Sexual stage-specific A-to-I mRNA editing is mediated by tRNA-editing enzymes in fungi.

A-to-I RNA editing catalyzed by adenosine-deaminase-acting-on-RNA (ADARs) was assumed to be unique to metazoans because fungi and plants lack ADAR homologs. However, genome-wide messenger RNA (mRNA) editing was found to occur specifically during sexual reproduction in filamentous ascomycetes. Because systematic characterization of adenosine/cytosine deaminase genes has implicated the involvement of TAD2 and TAD3 orthologs in A-to-I editing, in this study, we used genetic and biochemical approaches to characterize the role of FgTAD2, an essential adenosine-deaminase-acting-on-tRNA (ADAT) gene, in mRNA editing in Fusarium graminearum. FgTAD2 had a sexual-stage-specific isoform and formed heterodimers with enzymatically inactive FgTAD3. Using a repeat-induced point (RIP) mutation approach, we identified 17 mutations in FgTAD2 that affected mRNA editing during sexual reproduction but had no effect on transfer RNA (tRNA) editing and vegetative growth. The functional importance of the H352Y and Q375*(nonsense) mutations in sexual reproduction and mRNA editing were confirmed by introducing specific point mutations into the endogenous FgTAD2 allele in the wild type. An in vitro assay was developed to show that FgTad2-His proteins purified from perithecia, but not from vegetative hyphae, had mRNA editing activities. Moreover, the H352Y mutation affected the enzymatic activity of FgTad2 to edit mRNA but had no effect on its ADAT activity. We also identified proteins co-purified with FgTad2-His by mass spectrometry analysis and found that two of them have the RNA recognition motif. Taken together, genetic and biochemical data from this study demonstrated that FgTad2, an ADAT, catalyzes A-to-I mRNA editing with the stage-specific isoform and cofactors during sexual reproduction in fungi.

Impact of Gibberella Ear Rot on Grain Quality and Yield Components in Maize as Influenced by Hybrid Reaction.

The impact of Gibberella ear rot (GER; caused by Fusarium graminearum) on deoxynivalenol (DON) contamination of grain and yield components in maize were investigated using data from 30 environments in Ohio (3 years by 10 locations). Fifteen hybrids, later classified as susceptible (SU), moderately susceptible (MS), or moderately resistant (MR), based on the magnitude of differences in mean arcsine square-root-transformed GER severity (arcSEV) and log-transformed DON (logDON) relative to a reference SU check, were planted in each environment, and 10 ears per hybrid were inoculated with a spore suspension of F. graminearum. Relationships between GER severity and DON were well described by a Kono-Sugino-type nonlinear equation. Estimated parameters representing height (A) and steepness (ß) of the curves were significantly higher for SU than MS and MR hybrids but A was not significantly different between MS and MR. Results from a surrogacy analysis showed that GER was a moderate trial- and individual-level surrogate for DON. Both grain weight per ear and ear diameter decreased with increasing arcSEV but the regression slopes varied among resistance classes. The rates of reduction in both yield components per unit increase in arcSEV were significantly greater for SU than for MS and MR. An estimated 50% reduction in grain weight occurred at 62% GER severity for SU, compared with 77% severity for MS and 83% for MR. These results show that GER severity can be used as a surrogate for early estimation of DON contamination and yield loss to help guide grain handling and marketing decisions.

Stage-Specific Genetic Interaction between FgYCK1 and FgBNI4 during Vegetative Growth and Conidiation in Fusarium graminearum.

