HapX-mediated H2B deub1 and SreA-mediated H2A.Z deposition coordinate in fungal iron resistance.

Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.

SUMOylation regulates low-temperature survival and oxidative DNA damage tolerance in Botrytis cinerea.

SUMOylation as one of the protein post-translational modifications plays crucial roles in multiple biological processes of eukaryotic organisms. Botrytis cinerea is a devastating fungal pathogen and capable of infecting plant hosts at low temperature. However, the molecular mechanisms of low-temperature adaptation are largely unknown in fungi. Combining with biochemical methods and biological analyses, we report that SUMOylation regulates pathogen survival at low temperature and oxidative DNA damage response during infection in B. cinerea. The heat shock protein (Hsp70) BcSsb and E3 ubiquitin ligase BcRad18 were identified as substrates of SUMOylation; moreover, their SUMOylation both requires a single unique SUMO-interacting motif (SIM). SUMOylated BcSsb regulates ß-tubulin accumulation, thereby affecting the stability of microtubules and consequently mycelial growth at low temperature. On the contrary, SUMOylated BcRad18 modulates mono-ubiquitination of the sliding clamp protein proliferating cell nuclear antigen (PCNA), which is involved in response to oxidative DNA damage during infection. Our study uncovers the molecular mechanisms of SUMOylation-mediated low-temperature survival and oxidative DNA damage tolerance during infection in a devastating fungal pathogen, which provides novel insights into low-temperature adaptation and pathogenesis for postharvest pathogens as well as new targets for inhibitor invention in disease control.

SUMOylation regulates pre-mRNA splicing to overcome DNA damage in fungi.

Pathogenic fungi are subject to DNA damage stress derived from host immune responses during infection. Small ubiquitin-like modifier (SUMO) modification and precursor (pre)-mRNA splicing are both involved in DNA damage response (DDR). However, the mechanisms of how SUMOylation and splicing coordinated in DDR remain largely unknown. Combining with biochemical analysis, RNA-Seq method, and biological analysis, we report that SUMO pathway participates in DDR and virulence in Fusarium graminearum, a causal agent of Fusarium head blight of cereal crops world-wide. Interestingly, a key transcription factor FgSR is SUMOylated upon DNA damage stress. SUMOylation regulates FgSR nuclear-cytoplasmic partitioning and its phosphorylation by FgMec1, and promotes its interaction with chromatin remodeling complex SWI/SNF for activating the expression of DDR-related genes. Moreover, the SWI/SNF complex was found to further recruit splicing-related NineTeen Complex, subsequently modulates pre-mRNA splicing during DDR. Our findings reveal a novel function of SUMOylation in DDR by regulating a transcription factor to orchestrate gene expression and pre-mRNA splicing to overcome DNA damage during the infection of F. graminearum, which advances the understanding of the delicate regulation of DDR by SUMOylation in pathogenic fungi, and extends the knowledge of cooperation of SUMOylation and pre-mRNA splicing in DDR in eukaryotes.

Fungicide Resistance: Progress in Understanding Mechanism, Monitoring, and Management.

Fungicide treatments are often essential for maintaining healthy crops and to achieve reliable and high-quality yields. However, continued use of fungicides with the same modes of action can lead to development of fungicide resistance, which has emerged in various plant pathogens and is a serious threat to effective crop protection. Exploration of resistance mechanisms is critical for resistance monitoring and management. This brief review summarizes advances during the past five decades in understanding the molecular resistance mechanisms of plant pathogenic fungi and oomycetes to major classes of fungicides, including benzimidazoles, myosin inhibitors, sterol demethylation inhibitors, quinone outside inhibitors, succinate dehydrogenase inhibitors, anilinopyrimidines, carboxylic acid amides, and oxysterol-binding protein homolog inhibitors. Based on known resistance mechanisms, PCR- and loop-mediated isothermal amplification-based approaches have been developed to allow high-throughput monitoring and early/rapid detection of emerging resistance. Classical principles in fungicide resistance management are also summarized, including using different modes of action of fungicides, limiting the number of applications of the chemicals with site-specific modes of action, and avoidance of their eradicant use. Future studies on novel strategies of disease management, including development of epigenetics- and RNA-based fungicides, will provide valuable knowledge for management of fungicide resistance.

The Importin FgPse1 Is Required for Vegetative Development, Virulence, and Deoxynivalenol Production by Interacting with the Nuclear Polyadenylated RNA-Binding Protein FgNab2 in Fusarium graminearum.

