Structural and functional characterization of TRI3 trichothecene 15‐O‐acetyltransferase from Fusarium sporotrichioides

Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13‐epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15‐O‐trichothecene acetyltransferase isolated from F. sporotrichioides and the “in vivo” characterization of Δtri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15‐decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15‐decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3‐O‐trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.     

Use of the volatile trichodiene to reduce Fusarium head blight and trichothecene contamination in wheat

Fusarium graminearum is the primary cause of Fusarium head blight (FHB), one of the most economically important diseases of wheat worldwide. FHB reduces yield and contaminates grain with the trichothecene mycotoxin deoxynivalenol (DON), which poses a risk to plant, human and animal health. The first committed step in trichothecene biosynthesis is formation of trichodiene (TD). The volatile nature of TD suggests that it could be a useful intra or interspecies signalling molecule, but little is known about the potential signalling role of TD during F. graminearum-wheat interactions. Previous work using a transgenic Trichoderma harzianum strain engineered to emit TD (Th + TRI5) indicated that TD can function as a signal that can modulate pathogen virulence and host plant resistance. Herein, we demonstrate that Th + TRI5 has enhanced biocontrol activity against F. graminearum and reduced DON contamination by 66% and 70% in a moderately resistant and a susceptible cultivar, respectively. While Th + TRI5 volatiles significantly influenced the expression of the pathogenesis-related 1 (PR1) gene, the effect was dependent on cultivar. Th + TRI5 volatiles strongly reduced DON production in F. graminearum plate cultures and downregulated the expression of TRI genes. Finally, we confirm that TD fumigation reduced DON accumulation in a detached wheat head assay.     

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.     

New tricks of an old enemy: isolates of Fusarium graminearum produce a type A trichothecene mycotoxin

The ubiquitous filamentous fungus Fusarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F. graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX‐2) was characterized by liquid chromatography‐tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX‐2 as 3α‐acetoxy‐7α,15‐dihydroxy‐12,13‐epoxytrichothec‐9‐ene. Compared with the well‐known 3‐acetyl‐deoxynivalenol (3‐ADON), it lacks the keto group at C‐8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10‐fold higher contamination with its deacetylated form, named NX‐3, (up to 540 mg kg^?1) compared with NX‐2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga Chlamydomonas reinhardtii. NX‐3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX‐2 is far less toxic, similar to 3‐ADON. Genetic analysis revealed a different TRI1 allele in the N‐isolates, which was verified to be responsible for the difference in hydroxylation at C‐8.     

Isolates of Fusarium graminearum collected 40–320?meters above ground level cause Fusarium head blight in wheat and produce trichothecene mycotoxins

The aerobiology of fungi in the genus Fusarium is poorly understood. Many species of Fusarium are important pathogens of plants and animals and some produce dangerous secondary metabolites known as mycotoxins. In 2006 and 2007, autonomous unmanned aerial vehicles (UAVs) were used to collect Fusarium 40–320 m above the ground at the Kentland Farm in Blacksburg, Virginia. Eleven single-spored isolates of Fusarium graminearum (sexual stage Gibberella zeae) collected with autonomous UAVs during fall, winter, spring, and summer months caused Fusarium head blight on a susceptible cultivar of spring wheat. Trichothecene genotypes were determined for all 11 of the isolates; nine isolates were DON/15ADON, one isolate was DON/3ADON, and one isolate was NIV. All of the isolates produced trichothecene mycotoxins in planta consistent with their trichothecene genotypes. To our knowledge, this is the first report of a NIV isolate of F. graminearum in Virginia, and DON/3ADON genotypes are rare in populations of the fungus recovered from infected wheat plants in the eastern United States. Our data are considered in the context of a new aerobiological framework based on atmospheric transport barriers, which are Lagrangian coherent structures present in the mesoscale atmospheric flow. This framework aims to improve our understanding of population shifts of F. graminearum and develop new paradigms that may link field and atmospheric populations of toxigenic Fusarium spp. in the future.     

