text:
- Original article
- Open Access
OsHLH61-OsbHLH96 influences rice defense to brown planthopper through regulating the pathogen-related genes
Rice volume 12, Article number: 9 (2019)
In plants, basic helix-loop-helix (bHLH) proteins form the largest transcription factor (TF) family. Among them, HLH proteins are a small group of atypical members that lack the basic domain, and form dimers with bHLH proteins. Although bHLH proteins have been proved to play important roles in plant development and physiology, the function of HLH proteins is rarely studied, not to mention in plant biotic resistance. Brown planthopper (BPH) is a kind of rice-specific insect that causes devastating yield losses each year.
In this study, we identified OsHLH61 gene that encodes HLH protein. OsHLH61 gene could be highly induced by BPH infestation. Furthermore, Methyl Jasmonic acid (Me-JA) and cis-12-oxo- phytodienoic acid (OPDA) induced expression of OsHLH61, while SA repressed it. We knocked down expression of OsHLH61 by RNA interference (RNAi), the transgenic plants were susceptible to BPH infestation. RNA-seq analysis revealed that some pathogen-related (PR) genes in the Salicylic acid (SA) signaling pathway that mediate plant immunity were obviously down-regulated in the OsHLH61 RNAi plants. Meanwhile, yeast two-hybrid assay and bimolecular luciferase complementation (BiLC) analysis identified bHLH096 to be an interacting factor of OsHLH61. Also, some PR genes were down-regulated in the OsbHLH96 over expressing lines. Expression of OsbHLH96 was inhibited. Besides, OsbHLH96 might interact with Jasmonate Zim-Domain3 (OsJAZ3).
Altogether, we identified an OsHLH61–OsbHLH96 complex that might mediate defense to BPH through regulating PR genes. And OsHLH61–OsbHLH96 might be important in mediating SA and JA signaling crosstalk.
During their sessile growth, plants need to deal with various environmental stresses caused by biotic and abiotic factors. In thousands of years’ evolution, plants respond to these stresses through activation of series of responding molecules (Baniwal et al. 2004; Sunkar 2010). These defensing elements include TFs (Shi et al. 2018; Viana et al. 2018; Xiao et al. 2013), chaperone (Attallah et al. 2007), mitogen-activated protein kinase (Liang and Zhou, 2018), reactive oxygen species (ROS) (Miller et al. 2008), plant hormones (Bari and Jones, 2009; Peleg and Blumwald, 2011; Santino et al. 2013) and even sugar (Wingler and Roitsch, 2008).
bHLH proteins are the largest TF family in plants that function extensively in plant development and defensive response through crosstalk among different signaling pathways (Ezer et al. 2017; Kazan and Manners, 2013). The bHLH protein Myelocytomatosis protein 2 (MYC2) is involved in JA-regulated plant development (Dombrecht et al. 2007), root formation (Chen et al. 2011), insect resistance (Schweizer et al. 2013) and pathogen response (Kazan and Manners, 2013), and is becoming a master regulator of JA-mediated responses. Rice OsbHLH148 is involved in JA signaling by interacting with OsJAZ and mediates drought tolerance (Seo et al. 2011). However, HLH proteins are a small group of atypical members in the bHLH family that lack the basic domain and usually form dimers or multimers with bHLH proteins and inhibit their transcriptional activity (Carretero-Paulet et al. 2010; Li et al. 2006). In rice, positive regulator of grain length 2 (PGL2) regulates grain weight by influencing cell elongation and interacting with a bHLH protein Antagonist of Pgl1 (APG) (Heang and Sassa, 2012). In Arabidopsis, HLH proteins ATBS1-interacting factors (AIFs) and ILI1 binding bHLH (IBH1) respectively interact with paclobutrazol resistance1 (PREs) and activators for cell elongation 1 (ACE1) to form HLH-bHLH complex and mediate brassinosteroid (BR) signaling and cell elongation (Wang et al. 2009), and this regulating pathway is conserved in rice (Zhang et al. 2009). A recent study in rice indicates that a HLH protein BR upregulated 1-like (OsBUL1) regulates cell elongation by forming an OsBUL1 complex1 (Jang et al. 2017). Nevertheless, it is not clear whether HLH proteins function in stress response.
The brown planthopper (BPH) is a rice-specific herbivore, which causes severe yield losses each year in rice planting areas throughout Asia (Cheng et al. 2013; Flowers 2004). JA and SA mediated signaling pathways have been extensively identified in plant stress response against pathogen and insect (Berens et al. 2017). Generally, SA is proved to positively regulate rice defense to BPH (Yang and Zhang, 2016). Although the role of JA in BPH resistance is still controversy (Yang and Zhang, 2016), increasing evidence supports the negative role of JA in BPH resistance. For example, silencing of herbivore-induced rice type 2 13-lipoxygenase (OsHI-LOX) in JA synthesis is resistant to BPH (Zhou et al. 2009), and gain of function of 9-lipoxygenase gene (Osr9-LOX1) is favorable for the survival of the BPH larva (Zhou et al. 2014). The resistance genes Bph14 and Bph29 could both increase expression of genes in SA pathway and suppress genes in the JA pathway (Du et al. 2009; Wang et al. 2015). However, Bph6 and Bph9 could simultaneously activate SA and JA signaling pathway (Zhao et al. 2016; Guo et al. 2018), indicating that the specific role of JA in different background might also influence by the resistance genes. Nevertheless, the crosstalk between JA and SA in mediating BPH resistance is still unclear.
PR genes are key factors in the immune pathway and function as markers for systemic acquired resistance (SAR) response (Glazebrook 2005). Expression of several PR1 genes is significantly induced by rice blast (Mitsuhara et al. 2008); and transgenic tobacco plants expressing high level of PR-la protein are resistant to pathogens (Alexander et al. 1993). Some PR1 proteins are proved to be antifungal (Niderman et al. 1995). Expression of some PR genes in SA signaling transduction is greatly influenced by the >non-expressor of pathogenesis-related genes 1(OsNPR1) in mediating resistance to bacterial and rice blast (Sugano et al. 2010). Arabidopsis monomer NPR1 can enter into the nucleus and interact with TGACG motif-binding factor (TGA), which directly regulates the transcription of some PR genes (Chern et al. 2014; Despres et al. 2000; Zhang et al. 2003). In rice, rTGA2.2 could interact with OsNPR1 (Chern et al. 2005), and rTGA 2.1 negatively regulates bacterial diseases and PR10 gene (Fitzgerald et al. 2005). Transgenic rice lines carrying the OsAOS2 gene under the control of a strong, pathogen-inducible promoter exhibit enhanced activation of some PR genes such as PR1a, PR3, and PR5, and increase resistance to rice blast (Mei et al. 2006). Also, some PR genes could be induced by BPH (Hu et al. 2017).
