The tomato resistance gene Bs4 suppresses leaf watersoaking phenotypes induced by AvrHah1, a transcription activator-like effector from tomato-pathogenic xanthomonads.

The Xanthomonas transcription activator-like effector (TALE) protein AvrBs3 transcriptionally activates the executor-type resistance (R) gene Bs3 from pepper (Capsicum annuum), thereby triggering a hypersensitive cell death reaction (HR). AvrBs3 also triggers an HR in tomato (Solanum lycopersicum) upon recognition by the nucleotide-binding leucine-rich repeat (NLR) R protein Bs4. Whether the executor-type R protein Bs3 and the NLR-type R protein Bs4 use common or distinct signalling components to trigger an HR remains unclear. CRISPR/Cas9-mutagenesis revealed, that the immune signalling node EDS1 is required for Bs4- but not for Bs3-dependent HR, suggesting that NLR- and executor-type R proteins trigger an HR via distinct signalling pathways. CRISPR/Cas9-mutagenesis also revealed that tomato Bs4 suppresses the virulence function of both TALEs, the HR-inducing AvrBs3 protein and of AvrHah1, a TALE that does not trigger an HR in tomato. Analysis of AvrBs3- and AvrHah1-induced host transcripts and disease phenotypes in CRISPR/Cas9-induced bs4 mutant plants indicates that both TALEs target orthologous transcription factor genes to promote disease in tomato and pepper host plants. Our studies display that tomato mutants lacking the TALE-sensing Bs4 protein provide a novel platform to either uncover TALE-induced disease phenotypes or genetically dissect components of executor-triggered HR.

Exploiting the sequence diversity of TALE-like repeats to vary the strength of dTALE-promoter interactions.

Designer transcription activator-like effectors (dTALEs) are programmable transcription factors used to regulate user-defined promoters. The TALE DNA-binding domain is a tandem series of amino acid repeats that each bind one DNA base. Each repeat is 33-35 amino acids long. A residue in the center of each repeat is responsible for defining DNA base specificity and is referred to as the base specificying residue (BSR). Other repeat residues are termed non-BSRs and can contribute to TALE DNA affinity in a non-base-specific manner. Previous dTALE engineering efforts have focused on BSRs. Non-BSRs have received less attention, perhaps because there is almost no non-BSR sequence diversity in natural TALEs. However, more sequence diverse, TALE-like proteins are found in diverse bacterial clades. Here, we show that natural non-BSR sequence diversity of TALEs and TALE-likes can be used to modify DNA-binding strength in a new form of dTALE repeat array that we term variable sequence TALEs (VarSeTALEs). We generated VarSeTALE repeat modules through random assembly of repeat sequences from different origins, while holding BSR composition, and thus base preference, constant. We used two different VarSeTALE design approaches combing either whole repeats from different TALE-like sources (inter-repeat VarSeTALEs) or repeat subunits corresponding to secondary structural elements (intra-repeat VarSeTALEs). VarSeTALE proteins were assayed in bacteria, plant protoplasts and leaf tissues. In each case, VarSeTALEs activated or repressed promoters with a range of activities. Our results indicate that natural non-BSR diversity can be used to diversify the binding strengths of dTALE repeat arrays while keeping target sequences constant.

Does Bilateral ITA Grafting Increase Perioperative Complications? Outcome of 6,476 Patients with Bilateral versus 5,020 Patients with Single ITA Bypass.

OBJECTIVES: Despite the superior patency of internal thoracic artery (ITA) grafting compared with saphenous veins, frequency of bilateral ITA (BITA) grafting in Europe is still approximately 10%. The aim of the present study was to compare the early outcome of patients receiving either BITA or single ITA (SITA) grafting. METHODS: A total of 11,496 patients with isolated coronary artery bypass grafting (CABG), operated between January 1996 and December 2012, were analyzed retrospectively; 0.6476 patients (mean age 65.2 years, 81.3% males) received BITA and 5,020 patients (mean age 66.6 years, 76.7% males) SITA grafting. Mean body mass index (BMI) was 27.2 versus 27.4, p = 0.017. Incidence of diabetes was 28.9 versus 28.4%, p = 0.08. Ejection fraction (EF) > 50 was 71.3% (BITA) versus 66.3% (SITA), p < 0.001. Elective operations were performed in 88.4% (BITA) versus 83.3% (SITA), and urgent/emergent surgery was necessary in 11.6% (BITA) versus 16.7% (SITA), p < 0.001. RESULTS: Number of grafts was 3.76 (BITA) versus 3.06, p < 0.001. Duration of surgery (194.4 vs. 180.4 minutes) as well as X-clamp time (60.4 vs. 51.7 minutes) was prolonged for BITA, p < 0.001. Perioperative infarction rate revealed 3.2% (BITA) versus 3.6%, p = 0.54. Frequency of rethoracotomy due to bleeding was higher in the BITA group (3.8 vs. 2.1%), p < 0.001. Sternal instabilities occurred in 2.3% (BITA) versus 2.2%, p = 0.749. Duration of mechanical ventilation < 12 hours was 74.6 versus 77.1%, p = 0.09 and duration of in-hospital stay was 10.5 versus 10.4 days, p = 0.68. Thirty-day mortality was 2.4% (BITA) versus 3.0%, p = 0.09. Multivariate analysis identified prolonged duration of surgery, BMI > 30, emergent operations, advanced age, and BITA grafting as predictor for sternal instabilities. EF < 30%, advanced age plus emergency were associated with increased 30-day mortality. CONCLUSION: CABG using BITA can be performed routinely with good clinical results and low mortality. Compared with SITA grafting, bleeding complications were enhanced.

Heat shock factor HSFB2a involved in gametophyte development of Arabidopsis thaliana and its expression is controlled by a heat-inducible long non-coding antisense RNA.