CK1 casein kinases are well conserved in filamentous fungi. However, their functions are not well characterized in plant pathogens. In Fusarium graminearum, deletion of FgYCK1 caused severe growth defects and loss of conidiation, fertility, and pathogenicity. Interestingly, the Fgyck1 mutant was not stable and often produced fast-growing spontaneous suppressors. Suppressor mutations were frequently identified in the FgBNI4 gene by sequencing analyses. Deletion of the entire FgBNI4 or disruptions of its conserved C-terminal region could suppress the defects of Fgyck1 in hyphal growth and conidiation, indicating the genetic relationship between FgYCK1 and FgBNI4. Furthermore, the Fgyck1 mutant showed defects in polarized growth, cell wall integrity, internalization of FgRho1 and vacuole fusion, which were all partially suppressed by deletion of FgBNI4. Overall, our results indicate a stage-specific functional relationship between FgYCK1 and FgBNI4, possibly via FgRho1 signaling for regulating polarized hyphal growth and cell wall integrity.

Recombinant Lipase from Gibberella zeae Exhibits Broad Substrate Specificity: A Comparative Study on Emulsified and Monomolecular Substrate.

Using the classical emulsified system and the monomolecular film technique, the substrate specificity of recombinant Gibberella zeae lipase (rGZEL) that originates from Gibberella zeae was characterized in detail. Under the emulsified reaction system, both phospholipase and glycolipid hydrolytic activities were observed, except for the predominant lipase activity. The optimum conditions for different activity exhibition were also determined. Compared with its lipase activity, a little higher ratio of glycolipid hydrolytic activity (0.06) than phospholipase activity (0.02) was found. rGZEL preferred medium chain-length triglycerides, while lower activity was found for the longer-chain triglyceride. Using the monomolecular film technique, we found that the preference order of rGZEL to different phospholipids was 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) > 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (PG) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > l-α-phosphatidylinositol (PI) > cardiolipin (CL) > 3-sn-phosphatidic acid sodium salt (PA) > l-α-phosphatidylethanolamine (PE), while no hydrolytic activity was detected for sphingomyelin (SM). Moreover, rGZEL showed higher galactolipase activity on 1,2-distearoyimonoglactosylglyceride (MGDG). A kinetic study on the stereo- and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers (DDGs). rGZEL presented higher preference for distal DDG enantiomers than adjacent ester groups, however, no hydrolytic activity to the sn-2 position of diglyceride analogs was found. Furthermore, rGZEL preferred the R configuration of DDG enantiomers. Molecular docking results were in concordance with in vitro tests.

A Gin4-Like Protein Kinase GIL1 Involvement in Hyphal Growth, Asexual Development, and Pathogenesis in Fusarium graminearum.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) on wheat and barley. In a previous study, a GIN4-like protein kinase gene, GIL1, was found to be important for plant infection and sexual reproduction. In this study we further characterized the functions of GIL1 kinase in different developmental processes. The Δgil1 mutants were reduced in growth, conidiation, and virulence, and formed whitish and compact colonies. Although phialide formation was rarely observed in the mutants, deletion of GIL1 resulted in increased hyphal branching and increased tolerance to cell wall and cell membrane stresses. The Δgil1 mutants produced straight, elongated conidia lacking of distinct foot cells and being delayed in germination. Compared with the wild type, some compartments in the vegetative hyphae of Δgil1 mutants had longer septal distances and increased number of nuclei, suggesting GIL1 is related to cytokinesis and septation. Localization of the GIL1-GFP fusion proteins to the septum and hyphal branching and fusion sites further supported its roles in septation and branching. Overall, our results indicate that GIL1 plays a role in vegetative growth and plant infection in F. graminearum, and is involved in septation and hyphal branching.

Distinct cell cycle regulation during saprophytic and pathogenic growth in fungal pathogens.

