WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

<p>Abstract</p> <p>Background</p> <p>Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the <it>ICS1 </it>gene. Also <it>AVR</it>_{<it>PPHB </it>}<it>SUSCEPTIBLE 3 </it>(<it>PBS3</it>) plays an important role in SA metabolism, as <it>pbs3 </it>mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of <it>ICS1 </it>and <it>PBS3</it>.</p> <p>Results</p> <p>Expression studies with <it>ICS1 promoter::β-glucuronidase </it>(<it>GUS</it>) genes in <it>Arabidopsis thaliana </it>protoplasts cotransfected with <it>35S::WRKY28 </it>showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous <it>ICS1 </it>and <it>PBS3 </it>genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the <it>ICS1 </it>promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the <it>ICS1 </it>promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the <it>ICS1 </it>promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA.</p> <p>Conclusions</p> <p>The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of <it>ICS1 </it>and <it>PBS3</it>, respectively, and support this <it>in silico </it>screening as a powerful tool for identifying new components of stress signaling pathways.</p

Prospecting for Genes involved in transcriptional regulation of plant defenses, a bioinformatics approach

<p>Abstract</p> <p>Background</p> <p>In order to comprehend the mechanisms of induced plant defense, knowledge of the biosynthesis and signaling pathways mediated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) is essential. Potentially, many transcription factors could be involved in the regulation of these pathways, although finding them is a difficult endeavor. Here we report the use of publicly available Arabidopsis microarray datasets to generate gene co-expression networks.</p> <p>Results</p> <p>Using 372 publicly available microarray data sets, a network was constructed in which Arabidopsis genes for known components of SA, JA and ET pathways together with the genes of over 1400 transcription factors were assayed for co-expression. After determining the Pearson Correlation Coefficient cutoff to obtain the most probable biologically relevant co-expressed genes, the resulting network confirmed the presence of many genes previously reported in literature to be relevant for stress responses and connections that fit current models of stress gene regulation, indicating the potential of our approach. In addition, the derived network suggested new candidate genes and associations that are potentially interesting for future research to further unravel their involvement in responses to stress.</p> <p>Conclusions</p> <p>In this study large sets of stress related microarrays were used to reveal co-expression networks of transcription factors and signaling pathway components. These networks will benefit further characterization of the signal transduction pathways involved in plant defense.</p

MRI versus mammography for breast cancer screening in women with familial risk (FaMRIsc): a multicentre, randomised, controlled trial

Background: Approximately 15% of all breast cancers occur in women with a family history of breast cancer, but for whom no causative hereditary gene mutation has been found. Screening guidelines for women with familial risk of breast cancer differ between countries. We did a randomised controlled trial (FaMRIsc) to compare MRI screening with mammography in women with familial risk. Methods: In this multicentre, randomised, controlled trial done in 12 hospitals in the Netherlands, women were eligible to participate if they were aged 30–55 years and had a cumulative lifetime breast cancer risk of at least 20% because of a familial predisposition, but were BRCA1, BRCA2, and TP53 wild-type. Participants who were breast-feeding, pregnant, had a previous breast cancer screen, or had a previous a diagnosis of ductal carcinoma in situ were eligible, but those with a previously diagnosed invasive carcinoma were excluded. Participants were randomly allocated (1:1) to receive either annual MRI and clinical breast examination plus biennial mammography (MRI group) or annual mammography and clinical breast examination (mammography group). Randomisation was done via a web-based system and stratified by centre. Women who did not provide consent for randomisation could give consent for registration if they followed either the mammography group protocol or the MRI group protocol in a joint decision with their physician. Results from the registration group were only used in the analyses stratified by breast density. Primary outcomes were number, size, and nodal status of detected breast cancers. Analyses were done by intention to treat. This trial is registered with the Netherlands Trial Register, number NL2661. Findings: Between Jan 1, 2011, and Dec 31, 2017, 1355 women provided consent for randomisation and 231 for registration. 675 of 1355 women were randomly allocated to the MRI group and 680 to the mammography group. 218 of 231 women opting to be in a registration group were in the mammography registration group and 13 were in the MRI registration group. The mean number of screening rounds per woman was 4·3 (SD 1·76). More breast cancers were detected in the MRI group than in the mammography group (40 vs 15; p=0·0017). Invasive cancers (24 in the MRI group and eight in the mammography group) were smaller in the MRI group than in the mammography group (median size 9 mm [5–14] vs 17 mm [13–22]; p=0·010) and less frequently node positive (four [17%] of 24 vs five [63%] of eight; p=0·023). Tumour stages of the cancers detected at incident rounds were significantly earlier in the MRI group (12 [48%] of 25 in the MRI group vs one [7%] of 15 in the mammography group were stage T1a and T1b cancers; one (4%) of 25 in the MRI group and two (13%) of 15 in the mammography group were stage T2 or higher; p=0·035) and node-positive tumours were less frequent (two [11%] of 18 in the MRI group vs five [63%] of eight in the mammography group; p=0·014). All seven tumours stage T2 or higher were in the two highest breast density categories (breast imaging reporting and data system categories C and D; p=0·0077) One patient died from breast cancer during follow-up (mammography registration group). Interpretation: MRI screening detected cancers at an earlier stage than mammography. The lower number of late-stage cancers identified in incident rounds might reduce the use of adjuvant chemotherapy and decrease breast cancer-related mortality. However, the advantages of the MRI screening approach might be at the cost of more false-positive results, especially at high breast density. Funding: Dutch Government ZonMw, Dutch Cancer Society, A Sister's Hope, Pink Ribbon, Stichting Coolsingel, J&T Rijke Stichting