Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.
Citation: Wang G, Wang C, Hou R, Zhou X, Li G, Zhang S, et al. (2012) The AMT1 Arginine Methyltransferase Gene Is Important for Plant Infection and Normal Hyphal Growth in Fusarium graminearum. PLoS ONE 7(5): e38324. https://doi.org/10.1371/journal.pone.0038324
Editor: Yin-Won Lee, Seoul National University, Republic of Korea
Received: March 26, 2012; Accepted: May 3, 2012; Published: May 31, 2012
Copyright: © 2012 Wang 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 work was supported by the National Major Project of Breeding for New Transgenic Organisms (2012ZX08009003) and a grant from the National Research Initiative of the United States Department of Agriculture (USDA) CSREES (#2007-35319-102681). Dr. Wang and Dr. Li were partially supported by China Scholarship Council fellowships. 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.
In eukaryotic organisms, reversible phosphorylation of proteins by protein kinase and phosphatase is well known to regulate various growth and development processes. Protein methylation is another form of post-translational modifications that also play regulatory roles in various processes, including nucleo-cytoplasmic transport of proteins, transcriptional activation and elongation, mRNA precursors splicing, and signal transduction , , , . The majority of protein methylation occurred at the arginine residues are catalyzed by protein arginine methyltransferases (PRMTs), which are divided into four major classes. Type I and type II PRMTs catalyze asymmetric and symmetric ω NG, NG-dimethylation of arginine residues, respectively . Whereas type III PRMTs catalyze ω NG monomethylation of arginines, type IV PRMTs catalyze the formation of δ NG-monomethylarginine. In human, type I PRMTs include PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8. PRMT5, PRMT7, and PRMT9 are type II PRMTs . Whereas PRMT1, PRMT3, and PRMT5 are well conserved in eukaryotic organisms, PRMT2, PRMT8, and PRMT9 lack distinct orthologs in unicellular eukaryotes and may be required for tissue-specific functions in multicellular organisms , .
The budding yeast Saccharomyces cerevisiae has only three PRMT genes, HMT1, RMT2, and HSL7 . HMT1 (type I) is the major arginine methyltransferase and possesses similar functions of mammalian PRMT1. HMT1 is not essential for cell growth in yeast. However, deletion of HMT1 is synthetically lethal with mutations in the NPL3 or CBP80 genes . RMT2 is a type IV PRMT gene that is found in fungi and plants but not in protozoa and human . The HSL7 gene (type II) is orthologous to human PRMT5 . In Arabidopsis, many RNA binding or processing proteins are methylated by AtPRMT5. Mutations in the AtPRMT5 gene affected RNA splicing in hundreds of genes involved in different biological processes and causes pleiotropic developmental defects, such as late flowering .
In S. cerevisiae, Hmt1 is a non-essential member of the heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in mRNA biogenesis . It confers SAM-dependent methylation to components of hnRNPs, which often contain C-terminal RGG-rich repeats as the sites of arginine methylation . Hrp1, Nab2, and Npl3 are among the most studied hnRNPs that are methylated by Hmt1. Methylation by Hmt1 is important for their export from the nucleus , , . Hrp1 is involved in the processing of 3′ ends of pre-mRNA, mRNA polyadenylation, and the nonsense mediated decay pathway , , . The Nab2 protein is required for the export of poly(A) RNA and poly(A) tail length control , . Npl3 has been implicated in transcription elongation and termination . Methylation by Hmt1 in the nucleus and phosphorylation by the SR protein kinase Sky1 in the cytoplasm regulate the nucleo-cytoplasmic transport of Npl3 .
Orthologs of yeast PRMT genes are well conserved in filamentous plant pathogenic ascomycetes. However, none of them have been experimentally characterized for their biological functions in plant pathogenic ascomycetes. F. graminearum is a major causal agent of wheat and barley head blight or scab worldwide , . Fusarium head blight (FHB) poses as a serious problem in wheat production by causing severe yield losses and contamination of infested kernels with harmful mycotoxins, including deoxynivalenol (DON) and zearalenone , . Because of the importance of PRMT genes in eukaryotes , , in this study we identified and functionally characterized all of the four predicted PRMT genes in F. graminearum. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant was significantly reduced in virulence and DON production in infection assays with flowering wheat heads. Our results indicate that AMT1, like its ortholog HMT1 in yeast, is the predominant arginine methyltransferase in F. graminearum. Although dispensable for sexual and asexual reproduction, AMT1 is important for normal growth rate, stress responses, plant infection, and nucleo-cytoplasmic transport of FgHrp1.
Identification of the HMT1 ortholog, AMT1, in F. graminearum
The genome of F. graminearum contains four PRMT genes, FGSG_01134 (XP_381310), FGSG_10718 (XP_390894), FGSG_00501 (XP_380677), and FGSG_10756 (XP_390932) that are named AMT1-AMT4 (for arginine methyltransferase genes) in this study. FGSG_01134 (AMT1) is orthologous to HMT1, which is the main arginine methyltransferase gene in S. cerevisiae. The 345-amino acid protein encoded by HMT1 has a typical arginine methyltransferase domain. FGSG_10718 (AMT2) encodes a PRMT3-like protein that also shares significant homology with yeast HMT1. FGSG_00501 (AMT3) and FGSG_10756 (AMT4) are orthologous to yeast RMT2 and HSL7, respectively (Figure S1). Unlike the budding yeast, filamentous ascomycetes such as Magnaporthe oryzae and Aspergillus nidulans (Figure S1) have four PRMT genes.
Generation of amt1 deletion mutants
The AMT1 gene replacement construct (Fig. 1A) was generated with the split-marker approach and transformed into the wild-type strain PH-1. Putative Δamt1 mutants were identified by PCR and confirmed by Southern blot analysis (Fig. 1B). In the wild type, a 7.0-kb BamHI band was detected with an AMT1 fragment amplified with primers AMT1/5F and AMT1/6R (Table S2) as the probe A (Fig. 1B). The same probe had no hybridization signal in transformants M1, M2, and M3 (Table 1). When probed with a fragment of the hph gene, PH-1 had no hybridization signals. Transformants M1 and M2 had a 6.4-kb band (Fig. 1B), which is similar to the expected size derived from the gene replacement event (Fig. 1A). Transformant M3 had a weak 6.4-kb band but a strong 10-kb band, suggesting that besides targeted homologous recombination, multiple copies of the AMT1 gene replacement construct were integrated ectopically during transformation. Therefore, only transformants M1 and M2 were the expected amt1 deletion mutants with no additional integration events. Mutants M1 and M2 had the same phenotype although only data with mutant M2 were described below.
