Immunocytochemical Evidence for the Presence of Barley Thionins in Rachis and Stalk of Wheat: Decrease of Antibody Binding at Fusarium culmorum Penetration Sites

In thin sections of wheat rachis and stem, embedded in Spurr's resin, prefixed with glutaraldehyde and postfixed with osmium, large amounts of barley thionins were localized in cell walls with anti-thionin-protein A/gold. Reduced binding to thionin was detected in cell walls penetrated by Fusarium culmorum, suggesting that cell wall thionins were degraded by the fungus.     

Immunocytochemical Localization of Cell Wall-Bound Thionins and Hydroxyproline-Rich Glycoproteins in Fusarium culmorum-Infected Wheat Spikes

Abstract In the present study, a rabbit polyclonal antiserum against cell wall‐bound thionins from barley leaf and a mouse monoclonal antibody against hydroxyproline‐rich glycoproteins (HRGP) from maize were used to investigate the subcellular localization of thionins and HRGP or extensins in Fusarium culmorum‐infected wheat spikes by means of the immunogold labelling technique. The proteins were localized in cell walls of different tissues including the lemma, ovary and rachis, while the cytoplasm and organelles in these tissues showed almost no labelling. However, accumulation of thionins and HRGP in infected wheat spikes of resistant wheat cultivars differed distinctly from those of susceptible cultivars. Compared with the healthy tissues, labelling densities for the two types of proteins in cell walls of the infected lemma, ovary and rachis increased only slightly in the susceptible cultivar Agent, while in cell walls of infected tissues of the resistant cultivar Arina labelling densities of thionins and HRGP increased markedly. These findings indicated that accumulation of thionins and HRGP in cell walls of infected resistant wheat spikes may be involved in defence responses to infection and in spreading of F. culmorum.     

Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis

The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad‐spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH‐1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co‐expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.     

Cultivar-related differences in the distribution of cell-wall-bound thionins in compatible and incompatible interactions between barley and powdery mildew

Leaf-specific thionins of barley (Hordeum vulgare L.) have been identified as a novel class of cell-wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The distribution of these polypeptides has been studied in the host-pathogen system of barley and Erisyphe graminis DC.f.sp. hordei Marchal (powdery mildew). Immunogold-labelling of thionins in several barley cultivars indicates that resistance or susceptibility may be attributed to the presence or absence of thionins at the penetration site in walls and papillae of epidermal leaf cells.All of the leaf-specific thionin genes are confined to the distal end of the short arm of chromosome 6 of barley. None of the genes for cultivarspecific resistance to powdery mildew which have previously been mapped on barley chromosomes are found close to this locus.     

Shotgun Analysis of the Secretome of Fusarium graminearum

Fusarium head blight, caused predominately by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. To characterize the profile of proteins secreted by F. graminearum, the extracellular proteins were collectively obtained from F. graminearum culture supernatants and evaluated using one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry. A total of 87 proteins have been identified, of which 63 were predicted as secretory proteins including those with known functions. Meanwhile, 20 proteins that are not homologous to genomic sequences with known functions have also been detected. Some of the identified proteins are possible virulence factors and may play extracellular roles during F. graminearum infection. This study provides a valuable dataset of F. graminearum extracellular proteins, and a better understanding of the virulence mechanisms of the pathogen.     

Molecular characterisation of a novel plant homeobox gene expressed in the maturing xylem zone of Populus tremula × tremuloides

The Arabidopsis Thi2.1 thionin gene was cloned and sequenced. The promoter was fused to the uidA gene and stably transformed into Arabidopsis to study its regulation. GUS expression levels correlated with the steady-state levels of Thi2.1 mRNA, thus demonstrating that the promoter is sufficient for the regulation of the Thi2.1 gene. The sensitivity of the Thi2.1 gene to methyl jasmonate was found to be developmentally determined. Systemic and local expression could be induced by wounding and inoculation with Fusarium oxysporum f sp. matthiolae. A deletion analysis of the promoter identified a fragment of 325 bp upstream of the start codon, which appears to contain all the elements necessary for the regulation of the Thi2.1 gene. These results support the view that thionins are defence proteins, and indicate the possibility that resistance of Arabidopsis plants to necrotrophic fungal pathogens is mediated through the octadecanoid pathway.     