In a number of dimorphic and hemibiotrophic pathogens, cell cycle regulation has been shown to be important for morphological changes related to infectious growth or infection-related morphogenesis. However, the role of mitotic CDK kinase Cdc2, the key regulator of cell cycle, in pathogenic growth is not clear, because most fungal pathogens have a single CDC2 gene that is essential for cell cycle progression and viability. Interestingly, the wheat scab fungus Fusarium graminearum has two CDC2 genes. Although CDC2A and CDC2B have redundant functions in vegetative growth and asexual production, only CDC2A is required for invasive growth and plant infection. In this study, we showed that Cdc2A and Cdc2B interacted with each other and may form homo- and heterodimers in vegetative hyphae. We also identified sequence and structural differences between Cdc2A and Cdc2B that may be related to their functional divergence. These results, together with earlier studies with cyclins, important for differentiation and infection in Candida albicans and Ustilago maydis, indicated that dimorphic and hemibiotrophic fungal pathogens may have stage-specific cyclin-CDK combinations or CDK targets during saprophytic and pathogenic growth.

High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production.

Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes.

Germination of Fusarium graminearum Ascospores and Wheat Infection are Affected by Dry Periods and by Temperature and Humidity During Dry Periods.

The effects of temperature and relative humidity (RH) on germination of Fusarium graminearum ascospores, and of dry periods (DP) of different lengths and of temperature and RH during DP on ascospore survival were studied both in vitro and in planta. Optimal temperatures for ascospore germination at 100% RH were 20 and 25°C; germination was ≤5% when ascospores were incubated at 20°C and RH ≤ 93.5%. Viable ascospores were found at all tested combinations of DP duration (0 to 48 h) × temperature (5 to 40°C) or RH (32.5 to 100% RH). Germination declined as DP duration and temperature increased. Germination was lower for ascospores kept at 65.5% RH during the DP than at 76.0, 32.5, or 93.5% RH. Equations were developed describing the relationships between ascospore germination, DP duration and temperature or RH during DP. Durum wheat spikes were inoculated with ascospores and kept dry for 0 to 48 h at approximately 15°C and 65% RH; plants were then kept in saturated atmosphere for 48 h to favor infection. Fungal biomass, measured as F. graminearum DNA by quantitative polymerase chain reaction, declined as DP increased to 24 and 48 h at 3 and 9 days postinfection but not in spikes at maturity.

Inhibition of Fusarium graminearum growth and mycotoxin production by phenolic extract from Spirulina sp.

Fusarium graminearum is a fungal species complex pathogenic occurring worldwide, mainly associated with cereal crops. The most important Fusarium mycotoxins are fumonisins, zearalenone and trichothecenes. The availability of efficient control measures that are less harmful to both the environment and the consumers is urgent. For such, phenolic acids (PAs) from natural sources are known to reduce fungal contaminations. This work aimed to identify the PAs present in a culture extract of Spirulina algae (strain LEB-18) and evaluate its effect on mycelial growth rate, glucosamine level, amylase activity and mycotoxin production by four strains of two lineages of F. graminearum. Results showed that amendment of potato dextrose media with LEB-18 extract (3% w/v), which was mainly composed by gallic acid, greatly reduced radial growth of fungal colonies compared to media containing a single PA and the control. Also, average reductions of 40% and 62% in the glucosamine levels and the amylase activity were observed. In general, the LEB-18 extract and the PAs reduced mycotoxin concentration, with an average reduction of 68% for the trichothecene mycotoxins deoxynivalenol and nivalenol.

Comparative secretome analysis of Fusarium graminearum and two of its non-pathogenic mutants upon deoxynivalenol induction in vitro.

To understand early events in plant-pathogen interactions, it is necessary to explore the pathogen secretome to identify secreted proteins that help orchestrate pathology. The secretome can be obtained from pathogens grown in vitro, and then characterized using standard proteomic approaches based on protein extraction and subsequent identification of tryptic peptides by LC-MS. A subset of the secretome is composed of proteins whose presence is required to initiate infection and their removal from the secretome would result in pathogens with reduced or no virulence. We present here comparative secretome from Fusarium graminearum. This filamentous fungus causes Fusarium head blight on wheat, a serious cereal disease found in many cereal-growing regions. Affected grain is contaminated with mycotoxins and cannot be used for food or feed. We used label-free quantitative MS to compare the secretomes of wild-type with two nonpathogenic deletion mutants of F. graminearum, Δtri6, and Δtri10. These mutations in mycotoxin-regulating transcription factors revealed a subset of 29 proteins whose relative abundance was affected in their secretomes, as measured by spectral counting. Proteins that decreased in abundance are potential candidate virulence factors and these included cell wall-degrading enzymes, metabolic enzymes, pathogenesis-related proteins, and proteins of unknown function.