A. The AMT1 locus and gene replacement construct. The AMT1 and hph genes are marked with empty and black arrows, respectively. 1F, 2R, 3F, and 4R are primers used to amplify the flanking sequences. BamHI (B). B. Southern blot analysis with the wild type (PH-1) and Δamt1 transformants (M1, M2, and M3). All DNA samples were digested with BamHI. The blots were hybridized with probe A (left) amplified with primers AMT1/5F and AMT1/6R and probe B (right) amplified with H852 and H850. C. Colony morphology of the PH-1, Δamt1 mutant M2, and Δamt1/AMT1 transformant C2 cultures grown on CM. Photographs were taken after incubation for 3 days.
When assayed for growth on CM medium, the Δamt1 mutant produced less aerial hyphae than the wild type (Fig. 1C) and had approximately 24% reduction in growth rate (Table 2). It was also reduced in aerial hyphal growth and growth rate on PDA, 5×YEG, and YEPD plates (Figure S2). When the wild-type AMT1 allele was transformed into the Δamt1 mutant, defects in hyphal growth and other phenotypes described below were rescued in the resulting Δamt1/AMT1 transformant C2 (Table 2). These results indicate that deletion of AMT1 is directly responsible for the growth defects observed in the mutant and AMT1 is important for normal vegetative growth in F. graminearum.
AMT1 is dispensable for sexual and asexual reproduction
The Δamt1 mutant was normal in sexual reproduction (Figure S3). On mating plates, abundant perithecia were formed by the Δamt1 mutant. Ascospore cirrhi were observed on top of mature perithecia 3 weeks after fertilization (Figure S3). The Δamt1 mutant also had no obvious defects in conidiation (Table 2) and conidium germination. The morphology of Δamt1 ascospores and conidia was normal. These data indicates that although the Δamt1 mutant was slightly reduced in vegetative growth, AMT1 is dispensable for sexual and asexual reproduction.
The Δamt1 mutant is significantly reduced in virulence
In infection assays with flowering wheat heads, the Δamt1 mutant caused typical scab symptoms in the inoculated kernels and was able to spread to nearby spikeletes (Fig. 2A). However, it was significantly reduced in virulence compared to PH-1 (Fig. 2A). The average disease index, a measurement for virulence by counting diseased spikeletes per wheat head, of the Δamt1 mutant and PH-1 was 4.3 and 13.8, respectively (Table 2), indicating that the Δamt1 mutant was defective in disease spreading.
A. Flowering wheat heads were drop-inoculated with conidia from the wild-type (PH-1), Δamt1 mutant (M2), and Δamt1/AMT1 (C2) strains. Black dots mark the inoculated spikeletes. Photographs were taken 14 days post-inoculation (dpi). B. Corn silks were inoculated with blocks of cultures of PH-1, Δamt1 mutant M2, and Δamt1/AMT1 transformant C2. Photographs were taken 6 dpi.
Corn also is a host to F. graminearum. In infection assays with corn silks, the Δamt1 mutant caused only limited discoloration near the inoculation sites. Under the same conditions, extensive discoloration was observed in corn silks inoculated with PH-1 (Fig. 2B), confirming that AMT1 is important for virulence in F. graminearum.
To characterize the defects of the Δamt1 mutant in plant infection, inoculated flowering wheat heads were sampled, fixed, and examined for hyphal growth. At 48 h post-inoculation (hpi), fungal growth was observed on the surface and inside glume tissues inoculated with the wild type (Fig. 3). In wheat heads inoculated with the Δamt1 mutant, fungal growth was abundant on the surface and rarely in glume tissues (Fig. 3). At 120 hpi, the wild type had colonized the vascular and other tissues of the rachis and produced abundant intracellular hyphae (Fig. 3). In contrast, fungal growth was limited or much sparse in the rachis near the spikeletes inoculated with the Δamt1 mutant (Fig. 3), indicating that AMT1 is important for invasion and spreading in plant tissues. These observations are consistent with reduced virulence of the Δamt1 mutant.
A. Colonization of glume tissues by the wild-type (PH-1), Δamt1 mutant (M2), and Δamt1/AMT1 (C2) strains 48 hpi. B. The rachises directly beneath the inoculated spikeletes were examined 120 hpi. Hyphae growth (marked with arrows) was abundant in plant tissues inoculated with the wild type and Δamt1/AMT1 strains but scarce in samples inoculated with Δamt1 mutant. Bar = 50 µm.
The Δamt1 mutant has increased sensitivity to oxidative and membrane stresses
To determine whether the Δamt1 mutant was defective in stress responses, we assayed its growth on PDA plates with 0.05% H2O2, 0.01%SDS, and 0.7 M NaCl. It appears that AMT1 is dispensable for responses to hyperosmotic stress because the wild type and Δamt1 mutant stains had no obvious difference in vegetative growth on PDA plates with 0.7 M NaCl (Fig. 4). However, AMT1 is likely involved in responses to oxidative and membrane stresses. In the presence of 0.05% H2O2 or 0.01%SDS, the Δamt1 mutant was more significantly reduced in growth rate than the wild type (Fig. 4).
Subcellular localization of AMT1-GFP fusion
To determine the expression and localization of AMT1, we generated an AMT1-GFP fusion construct and transformed it into the Δamt1 mutant. After screened by PCR and confirmed by Southern blot analysis, transformant Y5 (Table 1) was identified as one of the transformants expressing the AMT1-GFP construct under the control of its native promoter. Similar to the complemented strain C2, defects of the Δamt1 mutant were rescued in the Δamt1/AMT1-GFP transformant. GFP signals were present in both cytoplasm and nuclei in conidia and 7 h germlings of transformant Y5 (Fig. 5). However, nuclei had stronger fluorescence signals than the cytoplasm, indicating that majority of Amt1-GFP proteins localized to the nucleus.
Conidia (A) and 7 h germlings (B) of the Δamt1/AMT1-GFP transformant (Y5) were examined by phase contrast (DIC) or epifluorescence (GFP) microscopy. GFP signals were present in both the cytoplasm and nucleus but were stronger in the nucleus. Bar = 50 µm.
