of the Δmyt3-com and MYT3-oe strains was similar to that of the wild type (Table 2). When MM was supplemented with ammonium instead of nitrate as a nitrogen source, the Δmyt3 strain grew better than the wild type, Δmyt3-com, and MYT3-oe strains (Figure 4A). Additionally, expression level of nitrate reductase gene (FGSG_01947) homologous to niaD of F. fujikuroi [48] was significantly decreased in the Δmyt3 strain grown in MM + nitrate (Figure 4B). Although the expression of this gene from the Δmyt3 strain grown in MM + ammonium was slightly decreased in comparison to the wild type and Δmyt3-com, the magnitude of the induction was comparably low in all strains grown MM + ammonium compared to the MM + nitrate (Figure 4B). Together, these results indicate that MYT3 is involved in nitrogen metabolic pathway by regulating the expression of nitrate reductase gene.
(A) Vegetative growth of each strain on complete media (CM), minimal media (MM) containing nitrate, and MM containing ammonium. Photographs were taken after 5 days (CM, MM + nitrate) and 7 days (MM + ammonium) of incubation. WT, wild-type strain Z-3639; Δmyt3, MYT3 deletion mutant; Δmyt3-com, complemented strain of the Δmyt3; MYT3-oe, overexpressed strain of MYT3. (B) To measure expression of nitrate reductase gene, all strains were grown in CM media for 48 h and then washed with water. The resulting mycelia were transferred into MM+ nitrate or MM + ammonium, and total RNA was extracted 3 h after resuspension.
The Δmyt3 strain produced approximately 50-fold less conidia compared to the wild type 5 days after inoculation into CMC media, but there was no significant difference in conidiation among the wild type, Δmyt3-com, and MYT3-oe strains (Table 2). In addition, the germination rate of the Δmyt3 strain on CM (Figure 5A) and MM including nitrate (Figure 5B) was markedly slower than that of other strains. Taken together, these results represent that deletion of MYT3 contributes to the impairment of fungal development and growth.
(A) Numbers of germinated conidia in CM were counted 0, 4, and 8 h after inoculation. (B) Numbers of germinated conidia on MM containing nitrate were counted 0, 4, 8, 16 and 24 h after inoculation. Each experiment was performed in triplicate two independent times. WT, wild-type strain Z-3639; Δmyt3, MYT3 deletion mutant; Δmyt3-com, complemented strain of the Δmyt3; MYT3-oe, overexpressed strain of MYT3.
MYT3 is involved in pathogenicity during infection of wheat
Our observations that the Δmyt3 strain grows slowly on CM and MM (Table 2) led us to question whether the deletion of MYT3 affects F. graminearum pathogenicity during wheat infection. To evaluate pathogenicity on flowering wheat heads, conidial suspensions of each strain were point-inoculated on a spikelet and incubated in a greenhouse. The wild-type strain induced normal head blight symptoms, which manifests as discoloration, at 14 days after inoculation (Figure 6A). In contrast, the Δmyt3 strain was restricted to infection sites, which were unable to spread from the rachis to reach adjacent spikelets on the head (Figure 6A). Consequently, the average disease index of the Δmyt3 strain was approximately 10-fold lower than the wild-type level (Table 2). The infections by Δmyt3-com and MYT3-oe strains displayed the similar levels of pathogenicity to the wild-type strain (Figure 6A and Table 2). Thus, our results reveal that MYT3 plays an important role in the progression of head blight after initial colonization.
(A) Flowering wheat head were inoculated with conidia of the wild-type (WT) Z-3639, Δmyt3 mutant, complemented strain Δmyt3-com, and overexpressed strain MYT3-oe, respectively. Mock indicates a negative control inoculated with 0.01% Tween 20. Wheat heads were photographed 14 days after inoculation. (B) Analysis of total trichothecenes (deoxynivalenole and 15-acetyl-deoxynivalenol) was performed on each strain grown in minimal liquid medium supplemented with 5 mM agmatine (MMA). The measured amount of trichothecenes was normalized to the biomass of each strain. (C) Expression of TRI5 and TRI6 in the wild type, Δmyt3, MYT3-oe strains. Transcript levels were analyzed by qRT-PCR from cultures grown in MMA for 4 days and presented as relative transcript level compared to a control gene, CYP1.
