Two mitogen-activated protein kinase signalling cascades mediate basal resistance to antifungal plant defensins in Fusarium graminearum

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of Fusarium graminearum, which causes head blight disease in cereals. In order to determine the signalling cascades that are modulated by these defensins, we have isolated several insertional mutants of F. graminearum that exhibit hypersensitivity to MsDef1, but not to MtDef4. The molecular characterization of two of these mutants, designated enhanced sensitivity to defensin (esd), has revealed that the Mgv1 and Gpmk1 MAP kinase signalling cascades play a major role in regulating sensitivity of F. graminearum to MsDef1, but not to MtDef4. The Hog1 MAP kinase signalling cascade, which is responsible for adaptation of this fungus to hyperosmotic stress, does not participate in the fungal response to these defensins. Significantly, the esd mutants also exhibit hypersensitivity to other tested defensins and are highly compromised in their pathogenesis on wheat heads and tomato fruits. The studies reported here for the first time implicate two MAP kinase signalling cascades in a plant defensin-mediated alteration of fungal growth. Based on our findings, we propose that specific MAP kinase signalling cascades are essential for protection of a fungal pathogen from the antimicrobial proteins of its host plant.     

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue.     

Morphogenesis in germinating Fusarium graminearum macroconidia

Fusarium graminearum (teleomorph Gibberella zeae) is a significant pathogen of wheat and corn. F. graminearum forms multicellular macroconidia that play an important role in dissemination of the disease. The spatial pattern of morphogenesis in germinating macroconidia is described. Germ tubes preferentially emerge from the apical cells in a bipolar pattern that appears to be common to filamentous fungi. Chitin deposition occurs at two locations: the spore apices and cortical regions of macroconidial cells that subsequently produce a germ tube. The spatial pattern of morphogenesis requires the presence of functional microtubules, which may be responsible for the transport of key polarity factors to specific sites. These observations suggest that F. graminearum possesses a regulatory system that marks germ tube emergence sites. Perturbation of this system may represent an effective approach for inhibiting colonization of host plant surfaces.     

BDM1, a phosducin-like gene of Fusarium graminearum, is involved in virulence during infection of wheat and maize

Fusarium graminearum is a common pathogen of wheat and maize throughout the world. Despite recent advances in the elucidation of the genetic basis of virulence, significant gaps in the regulatory network underlying pathogenesis remain to be filled. In particular, little is known at the molecular level about the overlap among mechanisms of pathogenicity on maize and wheat. G-protein signalling has been implicated in pathogenesis in F. graminearum, although the underlying mechanisms are not fully understood. In this study, we investigated the involvement of a putative phosducin-like gene (BDM1) in growth, development and pathogenesis in F. graminearum. Targeted deletion of BDM1 revealed roles in sexual and asexual sporulation, germ tube development, hyphal branching and mycelial morphology. During pathogenesis, BDM1 is required for wild-type levels of colonization of maize silk tissue and stalks, but is dispensable for the colonization of kernels. The deletion of BDM1 also reduced the virulence of F. graminearum during the infection of wheat seedlings and heads, resulting in a significant reduction in fungal biomass and a delayed spread of visual symptom expression (i.e. bleaching in heads). Furthermore, BDM1 is required for wild-type levels of deoxynivalenol biosynthesis during the infection of wheat heads and maize silks. In summation, BDM1 is one of the few genes characterized to date in F. graminearum involved in virulence during infection of both maize and wheat. Thus, the functional characterization of BDM1 has established a new regulatory link between pathogenesis in maize and wheat, and provides a genetic resource through which the regulatory networks underlying virulence in F. graminearum can be further elucidated.     

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.     

Pseudomonas fluorescens CHA0 maintains carbon delivery to Fusarium graminearum-infected roots and prevents reduction in biomass of barley shoots through systemic interactions

Soil bacteria such as pseudomonads may reduce pathogen pressure for plants, both by activating plant defence mechanisms and by inhibiting pathogens directly due to the production of antibiotics. These effects are hard to distinguish under field conditions, impairing estimations of their relative contributions to plant health. A split-root system was set up with barley to quantify systemic and local effects of pre-inoculation with Pseudomonas fluorescens on the subsequent infection process by the fungal pathogen Fusarium graminearum. One root half was inoculated with F. graminearum in combination with P. fluorescens strain CHA0 or its isogenic antibiotic-deficient mutant CHA19. Bacteria were inoculated either together with the fungal pathogen or in separate halves of the root system to separate local and systemic effects. The short-term plant response to fungal infection was followed by using the short-lived isotopic tracer (11)CO(2) to track the delivery of recent photoassimilates to each root half. In the absence of bacteria, fungal infection diverted carbon from the shoot to healthy roots, rather than to infected roots, although the overall partitioning from the shoot to the entire root system was not modified. Both local and systemic pre-inoculation with P. fluorescens CHA0 prevented the diversion of carbon as well as preventing a reduction in plant biomass in response to F. graminearum infection, whereas the non-antibiotic-producing mutant CHA19 lacked this ability. The results suggest that the activation of plant defences is a central feature of biocontrol bacteria which may even surpass the effects of direct pathogen inhibition.     

Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from maize in Nepal and identification of a putative new lineage

On smallholder farms in the foothills of the Himalayan Mountains in Nepal, fungi of the Fusarium graminearum clade cause Gibberella ear rot of maize and contamination with the 8-ketotrichothecenes nivalenol and deoxynivalenol. Previous DNA marker analyses of the F.?graminearum clade from maize in Nepal found a high level of genetic diversity but were limited in detail or scope. The present study incorporated a collection of 251 field strains from a wide geographic distribution in Nepal and utilized sequencing of the MAT1-1-3 gene of the mating type locus to determine the number and frequency of lineages and species of the F. graminearum clade. The frequency of nivalenol and deoxynivalenol chemotypes was determined by chemical analysis and by TRI13 deletion-marker analysis. We found that Gibberella ear rot of maize in Nepal is associated with a complex of species of the F. graminearum clade - mainly Fusarium asiaticum and Fusarium meridionale, but also Fusarium boothii and a putative new lineage, which we have designated the 'Nepal lineage'. Fusarium graminearum sensu stricto, which dominates in maize elsewhere in Asia and worldwide, was not detected in Nepal. Although nivalenol production has been associated experimentally with lower virulence in maize ear rot and wheat head blight, this collection of the F. graminearum clade from maize in Nepal is dominated (4:1) by nivalenol producers, suggesting that traits other than crop plant pathogenesis affect population structure in this complex agroecosystem.     

Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat

The fungus Cochliobolus sativus is the main pathogen of common root rot, a serious soil-borne disease of wheat (Triticum aestivum L.). The fungus Fusarium graminearum is the primary pathogen of Fusarium head blight, a devastating disease of wheat worldwide. In this study, the wheat lipid transfer protein gene, TaLTP5, was cloned and evaluated for its ability to suppress disease development in transgenic wheat. TaLTP5 expression was induced after C. sativus infection. The TaLTP5 expression vector, pA25-TaLTP5, was constructed and bombarded into Chinese wheat variety Yangmai 18. Six TaLTP5 transgenic wheat lines were established and characterized. PCR and Southern blot analyses indicated that the introduced TaLTP5 gene was integrated into the genomes of six transgenic wheat lines by distinct patterns, and heritable. RT-PCR and real-time quantitative RT-PCR revealed that the TaLTP5 gene was over-expressed in the transgenic wheat lines compared to segregants lacking the transgene and wild-type wheat plants. Following challenge with C. sativus or F. graminearum, all six transgenic lines overexpressing TaLTP5 exhibited significantly enhanced resistance to both common root rot and Fusarium head blight compared to the untransformed wheat Yangmai 18.     

RAS2 regulates growth and pathogenesis in Fusarium graminearum

Fusarium graminearum is a ubiquitous pathogen of cereal crops, including wheat, barley, and maize. Diseases caused by F. graminearum are of particular concern because harvested grains frequently are contaminated with harmful mycotoxins such as deoxynivalenol (DON). In this study, we explored the role of Ras GTPases in pathogenesis. The genome of F. graminearum contains two putative Ras GTPase-encoding genes. The two genes (RAS1 and RAS2) showed different patterns of expression under different conditions of nutrient availability and in various mutant backgrounds. RAS2 was dispensable for survival but, when disrupted, caused a variety of morphological defects, including slower growth on solid media, delayed spore germination, and significant reductions in virulence on wheat heads and maize silks. Intracellular cAMP levels were not affected by deletion of RAS2 and exogenous treatment of the ras2 mutant with cAMP did not affect phenotypic abnormalities, thus indicating that RAS2 plays a minor or no role in cAMP signaling. However, phosphorylation of the mitogen-activated protein (MAP) kinase Gpmk1 and expression of a secreted lipase (FGL1) required for infection were reduced significantly in the ras2 mutant. Based on these observations, we hypothesize that RAS2 regulates growth and virulence in F. graminearum by regulating the Gpmk1 MAP kinase pathway.     