Amt1 influences the nuclear transport of FgHrp1
In S. cerevisiae, Hmt1 is involved in the regulation of nucleo-cytoplasmic transport of hnRNP components, including Hrp1 and Nab2 . Orthologs of HRP1 and NAB2 in F. graminearum are FGSG_13728.3 and FGSG_01282.3, respectively. We constructed the FgHRP1-GFP and FgNAB2-GFP fusion constructs and transformed them into the wild-type and Δamt1 mutant strains. In the resulting transformants expressing the FgHRP1-GFP construct, the subcellular localization of FgHrp1-GFP fusion proteins differed significantly between the wild-type and Δamt1 mutant (Fig. 6). In the Δamt1 mutant, GFP signals were detected mainly in the nucleus. Each nucleus had one or more dots of bright GFP signals that may correspond to hnRNP particles associated with FgHrp1. In the wild type, GFP signals were primarily observed in the cytoplasm, indicating that deletion of AMT1 affected the nuclear export of FgHrp1 proteins. Fluorescent particles in the cytoplasm may represent protein complexes that are associated with FgHrp1 after its exit from the nucleus.
Fresh conidia were harvested from transformants of PH-1 (HP10) and Δamt1 mutant M2 (HA11) expressing the FgHRP1-GFP fusion construct and incubated in liquid YEPD medium for 12 h. Germ tubes were then stained with DAPI and examined by fluorescence microscopy with the GFP- and DAPI-specific filters. FgHrp1-GFP fusion proteins mainly localized to the nucleus in the Δamt1 mutant but to the cytoplasm in the wild type. Bar = 20 µm.
In contrast, FgNab2-GFP proteins were distributed mainly in the nucleus in both wild-type and Δamt1 mutant strains (Figure S4), indicating that AMT1 is not important for the subcellular localization of FgNab2 in F. graminearum. Therefore, arginine methylation may play different roles in the nucleo-cytoplasmic transport of different hnRNP components in F. graminearum and S. cerevisiae.
Functional characterization of the other three PRMT genes in F. graminearum
To determine the functions of other three putative PRMT genes, the split-marker approach was used to generate the AMT2 (FGSG_10718), AMT3 (FGSG_00501), and AMT4 (FGSG_10756) gene replacement constructs. The resulting PCR products were transformed into protoplasts of the wild-type strain PH-1. The Δamt2, Δamt3, and Δamt4 knockout mutants (Table 1) were identified by PCR and confirmed by Southern blot analyses. In comparison with the wild type, the Δamt2, Δamt3, and Δamt4 mutants had no obvious defects in vegetative growth, conidiation, and production of perithecia and ascospores (Figure S3; Figure S5). They also had similar growth rate with the wild type on PDA plates with 0.05% H2O2, 0.01% SDS, or 0.7 M NaCl, or 300 µg/ml Congo red (Figure S6). In infection assay with corn silks, the Δamt2 and Δamt3 mutants were as virulent as the wild type but the Δamt4 was slightly reduced in virulence (Fig. 7A; Table S1). These results indicate that AMT2, AMT3, and AMT4 genes are dispensable for vegetative growth, asexual and sexual reproduction, and stress responses. While AMT2 and AMT3 were dispensable for plant infection, AMT4 was required for full virulence.
The Δamt1 Δamt2 double mutant has similar defects in plant infection with the Δamt1 mutant
The AMT1 and AMT2 genes are the only two predicted type I arginine methyltransferase genes in F. graminearum. They both share sequence similarity with yeast Hmt1 (Figure S1). To determine their functional relationship, we generated the Δamt1 Δamt2 double mutant by deletion of AMT2 in the Δamt1 mutant M2. The resulting double mutant (Table 1), similar to the Δamt1 mutant, was normal in conidiation and sexual reproduction (Figure S3) but slightly reduced in vegetative growth (Figure S5). In plant infection assays, the Δamt1 Δamt2 had similar defects in virulence with the Δamt1 mutant (Fig. 7A). On PDA plates with 0.05% H2O2 or 0.01% SDS, the Δamt1 and Δamt1 Δamt2 mutants also had similar growth defects (Fig. 7B). Therefore, the Δamt1 and Δamt1 Δamt2 mutants had no significant differences in growth, stress responses, and virulence. These results suggest that AMT1 and AMT2 have no overlapping functions.
Deletion of AMT1 results in less than 2-fold changes in the expression of AMT2, AMT3, and AMT4
RNA samples were isolated from vegetative hyphae of PH-1 and Δamt1 mutant grown in liquid CM for 6 h. In comparison with the wild type, the expression levels of AMT2, AMT3, and AMT4 were reduced approximately 21%, 10%, and 44%, respectively, in the Δamt1 mutant (Fig. 8A). However, none of them had over 2-fold changes in the expression level between the wild type and Δamt1 mutant strains.
RNA samples were isolated from germlings of the wild-type (PH-1) and Δamt1 mutant strains grown in liquid YEPD for 6 h. The expression levels of (A) three other PRMT genes, AMT2, AMT3, and AMT4, and (B) three predicted genes located in the telomeric region of chromosome 4 (FGSG_14027, FGSG_11614, and FGSG_11613) were assayed by qRT-PCR.
Deletion of AMT1 affects the expression of genes adjacent to the telomere
Because deletion of HMT1 is known to affect the formation of silent chromatin , we assayed the expression of three genes, FGSG_14027, FGSG_11614, and FGSG_11613 that are within 10 kb from the telomeric repeat sequences (TTAGGG) on supercontig 14 (Chromosome 4, Fig. 8B). The current version of F. graminearum assembly contains no other telemetric repeat sequences. FGSG_14027 (795–1625) encodes a putative histone deacetylase gene orthologous to yeast HOS4. It is less than 1 kb away from the telomeric repeats. FGSG_11614 (3250–5086) and FGSG_11613 (5679–7261) encode hypothetical proteins that are conserved in filamentous ascomycetes but not in yeast. Whereas the expression level of FGSG_14027 and FGSG_11614 was increased over approximately 4- and 5-fold, respectively, expression of FGSG_11613 was slightly increased but not significantly in the Δamt1 mutant compared to the wild type (Fig. 8B). Since FGSG_11613 is more distal to the telomeric repeats than the other two genes, it is likely that silencing of genes adjacent to the telomere is affected by deletion of AMT1.
The expression and activation of Mgv1, Gpmk1, and Fghog1 MAP kinases in the amt1 mutant
Because PRMTs are known to affect signal transduction in mammalian cells , we assayed the phosphorylation of all three MAP kinases that have been characterized in F. graminearum and known to important for plant infection , , , , . In comparison with the wild type strain, the amt1 mutant was normal in the expression and phosphorylation levels of Mgv1 and Fghog1 (Fig. 9). For Gpmk1, the expression level was normal but the phosphorylation level was slightly but not significantly reduced (Fig. 9). These data indicate that Amt1 does not significantly affect the expression and activation of these MAP kinases in F. graminearum.