Karyopherins are involved in transport through nuclear pore complexes. Karyopherins are necessary for nuclear import and export pathways and bind to their cargos. Polyadenylation of messenger RNA (mRNA) is necessary for various biological processes, regulating gene expression in eukaryotes. Until now, the association of karyopherin with mRNA polyadenylation has been less understood in plant pathogenic fungi. In our study, we focused on the biological functions of the karyopherin FgPse1 in Fusarium graminearum. The results showed that FgPse1 is involved in mycelial growth, asexual reproduction, virulence, and deoxynivalenol (DON) production. Co-immunoprecipitation and bimolecular fluorescence complementation showed that FgPse1 interacts with the nuclear polyadenylated RNA-binding protein FgNab2. Moreover, a fluorescence localization assay indicated that FgPse1 is necessary for the nuclear import of FgNab2. The nuclear import of FgNab2 regulates the expression of FgTri4, FgTri5, and FgTri6, which are essential for DON production. Thus, ΔFgPse1 and ΔFgNab2 showed consistent defects in DON production. In summary, our data indicated that FgPse1 is necessary for mycelial growth, virulence, and DON production, interacting with FgNab2 in F. graminearum. These results contribute to our understanding of the functions of importins in phytopathogenic fungi.

A fungal ABC transporter FgAtm1 regulates iron homeostasis via the transcription factor cascade FgAreA-HapX.

Iron homeostasis is important for growth, reproduction and other metabolic processes in all eukaryotes. However, the functions of ATP-binding cassette (ABC) transporters in iron homeostasis are largely unknown. Here, we found that one ABC transporter (named FgAtm1) is involved in regulating iron homeostasis, by screening sensitivity to iron stress for 60 ABC transporter mutants of Fusarium graminearum, a devastating fungal pathogen of small grain cereal crops worldwide. The lack of FgAtm1 reduces the activity of cytosolic Fe-S proteins nitrite reductase and xanthine dehydrogenase, which causes high expression of FgHapX via activating transcription factor FgAreA. FgHapX represses transcription of genes for iron-consuming proteins directly but activates genes for iron acquisition proteins by suppressing another iron regulator FgSreA. In addition, the transcriptional activity of FgHapX is regulated by the monothiol glutaredoxin FgGrx4. Furthermore, the phosphorylation of FgHapX, mediated by the Ser/Thr kinase FgYak1, is required for its functions in iron homeostasis. Taken together, this study uncovers a novel regulatory mechanism of iron homeostasis mediated by an ABC transporter in an important pathogenic fungus.

Plant defense compound triggers mycotoxin synthesis by regulating H2B ub1 and H3K4 me2/3 deposition.

Fusarium graminearum produces the mycotoxin deoxynivalenol (DON) which promotes its expansion during infection on its plant host wheat. Conditional expression of DON production during infection is poorly characterized. Wheat produces the defense compound putrescine, which induces hypertranscription of DON biosynthetic genes (FgTRIs) and subsequently leads to DON accumulation during infection. Further, the regulatory mechanisms of FgTRIs hypertranscription upon putrescine treatment were investigated. The transcription factor FgAreA regulates putrescine-mediated transcription of FgTRIs by facilitating the enrichment of histone H2B monoubiquitination (H2B ub1) and histone 3 lysine 4 di- and trimethylations (H3K4 me2/3) on FgTRIs. Importantly, a DNA-binding domain (bZIP) specifically within the Fusarium H2B ub1 E3 ligase Bre1 othologs is identified, and the binding of this bZIP domain to FgTRIs depends on FgAreA-mediated chromatin rearrangement. Interestingly, H2B ub1 regulates H3K4 me2/3 via the methyltransferase complex COMPASS component FgBre2, which is different from Saccharomyces cerevisiae. Taken together, our findings reveal the molecular mechanisms by which host-generated putrescine induces DON production during F. graminearum infection. Our results also provide a novel insight into the role of putrescine during phytopathogen-host interactions and broaden our knowledge of H2B ub1 biogenesis and crosstalk between H2B ub1 and H3K4 me2/3 in eukaryotes.

The RasGEF FgCdc25 regulates fungal development and virulence in Fusarium graminearum via cAMP and MAPK signalling pathways.

