Transcriptome profiling on the response of Mycosphaerella graminicola isolates to an azole fungicide using cDNA arrays

Resistance to azole antifungals is a major problem in the control of diseases caused by fungal pathogens of both humans and plants. Potential for the development of azole resistance in the wheat leaf blotch pathogen Mycosphaerella graminicola, the causal agent of the most economically significant foliar disease of wheat in north-western Europe, is now of particular concern after the recent emergence of widespread resistance to quinone outside inhibitor fungicides. Using a cDNA microarray representing around 25% of the genome, we have profiled the transcriptional response of M. graminicola to epoxiconazole, currently the most widely used azole fungicide on cereal crops. By comparing the transcription profiles of two M. graminicola isolates with contrasting sensitivities to epoxiconazole we show qualitative and quantitative differences in differentially expressed genes, including those involved in ergosterol biosynthesis, mitochondrial respiration and transport mechanisms. This represents the first study investigating the response of a plant pathogenic fungus to a fungicide using cDNA microarray technology

Fungicide-Driven Evolution and Molecular Basis of Multidrug Resistance in Field Populations of the Grey Mould Fungus Botrytis cinerea

The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum * §

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics

Cosmic ray test system for the ATLAS thin gap chamber modules at KOBE

Thin gap chamber modules giving function of forward muon trigger to the ATLAS detector in the LHC experiment have been constructed at High Energy Research Organization (KEK) in Japan and their performances have been checked at Kobe University. A large-scale test system specially designed for measuring uniformity of the detection efficiencies and the timing resolution of 8 TGC modules at the same time was successfully operated. Each TGC module had 72 anode wire channels and 64 cathode strip channels (in total 1088 readout channels for 8 modules). Drift tubes consisted of 12 layers (total 428 tubes), between which the TGC modules are put, determined trajectories of cosmic rays. Hit pattern and timing of all detector signals (Trigger counter. Drift tubes and TGCs) were measured by using VME modules. In regular data acquisition situation, i.e. about effective 19 Hz trigger rate from scintillation counters and 73% tracking efficiency by the drift tubes, the detection efficiency of each layer by 5 mm * 5 mm region of 8 TGC modules (24 layers) was measured using about 7 million cosmic ray tracks per module in 10 days. 306 modules were already inspected at June 2003. (7 refs)