Total proteins were isolated from the wild-type (PH-1) and amt1 mutant (M2) strains. When detected with an anti-TpEY antibody, the phosphorylation levels of Mgv1 had no significant changes in the amt1 mutant in comparison with the wild type. The phosphorylation of FgHog1 detected with an anti-TpGY antibody also appeared to be normal in the amt1 mutant. Detection with a monoclonal anti-actin antibody showed equal amount of proteins.
Methylation of the arginine residues by arginine methyltransferases plays important roles in various cellular processes in eukaryotic organisms such as nucleo-cytoplasmic transport and mRNA biogenesis , , . The genome of F. graminearum contains four predicted arginine methyltransferase genes that belong to the type I, type II, and type VI of PRMTs , . Phenotype analyses with targeted deletion mutants of these PRMT genes indicated that only the Δamt1 mutant had obvious defects in growth and plant infection. Mutants deleted of the other three PRMT genes had no significant phenotypes. Therefore, similar to its ortholog in yeast, AMT1 must be the predominant arginine methyltransferase in F. graminearum. In A. nidulans, three AMT1 genes, rmtA, rmtB, and rmtC that are orthologous to AMT1, AMT2, and AMT4, respectively, have been characterized. None of the rmtA, rmtB, and rmtC deletion mutants had obvious defects in vegetative growth, sexual, and asexual reproduction on normal growth conditions , suggesting that A. nidulans may lack a predominant PRMT gene.
AMT2 encodes a predicted type I PRMT protein that shares significant sequence similarity to PRMT3 in human. Its orthologs are well conserved in filamentous fungi, including M. oryzae, A. nidulans, and Neurospora crassa and the fission yeast Schizosaccharomyces pombe. However, S. cerevisiae and Candida albicans lack a distinct ortholog of AMT2, suggesting that this gene may have been lost in some Saccharomycetales species during evolution. As the only other predicted type I PRMT gene in F. graminearum, AMT2 shares limited homology with AMT1 and yeast HMT1. Although Amt2 has a C2H2 zinc finger domain that is absent in Amt1, deletion of the AMT2 gene had no obvious phenotypic changes. In A. nidulans, deletion of the rmtB gene also lacked any detectable phenotype . To determine the relationship between AMT1 and AMT2, we deleted the AMT2 gene in the Δamt1 mutant. The Δamt1 mutant and the Δamt1 Δamt2 double mutant had no significant differences in the phenotypes assayed, including growth rate, sensitivities to oxidative stress, and virulence. Deletion of AMT1 also had no significant impact on the expression level of AMT2 (Fig. 8A). Therefore, it is unlikely for AMT2 to have overlapping functions with AMT1 in F. graminearum.
For the other two PRMT genes in F. graminearum, AMT3 and AMT4 are orthologous to the RMT2 and HSL7 genes in yeast, respectively. Rmt2 and its related PRMT genes are specific to fungi and plants . In yeast, Rmt2 specifically methylates ribosomal protein Rpl12 (L12) on Arg67 . The rmt2 mutant is defective in δ NG -methylarginine modifications but normal in growth and reproduction . In C. albicans, the rmt2/rmt2 mutant grew as robustly as the reconstituted or heterozygous strains in rich media but the level of δ NG-monomethylarginine is reduced . However, no data on virulence of the mutant were presented. The genome of A. nidulans contains the ortholog of AMT3 (Figure S1) but it has not been experimentally characterized for its biological function .
In S. cerevisiae, Hsl7 is required along with Hsl1 kinase for bud neck recruitment, phosphorylation, and degradation of Swe1 . The Δhsl7 mutant produces elongated, anucleate buds and has increased sensitivity to Calcofluor and CaCl2 . In F. graminearum, the AMT4 deletion mutant had no obvious defects but the rmtC mutant of A. nidulans had increased sensitivity to oxidative stress and elevated temperatures . In U. maydis, the Hsl7 ortholog was identified as a Smu1 PAK kinase interacting protein. It regulates cell length and the filamentous response to solid SLAD (synthetic low ammonia plus 2% dextrose) but is dispensable for plant infection. Although the amt3 and amt4 mutants of F. graminearum had no obvious defects in phenotypes assayed in this study, AMT3 and AMT4 genes are well conserved in filamentous fungi . It is likely that they are functional in some biological processes that remain to be characterized in F. graminearum.
In S. cerevisiae, Hmt1 is a non-essential component of the hnRNP complex . Hmt1 affects the nucleo-cytoplasmic transport of other hnRNP components that are important for mRNA biogenesis. In F. graminearum, the Δamt1 mutant was reduced approximately 24% in vegetative growth but normal in conidiation and ascospore production. If it also is a component of hnRNP in F. graminearum, Amt1 may be dispensable for mRNA processing of genes that are important for sexual reproduction and conidiation. In Arabidopsis, the AtPRMT5 gene only affects RNA splicing in a subset of genes . It is likely that only subsets of genes important for vegetative growth and plant infection (infectious growth) are affected in the Δamt1 mutant in F. graminearum. AMT1 appears to play no or only minor roles in genes involved in sexual and asexual reproduction.
In infection assays with flowering wheat heads and corn silks, the Δamt1 mutant was significantly reduced in virulence. Although AMT1 orthologs are well conserved, none of them have been characterized in plant pathogenic ascomycetes. In the human pathogen Candida albicans, deletion of CaHMT1 affects the expression and localization of NPL3 . However, the function of CaHMT1 in virulence has not been reported. One common stress faced by hyphae of necrotrophic fungi in planta is reactive oxygen species (ROS) generated during oxidative burst , . The Δamt1 mutant, similar to the rmtA mutant of A. nidulans , had increased sensitivity to H2O2. It also had a slightly reduced growth rate and increased sensitivity to membrane stress. All of these defects may contribute to the defects of the Δamt1 mutant in plant infection. In addition, in diseased wheat kernels, the Δamt1 mutant was reduced in the production of DON, which is a well-characterized virulence factor in F. graminearum , . However, reduced DON production in infested plant tissues may be related to reduced fungal biomass of the Δamt1 mutant.
In S. cerevisiae, two of the well-characterized hnRNP components are Hrp1 and Nab2 , . HRP1 is an essential gene that encodes a RRM-containing protein required for the cleavage and polyadenylation of pre-mRNA at the 3′-ends . Nab2 is a nuclear polyA RNA-binding protein required for nuclear mRNA export and poly(A) tail length control. Methylation by Hmt1 regulates the shuttle of Hrp1 and Nab2 between the nucleus and cytoplasm. Hrp1 and Nab2 fail to exit the nucleus in cells lacking Hmt1 , . In F. graminearum, FgHrp1-GFP fusion proteins mainly localized to the cytoplasm in the wild-type strain. In the Δamt1 mutant, FgHrp1-GFP proteins were accumulated in the nucleus (Fig. 5), suggesting that Amt1 is required for the nucleo-cytoplasm transport of FgHrp1. However, in transformants expressing the FgNAB2-GFP fusion construct, GFP signals mainly localized to the nucleus in both the wild type and Δamt1 mutant. The localization and nucleo-cytoplamic transport of FgNab2 appears to be independent of Amt1. These results indicate that methylation by this PRMT and nucleo-cytoplasmic transport of hnRNP components may be different between S. cerevisiae and F. graminearum.