Previously, we revealed that over-expression of allene oxide cyclase (AOC) gene increased resistance to BPH in a JA-independent manner (Guo et al. 2014). To further reveal the downstream genes in AOC-mediated BPH resistance, we identified OsHLH61, which was up-regulated in AOC over-expressing plants. Function analysis revealed that OsHLH61 positively regulated BPH resistance by influencing expression of PR1, PR5 and PR10 genes. OsbHLH96 was proved to be the interacting factor of OsHLH61, and OsJAZ3 might interact with OsbHLH96. Meanwhile, OsbHLH96 could regulate the expression of PR genes negatively. Therefore, we revealed the role of OsHLH61–OsbHLH96 complex in defense to BPH and added new points in SA and JA crosstalk in defending against BPH.
Homologous comparison and phylogenetic analysis of the OsHLH61 sequence
We identified OsHLH61 (Os07g0676600) gene in AOC over-expressing plants (Guo et al. 2014), and then cloned it. The coding region of OsHLH61 gene contains 474 base pairs. The encoded protein OsHLH61 contains an atypical HLH domain in the 2–50 amino acids and belongs to group D (Atchley and Fitch, 1997), which lacks the basic domain, and so that could not directly bind to DNA (Li et al. 2006). Furthermore, members in group D could negatively regulate the bHLH proteins and repress their transcriptional activation (Sun et al. 1991). To further analyze OsHLH61, we selected some homologous genes in monocotyledonous plants Oryza brachyantha and Zea mays, and dicotyledonous plants Cajanuy caja, Theobroma cacao and Gossyplum arboretum, and performed sequence alignment. It was revealed that the HLH domains were highly conserved; and the α-helix in the HLH domain each had two conserved sites, Leu12 and Leu44 (Fig. 1a), which have been proved to be pivotal for dimerization (Brownlie et al. 1997; Carretero-Paulet et al. 2010). analysis using Neighbor-Joining, and revealed that OsHLH61 is nearest to the ObSCREAM2-like protein in wild rice (Fig. 1b).
Expression profile of OsHLH61 and subcellular localization of OsHLH61 protein
To verify the response of OsHLH61 to BPH, we checked expression of OsHLH61 using a quantitative reverse transcriptase PCR (qRT–PCR) after BPH infestation, it was revealed that OsHLH61 was induced as early as 3 h, and reached a peak at 7 h (Fig. 2a). So that OsHLH61 can be induced by BPH infestation.
Next, we checked the expression of OsHLH61 in different tissues, it was revealed that OsHLH61 expressed highly in the leaf sheath (Fig. 2b), where BPH feeds. Furthermore, we constructed an OsHLH61p::GUS plants to monitor the tissues where OsHLH61 proteins expressed. In consistence, GUS signal was concentrated in the leaf sheath (Fig. 2c). Besides, GUS signal was also detected in the young stem, and the initiation sites of root hair (Fig. 2c).
Since expression of OsHLH61 was influenced by AOC, which function in JA and OPDA biosynthesis (Guo et al. 2014), we further checked if OsHLH61 was responsive to MeJA and OPDA, it was revealed that OsHLH61 could be induced by both MeJA and OPDA (Fig. 2d). Meanwhile, under SA treatment, OsHLH61 was down-regulated at 1 h (Fig. 2e).
To further study the molecular basis of the OsHLH61 function, we checked the subcellular localization of the OsHLH61 protein. OsHLH61 localized ubiquitously in the protoplast, with the exception to the chloroplast (Fig. 2f), this was in consistence with that of OsBUL1 in rice (Jang et al. 2017). So that, HLH protein might be ubiquitously localized, different from bHLH protein, which are localized in the nucleus (Cui et al. 2016; Xu et al. 2014).
Knockdown of OsHLH61 rendered the plant more susceptible to BPH
To investigate the genetic function of OsHLH61, we constructed RNAi plants (HLHR) of OsHLH61 by transforming the RNAi plasmid into wild type (WT) ZH11. We chose 5 lines with obvious down-regulation of OsHLH61 (Fig. 3a) for further analysis. Expression of HLH61-like (OsbHLH166, Os03g0338400), the homologous gene of OsHLH61 in rice, was not influenced (Additional file 1: Figure S1a), indicating the specific down-regulation of OsHLH61 in these HLHR plants. Then we performed individual analysis and revealed that the HLHR plants were more susceptible to BPH infestation (Fig. 3b). In consistence, the mortality of the HLHR plants was much higher than the WT after BPH infestation (Fig. 3c).
Besides, knock down of OsHLH61 influenced rice development. The leaves of the HLHR plants curled adaxially (Additional file 1: Figure S1b) and the tiller number increased (Additional file 1: Figure S1c). In addition, the fertility of the HLHR plants was reduced (Additional file 1: Figure S1 d, e).
OsHLH61 interacted with OsbHLH96
Since OsHLH61 was an atypical bHLH protein that needs to form HLH-bHLH complex in functioning, we tried to search the possible interacting bHLH protein of OsHLH61. There are 177 bHLH proetins in rice, with 26 atypical HLHs and 151 typical bHLHs (Carretero-Paulet et al. 2010; Li et al. 2006). We screened a yeast library and identified one bHLH protein, OsbHLH96, to interact with OsHLH61 in yeast two-hybrid analysis (Fig. 4a). BILC assay verified the interaction between OsHLH61 and OsbHLH96 (Fig. 4b). We found that OsHLH61 could not form homodimers in yeast two-hybrid test (Fig. 4a), but OsbHLH96 could (Fig. 4c). So that OsbHLH96 is the interacting protein of OsHLH61.
It is reported that AtMYC2 (Thireault et al. 2015) and OsbHLH148 (Seo et al., 2011) can respectively interact with JAZs in functioning. We wondered if OsbHLH96 could interact with OsJAZs, so that we checked OsJAZ1, OsJAZ3, OsJAZ5, OsJAZ7, OsJAZ9, OsJAZ11 and OsJAZ12, and revealed that only OsJAZ3 could interact with OsbHLH96 in yeast two-hybrid analysis (Fig. 4c).
Some PRs were significantly down-regulated in OsHLH61 RNAi plants
