A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

A triglyceride lipase gene FgLip1 was identified in the genome of <i>Fusarium graminearum</i> strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions <i>in vitro</i>. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5 upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into <i>F. graminearum</i>. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.

1. Fusarium roseum �Graminearum� was the predominant fungus associated with stalk rot of maize in eastern Australia in the 1972, 1973 and 1974 growing seasons. All isolates of this pathogen were of the Group 2 type. Thus Group 2 contrasts with Group I which is normally isolated :Erora crown rot of wheat and grasses. Other fungi isolated in order of frequency were Diplodia maydis, F. rnoniliforme �Subglutinans�, Bipolaris sorokiniana, Nigrospora oryzac, F. roseum �Semitectum�, F. moniliforme, F. roseum �Equiseti�, F. roseum �Concolor�, Macrophomina phaseolina, Rhizoctonia sp., F. roseum �Acuminatum�, F. oxysporum, F. solani, F. tricinctum and F. roseum �Heterosporum�. The relative isolation frequencies of the fungi varied according to the seasonal conditions. Stalk rots were not of major importance in 1973, a relatively dry growing season. However, in 1974, a wet growing season, stalk rot diseases were common in all areas investigated. 2. Isolates of F. roseum �Graminearum�,derived mainly from wheat and maize but also from other sources and from various regions of eastern Australia, were examined for perithecia formation, colony characteristics, fertility, colony growth, conidia production and conidia size. The distribution of the fungus in field colonized maize and wheat plants was also studied. The Group 1 isolates did not produce perithecia, were heterothallic and very infertile, had a mean colony growth of 4.4 cm per 3 days (range, 3.9- 5.1) and produced relatively large numbers of conidia. In contrast, Group 2 isolates were homothallic and produced perithecia readily, had a mean colony growth of 5.4 cm per 3 days (range, 4.7�6.1) and produced relatively low numbers of conidia. Group 1 isolates were found to be commonly associated with crowns and roots of plants and Group 2 isolates were commonly associated with aerial plant parts. 3. The ability of a number of Group 1 and Group 2 isolates to produce the fungal hormone, zearalenone was assessed. Group I isolates produced three to four times more zearalenone than Group 2 isolates. In addition, a. culture which had previously produced perithecia but had lost that ability following numerous transfers, produced no detectable zearalenone. The results provided good evidence that the observed difference in perithecia formation was directly related to the ability to produce zearalenone. 4. The pathogenicity to wheat, maize and carnations of Group 1 isolates from crown rot affected wheat plants and Group 2 isolates from stalk rot affected maize plants was tested. Pathogenicity of 11 other isolates from teosinte, carnations, pearl millet, wheat and barley scab, banana, ginger and common wheat grass was also assessed. The results indicated that pathogenic specialization exists within F. roseum �Graminearum�. Wheat isolates were the most pathogenic to wheat, carnation isolates were the most pathogenic to carnations and all maize isolates were pathogenic to maize while those from wheat and common wheat grass were not as pathogenic to maize. Moreover, Group 2 isolates were more pathogenic when inoculated in aerial plant parts, and the Group I isolates were more pathogenic when inoculated in plant parts in soil. Inoculations on wheat seedlings in sterile field soil demonstrated that the inherent pathogenicity to wheat seedlings of isolates from wheat and maize were similar. 5. Some factors which could contribute to the observed pathogenic differences between isolates from wheat and maize to wheat seedlings in field soil were examined. Conidia volume, germination rate and inherent germinability in the soil were studied. The Group I isolates had the largest volume, the most rapid germination and the highest inherent germinability. Pathogenicity was positively correlated with conidium volume and inherent germinability. In addition, the inherent germinability and conidium volume were positively correlated. Thus, it was established that pathogenic behaviour of conidia of Group 1 and Group 2 reflected differences in conidia morphology.

Universiẗat, Diss., 2003--Münster (Westfalen).

No description available.

Fusarium graminearum is responsible for causing Fusarium head blight in cereals and maize imposing a significant impact in Canadian agriculture. While a handful of secondary metabolites produced by F. graminearum are recognized as contributors to disease virulence, the functions of numerous molecular products arising from biosynthetic gene clusters expressed during infection remain undiscovered. Presented here are the results of CRISPR-Cas9 mediated gene-deletion experiments disrupting core biosynthetic genes from four biosynthetic gene clusters with reported in-planta transcription: C08, C16, C13 and C70. Both wheat head infection assays and coleoptile infection assays were used to evaluate the pathology phenotypes of transformant strains illustrating potential links between C16 and pathogenicity. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products and attempt to identify unknown secondary metabolites of the biosynthetic gene clusters. While C08 secondary metabolite remained elusive, confirmation of C16 secondary metabolites led to hypotheses regarding their potential connections to the inhibition of plant immune response and untargeted secondary metabolite profiling of the C13/C70 transformant strains suggests that this BGC may have significant implications for global secondary metabolite production.

No description available.