Materials and Methods
Strains and culture conditions
The wild-type strain PH-1 and all the transformants of F. graminearum generated in this study were routinely cultured on PDA agar plates . Growth rate and conidiation were assayed as described , . DNA and RNA were extracted from vegetative hyphae harvested from liquid YEPD (1% yeast extract, 2% peptone, 2% glucose). Sexual reproduction, and protoplast preparation, and PEG-mediated transformation were performed as described . Hygromycin B (Calbiochem, La Jolla, CA) and geneticin (Sigma, St. Louis, MO) were added to the final concentration of 300 and 350 µg/ml, respectively, to the TB3 medium for transformant selection. To test sensitivity against various stresses, vegetative growth was assayed on PDA plates with 0.05% H2O2 (v/v), 0.01% SDS (w/v), or 0.7 M NaCl as described , .
Generation of Δamt1, Δamt2, Δamt3, Δamt4, and Δamt1 Δamt2 mutants
All the mutants were generated with the split-marker approach . For AMT1, the 0.83-kb upstream and 0.65-kb downstream flanking sequences were amplified with primer pairs AMT1/1F- 2R and AMT1/3F-4R, respectively (Fig. 1A and Table S2). The resulting PCR products were connected to the hygromycin phosphotransferase (hph) fragments amplified with primers HY/R-YG/F and HYG/F-HYG/R by overlapping PCR and transformed into protoplasts of PH-1 as described , . Hygromycin-resistant transformants were screened for Δamt1 mutants by PCR with primer pairs AMT1F5-R6, AMT1F7-H855R, and H856F-AMT1R8 (Table S2). Putative Δamt1 mutants were then analyzed by Southern blot hybridizations to confirm the gene replacement event. The same approach was used to generate the Δamt2, Δamt3, and Δamt4 mutants. To generate the Δamt1 Δamt2 double mutant, the AMT2 gene replacement construct generated with the neomycin resistance gene (NEOR) was transformed into the Δamt1 mutant M2.
Complementation of the Δamt1 mutant
A fragment containing the entire AMT1 gene and its promoter and terminator sequences was amplified with primers AMT1-CM/F and AMT1-CM/R (Table S2), digested with PstI and BamHI, and cloned between the PstI and BamHI sites of the NEOR vector pHZ100 . The resulting construct, pAMT1, was transformed into protoplasts of the Δamt1 mutant M2. The Δamt1/AMT1 transformants were confirmed by PCR and Southern blot analyses.
Generation of AMT1-GFP, HRP1-GFP, and NAB2-GFP fusion constructs
To generate the AMT1-GFP fusion, PCR products amplified with primers AMT1-YA/F and AMT1-YA/R (Table S2) were cloned into pFL2 by the yeast gap repair approach , . The same approach was used to generating the HRP1-GFP and NAB2-GFP fusion constructs. All GFP fusion constructs were verified by sequencing analysis and transformed into protoplasts of PH-1 or the Δamt1 mutant M2. G418-resistant transformants harboring the transforming AMT1-GFP, HRP1-GFP, or NAB2-GFP construct were identified by PCR and confirmed by the presence of GFP signals.
Infection and DON production assays
For infection assays with flowering wheat heads of cultivars XiaoYan 22 or Norm, conidia were harvested from 5-day-old CMC cultures and re-suspended in sterile distilled water to 2.0×105 conidia/ml. The fifth spikelet from the base of the spike was inoculated with 10 µl of the conidial suspension as described . Inoculated wheat heads were capped with a plastic bag to keep humidity for 48 h. After removing the bags, wheat plants were cultured for another 12 days before examination for symptomatic spikeletes. Infested kernels were harvested and assayed for DON production as described . For microscopic examinations, glumes and rachises were collected from inoculated spikeletes and embedded in Spurr resins . Thick sections (1 µm) were collected and placed on glass slides. After staining with aqueous 0.5% (w/v) toluidine blue, sections were examined and photographed with an Olympus BX-51 microscope (Olympus Corporation, Japan). Infection assays with corn silks of cultivar Pioneer 2375 were conducted as described .
RNA samples were isolated from 6 h germlings grown in liquid YEPD medium with the TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized with the Fermentas 1st cDNA synthesis kit (Hanover, MD). All qRT-PCR reactions were performed with the Bio-Rad C1000 qRT-PCR machine. Primers used for qRT-PCR analysis were listed in Table S2. Relative expression levels of each gene were calculated by the 2−ΔΔCt method  with the F. graminearum GAPDH gene  as the endogenous reference. Data from three biological replicates were used to calculate the mean and standard deviation.
Western blot analysis
Total proteins were isolated from 24 h germlings grown in CM, separated on a 12.5% SDS-PAGE, and transferred to nitrocellulose membranes for western blot analysis as described , . TEY-phosphorylation of Mgv1 and Gpmk1 and TGY- phosphorylation of FgHog1 were detected with the PhophoPlus p44/42 and p38 MAP kinase antibody kits (Cell Signaling Technology, Danvers, MA) following the manufacturer's instructions .
Phylogenetic analysis of fungal PRMTs. The amino acid sequences encoded by PRMT genes from Fusarium graminearum, Candida albicans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Magnaporthe oryzae, Neurospora crassa, Aspergillus nidulans, and Homo sapiens were analyzed by the DNAman5.0 program to create the dendrogram. The branch length is proportional to the mean number of differences per residue along each branch. All of the filamentous ascomycetes analyzed have four PRMT genes. Whereas three of them are orthologous to human PRMT1, PRMT3, and PRMT5, the fourth one is specific to fungi and plants. Scale bar is equal to 5% sequence divergence.
Cultures of the wild type and Δ amt1 mutant M2 grown on PDA, 5×YEG, and YEPD plates.
Perithecia and cirrhi produced by the wild-type strain (PH-1) and the Δamt1 (M2), Δamt2 (KS2), Δamt3 (KT3), Δamt4 (KF4), and Δamt1 Δamt2 (DM7) mutants. Photographs were taken 14 days after fertilization.