Differences in susceptibility of winter wheat varieties for Fusarium species under Belgian growing conditions

Fusarium head blight is an important disease of cereal crops caused by Fusarium species. It causes not only a reduction in yield, but most Fusarium species (F. graminearum. F. culmorum, F. avenaceum. F. poae) produce also a range of toxic metabolites such as deoxynivalenol (DON) and zearalenone (ZEA). The evaluation of Fusarium species was followed up under natural infection conditions during the growing seasons 2001--2002 and 2002--2003 in two varietal winter wheat experiments on the experimental farm of the Hogeschool Gent at Bottelare. Disease pressure, DON and ZEA content, different Fusarium species as well as growth and yield parameters were determined. In both years there were significant differences between the varieties concerning the susceptibility to Fusarium and the DON content. ZEA was not found in the kernels. The mean deoxynivalenol (DON) content was in 2002 (1,126 mg/kg) higher than in 2003 (0.879 mg/kg) although the mean disease severity was bigger in 2003 than in 2002 what means that the DON content was not always correlated with the disease severity. The Fusarium species most frequently identified in our two field trials (Bottelare) were F. graminearum and F. culmorum Varietal differences in susceptibility to Fusarium species and DON contamination could be detected.     

Gibberella zeae ascospore production and collection for microarray experiments

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004). The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection. These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.     

Overexpression of NRPS4 leads to increased surface hydrophobicity in fusarium graminearum

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.     

A jacalin-related lectin-like gene in wheat is a component of the plant defence system

Jacalin-related lectins (JRLs) are a subgroup of proteins with one or more jacalin-like lectin domains. Although JRLs are often associated with biotic or abiotic stimuli, their biological functions in plants, as well as their relationships to plant disease resistance, are poorly understood. A mannose-specific JRL (mJRL)-like gene (TaJRLL1) that is mainly expressed in stem and spike and encodes a protein with two jacalin-like lectin domains was identified in wheat. Pathogen infection and phytohormone treatments induced its expression; while application of the salicylic acid (SA) biosynthesis inhibitor paclobutrazol and the jasmonic acid (JA) biosynthesis inhibitor diethyldithiocarbamic acid, respectively, substantially inhibited its expression. Attenuating TaJRLL1 through virus-induced gene silencing increased susceptibility to the facultative fungal pathogen Fusarium graminearum and the biotrophic fungal pathogen Blumeria graminis. Arabidopsis thaliana transformed with TaJRLL1 displayed increased resistance to F. graminearum and Botrytis cinerea. JA and SA levels in transgenic Arabidopsis increased significantly. A loss or increase of disease resistance due to an alteration in TaJRLL1 function was correlated with attenuation or enhancement of the SA- and JA-dependent defence signalling pathways. These results suggest that TaJRLL1 could be a component of the SA- and JA-dependent defence signalling pathways.     

Molecular characterization of a Fus3/Kss1 type MAPK from Puccinia striiformis f. sp. tritici, PsMAPK1

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes the destructive wheat stripe rust disease worldwide. Due to the lack of reliable transformation and gene disruption method, knowledge about the function of Pst genes involved in pathogenesis is limited. Mitogen-activated protein kinase (MAPK) genes have been shown in a number of plant pathogenic fungi to play critical roles in regulating various infection processes. In the present study, we identified and characterized the first MAPK gene PsMAPK1 in Pst. Phylogenetic analysis indicated that PsMAPK1 is a YERK1 MAP kinase belonging to the Fus3/Kss1 class. Single nucleotide polymerphisms (SNPs) and insertion/deletion were detected in the coding region of PsMAPK1 among six Pst isolates. Real-time RT-PCR analyses revealed that PsMAPK1 expression was induced at early infection stages and peaked during haustorium formation. When expressed in Fusarium graminearum, PsMAPK1 partially rescued the map1 mutant in vegetative growth and pathogenicity. It also partially complemented the defects of the Magnaporthe oryzae pmk1 mutant in appressorium formation and plant infection. These results suggest that F. graminearum and M. oryzae can be used as surrogate systems for functional analysis of well-conserved Pst genes and PsMAPK1 may play a role in the regulation of plant penetration and infectious growth in Pst.     

Glucosylceramide synthase is essential for alfalfa defensin-mediated growth inhibition but not for pathogenicity of Fusarium graminearum

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The DeltaFggcs1-null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The DeltaFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild-type PH-1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1-mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host-dependent.