Flippases play specific but distinct roles in the development,pathogenicity, and secondary metabolism of Fusarium graminearum

The membrane trafficking system is important for compartmentalization of the biosynthesis pathway and secretion of deoxynivalenol (DON) mycotoxin (a virulence factor) in Fusarium graminearum. Flippases are transmembrane lipid transporters and mediate a number of essential physiological steps of membrane trafficking, including vesicle budding, charging, and protein diffusion within the membrane. However, the roles of flippases in secondary metabolism remain unknown in filamentous fungi. Herein, we identified five flippases (FgDnfA, FgDnfB, FgDnfC1, FgDnfC2, and FgDnfD) in F. graminearum and established their specific and redundant functions in the development and pathogenicity of this phytopathogenic fungus. Our results demonstrate that FgDnfA is critical for normal vegetative growth while the other flippases are dispensable. FgDnfA and FgDnfD were found crucial for the fungal pathogenesis, and a remarkable reduction in DON production was observed in ΔFgDNFA and ΔFgDNFD. Deletion of the FgDNFB gene increased DON production to about 30 times that produced by the wild type. Further analysis showed that FgDnfA and FgDnfD have positive roles in the regulation of trichothecene (TRI) genes (TRI1, TRI4, TRI5, TRI6, TRI12, and TRI101) expression and toxisome reorganization, while FgDnfB acts as a negative regulator of DON synthesis. In addition, FgDnfB and FgDnfD have redundant functions in the regulation of phosphatidylcholine transport, and double deletion of FgDNFB and FgDNFD showed serious defects in fungal development, DON synthesis, and virulence. Collectively, our findings reveal the distinct and specific functions of flippase family members in F. graminearum and principally demonstrate that FgDnfA, FgDnfD, and FgDnfB have specific spatiotemporal roles during toxisome biogenesis.     

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.     

FgVELB is associated with vegetative differentiation, secondary metabolism and virulence in Fusarium graminearum

The velvet complex containing VeA, VelB and LaeA has been showed to play critical roles in the regulation of secondary metabolism and diverse cellular processes in Aspergillus spp. In this study, we identified FgVelB, a homolog of Aspergillus nidulans VelB, from Fusarium graminearum using the BLASTP program. Disruption of FgVELB gene led to several phenotypic defects, including suppression of aerial hyphae formation, reduced hyphal hydrophobicity and highly increased conidiation. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents, which may be related to a high level of glycerol accumulation in the mutant. Additionally, the mutant exhibited increased sensitivity to the phenylpyrrole fungicide fludioxonil. Ultrastructural and histochemical analyses revealed that conidia of FgVELB deletion mutant contained numerous lipid droplets. Pathogenicity assays showed FgVELB deletion mutant was impaired in virulence on flowering wheat head, which is consistent with the observation that FgVelB is involved in the regulation of deoxynivalenol biosynthesis in F. graminearum. All of the defects were restored by genetic complementation of the mutant with wild-type FgVELB gene. Yeast two hybrid assays showed that FgVelB does not interact with FgVeA. Taken together, results of this study indicated that FgVelB plays a critical role in the regulation of various cellular processes in F. graminearum.     

Transposon-tagging identifies novel pathogenicity genes in Fusarium graminearum

With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.     

The velvet gene, FgVe1, affects fungal development and positively regulates trichothecene biosynthesis and pathogenicity in Fusarium graminearum

