Pervasive fungicide resistance in Botrytis from strawberry in Norway: Identification of the grey mould pathogen and mutations

Control of grey mould, caused by Botrytis spp., is a major challenge in open field strawberry production. Botrytis was isolated from plant parts collected from 19 perennial strawberry fields with suspected fungicide resistance in the Agder region of Norway in 2016. Resistance to boscalid, pyraclostrobin and fenhexamid was high and found in 89.1%, 86.0% and 65.4% of conidia samples, respectively. Multiple fungicide resistance was common; 69.6% of conidia samples exhibited resistance to three or more fungicides. Botrytis group S and B. cinerea sensu stricto isolates were obtained from 19 and 16 fields, respectively. The sdhB, cytb, erg27 and mrr1 genes of a selection of isolates were examined for the presence of mutations known to confer fungicide resistance to boscalid, pyraclostrobin, fenhexamid and pyrimethanil plus fludioxonil, respectively. Allele-specific PCR assays were developed for efficient detection of resistance-conferring mutations in cytb. Among B. cinerea isolates, 84.7%, 86.3% and 61.3% had resistance-conferring mutations in sdhB, cytb and erg27, respectively. A triplet deletion in mrr1, resulting in ΔL497, commonly associated with the multidrug resistance phenotype MDR1h, was detected in 29.2% of Botrytis group S isolates. High frequencies of resistance to several fungicides were also detected in Botrytis from both imported and domestically produced strawberry transplants. Fungicide resistance frequencies were not different among fields grouped by level of grey mould problem assessed by growers, indicating factors other than fungicide resistance contributed to control failure, a fact that has important implications for future management of grey mould.publishedVersio

DNA copy number changes in high-grade malignant peripheral nerve sheath tumors by array CGH

<p>Abstract</p> <p>Background</p> <p>Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH).</p> <p>Results</p> <p>Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase-like 2 (<it>LOXL2</it>) was identified as a candidate target for the 8p23.1-p12 gain. Within 17q, the genes topoisomerase II-α (<it>TOP2A</it>), ets variant gene 4 (E1A enhancer binding protein, <it>E1AF</it>) (<it>ETV4</it>) and baculoviral IAP repeat-containing 5 (survivin) (<it>BIRC5</it>) showed increased expression in all samples compared to two benign tumors. Increased expression of these genes has previously been associated with poor survival in other malignancies, and for <it>TOP2A</it>, in MPNSTs as well. In addition, we have analyzed the expression of five micro RNAs located within the 17q23.2-q25.3 region, but none of them showed high expression levels compared to the benign tumors.</p> <p>Conclusion</p> <p>Our study shows the potential of using DNA copy number changes obtained by array CGH to predict the prognosis of MPNST patients. Although no clear correlations between the expression level and patient outcome were observed, the genes <it>TOP2A</it>, <it>ETV4 </it>and <it>BIRC5 </it>are interesting candidate targets for the 17q gain associated with poor survival.</p

Funn av soppmiddelresistent Botrytis i åkerbønne

publishedVersio

miR-486-5p expression is regulated by DNA methylation in osteosarcoma

Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. Results To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. Conclusions miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.publishedVersio

Endogenous transcript levels of mature miR-142-5p/3p and primary <i>mir-142</i> in cells of mesenchymal and hematopoietic origin, and after treatment with epigenetic modifiers.

<p>(<b>A</b>) Expression levels as determined by qPCR in tumorigenic cells (MG-63, OHS, IOR/OS14, U-2 OS, IOR/OS10, IOR/SARG, IOR/MOS and HOS), non-tumorigenic mesenchymal cells (iMSC#3, hMSCs and primary osteoblasts), and hematopoietic cells (K562 and PBPCs). Expression is depicted relative to the MG-63 cells (set to 1) and the numbers above the histograms represent the expression level of primary <i>mir-142</i>. <i>RNU44</i> or Glyceraldehyde 3-phosphate dehydrogenase (<i>GAPDH</i>) were used for normalization of mature and primary transcripts, respectively. iMSC#3, immortalized bone marrow-derived stromal cells; hMSCs, primary bone marrow-derived stromal cells; K562, K562 leukemia cells; PBPCs, peripheral blood progenitor cells. (<b>B</b>) Quantification of expression using qPCR after treatment with 5-Aza-2′-deoxycytidine (5-Aza). Expression is shown relative to untreated cells (set to 1). <i>RNU44</i> or <i>GAPDH</i> were used for normalization of mature and primary transcripts, respectively. (<b>C</b>) Expression levels of miR-142-3p, primary <i>mir-142</i> and mesoderm specific transcript (<i>MEST</i>) in MG-63 cells as determined by qPCR, after treatment with 5-Aza alone, a combination of 5-Aza and trichostatin A (TSA), or TSA alone. <i>RNU44</i> or <i>GAPDH</i> were used for normalization of mature and primary transcripts (including <i>MEST</i>), respectively. The error bars in all qPCR experiments show the standard deviation of technical replicates.</p

<i>In silico</i> identification of putative CGIs and characterization of the primary <i>mir-142</i> transcript.