MYT3 has an effect on the biosynthesis of trichothecenes
To determine whether MYT3 affects trichothecene biosynthesis, we measured trichothecene production by the wild type, Δmyt3, Δmyt3-com, and MYT3-oe strains grown on MMA. After normalizing to the amount of fungal biomass, the Δmyt3 strain produced approximately 10-fold less trichothecenes compared to the wild type (Figure 6B). Furthermore, the ability to produce trichothecenes was restored in the Δmyt3-com strain (Figure 6B). To confirm this observation, we investigated the expression of the trichothecene synthesis genes TRI5 (FGSG_03537) and TRI6 (FGSG_03536), which encode a trichodiene synthase and a major transcriptional regulator for trichothecene production, respectively [49]. In the Δmyt3 strain, the expression levels of TRI5 and TRI6 were reduced more than six-fold (Figure 6C) compared to the wild type and MYT3-oe strains. Together, these results show that the reduction in trichothecene biosynthesis is attributable to disruption of MYT3.
MYT3 is dispensable for male/female fertility in outcrosses
Self-fertility of the Δmyt3 strain was determined from the formation of perithecia and ascospores on carrot agar media. The wild-type strain began to produce protoperithecia 3 days after sexual induction. After an additional 3- or 4-day incubation, mature perithecia were formed; the perithecia contained asci, each with eight ascospores (Figure 7A). In contrast, the Δmyt3 strain only produced mycelia and/or conidia, and never produced fruiting bodies (Figure 7A). The sexual defects in the Δmyt3 strain were recovered in the Δmyt3-com strain.
(A) Strains grown on carrot agar media were self-fertilized. Photos were taken 9 days after sexual induction. Dissecting the perithecia showed the asci and ascospores of each strain (inset boxes). WT, wild-type strain Z-3639; Δmyt3, MYT3 deletion mutant; Δmyt3-com, complemented strain of the Δmyt3; MYT3-oe, overexpressed strain of MYT3. Scale bar = 500 μm. (B) Heterozygous outcrosses. The Δmyt3 strain, as either a female or male strain, was outcrossed with self-sterile Δmat2 strain. Photos were taken 9 days after outcrossing. Scale bar = 500 μm.
When a conidial suspension of the Δmyt3 strain was spermatized to a self-sterile Δmat2 strain, a deletion mutant of MAT1-2, no perithecia were formed on the outcrossed cultures, indicating that the Δmyt3 strain lost male fertility (Figure 7B). Furthermore, when the Δmyt3 strain was used as a female strain in outcrossing with the Δmat2 strain, no perithecia were found on the carrot agar media (Figure 7B). Taken together, these results show that deletion of MYT3 abolishes both male and female fertility.
Discussion
Previously, we reported that the Myb-like proteins MYT1 and MYT2 play important roles in female fertility and perithecium size during the sexual development of F. graminearum [12], [13]. Specifically, deleting MYT2 enhanced vegetative growth and increased the number of ascospores [13]. In addition, we observed that MYT2 displays differential expression in germinated hyphae compared to ascospores and conidia [13]. These results suggest that MYT2 suppresses cell proliferation in various developmental stages, as well as critical roles for Myb-like proteins. Except with MYT1 and MYT2, the F. graminearum genome has several Myb-like proteins whose biological functions remain unclear. As a series of Myb-like proteins in F. graminearum, we characterized the roles of MYT3 in aspects of fungal development and pathogenicity.
