Capping proteins regulate fungal development,DON-toxisome formation and virulence in Fusarium graminearum

Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.     

Microtubule-assisted mechanism for toxisome assembly in Fusarium graminearum

In Fusarium graminearum, a trichothecene biosynthetic complex known as the toxisome forms ovoid and spherical structures in the remodelled endoplasmic reticulum (ER) under mycotoxin-inducing conditions. Previous studies also demonstrated that disruption of actin and tubulin results in a significant decrease in deoxynivalenol (DON) biosynthesis in F. graminearum. However, the functional association between the toxisome and microtubule components has not been clearly defined. In this study we tested the hypothesis that the microtubule network provides key support for toxisome assembly and thus facilitates DON biosynthesis. Through fluorescent live cell imaging, knockout mutant generation, and protein–protein interaction assays, we determined that two of the four F. graminearum tubulins, α₁ and β₂ tubulins, are indispensable for DON production. We also showed that these two tubulins are directly associated. When the α₁–β₂ tubulin heterodimer is disrupted, the metabolic activity of the toxisome is significantly suppressed, which leads to significant DON biosynthesis impairment. Similar phenotypic outcomes were shown when F. graminearum wild type was treated with carbendazim, a fungicide that binds to microtubules and disrupts spindle formation. Based on our results, we propose a model where α₁–β₂ tubulin heterodimer serves as the scaffold for functional toxisome assembly in F. graminearum.     

SNARE protein FgVam7 controls growth,asexual and sexual development,and plant infection in Fusarium graminearum

Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins play critical and conserved roles in membrane fusion and vesicle transport of eukaryotic cells. Previous studies have shown that various homologues of SNARE proteins are also important in the infection of host plants by pathogenic fungi. Here, we report the characterization of a SNARE homologue, FgVam7, from Fusarium graminearum that causes head blight in wheat and barley worldwide. Phylogenetic analysis and domain comparison reveal that FgVam7 is homologous to Vam7 proteins of Saccharomyces cerevisiae (ScVam7), Magnaporthe oryzae (MoVam7) and several additional fungi by containing a PhoX homology (PX) domain and a SNARE domain. We show that FgVam7 plays a regulatory role in cellular differentiation and virulence in F. graminearum. Deletion of FgVAM7 significantly reduces vegetative growth, conidiation and conidial germination, sexual reproduction and virulence. The ΔFgvam7 mutant also exhibits a defect in vacuolar maintenance and delayed endocytosis. Moreover, the ΔFgvam7 mutant is insensitive to salt and osmotic stresses, and hypersensitive to cell wall stressors. Further characterization of FgVam7 domains indicate that the PX and SNARE domains are conserved in controlling Vam7 protein localization and function, respectively. Finally, FgVam7 has been shown to positively regulate the expression of several deoxynivalenol (DON) biosynthesis genes TRI5, TRI6 and TRI101, and DON production. Our studies provide evidence for SNARE proteins as an additional means of regulatory mechanisms that govern growth, differentiation and virulence of pathogenic fungi.     

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.     

The plasma membrane H+-ATPase FgPMA1 regulates the development,pathogenicity, and phenamacril sensitivity of Fusarium graminearum by interacting with FgMyo-5 and FgBmh2

Fusarium graminearum, as the causal agent of Fusarium head blight (FHB), not only causes yield loss, but also contaminates the quality of wheat by producing mycotoxins, such as deoxynivalenol (DON). The plasma membrane H⁺-ATPases play important roles in many growth stages in plants and yeasts, but their functions and regulation in phytopathogenic fungi remain largely unknown. Here we characterized two plasma membrane H⁺-ATPases: FgPMA1 and FgPMA2 in F. graminearum. The FgPMA1 deletion mutant (∆FgPMA1), but not FgPMA2 deletion mutant (∆FgPMA2), was impaired in vegetative growth, pathogenicity, and sexual and asexual development. FgPMA1 was localized to the plasma membrane, and ∆FgPMA1 displayed reduced integrity of plasma membrane. ∆FgPMA1 not only impaired the formation of the toxisome, which is a compartment where DON is produced, but also suppressed the expression level of DON biosynthetic enzymes, decreased DON production, and decreased the amount of mycelial invasion, leading to impaired pathogenicity by exclusively developing disease on inoculation sites of wheat ears and coleoptiles. ∆FgPMA1 exhibited decreased sensitivity to some osmotic stresses, a cell wall-damaging agent (Congo red), a cell membrane-damaging agent (sodium dodecyl sulphate), and heat shock stress. FgMyo-5 is the target of phenamacril used for controlling FHB. We found FgPMA1 interacted with FgMyo-5, and ∆FgPMA1 showed an increased expression level of FgMyo-5, resulting in increased sensitivity to phenamacril, but not to other fungicides. Furthermore, co-immunoprecipitation confirmed that FgPMA1, FgMyo-5, and FgBmh2 (a 14-3-3 protein) form a complex to regulate the sensitivity to phenamacril and biological functions. Collectively, this study identified a novel regulating mechanism of FgPMA1 in pathogenicity and phenamacril sensitivity of F. graminearum.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relationship between Fusarium graminearum and Alternaria alternata contamination and deoxynivalenol occurrence on Argentinian durum wheat

A mycological survey was carried out on durum wheat (Triticum durum) samples from the main production area of Argentina. The isolation frequency and relative density of species of dematiaceous fungi, and genus Fusarium were calculated. Alternaria alternata and Fusarium graminearum were the predominant fungal species. An analysis of deoxynivalenol (DON) natural contamination was also performed on a limited number of samples (60). DON contamination levels in positive samples ranged from 26 to 6400 μg/kg. The non-parametric techniques applied showed that there is a positive relationship between DON contamination and F. graminearum relative densities and a negative relationship between DON contamination and A. alternata relative densities. This revised version was published online in August 2006 with corrections to the Cover Date.     