Synthesis and bioactivities of novel thioether/sulfone derivatives containing 1,2,3-thiadiazole and 1,3,4-oxadiazole/thiadiazole moiety.

A series of new thioether/sulfone compounds containing 1,2,3-thiadiazole and 1,3,4-oxadiazole/1,3,4-thiadiazole moiety were synthesized, the structures of all products were confirmed by IR, (1)H NMR, (13)C NMR, and element analysis. Preliminary antifungal activity test showed that compound 8a exhibited moderate antifungal activity against Fusarium oxysporum at 50µg/mL. Preliminary antiviral activity results showed that compounds 7a, 7c, 7d, 8a, and 9a displayed high antiviral activity against tobacco mosaic virus. The present work demonstrates that thioether/sulfone heterocyclic derivatives could be considered as new lead compounds for antiviral studies.

Soil Fungistasis against Fusarium Graminearum under Different tillage Systems.

The establishment of the harmful pathogen Fusarium graminearum in different agroecosystems may strongly depend on the ability of the soils to suppress its development and survival. This study aimed to evaluate the influence of different soil tillage systems (i.e., conventional tillage, reduced tillage and no-tillage) on soil fungistasis against F. graminearum. Soil samples were collected three times during the plant growing season in 2016 and 2017 from a long-term, 20-year soil tillage experiment. The F. graminearum in the soil samples was quantified by real-time qPCR. The soil fungistasis was evaluated by the reduction in the radial growth of F. graminearum in an in vitro assay. The antagonistic activity of the soil bacteria was tested using the dual culture method. The F. graminearum DNA contents in the soils were negatively correlated with soil fungistasis (r = -0.649 *). F. graminearum growth on the unfumigated soil was reduced by 70-87% compared to the chloroform fumigated soil. After the plant vegetation renewal, the soil fungistasis intensity was higher in the conventionally tilled fields than in the no-tillage. However, no significant differences were obtained among the tillage treatments at the mid-plant growth stage and after harvesting. 23 out of 104 bacteria isolated from the soil had a moderate effect, and only 1 had a strong inhibitory effect on the growth of F. graminearum. This bacterium was assigned 100% similarity to the Bacillus amyloliquefaciens Hy7 strain (gene bank no: JN382250) according to the sequence of the 16S ribosome subunit coding gene. The results of our study suggest that the presence of F. graminearum in soil is suppressed by soil fungistasis; however, the role of tillage is influenced by other factors, such as soil biological activity, type and quantity of plant residues and environmental conditions.

Ursodeoxycholic acid production by Gibberella zeae mutants.

Ursodeoxycholic acid (UDCA) is a highly demanded pharmaceutical steroid widely used in medicine. An ascomycete Gibberella zeae VKM F-2600 is capable of producing UDCA by 7ß-hydroxylation of lithocholic acid (LCA). The present study is aimed at the improvement of the fungus productivity. The original procedures for the protoplast obtaining followed by UV mutagenesis and screening of ketoconazole-resistant mutant clones have been applied. The highest yield of G. zeae protoplasts was obtained when using the mycelium in the active growth phase, ammonium chloride as an osmotic stabilizer and treatment of the fungal cells by the lytic enzymes cocktail from Trichoderma hurzanium. The conditions for effective protoplast regeneration and the UV-mutagenesis were found to provide 6-12% survival rate of the protoplasts with superior number of possible mutations. Three of 27 ketoconazole-resistant mutant clones obtained have been selected due to their increased biocatalytic activity towards LCA. The mutant G. zeae M23 produced 26% more UDCA even at relatively high LCA concentration (4 g/L) as compared with parent fungal strain, and the conversion reached 88% (w/w). The yield of UDCA reached in this study prefers those ever reported. The results contribute to the knowledge on ascomycete mutagenesis, and are of importance for biotechnological production of value added cholic acids.