To analyze the genes involved in OsHLH61 functioning, we carried out a RNA-seq analysis of the HLHR-4 plants before and after BPH infestation. Three kind of samples, WT ZH11, HLHR-4 without BPH infestation, and HLHR-4 after BPH infestation for 12 h (named as ZH0, HR0 and HR12 respectively), were used for analysis. We found that most PR genes were down-regulated in HR0 compared with in ZH0, so that data of PR genes were extracted and displayed in heat map (Fig. 5a), it was revealed that most PR genes in HR0 were down-regulated. Because the expression of PR1a (Os07g0129200), PR5 (Os04g0689900) and PR10a (Os12g0555500) were influenced by BPH feeding (Hu et al. 2017), we further checked the expression of PR1a, PR5, PR10a, PR1-like (PR1L, Os07g0127500) and some PR1 (Os07g0125201, Os07g0125000, Os07g0124900) by qRT-PCR. It was revealed that all of them were down-regulated in the HR0 plants (Fig. 5b). Besides, expression of the OsHLH61-like was not influenced in RNA-seq analysis, further indicating that OsHLH61 was specifically down-regulated in the HLHR plants (data not shown).
Over-expression of OsbHLH96 down-regulated expression of some PR genes
Now that OsbHLH96 is the interacting protein of OsHLH61, we want to know if OsbHLH96 could regulate expression of PR genes. We constructed the over-expression lines of OsbHLH96 (bHOE) and selected two positive lines for further analysis (Fig. 6a). Through qRT-PCR analysis, it was revealed that PR1a, PR1L, PR5 and PR10a genes were down-regulated in OsbHLH96 over-expressing lines (Fig. 6b). Now that expression of PR genes was influenced by OsbHLH96, we further analyzed if there are any E-box motifs for bHLH protein binding in the promoters of the PR genes (Goossens et al. 2016). The promoters of PR1a, PR5, PR10a and AOS2 (Os03g0225900) have some E-box motifs (http://plantpan2.itps.ncku.edu.tw). Next, we performed Dual-Luc assay to determine whether OsbHLH96 could directly regulate PR genes. Neither the luciferase signals (Fig. 6c) nor the LUC/RLUC ratios (Fig. 6d) support a direct regulation. PR genes can be induced by SA (Agrawal et al., 2000), we chose PR1L to determine its expression under SA treatment. It was revealed that PR1L was induced by SA (Fig. 6e). However, OsbHLH96 was repressed by SA treatment (Fig. 6f).
In this study, we revealed that down-regulation of OsHLH61 showed significant sensitivity to BPH, and proved the function of HLH–bHLH heterodimer in BPH response. We showed that OsHLH61 form bHLH–HLH heterodimer with OsbHLH96 (Fig. 4a, b). In both OsHLH61 RNAi and OsbHLH96 over-expressing plants, PR genes were down-regulated (Fig. 5a, b; Fig. 6b). PR1a gene can be induced by small brown planthopper indigestion (Hao et al. 2011), and PR1b has been reported to be induced by brown planthopper indigestion (Hu et al. 2017). Some PR1 proteins are proved to be antifungal (Niderman et al. 1995). Our study indicated that down regulation of PR genes in OsHLH61 RNAi plants might account for their sensitivity to BPH (Fig. 3b).
PR genes are considered as markers for plant resistance (Glazebrook, 2005, Liu et al., 2018, Liu et al., 2013). Some PR genes were down-regulated in OsHLH61 RNAi plants (Fig. 5). OsHLH61 was induced by JA, OPDA, but repressed by SA (Fig. 2d, e), and up-regulated in AOC and OPR3 over-expressing lines (Guo et al., 2014), while the transcriptional level of AOC or OPR3 was unchanged in OsHLH61 RNAi plants (data not shown), demonstrating that OsHLH61 located downstream of both AOC and OPR3 in JA synthetic pathway. OsbHLH96 was inhibited by SA (Fig. 6g), while PR genes were induced by SA (Fig. 6f), together with the down-regulation of PR genes in the OsbHLH96 over-expressing lines (Fig. 6b), indicating OsbHLH96 to be a negative regulator of SA signaling pathway in mediating resistance. In this study, we proved that OsHLH61 could interact with OsbHLH96 (Fig. 4a, b). And OsJAZ3, a JA pathway repressor (Chini et al., 2007), might interact with OsbHLH96 (Fig. 4d). Therefore, the OsHLH61–OsbHLH96 complex might mediate the crosstalk between SA and JA in regulating BPH resistance.
Furthermore, OsJAZ3 interacts with OsCOI1a (Os01g0853400) in the presence of 120 μM coronatine (Seo et al., 2011). OsCOI1a-silenced plants are more sensitive to chewing insects (Ye et al., 2012), Meanwhile, OsCOI1a influence the crosstalk between JA and GA (Yang et al., 2012). So that OsHLH61-OsbHLH96-OsJAZ3 might form multimers in stress response and plant hormone crosstalk.
Some TFs regulate PR genes directly (Zhang et al., 2003; Chern et al., 2014). Although expression of some PR genes were down-regulated in the OsHLH61 RNAi plants and the OsbHLH96 over-expressing lines (Fig. 5; Fig. 6f), direct regulation of OsbHLH96 to the PR genes was not detected (Fig. 6c, d). There might be other TFs to regulate PR genes instead. In the OsHLH61 RNAi plants, expressions of more than 30 TF-encoding genes were influenced (Additional file 2: Table S2). These TFs might be powerful candidates for direct regulation of PR gene.
In this study, OsHLH61 RNAi plants were more susceptible to BPH than WT. OsHLH61 and OsbHLH96 can form heterodimer in functioning, and regulate the expression of PR genes positively and negatively respectively, their antagonism in regulating PR genes might be important to the understanding of the crosstalk between SA and JA signaling in mediating BPH resistance.
Materials and methods
Plant and insect
The WT rice variety is ZH11 (Oryza sativa L. subsp. japonica cv. Zhonghua No.11). Rice plants were grown in a greenhouse at 28 ± 2 °C with a 12-h light/12-h dark cycle, and 70%–80% relative humidity. The BPH population was originally obtained from rice fields in Songjiang, Shanghai, China, and maintained on plants in a climate-controlled room at 26 ± 2 °C, 12-h light/12-h dark cycle and 80% relative humidity, or in the field under natural condition.
BPH performance measurements
Individual plant test was carried out at seedling stage using at least six replicates of each cultivar or line as previously described (Wang et al. 2012; Zhao et al. 2016). Each seedling about 3-week-stage was put under a plastic cage (diameter 4 cm, height 8 cm, with a breather window) infested with 12–18 s-instar BPH nymphs. Plant damage levels were observed daily, and 6–9 days later, the plants were scored as susceptible (dead) or resistant (alive).
To construct the OsHLH61-RNAi plasmid, a 273-bp gene-specific fragment of the OsHLH61 coding sequence was amplified and cloned into PTCK303 vector in sense orientation by BamHI and KpnI, and in antisense orientation by SacI and SpeI.
To construct OsbHLH96 over-expression plasmid, the full-length OsbHLH96 was cloned into the BamHI and KpnI sites of pCambia1301-35SNOS vector, and got bHLH96OE plasmid.
Constructs used for rice protoplast transfection was generated with pA7-YFP, OsHLH61 cDNA sequence was cloned into the vector with pA7-HLH61-YFPF and.
pA7-HLH61-YFPR primers. For construction of OsHLH61p::GUS plasmid, a 2.27 kb promoter of OsHLH61 was cloned into p1300GUSNOS vector.