Ras GTPases act as molecular switches to control various cellular processes by coupling integrated signals in eukaryotes. Activities of Ras GTPases are triggered by Ras GTPase guanine nucleotide exchange factors (RasGEFs) in general, whereas the role of RasGEF in plant pathogenic fungi is largely unknown. In this study, we characterized the only RasGEF protein in Fusarium graminearum, FgCdc25, by combining genetic, cytological and phenotypic strategies. FgCdc25 directly interacted with RasGTPase FgRas2, but not FgRas1, to regulate growth and sexual reproduction. Mutation of the FgCDC25 gene resulted in decreased toxisome formation and deoxynivalenol (DON) production, which was largely depended on cAMP signalling. In addition, FgCdc25 indirectly interacted with FgSte11 in FgSte11-Ste7-Gpmk1 cascade, and the ΔFgcdc25 strain totally abolished the formation of infection structures and was nonpathogenic in planta, which was partially recovered by addition of exogenous cAMP. In contrast, FgCdc25 directly interplayed with FgBck1 in FgBck1-MKK1-Mgv1 cascade to negatively control cell wall integrity. Collectively, these results suggest that FgCdc25 modulates cAMP and MAPK signalling pathways and further regulates fungal development, DON production and plant infection in F. graminearum.

The MAPK kinase BcMkk1 suppresses oxalic acid biosynthesis via impeding phosphorylation of BcRim15 by BcSch9 in Botrytis cinerea.

The mitogen-activated protein kinase (MAPK) cassette of the cell wall integrity (CWI) pathway is primarily responsible for orchestrating changes of cell wall. However, functions of this cassette in other cellular processes are not well understood. Here, we found that the Botrytis cinerea mutant of MAPK kinase (BcMkk1) displays more serious defects in mycelial growth, conidiation, responses to cell wall and oxidative stresses, but possesses less reduced virulence than the mutants of its upstream (BcBck1) and downstream (BcBmp3) kinases. Interestingly, BcMkk1, but not BcBck1 and BcBmp3, negatively regulates production of oxalic acid (OA) and activity of extracellular hydrolases (EHs) that are proposed to be virulence factors of B. cinerea. Moreover, we obtained evidence that BcMkk1 negatively controls OA production via impeding phosphorylation of the Per-Arnt-Sim (PAS) kinase BcRim15 by the Ser/Thr kinase BcSch9. In addition, the fungal Pro40 homolog BcPro40 was found to interact simultaneously with three MAPKs, implying that BcPro40 is a scaffold protein of the CWI pathway in B. cinerea. Taken together, results of this study reveal that BcMkk1 negatively modulates virulence via suppressing OA biosynthesis in B. cinerea, which provides novel insight into conserved and species-specific functions of the MAPK kinase in fungi.

Lipid droplet biogenesis regulated by the FgNem1/Spo7-FgPah1 phosphatase cascade plays critical roles in fungal development and virulence in Fusarium graminearum.

Lipid droplets (LDs) control lipid metabolism in eukaryotic cells in general. However, the biogenesis regulation and biological functions of LDs are largely unknown in pathogenic fungi. Rapamycin treatment results in a significant increase of LD biogenesis in Fusarium graminearum. Molecular mechanisms of the target of rapamycin (TOR) pathway in regulating LD biogenesis and the functions of LD in virulence of F. graminearum were investigated in depth by combining genetic, cytological and phenotypic strategies. TOR in Fusarium graminearum (FgTOR) inhibition by rapamycin induces LD biogenesis through the FgPpg1/Sit4 signaling branch. FgPpg1 promotes phosphorylation of protein phosphatase FgNem1 by the protein kinase FgCak1. The phosphorylated FgNem1 dephosphorylates the phosphatidate phosphatase FgPah1. Dephosphorylated FgPah1 is active and stimulates LD biogenesis. Moreover, deletion of FgNem1/Spo7 or FgPah1 leads to serious defects in vegetative growth, sexual development and virulence. The results of this study provide novel insights into the regulatory mechanism and biological functions of the LDs in the devastating pathogenic fungus F. graminearum.

The Activators of Type 2A Phosphatases (PP2A) Regulate Multiple Cellular Processes Via PP2A-Dependent and -Independent Mechanisms in Fusarium graminearum.