<p>(<b>A</b>) Schematic representation of putative CGIs associated with <i>mir-142</i>. Each vertical line represents an individual CpG. Black, dark grey and light grey horizontal bars; CGIs predicted with CpG Island Explorer, CpG Island Searcher and CpGcluster, respectively. The numbers indicate the position relative to the <i>mir-142</i> precursor (<i>pre-mir-142</i>) (+1 to +87). O/E, observed/expected. (<b>B</b>) Schematic representation of the genomic <i>mir-142</i> region. The approximate 5′- end of <i>mir-142</i> was mapped using qPCR with assays specific for four different regions (depicted by vertical arrowheads). Grey and black arrowheads indicate increased expression or no change, respectively. The transcription start (TSS) and polyadenylation sites were identified using rapid amplification of cDNA ends. The approximate positions of the gene-specific primers (GSPs) and associated amplicons are indicated by horizontal arrowheads and attached lines, respectively. The putative CGIs are represented by horizontal bars. (<b>C</b>) Schematic representation of the 2 transcript variants of primary <i>mir-142</i> (<i>pri-mir-142</i>).</p

Reexpression of LSAMP inhibits tumor growth in a preclinical osteosarcoma model

Background Osteosarcomas are the most common primary malignant tumors of bone, showing complex chromosomal rearrangements with multiple gains and losses. A frequent deletion within the chromosomal region 3q13.31 has been identified by us and others, and is mainly reported to be present in osteosarcomas. The purpose of the study was to further characterize the frequency and the extent of the deletion in an extended panel of osteosarcoma samples, and the expression level of the affected genes within the region. We have identified LSAMP as the target gene for the deletion, and have studied the functional implications of LSAMP-reexpression. Methods LSAMP copy number, expression level and protein level were investigated by quantitative PCR and western blotting in an osteosarcoma panel. The expression of LSAMP was restored in an osteosarcoma cell line, and differences in proliferation rate, tumor formation, gene expression, migration rate, differentiation capabilities, cell cycle distribution and apoptosis were investigated by metabolic dyes, tumor formation in vivo, gene expression profiling, time-lapse photography, differentiation techniques and flow cytometry, respectively. Results We found reduced copy number of LSAMP in 45/76 osteosarcoma samples, reduced expression level in 25/42 samples and protein expression in 9/42 samples. By restoring the expression of LSAMP in a cell line with a homozygous deletion of the gene, the proliferation rate in vitro was significantly reduced and tumor growth in vivo was significantly delayed. In response to reexpression of LSAMP, mRNA expression profiling revealed consistent upregulation of the genes hairy and enhancer of split 1 (HES1), cancer/testis antigen 2 (CTAG2) and kruppel-like factor 10 (KLF10). Conclusions The high frequency and the specificity of the deletion indicate that it is important for the development of osteosarcomas. The deletion targets the tumor suppressor LSAMP, and based on the functional evidence, the tumor suppressor function of LSAMP is most likely exerted by reducing the proliferation rate of the tumor cells, possibly by indirectly upregulating one or more of the genes HES1, CTAG2 or KLF10. To our knowledge, this study describes novel functions of LSAMP, a first step to understanding the functional role of this specific deletion in osteosarcomas

Pervasive fungicide resistance in Botrytis from strawberry in Norway: Identification of the grey mould pathogen and mutations

Control of grey mould, caused by Botrytis spp., is a major challenge in open field strawberry production. Botrytis was isolated from plant parts collected from 19 perennial strawberry fields with suspected fungicide resistance in the Agder region of Norway in 2016. Resistance to boscalid, pyraclostrobin and fenhexamid was high and found in 89.1%, 86.0% and 65.4% of conidia samples, respectively. Multiple fungicide resistance was common; 69.6% of conidia samples exhibited resistance to three or more fungicides. Botrytis group S and B. cinerea sensu stricto isolates were obtained from 19 and 16 fields, respectively. The sdhB, cytb, erg27 and mrr1 genes of a selection of isolates were examined for the presence of mutations known to confer fungicide resistance to boscalid, pyraclostrobin, fenhexamid and pyrimethanil plus fludioxonil, respectively. Allele-specific PCR assays were developed for efficient detection of resistance-conferring mutations in cytb. Among B. cinerea isolates, 84.7%, 86.3% and 61.3% had resistance-conferring mutations in sdhB, cytb and erg27, respectively. A triplet deletion in mrr1, resulting in ΔL497, commonly associated with the multidrug resistance phenotype MDR1h, was detected in 29.2% of Botrytis group S isolates. High frequencies of resistance to several fungicides were also detected in Botrytis from both imported and domestically produced strawberry transplants. Fungicide resistance frequencies were not different among fields grouped by level of grey mould problem assessed by growers, indicating factors other than fungicide resistance contributed to control failure, a fact that has important implications for future management of grey mould