All the plasmids for genetic transformation were transformed into ZH11 using Agrobacterium-mediated method (Hiei et al., 1994). GUS activities were histochemically detected as described (Jefferson 1989).
For yeast two-hybrid analysis, the coding sequences of OsHLH61 and OsbHLH96 were cloned into pGADT7 and pGBKT7 vectors, the coding sequences of OsJAZ1, OsJAZ3, OsJAZ5, OsJAZ7, OsJAZ9, OsJAZ11 and OsJAZ12 were cloned into the pGADT7 vector. For BiLC analysis, the coding sequences of OsbHLH96 and OsHLH61 were cloned in-frame into the KpnI and SalI sites of 771-nluc and cluc-772 vector respectively.
For Dual-Luc analysis, the 2 kb promotor of OsPR1a, PR1L, OsPR5, OsPR10a and OsAOS2 were cloned into the KpnI and BamHI sites of pGreenII vector. The bHLH96OE plasmid and these plasmids were used for Dual-Luc analysis analyses (see the following).
Rice protoplast transformation
Rice protoplast transformation was performed by using polyethylene glycol -mediated transfections as described (Zhang et al. 2011). The YFP fluorescence signals for each combination were detected using an inverted confocal microscope (Olympus FV1000) 16 h after incubation. YFP fluorescent and chlorophyll auto-fluorescent signals were imaged at 514 nm, 527–532 nm and 650–798 nm respectively.
For BPH treatment, plants about 2-week-old were infested with second-instar BPH nymphs after starvation for 2 h at a rate of 5 insects per seedling, and stem were collected after 0, 3, 6, 12, 24 and 48 h. For phytohormone treatment, rice plants were sprayed with 4 mL of MeJA (400 μM), OPDA (100 μM), or SA (500 μM) solution in Dimethyl sulfoxide (DMSO), which was sprayed as control. Plant samples at 0, 0.5, 1.5, 3, 6, 12, 24 and 48 h were collected and stored at − 80 °C before RNA extraction.
Quantitative real-time PCR (qRT–PCR) analysis
Stem of 14-day-old WT plants were used to examine transcript levels of target genes before/after plants were infested with BPH. Three independent biological samples were used. Total RNA was isolated by using Trizo (Thermo). 1 μg of total RNA was reverse-transcribed using the First Strand cDNA Synthesis Kit (Toyobo), according to the manufacturer’s protocol. The qRT–PCR was performed with the SYBR Green Real-time PCR Master Mix Kit (Toyobo). Ubiquitin (Os03g0131300) was used as an internal standard to normalize cDNA concentrations.
Data differences in different lines or treatments were determined by analyzing variance followed by Student’s t-test. All tests were carried out with GraphPad Prizm (https://www.graphpad.com/scientific-software/prism/).
RNA-seq and analysis
Seedlings of non-infected HLHR4 and ZH11 plants, and of BPH infected HLHR4 plants were collected and RNAs extracted. Library was constructed, and sequencing was performed on a BGISEQ-500 and analysis was carried out under the help of Beijing Genomic Institution (www.genomics.org.cn, BGI, Shenzhen, China).
A phylogenetic tree was constructed using MEGA 6.0 (http://www.megasoftware.net/index.html) and the NJ method with the following parameters, Poisson correction, pairwise deletion, and bootstrap (500 replicates; random seed).
Yeast two hybrid screening
The yeast two-hybrid screening was carried out as described by Clontech (https://www.takarabio.com/products/protein-research/two-hybrid-and-one-hybrid-systems/yeast-two-hybrid-system/matchmaker-gold-yeast-two-hybrid-system). The vector pGBKT7-OsHLH61 was transformed into AH109 instead of Y2HGOLD. The mated culture was plated on the medium lacking leucine, tryptophan, histidine and adenine QDO (SD-LTHA) agar plates, the culture medium plates were incubated for 3–5 d, then put the colonies into new plates QDO for 3–5 d, yeast colony PCR were carried out and the PCR products sequenced.
Yeast two-hybrid assay was carried out using the lithium acetate/single-stranded carrier DNA/PEG method (Gietz and Schiestl, 2007).
Bimolecular luciferase complementation (BiLC) assay and dual-luciferase (dual-Luc) reporter assay
BiLC assay was carried out as described (Liu et al. 2018). OsbHLH96 was fused to nLUC, OsHLH61 was fused to cLUC. Both constructs were transformed into Agrobacterium strain GV3101. Overnight cultures were collected by centrifugation, re-suspended in MES buffer (10 mM MES pH 5.6, 10 mM MgCl2, 150 mM acetosyringone) to an OD600 of 1.0, mixed, nLUC and cLUC, bHLH96-nLUC and cLUC, nLUC and cLUC-HLH61 were used as control, and incubated at room temperature for 2–3 h. The Agrobacteria suspension was drawn into a 10 mL syringe (without the needle) and press carefully by hand into healthy leaves of 3-week-old N. benthamiana plants. The plants were left under continuous white light for 2 d after infiltration. 1 mM luciferin was infiltrated before the LUC signal was photographed with a cool CCD camera (Tanon 5200).
For Dual-Luc assay, OsbHLH96 and OsHLH61 were cloned into p1301-35SNOS vector, and the promotors of PR genes were cloned into pGreenII0800. Constructs for tests were then transformed into Agrobacterium strain GV3101 containing the pSoup-P19 plasmid (Hellens et al. 2005). LUC/RLUC ratio was measured using the Dual-Luciferase® Reporter Assay System kit (Promega).
The primer sequences used in this study were listed in Additional file 3: Table S2.
Sequences in this study can be found in the National Center for Biotechnology Information (NCBI) OsHLH61 (Os07g0676600); OsbHLH96(Os03g0188400); OsJAZ3(Os08g0428400); OsPR1a (Os07g0129200); PR1L (Os07g0127500); OsPR5 (Os04g0689900); OsPR10a(Os12g0555500); OsAOS2 (Os03g0225900); OsJAZ1(Os04g0653000); OsJAZ5(Os04g0395800); OsJAZ7(Os07g0615200); OsJAZ9(Os03g0180800);OsJAZ11(Os03g0180900); OsJAZ12(Os10g0392400); Ubiquitin(Os03g0131300).
Allene Oxide Synthase 2
Quantitative real-time PCR
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We are grateful to Dr. Hongtao Liu for providing the Dual-Luc system.
This work was supported by the National Transgenic Great Subject from the Ministry of Agriculture of China (2016ZX08009–003-001), the National Natural Science Foundation of China (31371949 and 31870232), and the grant from State Key Laboratory of Hybrid Rice (KF201805).
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Cite this article
Wang, M., Yang, D., Ma, F. et al. OsHLH61-OsbHLH96 influences rice defense to brown planthopper through regulating the pathogen-related genes. Rice 12, 9 (2019). https://doi.org/10.1186/s12284-019-0267-0
- Oryza sativa
- Transcription factor
- Brown planthopper
- PR gene