The Wor1-like Protein Fgp1 Regulates Pathogenicity,Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.     

Functional evaluation of a homologue of plant rapid alkalinisation factor (RALF) peptides in Fusarium graminearum

The cereal infecting fungus Fusarium graminearum is predicted to possess a single homologue of plant RALF (rapid alkalinisation factor) peptides. Fusarium mutant strains lacking FgRALF were generated and found to exhibit wildtype virulence on wheat and Arabidopsis floral tissue. Arabidopsis lines constitutively overexpressing FgRALF exhibited no obvious change in susceptibility to F. graminearum leaf infection. In contrast transient virus-mediated over-expression (VOX) of FgRALF in wheat prior to F. graminearum infection, slightly increased the rate of fungal colonisation of floral tissue. Ten putative Feronia (FER) receptors of RALF peptide were identified bioinformatically in hexaploid wheat (Triticum aestivum). Transient silencing of two wheat FER homoeologous genes prior to F. graminearum inoculation did not alter the subsequent interaction outcome. Collectively, our VOX results show that the fungal RALF peptide may be a minor contributor in F. graminearum virulence but results from fungal gene deletion experiments indicate potential functional redundancy within the F. graminearum genome. We demonstrate that virus-mediated over-expression is a useful tool to provide novel information about gene/protein function when results from gene deletion/disruption experimentation were uninformative.     

FGsub: <Emphasis Type="Italic">Fusarium graminearum</Emphasis> protein subcellular localizations predicted from primary structures

Background

The fungal pathogen Fusarium graminearum (telomorph Gibberella zeae) is the causal agent of several destructive crop diseases, where a set of genes usually work in concert to cause diseases to crops. To function appropriately, the F. graminearum proteins inside one cell should be assigned to different compartments, i.e. subcellular localizations. Therefore, the subcellular localizations of F. graminearum proteins can provide insights into protein functions and pathogenic mechanisms of this destructive pathogen fungus. Unfortunately, there are no subcellular localization information for F. graminearum proteins available now. Computational approaches provide an alternative way to predicting F. graminearum protein subcellular localizations due to the expensive and time-consuming biological experiments in lab.

Results

In this paper, we developed a novel predictor, namely FGsub, to predict F. graminearum protein subcellular localizations from the primary structures. First, a non-redundant fungi data set with subcellular localization annotation is collected from UniProtKB database and used as training set, where the subcellular locations are classified into 10 groups. Subsequently, Support Vector Machine (SVM) is trained on the training set and used to predict F. graminearum protein subcellular localizations for those proteins that do not have significant sequence similarity to those in training set. The performance of SVMs on training set with 10-fold cross-validation demonstrates the efficiency and effectiveness of the proposed method. In addition, for F. graminearum proteins that have significant sequence similarity to those in training set, BLAST is utilized to transfer annotations of homologous proteins to uncharacterized F. graminearum proteins so that the F. graminearum proteins are annotated more comprehensively.

Conclusions

In this work, we present FGsub to predict F. graminearum protein subcellular localizations in a comprehensive manner. We make four fold contributions to this filed. First, we present a new algorithm to cope with imbalance problem that arises in protein subcellular localization prediction, which can solve imbalance problem and avoid false positive results. Second, we design an ensemble classifier which employs feature selection to further improve prediction accuracy. Third, we use BLAST to complement machine learning based methods, which enlarges our prediction coverage. Last and most important, we predict the subcellular localizations of 12786 F. graminearum proteins, which provide insights into protein functions and pathogenic mechanisms of this destructive pathogen fungus.