Deletion of all three MAP kinase genes results in severe defects in stress responses and pathogenesis in Fusarium graminearum.

Mitogen-activated protein kinase (MAPK) cascades are activated by external stimuli and convert signals to cellular changes. Individual MAPKs have been characterized in a number of plant pathogenic fungi for their roles in pathogenesis and responses to biotic or abiotic stresses. However, mutants deleted of all the MAPK genes have not been reported in filamentous fungi. To determine the MAPK-less effects in a fungal pathogen, in this study we generated and characterized mutants deleted of all three MAPK genes in the wheat scab fungus Fusarium graminearum. The Gpmk1 mgv1 Fghog1 triple mutants had severe growth defects and was non-pathogenic. It was defective in infection cushion formation and DON production. Conidiation was reduced in the triple mutant, which often produced elongated conidia with more septa than the wild-type conidia. The triple mutant was blocked in sexual reproduction due to the loss of female fertility. Lack of any MAPKs resulted in an increased sensitivity to various abiotic stress including cell wall, osmotic, oxidative stresses, and phytoalexins, which are likely related to the defects of the triple mutant in environmental adaptation and plant infection. The triple mutant also had increased sensitivity to the biocontrol bacterium Bacillus velezensis and fungus Clonostachys rosea. In co-incubation assays with B. velezensis, the Gpmk1 mgv1 Fghog1 mutant had more severe growth limitation than the wild type and was defective in conidium germination and germ tube growth. In confrontation assays, the triple mutant was defective in defending against mycoparasitic activities of C. rosea and the latter could grow over the mutant but not wild-type F. graminearum. RNA-seq and metabolomics analyses showed that the MAPK triple mutant was altered in the expression of many ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes and the accumulation of metabolites related to arachidonic acid, linoleic acid, and alpha-linolenic acid metabolisms. Overall, as the first study on mutants deleted of all three MAPKs in fungal pathogens, our results showed that although MAPKs are not essential for growth and asexual reproduction, the Gpmk1 mgv1 Fghog1 triple mutant was blocked in plant infection and sexual reproductions. It also had severe defects in responses to various abiotic stresses and bacterial- or fungal-fungal interactions.

Enzymatic properties and expression patterns of five extracellular lipases of Fusarium graminearum in vitro.

Fusarium graminearum is a filamentous fungus that causes devastating diseases on plants of economic importance including maize, wheat, and barley. F. graminearum is able to utilize triglycerides as a carbon source during growth. Extracellular lipases are the preferred enzymes to catalyze the hydrolysis of fats and oils. Lipases are ubiquitous enzymes of considerable physiological significance and industrial potential. Previously, FGL1 was the first described F. graminearum extracellular lipase associated with virulence. We report the biochemical characterization of FGL1 and four new secreted lipases of F. graminearum. The lipase genes of F. graminearum wild-type strain 8/1 were sequenced, cloned and over-expressed in Pichia pastoris. We show that the lipases have their temperature optimum between 30 and 40°C and a pH optimum of ∼7. A broad range of lipase substrates, from C4 to C18 p-nitrophenyl esters, were hydrolyzed efficiently by the lipases, indicating the true lipolytic activity of the enzymes. Expression patterns of these lipases were also analyzed by semiquantitative RT-PCR in F. graminearum cultured in water supplemented with 2% (v/v) wheat germ oil at 28°C. Transcripts of all examined lipases are detectable and the genes are regulated differently under these culture conditions. Our data indicated that F. graminearum possesses a ubiquitous source of secreted lipases, which could be used for industrial intentions. We also provided the foundation of lipase expression in vitro, which is necessary for further characterization.