The Myb proteins belong to a large family of transcription factors that are essential for regulating genes for cellular processes. Extensive studies of Myb-like proteins in various eukaryotes have demonstrated that the roles of Myb proteins are functionally diverse. Myb proteins are involved in proliferation, differentiation, metabolic pathways, cell fate, and stress responses [15], [16], [17]. In this study, we found that MYT3 contains two Myb-like DNA binding domains, which are likely to build a helix-turn-helix structure with hydrophobic residues and conserved in the subphylum Pezizomycotina. However, due to the low sequence similarity with comparison of other Myb proteins identified previously, we cannot conclude whether MYT3 belongs to a certain subfamily of Myb proteins (e.g., R1, R2R3, or R1R2R3). Myb proteins have diverged substantially between organisms. For examples, 4R-Myb subfamily of Myb proteins was identified in plants, and consists of four repeats; one of the repeats contain R1/R2-like repeat [14]. The oomycetes P. infestans has an atypical class of Myb proteins that contain multi-domains, and neither of the tandem Myb domains resembles the R2 or R3 region of c-Myb. Additionally, several proteins with novel combinations of Myb domains were observed in this organism [21]. The F. graminearum genome is different from other eukaryotes in the total number of Myb proteins. The F. graminearum genome was reported to contain 19 Myb-like proteins [11], whereas Arabidopsis thaliana, Oryzae sativa, and P. sojae were predicted to express 198, 183, and 68 Myb-like proteins, respectively [28], [50]. In addition, most Myb proteins of F. graminearum have either one or two Myb-like DNA binding domains without the presence of a signature sequence, such as SHAQKY (data not shown) [51]. Taken together, Myb-like proteins in F. graminearum seem to be highly divergent within this species.
The regulatory functions of Myb proteins in response to nutritional stimuli, such as nitrogen and carbon, have been studied in other fungi. The deletion mutant of flbD, a Myb transcription factor, in A. nidulans showed an impaired ability to produce conidia under nitrogen starvation conditions [26]. In Schizosaccharomyces pombe, the Myb-type DNA binding protein Reb1 regulates the transcription of the ste9+ gene by binding its promoter, which is involved in G1 cell cycle arrest and sexual differentiation in response to nitrogen starvation [52]. Similar to the fungi, it was reported that CmMYB1, an R2R3-type MYB transcription factor, is responsible for the expression of nitrogen assimilation genes in a unicellular red alga Cyanidioschyzon merolae [53]. CmMYB1 expression was induced by nitrogen depletion, and the CmMYB1 mutant showed decreased cell viability after exposure to nitrogen-depleted conditions compared to the parental strain [53]. CmMYB1 specifically occupies the promoters of nitrogen assimilation genes only under nitrogen-depleted conditions, indicating that CmMYB1 is a central nitrogen regulator in C. merolae [53].
Related with the regulation of nitrogen metabolism, the mechanism mediated by global regulators AreA/Nit2 has been extensively studied in filamentous fungi [54], [55], [56]. The transcriptional regulator AreA activates the expression of nitrogen assimilatory genes such as nitrate reductase gene by binding its promoter when favored nitrogen sources, such as glutamine and ammonium, are not available [57], [58]. In this study, with our observations that growth defect of the Δmyt3 strain on MM and MM containing nitrate as a nitrogen source, we measured that expression of FgniaD, which encodes a nitrate reductase, was decreased in the Δmyt3 strain grown on MM + nitrate compared to the wild type. These observations gave rise to the hypothesis that MYT3 is involved in nitrogen assimilatory pathway. This hypothesis is supported by our observation that FgniaD has two putative Myb DNA-binding sequences at −47 and −227 bp upstream of the start codon, based on the binding sequences of 5′-Y(T/C)AACK(G/T)G-3′ as previously described [46]. This binding motif was shown to be responding to the Lys1575 and Asn1576 residues in the second Myb-like domain of MYT3, which has been implicated to participate in direct contact with DNA [46], [59]. However, further studies are needed to clarify whether Myb DNA-binding domains of MYT3 in F. graminearum bind to the FgniaD promoter.