The type 2A protein phosphatases (PP2As) are holoenzymes in all eukaryotes but their activators remain unknown in filamentous fungi. Fusarium graminearum contains three PP2As (FgPp2A, FgSit4, and FgPpg1), which play critical roles in fungal growth, development, and virulence. Here, we identified two PP2A activators (PTPAs), FgRrd1 and FgRrd2, and found that they control PP2A activity in a PP2A-specific manner. FgRrd1 interacts with FgPpg1, but FgRrd2 interacts with FgPp2A and very weakly with FgSit4. Furthermore, FgRrd2 activates FgPp2A via regulating FgPp2A methylation. Phenotypic assays showed that FgRrd1 and FgRrd2 regulate mycelial growth, conidiation, sexual development, and lipid droplet biogenesis. More importantly, both FgRrd1 and FgRrd2 interact with RNA polymerase II, subsequently modulating its enrichments at the promoters of mycotoxin biosynthesis genes, which is independent on PP2A. In addition, FgRrd2 modulates response to phenylpyrrole fungicide, via regulating the phosphorylation of kinase FgHog1 in the high-osmolarity glycerol pathway, and to caffeine, via modulating FgPp2A methylation. Taken together, results of this study indicate that FgRrd1 and FgRrd2 regulate multiple physiological processes via different regulatory mechanisms in F. graminearum, which provides a novel insight into understanding the biological functions of PTPAs in fungi.

The ATP-binding protein FgArb1 is essential for penetration, infectious and normal growth of Fusarium graminearum.

ATP-binding cassette (ABC) transporters act mainly to transport compounds across cellular membranes and are important for diverse biological processes. However, their roles in pathogenesis have not been well-characterized in Fusarium graminearum. Sixty F. graminearum ABC protein genes were functionally characterized. Among them, FgArb1 regulates normal growth and importantly is essential for pathogenicity. Thus, the regulatory mechanisms of FgArb1 in pathogenicity were analyzed in this study. FgArb1 interacts with the mitogen-activated protein kinase (MAPK) FgSte7, and partially modulates plant penetration by regulating the phosphorylation of FgGpmk1 (the downstream kinase of FgSte7). The FgArb1 mutant exhibited dramatically reduced infective growth within wounded host tissues, likely resulting from its increased sensitivity to oxidative stresses, defective cell wall integrity and reduced deoxynivalenol (DON) production. FgArb1 also is important for the production of sexual and asexual spores that are important propagules for plant infection. In addition, FgArb1 is involved in the regulation of protein biosynthesis through impeding nuclear export of small ribosomal subunit. Finally, acetylation modification at sites K28, K65, K341 and K525 in FgArb1 is required for its biological functions. Taken together, results of this study provide a novel insight into functions of the ABC transporter in fungal pathogenesis.

The FgSsb-FgZuo-FgSsz complex regulates multiple stress responses and mycotoxin production via folding the soluble SNARE Vam7 and ß2-tubulin in Fusarium graminearum.

Hsp70 proteins play important roles in protein folding in the budding yeast, but their functions in pathogenic fungi are largely unknown. Here, we found that Fusarium graminearum Hsp70 proteins FgSsb, FgSsz and their cochaperone FgZuo formed a complex. This complex was required for microtubule morphology, vacuole fusion and endocytosis. More importantly, the ß2-tubulin FgTub2 and SNARE protein FgVam7 were identified as targeting proteins of this complex. We further found that the complex FgSsb-FgZuo-FgSsz controlled sensitivity of F. graminearum to the antimicrotubule drug carbendazim and cold stress via regulating the folding of FgTub2. Moreover, this complex assisted the folding of FgVam7, subsequently modulated vacuole fusion and responses to heavy metal, osmotic and oxidative stresses. In addition, the deletion of this complex led to dramatically decreased deoxynivalenol biosynthesis. This study uncovers a novel regulating mechanism of Hsp70 in multiple stress responses in a filamentous fungus.

The microtubule end-binding protein FgEB1 regulates polar growth and fungicide sensitivity via different interactors in Fusarium graminearum.