domain: literature


text:
- The list below includes 604 free or cheap things to do in or near Palo Alto, California, including 126 different types of inexpensive activities like Parks, Nature & Wildlife Areas, Movie Theaters and Hiking Trails.
- From Stanford University to The Dish - Stanford Foothills, there are a variety of budget-friendly attractions in Palo Alto and in nearby cities within 25 miles like Pacifica, Half Moon Bay, San Jose and Los Altos.
67% 3 votes
401 Quarry Road, San Carlos, CA 94070 MoreLess Info
Filled with wall to wall trampolines, Rockin' Jump - The Ultimate Trampoline Park offers visitors several jumping arenas, from foam pits to a trampoline dodge ball zone. On Fridays, the park hosts a Rockin' Fridays night with a DJ.
50% 2 votes
2451 San Carlos Avenue, San Carlos, CA 94070 MoreLess Info
The San Carlos Avenue Neighborhood Park is a neighborhood park that includes an open turf area, a drinking fountain, picnic tables, benches, and barbecue stations. The park also contains a swing set and climbing obstacles for children.
Situated on 11.5 acres, Serra Park features a small creek, four picnic sites, a children's playground, skating rink and water play area. The park has open grass fields for recreational activities and provides access to adjacent sports fields.
78% 23 votes
550 E Remington Drive, Sunnyvale, CA 94087 MoreLess Info
Sunnyvale Community Center is a unique complex that features general recreation like gym programs and an indoor sports complex. The site also features a creative arts center and a performing arts center with a 200 seat theater.
93% 278 votes
8000 Patterson Ranch Road, Fremont, CA 94555 MoreLess Info
At the Coyote Hills Regional Park, visitors can go picnicking, camping, hiking, and bicycling. The location's staff also offers naturalist programs that cover subjects like Native American history and birds and butterflies.
Spanning a total of 5-acres of land, the Civic Center Park is a recreational park that includes multiple play structures for children of all ages. The park also has a basketball court, a par-3 golf course, and picnic tables.
Safari Run is a children's recreation center that specializes in hosting jungle themed parties and events for kids. The facility offers numerous play areas and attractions, as well as providing food and refreshments for party-goers.
Following a small brook, the Twin Pines Park offers roughly 10 acres of recreational activities. On the grounds of the park, there are multiple shaded picnic sites, open space trails, a children's playground, and an open use field.

domain: geography


Integral Group, a global network of ‘deep green’ engineers and consultants, is to rebrand as Introba, bringing all 1,100 employees in five countries under the same name.
The group currently operates under different names in Australia, the UK, Serbia, Canada, and the US.
Introba aims to transform the built environment by creating dynamic, living environments that are smart, resilient, and connected. It provides services including district planning, technical design, system integration, and performance optimisation through digital transformation and sustainable strategies.
Founded in 2008 by Kevin Hydes, Integral Group is driving the adoption of sustainable building design, moving beyond even green and net-zero buildings towards regenerative buildings – ones that actually help heal the environment rather than not harm it further.
“Integral Group, with an emphasis on decarbonisation and resilience, has grown into a global firm that offers complete green engineering solutions for some of the most challenging and exciting projects in the world. As one strong organisation, our combined businesses will empower our employees to uplift our shared communities, promote sustainability, and create smart, resilient, and connected built environments,” says Hydes, who will continue to play a crucial part in the leadership of strategic projects and activities as a speciality consultant.
Integral Group one of the pioneers of green building and net zero construction
Integral Group was a founding signatory of the World Green Building Council's Net Zero Carbon Commitment and Carbon Leadership Forum's MEP 2040 Challenge. The firm has also worked on over 100 Net Zero Energy buildings.
Recent projects include:
- Vancouver International Airport
- University of Melbourne
- Shepparton Art Museum
- George Mason University
- Sunshine Coast University Hospital
- Magic Johnson Park & Community Center
“The rebranding represents our reaffirmed dedication to providing our customers with deep technical expertise, cutting-edge innovations, and worldwide resources,” added Integral Group President Bill Overturf.
“Together, we can continue to improve the quality of life for our communities while promoting unity across our company by simplifying processes and procedures for employees to collaborate throughout all our offices.”