Considering that MYT3 is conserved in species of the subphylum Pezizomycotina of the Ascomycota, and that transcriptional/translational expression of MYT3 is increased during sexual development, we hypothesized that MYT3 plays an important role in sexual development. Most F. graminearum deletion mutants that have a sexual development defect exhibit an impairment in female fertility, but retain male fertility; for example, ZIF1 (b-ZIP transcription factor), FgVelB (velvet regulator), and FgflbA (regulator of G protein signaling) [7], [60], [61]. A MYT1 deletion mutant also resulted in a defect in female fertility, but retained male fertility [12]. Interestingly, the MYT3 deletion mutant lost both female and male fertility, and a complemented strain recovered all of the defects. To dissect these observations, we investigated whether deletion of MYT3 has an effect on vegetative compatibility. In this study, we generated nit1 and nitM mutants derived from the wild type and Δmyt3 strains (data not shown), respectively, and tested their ability to form heterokaryon by hyphal fusion from the pairing combinations (WT nitM × Δmyt3 nit1, WT nit1 × Δmyt3 nitM, and Δmyt3 nit1 × Δmyt3 nitM). However, all combinations produced fungal colonies (data not shown), indicating that the nit strains used in this study were in same vegetative compatibility group, and deletion of MYT3 did not cause vegetative incompatibility. Thus, these results suggest that MYT3 is not involved in heterokaryon formation through hyphal fusion.
We demonstrated that MYT3 plays an important role in F. graminearum on wheat heads. As previously mentioned, PsMYB1 is also involved in P. sojae pathogenesis of soybean, which silencing of PsMYB1 resulted in the impairment of zoosporogenesis and zoospore development [28]. In plants, several Myb transcription factors (e.g., AtMYB30, TaPIMP1, JAmyb, and BOS1) are involved in host resistance to pathogens and abiotic stress, which involves signaling pathways for plant defense molecules, such as jasmonate, salicylic acid, and abscisic acid [28], [62], [63], [64]. Our results also showed that the disease index of the Δmyt3 strain was approximately 10-fold less than the wild-type level, resulting from an inability of the Δmyt3 strain to spread from an inoculated spikelet to other spikelets. In addition, trichothecene production and TRI5 and TRI6 gene transcript levels were remarkably reduced in the Δmyt3 strain. This finding may also contribute to the reduced progression of plant disease because trichothecenes are an important virulence factor in F. graminearum [65]. Furthermore, we cannot exclude the possibility that MYT3 is required to respond to an environmental cue from the host that helps regulate pathogenesis: this possibility is supported by our observations of different growth pattern of the Δmyt3 strain to specific nitrogen source. Thus, considering involvement of MYT3 in pathogenesis, identification of target genes regulated by MYT3 will contribute critical information to help understand regulatory mechanisms of F. graminearum pathogenesis.
In summary, we examined the biological functions of MYT3, a Myb-like protein in F. graminearum that contains two Myb DNA-binding domains. Our results showed that MYT3 is localized in nuclei during developmental stages, affects various aspects of fungal development, such as conidiation, germination, and male/female fertility, and contributes to pathogenicity by either directly or indirectly influencing trichothecene biosynthesis. Our identification of these functions for MYT3 sheds further light on the complex roles of Myb-like proteins in fungal development and pathogenicity of F. graminearum.
Supporting Information
Figure S1.
Distribution and phylogenetic analysis of MYT3 homologs in fungi.
https://doi.org/10.1371/journal.pone.0094359.s001
(PDF)
Figure S2.
Strategy for fusion of GFP to MYT3.
https://doi.org/10.1371/journal.pone.0094359.s002
(PDF)
Figure S3.
Strategies for complementation and overexpression of MYT3.
https://doi.org/10.1371/journal.pone.0094359.s003
(PDF)
Author Contributions
Conceived and designed the experiments: YK HK HS YWL. Performed the experiments: YK HK HS GJC JCK YWL. Analyzed the data: YK HK HS GJC JCK YWL. Contributed reagents/materials/analysis tools: GJC JCK YWL. Wrote the paper: YK HK HS YWL.