In yeasts, the end-binding protein 1 (EB1) homologs regulate microtubule dynamics, cell polarization, and chromosome stability. However, functions of EB1 orthologs in plant pathogenic fungi have not been characterized yet. Here, we observed that the FgEB1 deletion mutant (ΔFgEB1) of Fusarium graminearum exhibits twisted hyphae, increased hyphal branching and curved conidia, indicating that FgEB1 is involved in the regulation of cellular polarity. Microscopic examination further showed that the microtubules of ΔFgEB1 exhibited less organized in comparison with those of the wild type. In addition, the lack of FgEB1 also altered the distribution of polarity-related class I myosin via the interaction with the actin. On the other hand, we identified four core septins as FgEB1-interacting proteins, and found that FgEB1 and septins regulated conidial polar growth in the opposite orientation. Interestingly, FgEB1 and FgKar9 constituted another complex that modulated the response to carbendazim, a microtubule-damaging agent specifically. In addition, the deletion of FgEB1 led to dramatically decreased deoxynivalenol (DON) biosynthesis. Taken together, results of this study indicate that FgEB1 regulates cellular polarity, fungicide sensitivity and DON biosynthesis via different interactors in F. graminarum, which provides a novel insight into understanding of the biological functions of EB1 in filamentous fungi.

Histone H3K4 methylation regulates hyphal growth, secondary metabolism and multiple stress responses in Fusarium graminearum.

Histone H3 lysine 4 methylation (H3K4me) is generally associated with actively transcribed genes in a variety of eukaryotes. The function of H3K4me in phytopathogenic fungi remains unclear. Here, we report that FgSet1 is predominantly responsible for mono-, di- and trimethylation of H3K4 in Fusarium graminearum. The FgSET1 deletion mutant (ΔFgSet1) was crippled in hyphal growth and virulence. H3K4me is required for the active transcription of genes involved in deoxynivalenol and aurofusarin biosyntheses. Unexpectedly, FgSet1 plays an important role in the response of F. graminearum to cell wall-damaging agents via negatively regulating phosphorylation of FgMgv1, a core kinase in the cell wall integrity pathway. In addition, ΔFgSet1 exhibited increased resistance to the transcription elongation inhibitor mycophenolic acid. Yeast two-hybrid assays showed that FgSet1 physically interacts with multiple proteins including FgBre2, FgSpp1 and FgSwd2. FgBre2 further interacts with FgSdc1. Western blotting analyses showed that FgBre2 and FgSdc1 are associated with H3K4me. Both proteins are also involved in regulating deoxynivalenol biosynthesis and in responses to mycophenolic acid and cell wall-damaging agents. Taken together, these data indicate that H3K4me plays critical roles not only in regulation of fungal growth and secondary metabolism but also in multiple stress responses in F. graminearum.

Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum.

Saccharomyces cerevisiae protein kinase Sch9 is one of the downstream effectors of the target of rapamycin (TOR) complex 1 and plays multiple roles in stress resistance, longevity and nutrient sensing. However, the functions of Sch9 orthologs in filamentous fungi, particularly in pathogenic species, have not been characterized to date. Here, we investigated biological and genetic functions of FgSch9 in Fusarium graminearum. The FgSCH9 deletion mutant (ΔFgSch9) was defective in aerial hyphal growth, hyphal branching and conidial germination. The mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall-damaging agents, and to rapamycin, while showing increased thermal tolerance. We identified FgMaf1 as one of the FgSch9-interacting proteins that plays an important role in regulating mycotoxin biosynthesis and virulence of F. graminearum. Co-immunoprecipitation and affinity capture-mass spectrometry assays showed that FgSch9 also interacts with FgTor and FgHog1. More importantly, both ΔFgSch9 and FgHog1 null mutant (ΔFgHog1) exhibited increased sensitivity to osmotic and oxidative stresses. This defect was more severe in the FgSch9/FgHog1 double mutant. Taken together, we propose that FgSch9 serves as a mediator of the TOR and high osmolarity glycerol pathways, and regulates vegetative differentiation, multiple stress responses and secondary metabolism in F. graminearum.

A small molecule species specifically inhibits Fusarium myosin I.

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease of cereal crops worldwide. Recently, a novel fungicide JS399-19 has been launched into the marketplace to manage FHB. It is compelling that JS399-19 shows highly inhibitory activity towards some Fusarium species, but not to other fungi, indicating that it is an environmentally compatible fungicide. To explore the mode of action of this species-specific compound, we conducted a whole-genome transcript profiling together with genetic and biochemical assays, and discovered that JS399-19 targets the myosin I of F. graminearum (FgMyo1). FgMyo1 is essential for F. graminearum growth. A point mutation S217L or E420K in FgMyo1 is responsible for F. graminearum resistance to JS399-19. In addition, transformation of F. graminearum with the myosin I gene of Magnaporthe grisea, the causal agent of rice blast, also led to JS399-19 resistance. JS399-19 strongly inhibits the ATPase activity of the wild-type FgMyo1, but not the mutated FgMyo1(S217L/E420K) . These results provide us a new insight into the design of species-specific antifungal compounds. Furthermore, our strategy can be applied to identify novel drug targets in various pathogenic organisms.