Deletion of AMT1 had no effects on the nucleo-cytoplasmic transport of FgNab2. In both transformants of PH-1 (NP12) and Δamt1 (NA14) mutant expressing the FgNAB2-GFP fusion construct, GFP signals mainly localized to the nucleus. Bar = 20 µm.
Three-day old PDA cultures of the wild-type strain (PH-1) and the Δ amt2 (KS2), Δ amt3 (KT3), Δ amt4 (KF4), and Δ amt1 Δ amt2 (DM7) mutants.
Assays for defects in stress responses. Cultures of the wild type (PH-1) and the Δamt2 (KS2), Δamt3 (KT3), and Δamt4 (KF4) mutants on PDA without or with 0.7 M NaCl, 300 µg/ml Congo red, 0.05% H2O2, or 0.01% SDS. Photographs were taken after incubation at 25°C for 3–5 days as labeled.
Disease index of AMTs mutants in the wheat head infection.
We thank Dr. Larry Dunkle at Purdue University for critical reading of this manuscript. We also thank Ms. Yimin Li for fruitful discussions.
Conceived and designed the experiments: JRX. Performed the experiments: GHW CFW RH XYZ GTL SJZ. Analyzed the data: GHW CFW. Contributed reagents/materials/analysis tools: GHW CFW. Wrote the paper: GHW CFW JRX.
- 1. Boisvert F, Chenard CA, Richard S (2005) Protein interfaces in signaling regulated by arginine methylation. Science's STKE 2005: re2. re2 p.F. BoisvertCA ChenardS. Richard2005Protein interfaces in signaling regulated by arginine methylation.Science's STKE 2005re2re2
- 2. Bachand F (2007) Protein arginine methyltransferases: From unicellular eukaryotes to humans. Eukaryot Cell 6: 889–898.F. Bachand2007Protein arginine methyltransferases: From unicellular eukaryotes to humans.Eukaryot Cell6889898
- 3. Bedford MT, Richard S (2005) Arginine methylation: An emerging regulator of protein function. Mol Cell 18: 263–272.MT BedfordS. Richard2005Arginine methylation: An emerging regulator of protein function.Mol Cell18263272
- 4. Yu MC, Bachand F, McBride AE, Komili S, Casolari JM, et al. (2004) Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors. Genes Dev 18: 2024–2035.MC YuF. BachandAE McBrideS. KomiliJM Casolari2004Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors.Genes Dev1820242035
- 5. Krause CD, Yang ZH, Kim YS, Lee JH, Cook JR, et al. (2007) Protein arginine methyltransferases: Evolution and assessment of their pharmacological and therapeutic potential. Pharmacol Therapeut 113: 50–87.CD KrauseZH YangYS KimJH LeeJR Cook2007Protein arginine methyltransferases: Evolution and assessment of their pharmacological and therapeutic potential.Pharmacol Therapeut1135087
- 6. Lee J, Sayegh J, Daniel J, Clarke S, Bedford MT (2005) PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280: 32890–32896.J. LeeJ. SayeghJ. DanielS. ClarkeMT Bedford2005PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family.J Biol Chem2803289032896
- 7. Scott HS, Antonarakis SE, Lalioti MD, Rossier C, Silver PA, et al. (1998) Identification and characterization of two putative human arginine methyltransferases (HRMT1L1 and HRMT1L2). Genomics 48: 330–340.HS ScottSE AntonarakisMD LaliotiC. RossierPA Silver1998Identification and characterization of two putative human arginine methyltransferases (HRMT1L1 and HRMT1L2).Genomics48330340
- 8. Sayegh J, Clarke SG (2008) Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast. Biochem Biophy Res Comm 372: 811–815.J. SayeghSG Clarke2008Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast.Biochem Biophy Res Comm372811815
- 9. McBride AE, Weiss VH, Kim HK, Hogle JM, Silver PA (2000) Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function – Cofactor binding and substrate interactions. J Biol Chem 275: 3128–3136.AE McBrideVH WeissHK KimJM HoglePA Silver2000Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function – Cofactor binding and substrate interactions.J Biol Chem27531283136
- 10. Pei YX, Niu LF, Lu FL, Liu CY, Zhai JX, et al. (2007) Mutations in the type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis. Plant Physiol 144: 1913–1923.YX PeiLF NiuFL LuCY LiuJX Zhai2007Mutations in the type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis.Plant Physiol14419131923
- 11. Shen EC, Henry MF, Weiss VH, Valentini SR, Silver PA, et al. (1998) Arginine methylation facilitates the nuclear export of hnRNP proteins. Genes Dev 12: 679–691.EC ShenMF HenryVH WeissSR ValentiniPA Silver1998Arginine methylation facilitates the nuclear export of hnRNP proteins.Genes Dev12679691
- 12. Green DM, Marfatia KA, Crafton EB, Zhang X, Cheng XD, et al. (2002) Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p. Jo Biol Chem 277: 7752–7760.DM GreenKA MarfatiaEB CraftonX. ZhangXD Cheng2002Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p.Jo Biol Chem27777527760
- 13. Kessler MM, Henry MF, Shen E, Zhao J, Gross S, et al. (1997) Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3′-end formation in yeast. Genes Dev 11: 2545–2556.MM KesslerMF HenryE. ShenJ. ZhaoS. Gross1997Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3′-end formation in yeast.Genes Dev1125452556
- 14. Gonzalez CI, Ruiz-Echevarria MJ, Vasudevan S, Henry MF, Peltz SW (2000) The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay. Mol Cell 5: 489–499.CI GonzalezMJ Ruiz-EchevarriaS. VasudevanMF HenrySW Peltz2000The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay.Mol Cell5489499
- 15. Gross S, Moore CL (2001) Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3 ′-end formation. Mol Cell Biol 21: 8045–8055.S. GrossCL Moore2001Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3 ′-end formation.Mol Cell Biol2180458055
- 16. Hector RE, Nykamp KR, Dheur S, Anderson JT, Non PJ, et al. (2002) Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export. EMBO J 21: 1800–1810.RE HectorKR NykampS. DheurJT AndersonPJ Non2002Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export.EMBO J2118001810
- 17. Wong CM, Tang HMV, Kong KYE, Wong GWO, Qiu HF, et al. (2010) Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p. Nucl Acids Res 38: 2217–2228.CM WongHMV TangKYE KongGWO WongHF Qiu2010Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p.Nucl Acids Res3822172228
- 18. Yun CY, Fu XD (2000) Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae. J Cell Biol 150: 707–717.CY YunXD Fu2000Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae.J Cell Biol150707717