References
- 1. Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal crops. Mol Plant Pathol 5: 515–525.
- 2. Guenther JC, Trail F (2005) The development and differentiation of Gibberella zeae (anamorph: Fusarium graminearum) during colonization of wheat. Mycologia 97: 229–237.
- 3.
Desjardins AE (2006) Fusarium mycotoxins chemistry, genetics, and biology. AE Desjardins, ed. editor. St. Paul, MN: APS Press.
- 4. Sutton JC (1982) Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum. Can J Plant Pathol 4: 195–209.
- 5. Paulitz TC (1996) Diurnal release of ascospores by Gibberella zeae in inoculated wheat plots. Plant Dis 80: 674–678.
- 6. Hou Z, Xue C, Peng Y, Katan T, Kistler HC, et al. (2002) A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection. Mol Plant-Microbe Interact 15: 1119–1127.
- 7. Lee J, Myong K, Kim J-E, Kim H-K, Yun S-H, et al. (2012) FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum. Microbiology 158: 1723–1733.
- 8. Shim W-B, Sagaram US, Choi YE, So J, Wilkinson HH, et al. (2006) FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F. graminearum. Mol Plant-Microbe Interact 19: 725–733.
- 9. Urban M, Mott E, Farley T, Hammond-Kosack K (2003) The Fusarium graminearum MAP1 gene is essential for pathogenicity and development of perithecia. Mol Plant Pathol 4: 347–359.
- 10. Yu H-Y, Seo J-A, Kim J-E, Han K-H, Shim W-B, et al. (2008) Functional analyses of heterotrimeric G protein Gα and Gβ subunits in Gibberella zeae. Microbiology 154: 392–401.
- 11. Son H, Seo Y-S, Min K, Park AR, Lee J, et al. (2011) A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum. PLoS Pathog 7: e1002310.
- 12. Lin Y, Son H, Lee J, Min K, Choi GJ, et al. (2011) A putative transcription factor MYT1 is required for female fertility in the ascomycete Gibberella zeae. PLoS ONE 6: e25586.
- 13. Lin Y, Son H, Min K, Lee J, Choi GJ, et al. (2012) A putative transcription factor MYT2 regulates perithecium size in the ascomycete Gibberella zeae. PLoS ONE 7: e37859.
- 14. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, et al. (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15: 573–581.
- 15. Oh IH, Reddy EP (1999) The myb gene family in cell growth, differentiation and apoptosis. Oncogene 18: 3017.
- 16. Ramsay RG, Gonda TJ (2008) MYB function in normal and cancer cells. Nat Rev Cancer 8: 523–534.
- 17. Ravaglia D, Espley RV, Henry-Kirk RA, Andreotti C, Ziosi V, et al. (2013) Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors. BMC Plant Biol 13: 68.
- 18. Beug H, von Kirchbach A, Doderlein G, Conscience JF, Graf T (1979) Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation. Cell 18: 375–390.
- 19. Gonda TJ, Gough NM, Dunn AR, de Blaquiere J (1985) Nucleotide sequence of cDNA clones of the murine myb proto-oncogene. EMBO J 4: 2003–2008.
- 20. Lipsick JS (1996) One billion years of Myb. Oncogene 12: 223–235.
- 21. Xiang Q, Judelson HS (2010) Myb transcription factors in the oomycete Phytophthora with novel diversified DNA-binding domains and developmental stage-specific expression. Gene 453: 1–8.
- 22. Anton IA, Frampton J (1988) Tryptophans in myb proteins. Nature 336: 719.
- 23. Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, et al. (1994) Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices. Cell 79: 639–648.
- 24. Howe KM, Reakes CF, Watson RJ (1990) Characterization of the sequence-specific interaction of mouse c-myb protein with DNA. EMBO J 9: 161–169.