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Ahh, Black Friday. The time of year when we, as Americans, are supposed to be thankful for what we have and who we have in our lives, so we spend the time trampling each other for TV deals at Walmart. Come to think of it, Black Friday might be the most American holiday of all. Cynicism aside, Black Friday is actually a good time to snag some great gifts, either for yourself or someone in your life, for great prices.
As car enthusiasts, and smart shoppers, Horatiu and I have been looking at some Black Friday deals for car enthusiasts and found some pretty good ones. So let’s take a look at some interesting deals for the gear head in your life.
Craftsman Mechanics 40-Piece Socket Set
If you’re a used BMW owner, which you likely are if you’re reading this blog, chances are you need some tools. Used Bimmers, especially older ones, need maintenance and, while it’s nice to just take it to the shop, many owners like to wrench their cars themselves (yours included). If you’re just starting out and need a basic set of tools, this Craftsman Mechanics 40-Piece Socket Set is worth a look.
It comes with a 3/8″ ratchet, an extension, a shallow set of sockets for both SAE and metric, as well as a deep set of sockets for both SAE and metric. It also comes with a spark plug socket, which is necessary for any self-wrenching gearhead, all for just $39.98, marked down from its original $60.00.
Ryobi Cordless Tire Inflator
Being able to inflate a tire on the road, or even just in your driveway but without the inconvenience of an extension cord, is great. A cordless tire inflator is one of those tools that you might not think is necessary but, once you use one, you never want to go back. This Ryobi cordless tire inflator can inflate tires up to 150 PSI, comes with a digital gauge, a comfortable pistol grip handle, comes with a few different nozzles, and even a little compartment to store said nozzles. All for just $28.
Of course, you’re going to need a Ryobi 18-volt battery, as this is a tool-only buy. However, the Ryobi battery platform is one of the more common tool battery platforms on the market and worth using, even for simple stuff like this.
Zojiroshi 16oz Stainless Steel Mug
Our very own Chuck Vossler did the most important test of 2021 — which is the best stainless steel coffee mug for your car? Of them all, there were two winners; the Yeti Rambler and the Zojiroshi. However, we’re going with the latter of which, as it’s thin enough to even fit into the pathetic cupholders of older BMWs and it’s currently on sale from $45.99 (which is pricey for a coffee mug) to $25.99.
In Chuck’s testing, the Zojiroshi also leaked the least amount of heat out over an hour and was the best fit for cupholders. If you want to upgrade your coffee intake (or that of your loved one’s) on the morning commute, this mug will do the trick on a budget.
Kershaw Drivetrain Drop Point Pocket Knife
I personally always keep a pocket knife on me. Not to be some try-hard Rambo wannabe but because knives genuinely come in handy all the time. That’s especially true while in the car, as a knife can be used to cut rubber hose in the event of a breakdown, cut a seatbelt in the event of an accident, or even just cut a zip-tie or box. This Kershaw Drivetrain Drop Point pocket knife was especially designed to be used for cars, thanks to its designated seatbelt cutter and even glass breaker.
The knife features a drop-point blade, Kershaw’s SpeedSafe assisted opening, a non-slip nylon handle, and D2 steel for the blade. I’ve had several Kershaw knives before and they always hold up well. This one is a great knife to keep in the car, in case of an emergency, and can be had for $42.25, down from $79.99.
Anker ROAV 800A Jump Starter
We’ve all been there; left the lights on and the next morning your battery is dead. Or maybe you’re battery’s just old and it dies while you’re at the office, leaving you stuck. This Anker ROAV 800A Jump Starter will have your car back up in running in minutes, without the help of another car. The days of using jumper cables and having to borrow someone else’s car to jump start your batter are over. Now, these tiny, light, and shockingly (pun intended) portable jump starters can do the trick on their own and they’re so much easier to use.
This one from Anker is capable of 800 amps, which is far more than enough to jump start even big diesel engines. It’s on sale right now from $99.99 down to $79.99.
The Motorized Coffee Club is an exclusive club where members receive coffee and access to special promotions at dozens of affiliate stores for car enthusiasts. This Black Friday, they offer a $1 Coffee Club Subscription and buy 1, get half off coffee and track mugs. Their coffee packs also come with some cool names: Deep Tracks, Tuned, Supercharged, Race Fuel, High Boost and more. Use code BMWBLOG for 15% OFF sitewide
Disclaimer: This post might contain affiliate links for Amazon

domain: hobby


text:
Editorial: A holistic remembrance
The world was left in shock when news of famed basketball player Kobe Bryantâ€™s death broke on Sunday, Jan. 26. Fans quickly took to social media to express their sorrows and give their condolences, celebrating his professional legacy and praising the positive impact he has had on millions.Â
However, over 10,000 people turned this public outpouring of emotion harmful by sending death threats and abusive messages to Washington Post reporter Felicia Sonmez when she tweeted a 2016 article written about the rape allegations made against Bryant shortly after the news of his sudden death. Sonmez was referencing the 2003 sexual assault case filed against Bryant, for which he settled out of court after the accuser agreed to drop the lawsuit in exchange for a public apology. In his statement, Bryant apologizes to his accuser, admitting that he understands â€śhow she feels that she did not consent to this encounter.â€ť As a result of her tweet, Sonmez received backlash from masses of people claiming it was inappropriate and was subsequently suspended by the Washington Post.
This decision was ultimately overturned after a petition signed by 300 of her fellow journalists called for her reinstitution. In spite of oneâ€™s partiality, there is value in recognizing shortcomings in public figures and understanding the consequences of certain actions regardless of how diminutive they seem in the face of an illustrious legacy. We, at the Hoofprint, believe that truth is a necessity that cannot be compromised for comfort.
First and foremost, it is a journalistâ€™s responsibility to represent all of the facts, no matter a personâ€™s status or fame, and this open-mindedness should be reflected in fans and civilians alike.Â
In covering Bryantâ€™s death and legacy, we should acknowledge his life in its totality â€” both good and bad. One should recognize his athletic achievements as well as advocacy for women athletes, but that should not negate his past nor excuse him from his sexual assault case.Â
We understand the need to remain sensitive to those affected by Bryantâ€™s death, especially those who knew him personally. Much of the criticism against bringing awareness to his sexual assault case stems from the argument that it is inappropriate to scrutinize his past so soon after his death. But, this begs the question: When is the right time?Â
In remembering a legacy, one cannot amplify someoneâ€™s achievements while silencing their malfeasance. Doing so would suggest that a certain level of public esteem exempts people of charges, even in the case of serious felonies such as rape.Â
Honoring oneâ€™s memory and acknowledging oneâ€™s mistakes should not be viewed as mutually exclusive. Rather, moral responsibility starts with seeking out truth for oneself and subsequently re-examining the legacy people leave.
By the Head Editorial Board

domain: law


text:
Talkhab-e Hamzeh Ali
|Talkhab-e Hamzeh Ali
تلخاب حمزه علي
|Time zone||IRST (UTC+3:30)|
|• Summer (DST)||IRDT (UTC+4:30)|
Talkhab-e Hamzeh Ali (Persian: تلخاب حمزه علي, also Romanized as Talkhāb-e Ḩamzeh ‘Alī) is a village in Kushk Rural District, Abezhdan District, Andika County, Khuzestan Province, Iran. At the 2006 census, its population was 101, in 18 families.
- Talkhab-e Hamzeh Ali can be found at GEOnet Names Server, at this link, by opening the Advanced Search box, entering "10878655" in the "Unique Feature Id" form, and clicking on "Search Database".
- "Census of the Islamic Republic of Iran, 1385 (2006)". Islamic Republic of Iran. Archived from the original (Excel) on 2011-11-11.
|This Andika County location article is a stub. You can help Wikipedia by expanding it.|


text:
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All people looking in Brisbane or more in Brisbane City, Brisbane City, Spring Hill, South Brisbane, South Brisbane


text:
You Searched For: Combined Gel Staining and Blot Processing Systems
Combined gel staining and blot processing systems simultaneously run and stain multiple specimen. The workstations program and store multiple application protocols allowing automated preruns, loads, and runs for maximum working efficiency. Able to control the temperature, voltage, and current parameters, the flexible machines guarantee that the same precise and consistent conditions will be applied to each sample. Eliminating extensive personnel involvement requirements, the combined gel staining and blot processing systems are more efficient choices than traditional methods.
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domain: chemistry