Trichothecenes are a group of toxic secondary metabolites produced mainly by Fusarium graminearum (teleomorph: Gibberella zeae) during the infection of crop plants, including wheat, maize, barley, oats, rye and rice. Some fungal genes involved in trichothecene biosynthesis have been shown to encode regulatory proteins. However, the global regulation of toxin biosynthesis is still enigmatic. In addition to the production of secondary metabolites belonging to the trichothecene family, F. graminearum produces the red pigment aurofusarin. The gene regulation underlying the production of aurofusarin is not well understood. The velvet gene (veA) is conserved in various genera of filamentous fungi. Recently, the veA gene from Aspergillus nidulans has been shown to be the key component of the velvet complex regulating development and secondary metabolism. Using blast analyses, we identified the velvet gene from F. graminearum, FgVe1. Disruption of FgVe1 causes several phenotypic effects. However, the complementation of this mutant with the FgVe1 gene restores the wild-type phenotypes. The in vitro phenotypes include hyperbranching of the mycelium, suppression of aerial hyphae formation, reduced hydrophobicity of the mycelium and highly reduced sporulation. Our data also show that FgVe1 modulates the production of the aurofusarin pigment and is essential for the expression of Tri genes and the production of trichothecenes. Pathogenicity studies performed on flowering wheat plants indicate that FgVe1 is a positive regulator of virulence in F. graminearum.     

蛋白激酶FgBud32参与禾谷镰刀菌的生长发育、致病和胁迫应答

由禾谷镰刀菌引起的小麦赤霉病是一种毁灭性的小麦真菌病害,在世界范围内造成小麦产量和质量的巨大损失。实验室前期在禾谷镰刀菌中共鉴定到116个蛋白激酶,其中FgBUD32基因的缺失会造成营养生长和有性生殖方面的严重缺陷,但其在禾谷镰刀菌中的详细功能尚未报道。本研究通过系统比较Fgbud32突变体与野生型PH-1及互补菌株的表型差异,对FgBud32在禾谷镰刀菌中的生物学功能进行了解析。研究结果显示Fgbud32突变体在多个表型方面存在缺陷,与野生型菌株以及互补菌株相比,其生长速率急剧下降,菌丝弯曲且分支减少;分生孢子的产量显著降低,形态变短,隔膜减少,萌发率降低且萌发速率延迟;在有性生殖时期不能产生子囊壳或子囊壳前体;对小麦穗和胚芽鞘的致病力以及DON毒素的合成能力均显著下降。进一步胁迫试验表明,FgBUD32基因的缺失导致禾谷镰刀菌对氧化胁迫(H₂O₂)以及DNA损伤胁迫(羟基脲和甲磺甲酯)的敏感性增加。此外,我们还发现FgBud32在细胞核和细胞质中均有定位,且在一定时期或条件下会从细胞质向细胞核内聚集。综上所述,FgBUD32基因参与了禾谷镰刀菌的营养生长、极性生长、无性/有性生殖、DON毒素合成、致病以及对氧化胁迫和DNA损伤胁迫的应答等多种生命活动,但其具体的作用机制还有待深入研究。     

The ADP-ribosylation factor-like small GTPase FgArl1 participates in growth,pathogenicity and DON production in Fusarium graminearum

Fusarium graminearum is the main pathogen of Fusarium head blight (FHB) in wheat and related species, which causes serious production decreases and economic losses and produces toxins such as deoxynivalenol (DON), which endangers the health of humans and livestock. Vesicle transport is a basic physiological process required for cell survival in eukaryotes. Many regulators of vesicle transport are reported to be involved in the pathogenicity of fungi. In yeast and mammalian cells, the ADP-ribosylation factor-like small GTPase Arl1 and its orthologs are involved in regulating vesicular trafficking, cytoskeletal reorganization and other significant biological processes. However, the role of Arl1 in F. graminearum is not well understood. In this study, we characterized the Arl1-homologous protein FgArl1 in F. graminearum and showed that FgArl1 is located in the trans-Golgi apparatus. The deletion of FgARL1 resulted in a significant decrease in vegetative growth and pathogenicity. Further analyses of the ΔFgarl1 mutant revealed defects in the production of DON. Taken together, these results indicate that FgArl1 is important in the development and pathogenicity of F. graminearum.     