     

Thionin Thi2.1 from Arabidopsis thaliana expressed in endothelial cells shows antibacterial, antifungal and cytotoxic activity

Thionins are plant antimicrobial peptides with antibacterial and antifungal activities. Thionin Thi2.1 cDNA from Arabidopsis thaliana was expressed in BVE-E6E7 bovine endothelial cell line and its activity was evaluated against Escherichia coli, Staphylococcus aureus, Candida albicans and different mammal cell lines. Total protein (2.5 mug) from conditioned medium (CM) of clone EC-Thi2.1 inhibited the growth of E. coli, S. aureus (>90%) and C. albicans strains (>80%) in relation to the CM from control cells. Also, CM of EC-Thi2.1 inhibited the viability of several transformed and normal mammal cell lines (38-95%). These results suggest that thionin Thi2.1 is an antimicrobial peptide that could be use in the treatment of mammalian infectious diseases.     

cDNA-derived identification of novel thionin precursors in Viscum album that contain highly divergent thionin domains but conserved signal and acidic polypeptide domains

The existence of new thionin variants in Viscum album has been deduced from cDNA sequences. Unlike the viscotoxins and related thionins previously found in different members of the Viscaceae, these novel thionins contain eight rather than six cysteine residues. In this respect they resemble thionins described previously from various cereals and from Pyrularia pubera, which also contain eight cysteine residues at identical positions. All of the new thionins of V. album are encoded as higher-molecular-weight precursors consisting of a signal peptide, a thionin domain and an acidic polypeptide domain. While the deduced amino acid sequences of the thionin domains of different precursor molecules are highly divergent, the two other domains are conserved among all of the variants and are distinct from the corresponding domains of thionin precursors of other plant species.     

Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica

Most thionins of higher plants are toxic to various bacteria, fungi, and animal and plant cells. The only known exception is the seed-specific thionin, crambin, of the crucifer Crambe abyssinica. Crambin has no net charge, is very hydrophobic and exhibits no toxicity. In the present work, the organization of the crambin precursor polypeptide was deduced from cDNA sequences. The precursor shows a domain structure similar to that of the preproprotein of other thionins, which contains a signal peptide, a thionin domain and a C-terminal amino acid extension. Unlike the thionin precursors studied thus far, both the thionin domain and the C-terminal amino acid extension of the crambin precursor have no net charge and are hydrophobic, thus facilitating their interaction, by analogy to that proposed for the corresponding domains of other thionin precursors that have positive and negative charges. The existence of a large number of novel and highly variable thionin variants in Crambe abyssinica has been deduced from cDNA sequences that were amplified by the polymerase chain reaction (PCR) from RNA of seeds, leaves and cotyledons. While the deduced amino acid sequences of the thionin domains of most of these thionin precursor molecules are highly divergent, the two other domains are conserved. Most of the predicted thionin variants are positively charged. The presence of positively charged residues in the thionin domains consistently correlates with the presence of a negatively charged residue in the C-terminal amino acid extension of the various thionin precursors. The different thionin variants are encoded by distinct sets of genes and are expressed in an organ-specific manner.     

Sharing a Host Plant (Wheat [Triticum aestivum]) Increases the Fitness of Fusarium graminearum and the Severity of Fusarium Head Blight but Reduces the Fitness of Grain Aphids (Sitobion avenae)

We hypothesized that interactions between fusarium head blight-causing pathogens and herbivores are likely to occur because they share wheat as a host plant. Our aim was to investigate the interactions between the grain aphid, Sitobion avenae, and Fusarium graminearum on wheat ears and the role that host volatile chemicals play in mediating interactions. Wheat ears were treated with aphids and F. graminearum inoculum, together or separately, and disease progress was monitored by visual assessment and by quantification of pathogen DNA and mycotoxins. Plants exposed to both aphids and F. graminearum inoculum showed accelerated disease progression, with a 2-fold increase in disease severity and 5-fold increase in mycotoxin accumulation over those of plants treated only with F. graminearum. Furthermore, the longer the period of aphid colonization of the host prior to inoculation with F. graminearum, the greater the amount of pathogen DNA that accumulated. Headspace samples of plant volatiles were collected for use in aphid olfactometer assays and were analyzed by gas chromatography-mass spectrometry (GC-MS) and GC-coupled electroantennography. Disease-induced plant volatiles were repellent to aphids, and 2-pentadecanone was the key semiochemical underpinning the repellent effect. We measured aphid survival and fecundity on infected wheat ears and found that both were markedly reduced on infected ears. Thus, interactions between F. graminearum and grain aphids on wheat ears benefit the pathogen at the expense of the pest. Our findings have important consequences for disease epidemiology, because we show increased spread and development of host disease, together with greater disease severity and greater accumulation of pathogen DNA and mycotoxin, when aphids are present.     