Fusarium graminearum est un champignon filamenteux qui parasite les plantes céréalières et le maïs et provoque la fusariose de l’épi. Durant l’infection, ce champignon produit des mycotoxines de la famille des trichothécènes qui s’accumulent dans les grains. Les processus de décontamination existants ne permettent pas d’éliminer complètement les trichothécènes. Ainsi, le meilleur moyen pour éviter leur accumulation dans les grains serait de pouvoir limiter leur occurrence au champ en contrôlant leur biosynthèse. Bien que la voie de biosynthèse des trichothécènes et les gènes Tri qui y sont impliqués soient bien décrits, les connaissances de base sur les mécanismes de régulation de ces gènes restent trop restreintes.Dans la première partie de ce travail, l’effet du pH sur la régulation des gènes Tri et la production de trichothécène a été étudié. En premier lieu, nous avons démontré que, in vitro, un pH acide joue le rôle d’inducteur alors qu’un pH neutre ou alcalin bloque l’expression des gènes Tri et la production de trichothécène. Ensuite, FgPac1, l’homologue du gène pacC/RIM101 codant le facteur de régulation par le pH chez les champignons a été identifié dans le génome de F. graminearum. A l’aide de souches recombinantes, nous avons démontré que la forme mature de ce facteur réprime l’expression des gènes Tri à pH acide et réduit la virulence du champignon lors de l’infection d’épis de blé. Enfin, le transcriptome de F. graminearum en réponse au pH et le rôle de Pac1 dans cette réponse a été analysé.Dans la deuxième partie de ce travail, le gène velvet sensible à la lumière, a été identifié chez F. graminearum. Ce gène constitue la composante clef d’un complexe qui coordonne la perception de la lumière avec le développement mais aussi avec le métabolisme secondaire chez les champignons. L’inactivation de FgVe1 chez F. graminearum nous a permis de démontrer son rôle dans le développement et la production de spores. Elle a montré aussi que ce gène est nécessaire pour permettre l’expression des gènes Tri, la production de trichothécène et la pathogénicité in planta.L’ensemble de ce travail permet de mieux comprendre la régulation de la production de trichothécène chez F. graminearum et ouvre des perspectives qui permettront sans doute, à long terme, d’élaborer des stratégies de lutte contre l’accumulation de trichothécène au champ. / The filamentous fungus Fusarium graminearum infects cereals plants and corn and causes “Fusarium Head Blight”. During infection, it produces mycotoxins belonging to trichothecenes family which accumulate in the grains. The available decontamination processes do not fully eliminate the trichothecene. Hence, the best way to avoid their occurrence in the grains is to limit their accumulation in the field by controlling their biosynthesis. Although the Tri genes implicated in the trichothecene biosynthetic pathway are well described, the basic knowledge regarding their regulation is still too limited.In the first part of this work, the effect of the pH on Tri genes regulation and trichothecene production was studied. First, we demonstrated that, in vitro, acidic pH acts as an inducer while a neutral or alkaline pH blocks Tri genes expression and trichothecene production. Then, FgPac1, the homologue of the pacC/RIM101 gene encoding the fungal pH regulatory factor was identified. Using recombinant strains, we demonstrated that the mature form of this factor represses Tri gene expression at acidic pH and reduces virulence during infection of wheat spikes. Finally, we analyzed the transcriptome of F. graminearum in response to pH and investigated the role of Pac1 in this response.In the second part of this work, the light-responsive velvet gene was identified in F. graminearum. This gene is the key component of a complex coordinating light perception with development and secondary metabolism in fungi. The disruption of FgVe1 in F. graminearum demonstrated its role in development and spores production. It also showed that this gene is necessary for Tri gene expression, trichothecene production and pathogenicity in planta.Overall, this work allows a better understanding of trichothecene regulation in F. graminearum and provides novel perspectives to develop new strategies against trichothecene accumulation during cereal growing in the field.

Parmi les nombreux pathogènes fongiques susceptibles d’infecter les épis de maïs, les espèces appartenant au genre Fusarium sont particulièrement préoccupantes pour la filière maïsicole. La fusariose est susceptible d’induire des pertes de rendement considérables et est fréquemment associée à une contamination des épis par des mycotoxines. Un des leviers prometteur repose sur la sélection génétique de plantes résistantes à Fusarium et à l’accumulation de mycotoxines. Plusieurs Quantitative Trait Loci (QTL) ont été identifiés d’après la caractérisation moléculaire de la résistance à la fusariose chez le maïs. Cependant malgré les progrès des approches génétiques, les mécanismes moléculaires impliqués restent en grande partie inconnus. L’identification de métabolites majeurs associés à la résistance reste donc indispensable pour la création d’un outil d’aide à la sélection variétale. Une approche métabolomique combinant de la spectrométrie de masse et de la 1H-RMN a été mise au point pour identifier un ensemble de métabolites de défense, constitutifs ou induits par l’infection, susceptibles d’intervenir dans la résistance à Fusarium. Cette approche a été appliquée aux grains à deux stades de développement sur 20 variétés présentant des degrés de résistance contrastés inoculés ou non avec une souche de Fusarium graminearum toxinogène par le canal des soies. Les résultats obtenus mettent en évidence un panel de métabolites liés à la résistance ou la sensibilité des variétés de maïs. / Fusarium graminearum is the main causal agent of maize ear rot or Gibberella ear rot (GER), an important fungal disease affecting maize. GER leads to significant economic loss and serious health issues due to the ability of F. graminearum to produce mycotoxins such as type B trichothecenes. One promising approach to control Giberella Ear Rot and reduce mycotoxins contamination is to promote host-genetic resistance. Several Quantitative Trait Loci (QTLs) have been identified in maize. However molecular basis to resistance to Fusarium infection remains largely unknown and the success of selection for GER resistance is still challenging. Biochemical approaches can provide valuable insights in the mechanisms crops employ against F. graminearum and its production of mycotoxins. A biochemical profiling could actually be an efficient way to decipher plant-pathogen interactions and progress in screening resistant maize lines. This study aims to elucidate the metabolic profiling of F. graminearum resistance and toxin accumulation in kernels toward the combination of high resolution mass spectrometry and 1H NMR to identify a large set of metabolites, preformed, constitutive as well as inducible defense metabolites that could play a key role in GER resistance. This approach was applied to kernels harvested at two developmental stages. Twenty genotypes with contrasting levels of resistance were inoculated, or not, with a toxigenic Fusarium graminearum strain through the silk channel. The obtained data allowed highlighting a set of biochemical compounds linked to the resistance or susceptibility of maize genotypes

La Fusariose de l’épi (FHB) est une maladie importante des céréales et en particulier du blé tendre. Elle est causée par deux genres fongiques, le genre Fusarium et le genre Microdochium. L’espèce Fusarium graminearum est l’agent principal de cette maladie. Elle affecte non seulement les rendements et la qualité des grains chez le blé, mais elle cause un sérieux problème sanitaire via la production des mycotoxines. L’établissement de cette maladie requiert l’expression de gènes végétaux (facteurs de sensibilité) qui restent encore méconnus. Afin d’étudier les événements moléculaires qui participent à la mise en place de cette maladie sur un grain de blé tendre en développement et sensible au FHB, une cinétique d’infection de cinq points correspondant aux principaux stades développementaux du grain a été réalisée. Ensuite, deux approches, la protéomique comparée et la transcriptomique comparée à l’aide de puces à ADN, ont été utilisées pour répondre à ces questions. L’analyse protéomique a permis d’identifier 73 protéines différentiellement régulées appartenant à 5 grands groupes fonctionnels alors que l’approche transcriptomique a mis en évidence 1309 gènes répartis dans 16 groupes fonctionnels différents. Ces deux approches ont montré que l’infection ne bloque pas le développement du grain, mais elle induit des changements importants dans le métabolisme primaire notamment sur la synthèse de l’amidon et des protéines de réserve. Elle a également montré que la réponse du grain au FHB est liée au stade développemental du grain. Cette étude apporte de nouveaux éléments nécessaires à la compréhension de la sensibilité du blé tendre à la Fusariose de l’épi. Cette sensibilité du grain de blé se caractérise principalement par l’induction des mécanismes de détoxification des mycotoxines, la mise en place des mécanismes du détournement du métabolisme carboné de l’hôte par l’agent pathogène et le contrôle de la mort cellulaire programmée des cellules végétales. Enfin, l’étude a permis d’établir une liste d’au moins 100 gènes candidats potentiellement impliqués dans la sensibilité du blé au FHB. / Fusarium head blight (FHB) is an important disease of cereals, particularly of wheat. It is caused by two fungal genera, the genus Fusarium and genus Microdochium. The species Fusarium graminearum is the principal agent of this disease. This disease affects not only yield and grain quality in wheat, but it causes serious health problem through the production of mycotoxins. The establishment of this disease requires the expression of plant genes (susceptibility factors) that are still unknown. To study the molecular events involved in the development of this disease in the susceptible wheat grain during its development, time course infection of five points corresponding to the main developmental stages of grain was performed. Then two approaches, proteomics and transcriptomics using DNA microarrays were used to answer to this question. Proteomic analysis identified 73 differentially regulated proteins belonging to five major functional groups while the transcriptomics data revealed 1309 genes involved in 16 distinct functional groups. Both approaches have shown that infection does not interrupt grain development, but induces significant changes in primary metabolism, mainly on the synthesis of starch and storage proteins. It also showed a link between FHB response and the grain development. This study provides new evidence necessary to understand the susceptibility response of wheat to FHB. The wheat grain susceptibility is mainly characterized by the induction of detoxifying mechanisms of mycotoxins, the diversion ofcarbon metabolism of the host by the pathogen and control of Programmed Cell Death (PCD) of plant cells. Finally, the study allowed the establishment of a list of at least 100 candidate genes potentially involved in the wheat susceptibility to FHB.