长江流域禾谷镰孢菌群部分菌株系统发育学、产毒素化学型及致病力研究

从采集自长江流域引起小麦赤霉病的禾谷镰孢菌群(Fusarium graminearum clade)菌株中选取了31株,扩增并测定了这些菌株的EF-1α(translation elongation factor)、PHO(phosphate permease)基因序列,利用相关软件进行了系统发育分析。对这些菌株的产毒素化学型进行了分子检测。同时,用两个小麦品种(扬麦158和安农8455)测定了菌株的致病力。系统发育分析表明绝大多数菌株与F.asiaticum聚为一枝,只有一个菌株11027与F.graminearum聚类。30株F.asiaticum中有24株产脱氧雪腐镰孢菌烯醇(deoxynivalenol,DON)和3-乙酰脱氧雪腐镰孢菌烯醇(3-acetyldeoxynivalenol,3-AcDON),另外6株产雪腐镰孢菌烯醇(Nivalenol,NIV)。一株F.graminearum菌株11027产脱氧雪腐镰孢菌烯醇(DON)和15-乙酰脱氧雪腐镰孢菌烯醇(15-acetyldeoxynivalenol,15-AcDON)。在扬麦158上,菌株间的致病力分化较为明显,产NIV毒素的菌株致病力普遍较弱,强致病力的菌株都产3-AcDON毒素。结果表明在我国长江流域,产3-AcDON毒素的F.asiaticum是引起小麦赤霉病的优势种群,中抗赤霉病的小麦品种扬麦158可以有效评价菌株的致病力强弱。     

MAPK pathway-related tyrosine phosphatases regulate development,secondary metabolism and pathogenicity in fungus Aspergillus flavus

Mitogen-activated protein kinase (MAPK) cascades are highly conserved in eukaryotic cells and are known to play crucial roles in the regulation of various cellular processes. However, compared with kinase-mediated phosphorylation, dephosphorylation catalysed by phosphatases has not been well characterized in filamentous fungi. In this study, we identified five MAPK pathway-related phosphatases (Msg5, Yvh1, Ptp1, Ptp2 and Oca2) and characterized their functions in Aspergillus flavus, which produces aflatoxin B₁ (AFB₁), one of the most toxic and carcinogenic secondary metabolites. These five phosphatases were identified as negative regulators of MAPK (Slt2, Fus3 and Hog1) pathways. Deletion of Msg5 and Yvh1 resulted in significant defects in conidiation, sclerotia formation, aflatoxin production and crop infection. Additionally, double knockout mutants (ΔMsg5/ΔPtp1, ΔMsg5/ΔPtp2 and ΔMsg5/ΔOca2) displayed similar defects to those observed in the ΔMsg5 single mutant, indicating that Msg5 plays a major role in the regulation of development and pathogenicity in A. flavus. Importantly, we found that the active site at C439 is essential for the function of the Msg5 phosphatase. Furthermore, the MAP kinase Fus3 was found to be involved in the regulation of development, aflatoxin biosynthesis and pathogenicity, and its conserved phosphorylation residues (Thr and Tyr) were critical for the full range of its functions in A. flavus. Overall, our results reveal that MAPK related tyrosine phosphatases play important roles in the regulation of development, secondary metabolism and pathogenicity in A. flavus, and could be developed as potential targets for preventing damage caused by this fungal pathogen.     

Inhibition of Fusarium graminearum growth and development by farnesol

The isoprenoid farnesol was previously shown to induce morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. This study demonstrates that under similar liquid media growth conditions, farnesol also triggers apoptosis in the plant pathogenic fungus Fusarium graminearum. However, unlike A. nidulans, F. graminearum spores treated with farnesol exhibited altered germination patterns and most (>60%) lysed upon prolonged exposure. Given the economic importance of F. graminearum as a pathogen of small grains, this study proposes that farnesol may have potential value as an antifungal compound.     

Fusarium graminearum Tri12p influences virulence to wheat and trichothecene accumulation

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.