- 19. Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal crops. Mol Plant Pathol 5: 515–525.RS GoswamiHC Kistler2004Heading for disaster: Fusarium graminearum on cereal crops.Mol Plant Pathol5515525
- 20. McMullen M, Jones R, Gallenberg D (1997) Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Dis 81: 1340–1348.M. McMullenR. JonesD. Gallenberg1997Scab of wheat and barley: A re-emerging disease of devastating impact.Plant Dis8113401348
- 21. Seong K, Li L, Hou ZM, Tracy M, Kistler HC, et al. (2006) Cryptic promoter activity in the coding region of the HMG-CoA rediactase gene in Fusarium graminearum. Fungal Genet Biol 43: 34–41.K. SeongL. LiZM HouM. TracyHC Kistler2006Cryptic promoter activity in the coding region of the HMG-CoA rediactase gene in Fusarium graminearum.Fungal Genet Biol433441
- 22. Yu MC, Lamming DW, Eskin JA, Sinclair DA, Silver PA (2006) The role of protein arginine methylation in the formation of silent chromatin. Genes Dev 20: 3249–3254.MC YuDW LammingJA EskinDA SinclairPA Silver2006The role of protein arginine methylation in the formation of silent chromatin.Genes Dev2032493254
- 23. Ochiai N, Tokai T, Nishiuchi T, Takahashi-Ando N, Fujimura M, et al. (2007) Involvement of the osmosensor histidine kinase and osmotic stress-activated protein kinases in the regulation of secondary metabolism in Fusarium graminearum. Biochem Biophy Res Comm 363: 639–644.N. OchiaiT. TokaiT. NishiuchiN. Takahashi-AndoM. Fujimura2007Involvement of the osmosensor histidine kinase and osmotic stress-activated protein kinases in the regulation of secondary metabolism in Fusarium graminearum.Biochem Biophy Res Comm363639644
- 24. Hou ZM, Xue CY, Peng YL, 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.ZM HouCY XueYL PengT. KatanHC Kistler2002A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility, heterokaryon formation, and plant infection.Mol Plant-Microbe Interact1511191127
- 25. Wang C, Zhang S, Hou R, Zhao Z, Zheng Q, et al. (2011) Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum. PLoS pathogens 7: e1002460.C. WangS. ZhangR. HouZ. ZhaoQ. Zheng2011Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum.PLoS pathogens7e1002460
- 26. Jenczmionka NJ, Maier FJ, Losch AP, Schafer W (2003) Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head-blight disease of wheat, are regulated by the MAP kinase gpmk1. Curr Genet 43: 87–95.NJ JenczmionkaFJ MaierAP LoschW. Schafer2003Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head-blight disease of wheat, are regulated by the MAP kinase gpmk1.Curr Genet438795
- 27. 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.M. UrbanE. MottT. FarleyK. Hammond-Kosack2003The Fusarium graminearum MAP1 gene is essential for pathogenicity and development of perithecia.Mol Plant Pathol4347359
- 28. Cimato TR, Tang J, Xu Y, Guarnaccia C, Herschman HR, et al. (2002) Nerve growth factor-mediated increases in protein methylation occur predominantly at type I arginine methylation sites and involve protein arginine methyltransferase 1. J Neurosci Res 67: 435–442.TR CimatoJ. TangY. XuC. GuarnacciaHR Herschman2002Nerve growth factor-mediated increases in protein methylation occur predominantly at type I arginine methylation sites and involve protein arginine methyltransferase 1.J Neurosci Res67435442
- 29. Zobel-Thropp P, Gary JD, Clarke S (1998) delta-N-methylarginine is a novel posttranslational modification of arginine residues in yeast proteins. J Biol Chem 273: 29283–29286.P. Zobel-ThroppJD GaryS. Clarke1998delta-N-methylarginine is a novel posttranslational modification of arginine residues in yeast proteins.J Biol Chem2732928329286
- 30. Bauer I, Graessle S, Loidl P, Hohenstein K, Brosch G (2010) Novel insights into the functional role of three protein arginine methyltransferases in Aspergillus nidulans. Fungal Genet Biol 47: 551–561.I. BauerS. GraessleP. LoidlK. HohensteinG. Brosch2010Novel insights into the functional role of three protein arginine methyltransferases in Aspergillus nidulans.Fungal Genet Biol47551561
- 31. McBride AE, Zurita-Lopez C, Regis A, Blum E, Conboy A, et al. (2007) Protein arginine methylation in Candida albicans: role in nuclear transport. Eukaryot Cell 6: 1119–1129.AE McBrideC. Zurita-LopezA. RegisE. BlumA. Conboy2007Protein arginine methylation in Candida albicans: role in nuclear transport.Eukaryot Cell611191129
- 32. Chern MK, Chang KN, Liu LF, Tam TCS, Liu YC, et al. (2002) Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2. J Biol Chem 277: 15345–15353.MK ChernKN ChangLF LiuTCS TamYC Liu2002Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2.J Biol Chem2771534515353
- 33. Niewmierzycka A, Clarke S (1999) S-adenosylmethionine-dependent methylation in Saccharomyces cerevisiae – Identification of a novel protein arginine methyltransferase. J Biol Chem 274: 814–824.A. NiewmierzyckaS. Clarke1999S-adenosylmethionine-dependent methylation in Saccharomyces cerevisiae – Identification of a novel protein arginine methyltransferase.J Biol Chem274814824
- 34. Kucharczyk R, Gromadka R, Migdalski A, Slonimski PP, Rytka J (1999) Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals. Yeast 15: 987–1000.R. KucharczykR. GromadkaA. MigdalskiPP SlonimskiJ. Rytka1999Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals.Yeast159871000
- 35. Ben Lovely C, Aulakh KB, Perlin MH (2011) Role of Hsl7 in morphology and pathogenicity and its interaction with other signaling components in the plant pathogen Ustilago maydis. Eukaryot Cell 10: 869–883.C. Ben LovelyKB AulakhMH Perlin2011Role of Hsl7 in morphology and pathogenicity and its interaction with other signaling components in the plant pathogen Ustilago maydis.Eukaryot Cell10869883
- 36. Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, et al. (2002) The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J Exp Bot 53: 1367–1376.GP BolwellLV BindschedlerKA BleeVS ButtDR Davies2002The apoplastic oxidative burst in response to biotic stress in plants: a three-component system.J Exp Bot5313671376