The tubulin cofactor A is involved in hyphal growth, conidiation and cold sensitivity in Fusarium asiaticum.

BACKGROUND: Tubulin cofactor A (TBCA), one of the members of tubulin cofactors, is of great importance in microtubule functions through participating in the folding of α/ß-tubulin heterodimers in Saccharomyces cerevisiae. However, little is known about the roles of TBCA in filamentous fungi. RESULTS: In this study, we characterized a TBCA orthologue FaTBCA in Fusarium asiaticum. The deletion of FaTBCA caused dramatically reduced mycelial growth and abnormal conidiation. The FaTBCA deletion mutant (ΔFaTBCA-3) showed increased sensitivity to low temperatures and even lost the ability of growth at 4°C. Microscopic observation found that hyphae of ΔFaTBCA-3 exhibited blebbing phenotypes after shifting from 25 to 4°C for 1- or 3-day incubation and approximately 72% enlarged nodes contained several nuclei after 3-day incubation at 4°C. However, hyphae of the wild type incubated at 4°C were phenotypically indistinguishable from those incubated at 25°C. These results indicate that FaTBCA is involved in cell division under cold stress (4°C) in F. asiaticum. Unexpectedly, ΔFaTBCA-3 did not exhibit increased sensitivity to the anti-microtubule drug carbendazim although quantitative real-time assays showed that the expression of FaTBCA was up-regulated after treatment with carbendazim. In addition, pathogenicity assays showed that ΔFaTBCA-3 exhibited decreased virulence on wheat head and on non-host tomato. CONCLUSION: Taken together, results of this study indicate that FaTBCA plays crucial roles in vegetative growth, conidiation, temperature sensitivity and virulence in F. asiaticum.

Functional analysis of the Fusarium graminearum phosphatome.

Phosphatases are known to play important roles in the regulation of various cellular processes in eukaryotes. However, systematic characterization of the phosphatome has not been reported in phytopathogenic fungi. The wheat scab fungus Fusarium graminearum contains 82 putative phosphatases. The biological functions of each phosphatase were investigated in this study. Although 11 phosphatase genes appeared to be essential, deletion mutants of the other 71 phosphatase genes were obtained and characterized for changes in 15 phenotypes, including vegetative growth, nutrient response and virulence. Overall, the deletion of 63 phosphatase genes resulted in changes in at least one of the phenotypes assayed. Interestingly, the deletion of four genes (Fg06297, Fg03333, Fg03826 and Fg07932) did not dramatically affect hyphal growth, but led to strongly reduced virulence. Western blot analyses showed that three phosphatases (Fg10516, Fg03333 and Fg12867) functioned as negative regulators of the mitogen-activated protein kinase signaling pathways. In addition, we found, for the first time, that FgCdc14 is dispensable for growth, but plays an important role in ribosome biogenesis. Overall, in this first functional characterization of the fungal phosphatome, phosphatases important for various aspects of hyphal growth, development, plant infection and secondary metabolism were identified in the phytopathogenic fungus F. graminearum.

The potential protein kinase A (Pka) phosphorylation site is required for the function of FgSge1 in Fusarium graminearum.

The new transcription factor Sge1 has garnered much attention in filamentous fungi recently, which highlights its role in pathogenicity, conidiation, and the production of secondary metabolites. In this study, we demonstrated that FgSge1 is localized in the nucleus in Fusarium graminearum using fluorescent protein GFP. Mutants containing a T67A mutation within the potential protein kinase A (Pka) phosphorylation site of FgSge1 exhibited a significant decrease in conidiation and dramatically impaired virulence on both wheat head and non-host tomato. These results indicated that the Pka phosphorylation site is required for the function of FgSge1 in F. graminearum. In addition, we characterized the FgSGE1 deletion mutants and found that the mutants showed increased sensitivity to osmotic stress mediated by NaCl or KCl, and to cell wall damaging agent congo red (CR). Real-time PCR assays revealed increased transcription levels of FgSGE1 with the treatment of NaCl or CR, and decreased FgSGE1 transcription in the FgOS-2 deletion mutant ΔFgOs-2. Based on the transcription levels, it can be concluded that FgSge1 is a downstream target of the mitogen-activated protein kinase FgOs-2.