- 25. Mateos L, Jimenez A, Revuelta JL, Santos MA (2006) Purine biosynthesis, riboflavin production, and trophic-phase span are controlled by a Myb-related transcription factor in the fungus Ashbya gossypii. Appl Environ Microbiol 72: 5052–5060.
- 26. Arratia-Quijada J, Sánchez O, Scazzocchio C, Aguirre J (2012) FlbD, a Myb transcription factor of Aspergillus nidulans, is uniquely involved in both asexual and sexual differentiation. Eukaryot Cell 11: 132–142.
- 27. Wieser J, Adams TH (1995) FlbD encodes a Myb-like DNA-binding protein that coordinates initiation of Aspergillus nidulans conidiophore development. Gene Dev 9: 491–502.
- 28. Zhang M, Lu J, Tao K, Ye W, Li A, et al. (2012) A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development. PLoS ONE 7: e40246.
- 29.
Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Ames, IA: Blackwell Pub.
- 30. Cappellini RA, Peterson JL (1965) Macroconidium formation in submerged cultures by a non-sporulating strain of Gibberella zeae. Mycologia 57: 962–966.
- 31. Gardiner DM, Kazan K, Manners JM (2009) Nutrient profiling reveals potent inducers of trichothecene biosynthesis in Fusarium graminearum. Fungal Genet Biol 46: 604–613.
- 32.
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
- 33. Sievers F, Wilm A, Dineen DG, Gibson TJ, Karplus K, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539.
- 34. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22: 195–201.
- 35. Lin K, Simossis VA, Taylor WR, Heringa J (2005) A simple and fast secondary structure prediction algorithm using hidden neural networks. Bioinformatics 21: 152–159.
- 36. Kosugi S, Hasebe M, Tomita M, Yanagawa H (2009) Systematic identification of yeast cell cycle-dependent nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci USA 106: 10171–10176.
- 37. Lee S, Son H, Lee J, Min K, Choi GJ, et al. (2011) Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae. Eukaryot Cell 10: 1043–1052.
- 38. Kim J-E, Han K-H, Jin J, Kim H, Kim J-C, et al. (2005) Putative polyketide synthase and laccase genes for biosynthesis of aurofusarin in Gibberella zeae. Appl Environ Microbiol 71: 1701–1708.
- 39. Yu J-H, Hamari Z, Han K-H, Seo J-A, Reyes-Dominguez Y, et al. (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41: 973–981.
- 40. Horwitz BA, Sharon A, Lu SW, Ritter V, Sandrock TM, et al. (1999) A G protein alpha subunit from Cochliobolus heterostrophus involved in mating and appressorium formation. Fungal Genet Biol 26: 19–32.
- 41. Son H, Lee J, Park AR, Lee Y-W (2011) ATP citrate lyase is required for normal sexual and asexual development in Gibberella zeae. Fungal Genet Biol 48: 408–417.
- 42. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
- 43. He J, Yang R, Zhou T, Tsao R, Young JC, et al. (2007) Purification of deoxynivalenol from Fusarium graminearum rice culture and mouldy corn by high-speed counter-current chromatography. J Chromatogr A 1151: 187–192.
- 44. Seo J-A, Kim J-C, Lee D-H, Lee Y-W (1996) Variation in 8-ketotrichothecenes and zearalenone production by Fusarium graminearum isolates from corn and barley in Korea. Mycopathologia 134: 31–37.
- 45. Lee J, Lee T, Lee Y-W, Yun S-H, Turgeon BG (2003) Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae. Mol Microbiol 50: 145–152.
- 46. Garzia A, Etxebeste O, Herrero-Garcia E, Ugalde U, Espeso EA (2010) The concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression and asexual development in Aspergillus nidulans. Mol Microbiol 75: 1314–1324.
