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Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific. [ABSTRACT FROM AUTHOR]

Fusarium graminearumis the causal agent of the Fusarium head blight (FHB) and a destructive pathogen of cereals accounting for high grain yield losses especially on wheat and maize. Like other fungal pathogens,F. graminearumsecretes various extracellular enzymes, which are hypothesized to be involved in host infection. Extracellular lipolytic activity ofF. graminearumwas strongly induced in culture by wheat germ oil; this allowed us to isolate, clone, and characterize a gene (FGL1) encoding a secreted lipase. Expression analysis indicated thatFGL1is induced by lipid-containing substrates and repressed by glucose.In planta,FGL1transcription was detected 1 day post-infection of wheat spikes. The function of theFGL1gene product was verified by specifically demonstrating lipase activity after expression in a heterologous host. Ebelactone B, a known lipase inhibitor, repressed the lipolytic activity of the enzyme. Disease severity was strongly reduced when wild-type conidia were supplemented with ebelactone B. Transformation-mediated disruption ofFGL1led to reduced extracellular lipolytic activity in culture and to reduced virulence to both wheat and maize. [ABSTRACT FROM AUTHOR]

Even closely related eukaryotic species may differ drastically in genome size. While insertion of retroelements represents a major source of genome enlargement, the mechanism mediating species‐ specific deletions is fairly obscure. We analyzed the formation of deletions during double‐strand break (DSB) repair in Arabidopsis thaliana and tobacco, two dicotyledonous plant species differing >20‐fold in genome size. DSBs were induced by the rare cutting restriction endonuclease I‐SceI and deletions were identified by loss of function of a negative selectable marker gene containing an I‐SceI site. Whereas the partial use of micro‐homologies in junction formation was similar in both species, in tobacco 40% of the deletions were accompanied by insertions. No insertions could be detected in Arabidopsis, where larger deletions were more frequent, indicating a putative inverse correlation between genome size and the average length of deletions. Such a correlation has been postulated before by a theoretical study on the evolution of related insect genomes and our study now identifies a possible molecular cause for the phenomenon, indicating that species‐specific differences in DSB repair might indeed influence genome evolution. [ABSTRACT FROM AUTHOR]

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome. [ABSTRACT FROM AUTHOR]

Summary During meiosis, homologous recombination occurs between allelic sequences. To evaluate the biological significance of such a pathway in somatic cells, we used transgenic tobacco plants with a restriction site for the rare cutting endonuclease I-Sce I within a negative selectable marker gene. These plants were crossed with two tobacco lines containing, in allelic position, either a deletion or an insertion within the marker gene that rendered both marker gene and restriction site inactive. After the double-strand break induction, we selected for repair events resulting in a loss of marker gene function. This loss was mostly due to deletions. We were also able to detect double strand break-induced allelic recombination in which the break was repaired by a faithful copying process from the homologue carrying the shortened transgene. The estimated frequency indicates that homologous recombination in somatic cells between allelic sites appears to occur at the same order of magnitude as between ectopic sites, and is thus far too infrequent to act as major repair pathway. As somatic changes can be transferred to the germ line, the prevalence of intrachromatid rearrangements over allelic recombination might be an indirect prerequisite for the enhanced genome plasticity postulated for plants. [ABSTRACT FROM AUTHOR]