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.     

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.     

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.     

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。     

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.     

Susceptibility of Kenyan Wheat Varieties to Head Blight, Fungal Invasion and Deoxynivalenol Accumulation Inoculated with Fusarium graminearum

Fifteen wheat varieties commercially grown in Kenya were tested for their susceptibility to head blight and mycotoxin accumulation after inoculation with Fusarium graminearum in pot experiments. The strains of the pathogen used had been isolated from wheat collected in different growing areas of Kenya. Head blight susceptibility was assessed as the percentage of spikelets bleached and area under disease progress curve; kernel colonization by fungal mycelium was determined as ergosterol content. All varieties were found to be moderately to highly susceptible. However, the varieties differed in head blight susceptibility (29–68% of spikelets bleached; mean 54%), fungal colonization (67–187  μ g/g ergosterol content; mean 111  μ g/g) and the resulting mycotoxin contamination [deoxynivalenol (DON) 5–31  μ g/g; mean 13.5  μ g/g]. Grain weight reductions due to head blight ranged from 23 to 57% (mean 44%). The varieties could be therefore divided into partially resistant and highly susceptible genotypes. The kernels of highly susceptible varieties had higher mycotoxin and ergosterol contents. However, the kernels of some varieties contained more fungal mycelium (ergosterol) without the corresponding high amounts of DON, suggesting that they possess some resistance to DON accumulation. Less susceptible varieties showed resistance to fungal spread, as indicated by a slow disease development and lower content of fungal biomass.     

DON生物合成的亚细胞定位和精准外排研究进展

单端孢霉烯B族毒素脱氧雪腐镰刀烯醇（deoxinivalenol, DON）是产毒镰刀菌在侵染小麦等作物过程中的一类重要的致病因子,可以帮助产毒镰刀菌在麦穗间扩展。DON会抑制蛋白质合成,对动物、微生物和寄主具有毒性（cytotoxicity and phytotoxicity）,然而产毒镰刀菌自身借助何种保护机制免受DON毒害目前研究甚少。DON毒害机制的研究对于镰刀菌毒素的持续防控和粮食安全、人民生命健康保障具有重要意义。综述了产毒镰刀菌DON合成解毒机制的最新研究进展,主要包括DON合成的亚细胞定位、合成基因簇内的外排蛋白和解毒基因作用方式,以期为有针对性地破解其解毒机制,设计研发高效靶向控毒技术的相关研究提供参考。     

Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum

The fungal pathogens Fusarium graminearum and F. culmorum cause ear blight disease on cereal crops worldwide. The disease lowers both grain quality and grain safety. Disease prevalence is increasing due to changes in cropping practices and the difficulties encountered by plant breeders when trying to introgress the polygene-based resistance. The molecular basis of resistance to Fusarium ear blight in cereal species is poorly understood. This is primarily due to the large size of cereal genomes and the expensive resources required to undertake gene function studies in cereals. We therefore explored the possibility of developing various model floral infection systems that would be more amenable to experimental manipulation and high-throughput gene function studies. The floral tissues of tobacco, tomato, soybean and Arabidopsis were inoculated with Fusarium conidia and this resulted in disease symptoms on anthers, anther filaments and petals in each plant species. However, only in Arabidopsis did this initial infection then spread into the developing siliques and seeds. A survey of 236 Arabidopsis ecotypes failed to identify a single genotype that was extremely resistant or susceptible to Fusarium floral infections. Three Arabidopsis floral mutants that failed to develop anthers and/or functional pollen (i.e. agamous-1, apetala1-3 and dad1) were significantly less susceptible to Fusarium floral infection than wild type. Deoxynivalenol (DON) mycotoxin production was also detected in Fusarium-infected flowers at >1 ppm. This novel floral pathosystem for Arabidopsis appears to be highly representative of a serious cereal crop disease.     

禾谷镰刀菌基因组中含寄主靶向模体分泌蛋白功能的初步分析

基于生物信息学的方法,以SignalP v3.0、TargetP v1.01、Big-PI Predictor和TMHMM v2.0四个分析软件对禾谷镰刀菌(Fusarium graminearum Schw.)全基因组的11 640个蛋白编码基因的N-端氨基酸序列进行信号肽分析,预测出606个潜在的分泌蛋白编码基因.通过对这些潜在的分泌蛋白进行MEME软件分析,发现其中有157个分泌蛋白的剪切点下游120氨基酸残基范围内具有一个保守的RXLX模体,其中有79个分泌蛋白具有可预测的功能性描述,包括FG00023.1具有与草酸盐氧化酶1有关的功能,FG01588.1具有与1,4-α-葡糖苷酶葡聚糖有关的功能,这些基因可作为禾谷镰刀菌致病相关的候选基因.其中FG04097.1编码的具有与丝氨酸型蛋白酶有关的功能在致病疫霉和疟原虫真核寄生物中具有相似保守的寄主靶标模体的效应蛋白中也观察到,但FG09127.1,FG05287.1编码的蛋白尚未被证明参与致病过程的研究报道.深入研究分泌蛋白将有助于明确植物与病原微生物互作的分子机制.利用禾谷镰刀菌基因组学研究成果,结合计算机技术和生物信息学的方法,分析其分泌蛋白组学,将有助于全面掌握其致病因子的结构与功能.对于这些蛋白功能的比较研究,将有利于对禾谷镰刀菌致病性的分子机制的探索,并为设计新的防治措施提供理论依据.     

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.     

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.     

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.