Nivalenol and 15‐acetyldeoxynivalenol Chemotypes of Fusarium graminearum Clade Species are Prevalent on Maize throughout China

Fusarium graminearum clade species are among the main causative agents of Gibberella ear rot (GER) in maize and responsible for the various trichothecene mycotoxins accumulated in contaminated maize grains. In this study, a total of 620 isolates from diseased maize ears collected from 59 districts in 19 provinces throughout China, previously identified morphologically as Fusarium graminearum clade, was genetically characterized at the species level based on SCAR (Sequence Characterized Amplified Region) and for their potential capability of mycotoxin production using the genetic chemotyping assay. The results showed that 359 isolates were F. asiaticum (SCAR 5), which consisted of 97% nivalenol (NIV)‐chemotypes, 0.8% 3‐acetyldeoxynivalenol (3‐ADON)‐producing isolates and 2.2% 15‐acetyldeoxynivalenol (15‐ADON) producers, whereas the remaining 261 isolates were identified as F. graminearum sensu stricto (SCAR 1), all of which produced 15‐ADON mycotoxins. This high proportion of NIV producers present in F. asiaticum is different from the chemotype patterns in F. asiaticum populations isolated from wheat and barley, where DON and its acetylated chemotypes were the predominant mycotoxins. Moreover, the majority of NIV producers (59.1%) and all the 3‐ADON‐producing strains were derived from the warmer regions in southern China, whereas most of the 15‐ADON‐producing strains (78.4%) were isolated from the colder regions in northern China. Our study is the first report of NIV chemotypes of F. asiaticum and 15‐ADON chemotypes of F. graminearum sensu stricto that were associated with the GER of maize in China.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity,production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum

Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS‐generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper‐EF‐hand‐containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial‐produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.     

Evidence that a secondary metabolic biosynthetic gene cluster has grown by gene relocation during evolution of the filamentous fungus Fusarium

Trichothecenes are terpene‐derived secondary metabolites produced by multiple genera of filamentous fungi, including many plant pathogenic species of Fusarium. These metabolites are of interest because they are toxic to animals and plants and can contribute to pathogenesis of Fusarium on some crop species. Fusarium graminearum and F. sporotrichioides have trichothecene biosynthetic genes (TRI) at three loci: a 12‐gene TRI cluster and two smaller TRI loci that consist of one or two genes. Here, comparisons of additional Fusarium species have provided evidence that TRI loci have a complex evolutionary history that has included loss, non‐functionalization and rearrangement of genes as well as trans‐species polymorphism. The results also indicate that the TRI cluster has expanded in some species by relocation of two genes into it from the smaller loci. Thus, evolutionary forces have driven consolidation of TRI genes into fewer loci in some fusaria but have maintained three distinct TRI loci in others.     

Variation in 8-ketotrichothecenes and zearalenone production by Fusarium graminearum isolates from corn and barley in Korea

A total of 214 Fusarium graminearum isolates were obtained from corn and barley which were collected from Kangwon province and the southern part of Korea, respectively, and were tested for 8-ketotrichothecenes and zearalenone (ZEA) production on rice grains. The incidences of trichothecene production by 105 isolates of F. graminearum from corn were 59.0% for deoxynivalenol (DON), 37.1% for 15-acetyldeoxynivalenol(15-ADON), 13.3% for 3-acetyldeoxynivalenol (3-ADON), 7.6% for 3,15-diacetyldeoxynivalenol (3,15-DADON), 20.0% for nivalenol (NIV), 6.7% for 4-acetylnivalenol (4-ANIV), and 1.0% for 4,15-diacetylnivalenol (4,15-DANIV). DON chemotypes frequently produced 15-ADON as the major isomer rather than 3-ADON and 9 of the 61 DON chemotypes produced low levels of NIV. On the other hand, the incidences of trichothecene production of 109 isolates by F. graminearum from barley were 24.8% for DON, 72.5% for NIV, 62.4% for 4-ANIV, and 10.1% for 4,15-DANIV. Of these isolates, 78 were NIV chemotypes and only one isolate produced DON and 3-ADON as major toxins. In addition, 26 of the 78 NIV chemotypes produced low levels of DON. ZEA was frequently produced by the trichothecene-producing isolates and the incidences of ZEA were 51.4% and 31.2% for the isolates from corn and barley, respectively. There was a great regional difference in trichothecene production by F. graminearum isolates between corn- and barley-producing areas in Korea.     

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.     

Bioprospecting for trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes with different abilities to modify the mycotoxin deoxynivalenol

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 10(4) M(-1)·s(-1) to 4.7 × 10(6) M(-1)·s(-1). Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study.     

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.     

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.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.     

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production

Background  

The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied.