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RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants. [ABSTRACT FROM AUTHOR]

Mitogen activated protein (MAP) kinases are key components in signalling networks. In Fusarium graminearum, one of the most devastating fungal plant pathogens, two MAP kinases are known to be involved in pathogenicity, Mgv1 and the Gibberella pathogenicity MAP kinase Gpmk1. Δgpmk1 mutants with a disrupted GPMK1 gene are unable to infect wheat spikelets. They exhibit altered secretion of several extracellular enzymes. Among those, the lipase FGL1 is known to be a major virulence factor of F. graminearum. FGL1 gene expression was decreased in Δgpmk1 strains during wheat head infection. To uncouple FGL1 expression from Gpmk1 activity, we generated Δgpmk1 strains that constitutively express FGL1 under control of the Cochliobolus heterostrophus glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter. The level of extracellular lipolytic activity of these strains in culture was comparable to the wild-type. These mutants showed fully restored conidiation and partial complementation of defects in development that were reported from the Δgpmk1 mutant. They also partially complemented the apathogenic disease phenotype of the Δgpmk1 mutants causing lesions in directly inoculated wheat spikelets. But in contrast to wild-type, their growth was restricted to directly inoculated spikelets. A barrier-like formation was observed at the rachis node. Based on these results, we could show that the lipase FGL1 is necessary but not sufficient to restore complete pathogenicity to the apathogenic Δgpmk1 mutant. Hence, we hypothesize that the MAP kinase Gpmk1 is involved in the regulation of additional factors required for complete virulence of F. graminearum. [ABSTRACT FROM AUTHOR]

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]

Abstract: Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3r gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast. [Copyright &y& Elsevier]

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]

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]