Exploring the influence of phospholipid monolayer conformation and environmental conditions on the interfacial binding of Gibberella Zeae lipase.

The involvement of different parameters on Gibberella zeae lipase (GZEL) membrane binding were characterized by using monomolecular film technology and circular dichroism spectroscopy. Among four kinds of phospholipid monolayers, 1,2­dimyristoyl­sn­glycero­3­phosphoethanolamine have the highest maximum insertion pressure (MIP) value. Comparing the GZEL adsorption to phosphatidylcholine monolayers with different acyl chains in sn-1 and sn-2 positions, the higher MIP values were found for 1,2­dilauroyl­sn­glycero­3­phosphocholine. Significantly improvement between 1,2­dioleoyl­sn­glycero­3­phosphocholine and 1,2­distearoyl­sn­glycero­3­phosphocholine suggested that the presence of fatty acid unsaturation may affect protein adsorption by changing the chemical structure in each phospholipid. The MIP value was shown higher (48.6 mN m-1) at pH 5 and pH 6 (47.5 ±â€¯1.9 mN m-1) but decreased significantly (34.2 mN m-1) at pH 9. This may indicate that the proportion of helices in the protein decreases with the alteration of the catalytic center, thus affecting the binding of the protein to its substrate. The MIP values obviously decreased with increasing salt ion concentration, suggesting that excessive salt ion concentration may destabilize the secondary and tertiary structures of the protein, thereby affecting the characteristics of its adsorption at the interfaces. Present studies improve our understanding on the protein-membrane interaction of this enzyme.

Unbalanced Roles of Fungal Aggressiveness and Host Cultivars in the Establishment of the Fusarium Head Blight in Bread Wheat.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is the foremost destructive disease of cereals worldwide. Effector-like molecules produced by F. graminearum play key roles in the infection process and are assumed to be one of the essential components of the pathogen's aggressiveness. However, their nature and role in the disease are still largely misunderstood. As a mean to provide relevant information about the molecular determinism of F. graminearum aggressiveness, we surveyed three F. graminearum strains on three wheat cultivars contrasted by their susceptibility to FHB. F. graminearum strains revealed large differences in aggressiveness which were mostly unchanged when facing hosts of contrasted susceptibility, suggesting that their behavior rely on intrinsic determinants. Surveying the fungal mass progress and the mycotoxin production rate in the spikes did not evidence any simple relationship with aggressiveness differences, while clues were found through a qualitative and quantitative characterization of the three strain proteomes established in planta especially with regards to early synthesized putative effectors. Independently of the wheat cultivar, the three F. graminearum strains produced systematically the same protein set during the infection but substantial differences in their abundance enabled the categorization of fungal aggressiveness. Overall, our findings show that the contrasts in F. graminearum aggressiveness were not based on the existence of strain-specific molecules but rather on the ability of the strain to ensure their sufficient accumulation. Protein abundance variance was mostly driven by the strain genetics and part was also influenced by the host cultivar but strain by cultivar interactions were marginally detected, depicting that strain-specific protein accumulations did not depend on the host cultivar. All these data provide new knowledge on fungal aggressiveness determinants and provide a resourceful repertoire of candidate effector proteins to guide further research.

Function of C-terminal peptides on enzymatic and interfacial adsorption properties of lipase from Gibberella zeae.