text:
Tuesday, December 25, 2012
A Lie Travels Half-way Around the World Before Truth Laces Up Its Boots
Scientists Report Faster Warming in Antarctica
So one might believe from this headline that the story is thus-- scientists have measured the temperature in Antarctica and found a warming trend from those actual measurements. Even the early body of the story would cause one to believe that.
A paper released Sunday by the journal Nature Geoscience reports that the temperature at a research station in the middle of West Antarctica has warmed by 4.4 degrees Fahrenheit since 1958. That is roughly twice as much as scientists previously thought and three times the overall rate of global warming, making central West Antarctica one of the fastest-warming regions on earth.You have to read on to find out what really happened.
The new research is an attempt to resolve a scientific controversy that erupted several years ago about exactly how fast West Antarctica is warming. With few automated weather stations and even fewer human observers in the region, scientists have had to use statistical techniques to infer long-term climate trends from sparse data.A nearby area called the Antarctic Peninsula, which juts north from West Antarctica and for which fairly good records are available, was already known to be warming rapidly. A 2009 paper found extensive warming in the main part of West Antarctica, but those results were challenged by a group that included climate change contrarians.To try to get to the bottom of the question, David H. Bromwich of Ohio State University pulled together a team that focused on a single temperature record. At a lonely outpost called Byrd Station, in central West Antarctica, people and automated equipment have been keeping track of temperature and other weather variables since the late 1950s.It is by far the longest weather record in that region, but it had intermittent gaps and other problems that had made many researchers wary of it. The Bromwich group decided to try to salvage the Byrd record.They retrieved one of the sensors and recalibrated it at the University of Wisconsin. They discovered a software error that had introduced mistakes into the record and then used computerized analyses of the atmosphere to fill the gaps.
Much of the warming discovered in the new paper happened in the 1980s, around the same time the planet was beginning to warm briskly.
Oh, so there were no new measurements. As an unknown blogger at Watt's Up With That eloquently wrote:
They can’t find any recent warming, so they took a broken sensor with “intermittent gaps and other problems”, “recalibrated” it, “used computerized analyses of the atmosphere to fill the gaps” and “discovered” warming that “happened in the 1980s”.If that is valid science, I'm Lord Kelvin.
The fact is a very small part of Antarctica, the Antarctic Peninsula has warmed (relatively), that is, the temperatures went from on average -50 degrees C to an average -49 degrees C, over many decades. The overwhelming bulk of the southernmost continent is as cold or colder than "normal." The seas around the continent have undoubtedly been getting colder and are producing ever more sea ice than "normal." (This despite the fact that believers in man-caused global warming have repeatedly told us that the effect of our CO2 would be amplified at both poles). The sea ice around Antarctica has this past year been constantly above normal, sometimes well above it. It's difficult to square that fact with claims of warming, such as it is, anywhere on the continent itself.
The excuse the alarmist use for the Antarctic temperature record not rising is that the seas around the continent act as a heat sink for the amplified, CO2 caused heat in the troposphere. But if that is true, then the Antarctic sea ice should be decreasing with every warmer surrounding seas. That's not happening, thus, their panic to find warming, any warming, even doubtful reconstructed warming, in Antarctica.
"...it doesn't matter how beautiful your theory is, it doesn't matter how smart you are -- if it doesn't agree with experiment [or real world data], it's wrong."
UPDATE: Here is a temperature map of Antarctica with the "reconstructed" Byrd station data incorporated. We tend to believe what's easiest to believe. Is it easier to believe the "reconstruction" was mishandled or easier to believe that, for no apparent reason, one single area of Antarctica is much, much warmer than all the rest? I think you know the answer.

domain: news


Chevrolet Corsica Engine Accessories
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Anthony Wright is executive director of Health Access California, the statewide health care consumer advocacy coalition. He blogs daily at the Health Access Weblog and is a regular contributor to the Treatment.
While there are several benefits from health reform that kick in this year, the common understanding is that the core coverage expansions will not take place until 2014. However, starting this month, April 2010, states have the option of expanding Medicaid to low-income individuals early--and getting at least 50 percent matching funds from the federal government.
Most states are in deep budget crises, and probably won't consider expansion. They were already mulling cuts instead. Here in California, with the particularly tough economic downturn, Governor Schwarzenegger has actually proposed taking more than 1.5 million people off Medicaid and eliminating our version of the Children's Health Insurance Program, Healthy Families, which covers another million children. These cuts have thankfully been prevented by the "maintenance of effort" requirements in first the stimulus act and now the new health reform law. (Cuts to Medicaid benefits and other health and human service programs are still pending.)
As I wrote earlier, this is a turnaround for Governor Schwarzenegger, who had his own state-based health reform a few years ago which, like the new federal health reform, would have expanded Medicaid to 133 percent of the poverty level and brought in poor adults without children at home. Back then, he extolled the virtues of expanding Medicaid and bringing in those federal matching funds to our state's health system, money that otherwise would be "left on the table" in D.C.
In fact, there's a powerful argument that states should expand, not cut, public insurance programs right now--despite the recession and budget deficits. The most obvious case is that there is greater need--the whole point of a social safety net is to be there during tough economic times, when people are less likely to have private, employer based coverage. But expanding coverage can also speed economic recovery. Too many elected leaders see Medicaid merely as a budget burden, rather than as a way to bring federal funds into their state economies and thus spur economic growth.
In fact, nothing else a state can do can have such a positive economic impact. Medicaid spending provides more “bang for the buck” economic impact than other state fiscal decisions, according to a recent University of California-Berkeley report. Put another way, spending (or cutting) a billion dollars in Medicaid will create (or eliminate) over 35,000 jobs. In comparison, an upper-income tax increase (or cut) has a much smaller 6,400 “jobs per billion” impact, and an oil severance tax even less.
Spending more on health and human services gives the economy a bigger boost not just because of the federal matching funds coming into the state, but also because of the “multiplier effect” it has on local economies. Health services, by definition, can’t be outsourced out-of-state. Also, the benefit goes to low-income patients and middle-income health workers. Both tend to spend the money immediately, recycling it into the economy.
Governors should not see Medicaid as a spending item that they want the “flexibility” to cut, or even just as a core safety-net program that it especially necessary during these tough times, but as the center of an economic recovery strategy. Hopefully health reform, through matching dollars or maintenance of effort requirements, will help not just expand Medicaid, but transform the thinking of state leaders in realizing the program's potential.