- 37. Torres MA, Jones JDG, Dangl JL (2006) Reactive oxygen species signaling in response to pathogens. Plant Physiol 141: 373–378.MA TorresJDG JonesJL Dangl2006Reactive oxygen species signaling in response to pathogens.Plant Physiol141373378
- 38. Proctor RH, Hohn TM, McCormick SP (1995) Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol Plant-Microbe Interact 8: 593–601.RH ProctorTM HohnSP McCormick1995Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene.Mol Plant-Microbe Interact8593601
- 39. Harris LJ, Desjardins AE, Plattner RD, Nicholson P, Butler G, et al. (1999) Possible role of trichothecene mycotoxins in virulence of Fusarium graminearum on maize. Plant Dis 83: 954–960.LJ HarrisAE DesjardinsRD PlattnerP. NicholsonG. Butler1999Possible role of trichothecene mycotoxins in virulence of Fusarium graminearum on maize.Plant Dis83954960
- 40. Zhou XY, Heyer C, Choi YE, Mehrabi R, Xu JR (2010) The CID1 cyclin C-like gene is important for plant infection in Fusarium graminearum. Fungal Genet Biol 47: 143–151.XY ZhouC. HeyerYE ChoiR. MehrabiJR Xu2010The CID1 cyclin C-like gene is important for plant infection in Fusarium graminearum.Fungal Genet Biol47143151
- 41. Ding SL, Mehrabi R, Koten C, Kang ZS, Wei YD, et al. (2009) Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum. Eukaryot Cell 8: 867–876.SL DingR. MehrabiC. KotenZS KangYD Wei2009Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum.Eukaryot Cell8867876
- 42. Li Y, Wang C, Liu W, Wang G, Kang Z, et al. (2011) The HDF1histone deacetylase gene is important for conidiation, sexual reproduction, and pathogenesis in Fusarium graminearum. Mol Plant-Microbe Interact 24: 487–496.Y. LiC. WangW. LiuG. WangZ. Kang2011The HDF1histone deacetylase gene is important for conidiation, sexual reproduction, and pathogenesis in Fusarium graminearum.Mol Plant-Microbe Interact24487496
- 43. Catlett NL, Lee B, Yoder OC, Turgeon BG (2003) Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genet Newsl 50: 9–11.NL CatlettB. LeeOC YoderBG Turgeon2003Split-marker recombination for efficient targeted deletion of fungal genes.Fungal Genet Newsl50911
- 44. 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.Y. WangW. LiuZ. HouC. WangX. Zhou2011A novel transcriptional factor important for pathogenesis and ascosporogenesis in Fusarium graminearum.Mol Plant-Microbe Interact24118128
- 45. Bluhm BH, Zhao X, Flaherty JE, Xu JR, Dunkle LD (2007) RAS2 regulates growth and pathogenesis in Fusarium graminearum. Molecular Plant-Microbe Interactions 20: 627–636.BH BluhmX. ZhaoJE FlahertyJR XuLD Dunkle2007RAS2 regulates growth and pathogenesis in Fusarium graminearum.Molecular Plant-Microbe Interactions20627636
- 46. Zhou X, Xu JR (2011) Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi. In: Xu JR, Bluhm B, editors. Fungal Genomics: Methods and Protocols. Heidelberg: Humana Press. pp. 199–212.X. ZhouJR Xu2011Efficient approaches for generating GFP fusion and epitope-tagging constructs in filamentous fungi.JR XuB. BluhmFungal Genomics: Methods and ProtocolsHeidelbergHumana Press199212
- 47. Bruno KS, Tenjo F, Li L, Hamer JE, Xu JR (2004) Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea. Eukaryot Cell 3: 1525–1532.KS BrunoF. TenjoL. LiJE HamerJR Xu2004Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea.Eukaryot Cell315251532
- 48. Gale LR, Chen LF, Hernick CA, Takamura K, Kistler HC (2002) Population analysis of Fusarium graminearum from wheat fields in eastern China. Phytopathology 92: 1315–1322.LR GaleLF ChenCA HernickK. TakamuraHC Kistler2002Population analysis of Fusarium graminearum from wheat fields in eastern China.Phytopathology9213151322
- 49. Kang ZS, Buchenauer H, Huang LL, Han QM, Zhang HC (2008) Cytological and immunocytochemical studies on responses of wheat spikes of the resistant Chinese cv. Sumai 3 and the susceptible cv. Xiaoyan 22 to infection by Fusarium graminearum. Euro J Plant Pathol 120: 383–396.ZS KangH. BuchenauerLL HuangQM HanHC Zhang2008Cytological and immunocytochemical studies on responses of wheat spikes of the resistant Chinese cv. Sumai 3 and the susceptible cv. Xiaoyan 22 to infection by Fusarium graminearum.Euro J Plant Pathol120383396
- 50. Seong K, Hou ZM, Tracy M, Kistler HC, Xu JR (2005) Random insertional mutagenesis identifies genes associated with virulence in the wheat scab fungus Fusarium graminearum. Phytopathology 95: 744–750.K. SeongZM HouM. TracyHC KistlerJR Xu2005Random insertional mutagenesis identifies genes associated with virulence in the wheat scab fungus Fusarium graminearum.Phytopathology95744750
- 51. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25: 402–408.KJ LivakTD Schmittgen2001Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method.Methods25402408
- 52. Pandolfi V, Jorge EC, Melo CMR, Albuquerque ACS, Carrer H (2010) Gene expression profile of the plant pathogen Fusarium graminearum under the antagonistic effect of Pantoea agglomerans. Genet Mol Res 9: 1298–1311.V. PandolfiEC JorgeCMR MeloACS AlbuquerqueH. Carrer2010Gene expression profile of the plant pathogen Fusarium graminearum under the antagonistic effect of Pantoea agglomerans.Genet Mol Res912981311
- 53. Ding S, Liu W, LLiuk A, Ribot C, Vallet J, et al. (2010) The Tig1 HDAC complex regulates infectious growth in the rice blast fungus Magnaporthe oryzae. Plant Cell 22: 2495–2508.S. DingW. LiuA. LLiukC. RibotJ. Vallet2010The Tig1 HDAC complex regulates infectious growth in the rice blast fungus Magnaporthe oryzae.Plant Cell2224952508
- 54. Liu W, Zhou X, Li G, Li L, Kong L, et al. (2011) Multiple plant surface signals are sensed by different mechanisms in the rice blast fungus for appressorium formation. PLoS Pathogens 7: e1001261.W. LiuX. ZhouG. LiL. LiL. Kong2011Multiple plant surface signals are sensed by different mechanisms in the rice blast fungus for appressorium formation.PLoS Pathogens7e1001261
- 55. Cuomo CA, Gueldener U, Xu JR, Trail F, Turgeon BG, et al. (2007) The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317: 1400–1402.CA CuomoU. GueldenerJR XuF. TrailBG Turgeon2007The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.Science31714001402