Heat stress transcription factors (HSFs) are central regulators of the heat stress response. Plant HSFs of subgroup B lack a conserved sequence motif present in the transcriptional activation domain of class A-HSFs. Arabidopsis members were found to be involved in non-heat shock functions. In the present analysis we investigated the expression, regulation and function of HSFB2a. HSFB2a expression was counteracted by a natural long non-coding antisense RNA, asHSFB2a. In leaves, the antisense RNA gene is only expressed after heat stress and dependent on the activity of HSFA1a/HSFA1b. HSFB2a and asHSFB2a RNAs were also present in the absence of heat stress in the female gametophyte. Transgenic overexpression of HSFB2a resulted in a complete knock down of the asHSFB2a expression. Conversely, asHSFB2a overexpression leads to the absence of HSFB2a RNA. The knockdown of HSFB2a by asHSFB2a correlated with an improved, knockdown of asHSFB2a by HSFB2a overexpression with an impaired biomass production early in vegetative development. In both cases the development of female gametophytes was impaired. A T-DNA knock-out line did not segregate homozygous mutant plants, only heterozygots hsfB2a-tt1/+ were viable. Approximately 50% of the female gametophytes were arrested in early development, before mitosis 3, resulting in 45% of sterile ovules. Our analysis indicates that the "Yin-Yang" regulation of gene expression at the HSFB2a locus influences vegetative and gametophytic development in Arabidopsis.

Identification of novel heat shock factor-dependent genes and biochemical pathways in Arabidopsis thaliana.

In order to assess specific functional roles of plant heat shock transcription factors (HSF) we conducted a transcriptome analysis of Arabidopsis thaliana hsfA1a/hsfA1b double knock out mutants and wild-type plants. We used Affymetrix ATH1 microarrays (representing more than 24 000 genes) and conducted hybridizations for heat-treated or non-heat-treated leaf material of the respective lines. Heat stress had a severe impact on the transcriptome of mutant and wild-type plants. Approximately 11% of all monitored genes of the wild type showed a significant effect upon heat stress treatment. The difference in heat stress-induced gene expression between mutant and wild type revealed a number of HsfA1a/1b-regulated genes. Besides several heat shock protein and other stress-related genes, we found HSFA-1a/1b-regulated genes for other functions including protein biosynthesis and processing, signalling, metabolism and transport. By screening the profiling data for genes in biochemical pathways in which known HSF targets were involved, we discovered that at each step in the pathway leading to osmolytes, the expression of genes is regulated by heat stress and in several cases by HSF. Our results document that in the immediate early phase of the heat shock response HSF-dependent gene expression is not limited to known stress genes, which are involved in protection from proteotoxic effects. HsfA1a and HsfA1b-regulated gene expression also affects other pathways and mechanisms dealing with a broader range of physiological adaptations to stress.

Galactinol synthase1. A novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis.

Heat shock factors (HSFs) are transcriptional regulators of the heat shock response. The major target of HSFs are the genes encoding heat shock proteins (HSPs), which are known to have a protective function that counteracts cytotoxic effects. To identify other HSF target genes, which may be important determinants for the generation of stress tolerance in Arabidopsis, we screened a library enriched for genes that are up-regulated in HSF3 (AtHsfA1b)-overexpressing transgenic plants (TPs). Galactinol synthase1 (GolS1) is one of the genes that is heat-inducible in wild type, but shows constitutive mRNA levels in HSF3 TPs. The generation and analysis of TPs containing GolS1-promoter::beta-glucuronidase-reporter gene constructs showed that, upon heat stress, the expression is transcriptionally controlled and occurs in all vegetative tissues. Functional consequences of GolS1 expression were investigated by the quantification of raffinose, stachyose, and galactinol contents in wild type, HSF3 TPs, and two different GolS1 knockout mutants (gols1-1 and gols1-2). This analysis demonstrates that (1) raffinose content in leaves increases upon heat stress in wild-type but not in the GolS1 mutant plants; and (2) the level of raffinose is enhanced and stachyose is present at normal temperature in HSF3 TPs. These data provide evidence that GolS1 is a novel HSF target gene, which is responsible for heat stress-dependent synthesis of raffinose, a member of the raffinose family oligosaccharides. The biological function of this osmoprotective substance and the role of HSF-dependent genes in this biochemical pathway are discussed.

Generation of dominant-negative effects on the heat shock response in Arabidopsis thaliana by transgenic expression of a chimaeric HSF1 protein fusion construct.

Upon heat stress, heat shock factors (HSFs) control the expression of heat shock protein (HSP) genes by transcriptional activation. The perplexing multiplicity of HSF genes in Arabidopsis- 21 potential genes have been identified - renders it difficult to identify mutant phenotypes. In this study, we have attempted to generate a transdominant-negative mutant of HSF by transgenic expression of a protein fusion construct, EN-HSF1, consisting of the Drosophila engrailed repressor domain (EN) and the complete Arabidopsis AtHSF1. Transgenic lines were screened for impaired ability to induce high levels of low-molecular-weight heat shock proteins (sHSPs). Two lines, EH14-6 and EH16-3, which showed quantitative differences in the expression of EN-HSF1, were further analysed for induction of thermotolerance and heat-stress-dependent mRNAs of a number of different HSF target genes encoding different HSP and HSF. The mRNA levels of all genes tested were moderately downregulated in EH14-6 but strongly reduced in EH16-3 plants compared to wild-type (Wt) and HSF1-overexpressing control plants. The inhibition of the induction of heat shock response correlated with impaired basal and acquired thermotolerance of the EH16-3 line. The kinetics of HSP expression suggest that the negative effect of EN-HSF1 is stronger in the early phase of the heat shock response, and that the reduction in mRNA levels is partially compensated at the translational level.