In Planta Stage-Specific Fungal Gene Profiling Elucidates the Molecular Strategies of Fusarium graminearum Growing inside Wheat Coleoptiles

The ascomycete Fusarium graminearum is a destructive fungal pathogen of wheat (Triticum aestivum). To better understand how this pathogen proliferates within the host plant, we tracked pathogen growth inside wheat coleoptiles and then examined pathogen gene expression inside wheat coleoptiles at 16, 40, and 64 h after inoculation (HAI) using laser capture microdissection and microarray analysis. We identified 344 genes that were preferentially expressed during invasive growth in planta. Gene expression profiles for 134 putative plant cell wall–degrading enzyme genes suggest that there was limited cell wall degradation at 16 HAI and extensive degradation at 64 HAI. Expression profiles for genes encoding reactive oxygen species (ROS)–related enzymes suggest that F. graminearum primarily scavenges extracellular ROS before a later burst of extracellular ROS is produced by F. graminearum enzymes. Expression patterns of genes involved in primary metabolic pathways suggest that F. graminearum relies on the glyoxylate cycle at an early stage of plant infection. A secondary metabolite biosynthesis gene cluster was specifically induced at 64 HAI and was required for virulence. Our results indicate that F. graminearum initiates infection of coleoptiles using covert penetration strategies and switches to overt cellular destruction of tissues at an advanced stage of infection.     

Leaf-specific thionins of barley-a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants

A novel class of highly abundant polypeptides with antifungal activity has been detected in cell walls of barley leaves. Similar polypeptides known as thionins occur not only in monocotyledonous but also in various dictoyledonous plants. The leaf-specific thionins of barley are encoded by a complex multigene family, which consists of at least 50-100 members per haploid genome. All of these genes are confined to chromosome 6. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that thionins are a naturally occurring, inducible plant protein possibly involved in the mechanism of plant defence against microbial infections.     

A Network Approach to Predict Pathogenic Genes for Fusarium graminearum

Fusarium graminearum is the pathogenic agent of Fusarium head blight (FHB), which is a destructive disease on wheat and barley, thereby causing huge economic loss and health problems to human by contaminating foods. Identifying pathogenic genes can shed light on pathogenesis underlying the interaction between F. graminearum and its plant host. However, it is difficult to detect pathogenic genes for this destructive pathogen by time-consuming and expensive molecular biological experiments in lab. On the other hand, computational methods provide an alternative way to solve this problem. Since pathogenesis is a complicated procedure that involves complex regulations and interactions, the molecular interaction network of F. graminearum can give clues to potential pathogenic genes. Furthermore, the gene expression data of F. graminearum before and after its invasion into plant host can also provide useful information. In this paper, a novel systems biology approach is presented to predict pathogenic genes of F. graminearum based on molecular interaction network and gene expression data. With a small number of known pathogenic genes as seed genes, a subnetwork that consists of potential pathogenic genes is identified from the protein-protein interaction network (PPIN) of F. graminearum, where the genes in the subnetwork are further required to be differentially expressed before and after the invasion of the pathogenic fungus. Therefore, the candidate genes in the subnetwork are expected to be involved in the same biological processes as seed genes, which imply that they are potential pathogenic genes. The prediction results show that most of the pathogenic genes of F. graminearum are enriched in two important signal transduction pathways, including G protein coupled receptor pathway and MAPK signaling pathway, which are known related to pathogenesis in other fungi. In addition, several pathogenic genes predicted by our method are verified in other pathogenic fungi, which demonstrate the effectiveness of the proposed method. The results presented in this paper not only can provide guidelines for future experimental verification, but also shed light on the pathogenesis of the destructive fungus F. graminearum.     

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.