- 47. Güldener U, Seong K-Y, Boddu J, Cho S, Trail F, et al. (2006) Development of a Fusarium graminearum Affymetrix GeneChip for profiling fungal gene expression in vitro and in planta. Fungal Genet Biol 43: 316–325.
- 48. Tudzynski B, Mende K, Weltring KM, Kinghorn JR, Unkles SE (1996) The Gibberella fujikuroi niaD gene encoding nitrate reductase: isolation, sequence, homologous transformation and electrophoretic karyotype location. Microbiology 142: 533–539.
- 49. Seong K-Y, Pasquali M, Zhou X, Song J, Hilburn K, et al. (2009) Global gene regulation by Fusarium transcription factors Tri6 and Tri10 reveals adaptations for toxin biosynthesis. Mol Microbiol 72: 354–367.
- 50. Yanhui C, Xiaoyuan Y, Kun H, Meihua L, Jigang L, et al. (2006) The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol Biol 60: 107–124.
- 51. Ehrenkaufer GM, Hackney JA, Singh U (2009) A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica. Cell Microbiol 11: 898–910.
- 52. Rodríguez-Sánchez L, Rodríguez-López M, García Z, Tenorio-Gómez M, Schvartzman JB, et al. (2011) The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress. J Cell Sci 124: 25–34.
- 53. Imamura S, Kanesaki Y, Ohnuma M, Inouye T, Sekine Y, et al. (2009) R2R3-type MYB transcription factor, CmMYB1, is a central nitrogen assimilation regulator in Cyanidioschyzon merolae. Proc Natl Acad Sci USA 106: 12548–12553.
- 54. Kudla B, Caddik MX, Langdon T, Martinez-Rossi NM, Bennett F, et al. (1990) The regulatory gene areA mediation nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9: 1355–1364.
- 55. Mihlan M, Homann V, Liu TW, Tudzynski B (2003) AREA directly mediates nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi, but its activity is not affected by NMR. Mol Microbiol 47: 975–991.
- 56. Todd RB, Fraser JA, Wong KH, Davis MA, Hynes MJ (2005) Nuclear accumulation of the GATA factor AreA in response to complete nitrogen starvation by regulation of nuclear export. Eukaryot Cell 4: 1646–1653.
- 57. Arst HN, Cove DJ (1973) Nitrogen metabolite repression in Aspergillus nidulans. Mol Gen Genet 126: 111–141.
- 58. Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol Rev 61: 17–32.
- 59. Tahirov TH, Sato K, Ichikawa-Iwata E, Sasaki M, Inoue-Bungo T, et al. (2002) Mechanism of c-Myb-C/EBP beta cooperation from separated sites on a promoter. Cell 108: 57–70.
- 60. Wang Y, Liu W, Hou Z, Wang C, Zhou X, et al. (2011) A novel transcriptional factor important for pathogenesis and ascosporogenesis in Fusarium graminearum. Mol Plant-Microbe Interact 24: 118–128.
- 61. Park AR, Cho A-R, Seo J-A, Min K, Son H, et al. (2012) Functional analyses of regulators of G protein signaling in Gibberella zeae. Fungal Genet Biol 49: 511–520.
- 62. Lee MW, Qi M, Yang Y (2001) A novel jasmonic acid-inducible rice myb gene associates with fungal infection and host cell death. Mol Plant-Microbe Interact 14: 527–535.
- 63. Mengiste T, Chen X, Salmeron J, Dietrich R (2003) The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell 15: 2551–2565.
- 64. Vailleau F, Daniel X, Tronchet M, Montillet JL, Triantaphylides C, et al. (2002) A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl Acad Sci USA 99: 10179–10184.
- 65. Proctor RH, Hohn TM, McCormick SP (1995) Reduced virulence of Gibberella zeae caused by disruption of a trichthecene toxin biosynthetic gene. Mol Plant-Microbe Interact 8: 593–601.
- 66. Bowden RL, Leslie JF (1999) Sexual recombination in Gibberella zeae. Phytopathology 89: 182–188.