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。     

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.     

Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12- and 1.79-fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.     

禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究

目的:旨在敲除禾谷镰刀菌Fusarium graminearum Fg PDE1基因,确定其缺失突变体表型,从而分析该基因的生物学功能。方法:应用Split-marker技术构建含有潮霉素基因敲除盒,通过PEG介导原生质体转化,PCR筛查抗潮霉素转化子以获得缺失突变体ΔFg PDE1,根据突变体表型变化及致病性的检测对Fg PDE1基因的功能进行分析。结果:采用Split-marker技术,成功构建了Fg PDE1基因敲除盒;PEG介导转化禾谷镰刀菌原生质体后成功获得转化子。经PCR筛查,得到3个PCR确认的敲除突变体;表型观察发现,ΔFg PDE1菌落的外型及菌落生长速度与野生型没有明显差异。孢子侵染西红柿果实实验证明:以西红柿为侵染宿主,相对于野生型,突变体致病性没有明显减弱;但突变体分生孢子产量显著下降。结论:Fg PDE1基因可能与禾谷镰刀菌分生孢子的形成有关。     

Impact of essential oils on growth rate, zearalenone and deoxynivalenol production by Fusarium graminearum under different temperature and water activity conditions in maize grain

AIMS: The effect of five essential oils (oregano, cinnamon, lemongrass, clove and palmarose) on growth rate, zearalenone (ZEA) and deoxynivalenol (DON) production by Fusarium graminearum strains was assessed. METHODS AND RESULTS: The influence of the essential oils was tested on irradiated maize at two concentrations (500 and 1000 mg kg-1), at different water activity (aw) (0.95 and 0.995) and temperature (20 and 30 degrees C) levels. At 0.995 aw all essential oils tested had an inhibitory effect on growth rate of F. graminearum at both temperatures studied. At this aw level, DON production in general was inhibited by all essential oils at 30 degrees C and, although palmarose and clove were the only essential oils with statistically significant inhibitory effect on ZEA production, an inhibitory trend was observed when cinnamon and oregano oils were added to maize grain. CONCLUSIONS: Antifungal and antimycotoxigenic activity of the essential oils assayed was shown to depend on environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: It is apparent that essential oils should be considered as alternative preharvest natural fungicides. Further investigation on natural maize grain might be useful to study the effectiveness of these essential oils in the presence of natural mycoflora of maize grain.     

Lactic acid bacteria in the inhibition of Fusarium graminearum and deoxynivalenol detoxification

Aims: Considering the agronomic and industrial damage that is caused by the fungus Fusarium graminearum, as well as the serious health risks it poses to humans and animals exposed to F. graminearum‐produced mycotoxin deoxynivalenol (DON), this study evaluated the ability of different lactic acid bacteria (LAB) strains to inhibit fungal development and remove DON in vitro. Methods and Results: The antagonistic effects of strains and commercial cultures of LAB were evaluated against F. graminearum IAPAR 2218 by the agar diffusion method. Additionally, the influence of the culture media, pH and the presence of lactic and acetic acid on these effects was tested. The capacity to remove DON by viable cells and heat‐inactivated cells was analysed in liquid media and quantified by high performance liquid chromatography (HPLC). All isolated strains and commercial cultures inhibited the fungus and removed DON. The pH and culture media concentration did not influence these abilities, but heat inactivation had a strong effect on the ability of bacteria to remove mycotoxin. Conclusions: The isolated bacteria are able to inhibit F. graminearum growth and remove DON in vitro. Significance and Impact of the Study: This study suggests potential application of the isolated LAB strains in the inhibition of F. graminearum IAPAR 2218 and DON removal in vitro.     

The STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of Fusarium graminearum

Striatin-interacting phosphatases and kinases (STRIPAKs) are evolutionarily conserved supramolecular complexes that control various important cellular processes such as signal transduction and development. However, the role of the STRIPAK complex in pathogenic fungi remains elusive. In this study, the components and function of the STRIPAK complex were investigated in Fusarium graminearum, an important plant-pathogenic fungus. The results obtained from bioinformatic analyses and the protein–protein interactome suggested that the fungal STRIPAK complex consisted of six proteins: Ham2, Ham3, Ham4, PP2Aa, Ppg1, and Mob3. Deletion mutations of individual components of the STRIPAK complex were created, and observed to cause a significant reduction in fungal vegetative growth and sexual development, and dramatically attenuae virulence, excluding the essential gene PP2Aa. Further results revealed that the STRIPAK complex interacted with the mitogen-activated protein kinase Mgv1, a key component in the cell wall integrity pathway, subsequently regulating the phosphorylation level and nuclear accumulation of Mgv1 to control the fungal stress response and virulence. Our results also suggested that the STRIPAK complex was interconnected with the target of rapamycin pathway through Tap42-PP2A cascade. Taken together, our findings revealed that the STRIPAK complex orchestrates cell wall integrity signalling to govern the fungal development and virulence of F. graminearum and highlighted the importance of the STRIPAK complex in fungal virulence.