BACKGROUND: The crystal structure of lipase from Gibberella zeae (GZEL) indicates that its C-terminal extension is composed of a loop and a α-helix. This structure is unique, possibly providing novel evidence on lipase mechanisms. METHODS: Two C-terminally truncated mutants (GZEL-Δ(α-helix) and GZEL-Δ(α-helix+loop)) were constructed. The role of these secondary structure segments on enzymatic activities and interfacial binding properties of GZEL was investigated by using conventional pH-stat method and monomolecular film techniques. In addition, inactive variants (Ser144Ala) of wild-type GZEL and two truncated mutants were constructed and produced specifically for interfacial binding experiments. RESULTS: Compared to the wild-type GZEL, lipase and phospholipase activities were significantly decreased in the two mutants. Deletion of the α-helix had great influence on the lipase activity of GZEL, resulting in residual 7.3% activity; the additional deletion of the loop led to 8.1% lipase activity. As for the phospholipase function, residual activities of 63.0% and 35.4% were maintained for GZEL-Δ(α-helix) and GZEL-Δ(α-helix+loop), respectively. Findings obtained with monomolecular film experiments further indicated that the reduction in phospholipase activity occurred with the anionic phospholipid as substrate, but was not seen with zwitterionic phospholipid. Results of the maximum insertion pressure, synergy factor and binding kinetic parameters documented that the α-helix structure of GZEL strongly influence the binding and insertion of enzyme to the phospholipid monolayer. Moreover, the interfacial binding function of α-helix was partly conformed by connecting to the C-terminal of Aspergillus oryzae lipase. GENERAL SIGNIFICANCE: Our results provide important information on the understanding of the structure-function relationship of GZEL.

High-resolution mapping of the recombination landscape of the phytopathogen Fusarium graminearum suggests two-speed genome evolution.

Recombination is a major evolutionary force, increasing genetic diversity and permitting efficient coevolution of fungal pathogen(s) with their host(s). The ascomycete Fusarium graminearum is a devastating pathogen of cereal crops, and can contaminate food and feed with harmful mycotoxins. Previous studies have suggested a high adaptive potential of this pathogen, illustrated by an increase in pathogenicity and resistance to fungicides. In this study, we provide the first detailed picture of the crossover events occurring during meiosis and discuss the role of recombination in pathogen evolution. An experimental recombinant population (n = 88) was created and genotyped using 1306 polymorphic markers obtained from restriction site-associated DNA sequencing (RAD-seq) and aligned to the reference genome. The construction of a high-density linkage map, anchoring 99% of the total length of the reference genome, allowed the identification of 1451 putative crossovers, positioned at a median resolution of 24 kb. The majority of crossovers (87.2%) occurred in a relatively small portion of the genome (30%). All chromosomes demonstrated recombination-active sections, which had a near 15-fold higher crossover rate than non-active recombinant sections. The recombination rate showed a strong positive correlation with nucleotide diversity, and recombination-active regions were enriched for genes with a putative role in host-pathogen interaction, as well as putative diversifying genes. Our results confirm the preliminary analysis observed in other F. graminearum strains and suggest a conserved 'two-speed' recombination landscape. The consequences with regard to the evolutionary potential of this major fungal pathogen are also discussed.

A Natural Mutation Involving both Pathogenicity and Perithecium Formation in the Fusarium graminearum Species Complex.

Members of the Fusarium graminearum species complex (Fg complex or FGSC) are the primary pathogens causing Fusarium head blight in wheat and barley worldwide. A natural pathogenicity mutant (strain 0225022) was found in a sample of the Fg complex collected in Japan. The mutant strain did not induce symptoms in wheat spikes beyond the point of inoculation, and did not form perithecia. No segregation of phenotypic deficiencies occurred in the progenies of a cross between the mutant and a fully pathogenic wild-type strain, which suggested that a single genetic locus controlled both traits. The locus was mapped to chromosome 2 by using sequence-tagged markers; and a deletion of ∼3 kb was detected in the mapped region of the mutant strain. The wild-type strain contains the FGSG_02810 gene, encoding a putative glycosylphosphatidylinositol anchor protein, in this region. The contribution of FGSG_02810 to pathogenicity and perithecium formation was confirmed by complementation in the mutant strain using gene transfer, and by gene disruption in the wild-type strain.