domain: economics


Some of this is the fault of the Indianapolis Colts. Their approach on Andrew Luck's recovery from shoulder surgery has been to hide him away like some museum antique in the process of being restored, the public unveiling delayed until all the cracks are properly repaired.
Here was coach Chuck Pagano's response when asked Wednesday if Luck would practice this week: "No."
Here was Pagano's response when asked Sept. 4 if Luck would be able to throw to receivers: "Anybody that comes off of PUP is eligible to practice."
Here was Pagano's response when asked Aug. 24 if he were surprised Luck was not yet practicing: "He's going through rehab. He's sticking with the process. When those docs and trainers tell us he's fine and he's ready to go, then you guys will see him. We'll all get to see him."
Luck is the cornerstone of the Colts franchise, a quarterback whose first three seasons all ended in the NFL playoffs, and the Pro Bowl. So when there is a vacuum of information created regarding such an important player, it seems almost inevitable it will be filled, quite possibly with a truckload of hooey.
But who saw this coming?
"I'm not sure if I want to use the word 'rumor' … there is sort of a general sense that you hear from people around the league, if you talk to people who cover the sport of football right now, that there is an ever-widening gap between Andrew Luck and the Indianapolis Colts. That his unhappiness and, perhaps, some familial unhappiness stems back beyond this. That maybe he felt he played when he wasn't 100 percent healthy.
"And there's some talk that - even that there's some talk with that enormous contract and his enormous talent that we could be seeing right before our eyes the end of the Andrew Luck era in Indianapolis."
Now, if all of that had not been said on ESPN's airwaves - television and radio - by veteran broadcaster Mike Greenberg, it might have been flushed down the nearest commode, where it belongs. But Greenberg said it, so it did not go unnoticed.
Here's the thing. I have Google on my computer. I'd imagine they have it at ESPN HQ, too, but I've not visited in more than a decade, so I cannot be 100 percent certain. But let's just assume they do, for the sake of argument. I typed "Andrew Luck contract" into my magical Google machine after learning of this kerfuffle, and here is what it told me:
Andrew Luck's contract with the Colts, which will pay him $19.4 million this year, does not expire until after the 2021 season. So that's 2017, 2018, 2019 - that's five more NFL seasons.
What are we doing here, people?
Let's suppose Luck is experiencing some degree of unhappiness with the Colts. That seems plausible, given they've done a poor job building him an offensive line, the team looks to be terrible and he currently is unavailable to help fix this mess. What are his options?
1. Free agency? Sure, when he's 32 years old.
2. Forcing a trade? OK, name the last in-his-prime, top-of-his-game, under-longterm-contract franchise quarterback who successfully engineered a deal that got him out of an undesirable situation. Excellent QBs are too rare, too valuable, to allow them to simply walk away to another team. And think about it: The people who let franchise legend Peyton Manning go near the end of his career in order to get their hands on Luck - and the club that once allowed itself to be steamrolled in the draft by John Elway - those people are not going to willingly blow up this deal. It would take a decade to recover the goodwill of the fan base.
3. Retirement? OK, so the guy who played last season with agony oozing from his shoulder is going to walk away from that competitive drive - and just under $52 million he'll earn in the next two seasons alone?
There is no avenue toward a divorce, presuming Luck is seeking one.
The agent who negotiated that fat contract for Luck, Will Wilson, got on the phone right quick Wednesday to columnist Bob Kravitz of WTHR.com. Kravitz didn't even have to call him.
"Quite simply and succinctly, there's no truth to that comment at all," Wilson told him. "Andrew did a long-term deal, and he did it for a reason: He wants to be with the Colts and he's committed to the Colts. It's a simple as that."
The oddest thing about the Greenberg report, if we're going to call it that, is that ESPN pays serious money to people like Adam Schefter to suss out such news, and they've got Mike Wells right there in Indianapolis. The Indianapolis Star has two excellent beat writers covering the Colts, Stephen Holder and Zak Keefer. Mike Chappell, now working for the city's Fox and CBS affiliates, jokes he has been covering the Colts since the Mayflower trucks arrived in 1984. And Kravitz is so wired to everything that happens with the team he happens to be the gentleman who broke the Deflategate story.
All the people with ears, and fingers, and no one heard enough to type anything similar to what Greenberg claimed to be common knowledge among NFL folks.
That's weird, isn't it?


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Midfielder Richie Wellens has signed a new three-year contract with Doncaster Rovers.
The 28-year-old joined Rovers last summer from Oldham Athletic and helped the club gain promotion to the Championship via the play-offs.
Wellens told the club's official website: "It's a good club. It's a club that's going places and I was more than happy to add on an extra two years to my current deal.
"The way we play football is one of the main reasons I wanted to stay.
"The manager is great in getting us to play the way he wants to play, he understands players very well. I'm more than happy here."
Meanwhile, Rovers boss Sean O'Driscoll has decided not to offer a contract to veteran defender Steven Pressley, who had been on trial at the Keepmoat Stadium.


Stephen Malkmus Biography »
Stephen Joseph Malkmus (born May 30, 1966) is an American musician best known as the lead singer and guitarist of the indie rock band Pavement. He currently performs with Stephen Malkmus and the Jicks. Still in Rock said that "Stephen Malkmus is one of only a handful of 90's songwriters whose music has overcome the ravages of time, alongside Thurston Moore, Robert Pollard, Doug Martsch, and a few others".
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The outgoing national security adviser stressed that Moscow’s actions should not go unanswered
Lieutenant General G. R. McMaster on Tuesday delivered a speech at the conference of the Atlantic Council on the occasion of the 100th anniversary of the partnership between the United States and the Baltic States, which also were made by the presidents of Latvia and Estonia and the Minister of foreign Affairs of Lithuania.
In his last speech as national security adviser McMaster has criticized Russia for “increasing aggression around the world.”
“We were not able to force Russia to pay dearly enough,” said he, adding that because of this “arrogance of the Kremlin’s growing.”
“Russia uses old and new forms of aggression with the purpose to undermine our open society,” the General said McMaster.
“Some countries have for too long closed its eyes,” he added, listing a number of Russia’s actions, including cyber attacks against the U.S. and its NATO allies and unsafe intercepts of US military aircraft and NATO.
The McMaster praised the reaction of the administration trump to Russia’s actions, especially coordinated expulsion of diplomats in response to the incident in Salisbury.
According to him, expelled diplomats helped to carry the Russian intervention in American politics, and their expulsion from the country, along with the closure of the Consulate in the US will limit the ability of Russia to disseminate propaganda and disinformation and interference in American elections.
“The US President, trump ordered to send dozens of employees of the Russian intelligence and to close the Russian Consulate in Seattle. This step will also help to protect our democratic institutions and processes, because these people organized the Russian campaign of propaganda, disinformation and political sabotage,” said McMaster.
He also gave a warning about those in the US who tend to “romanticize” such regimes as Russian, and think they have justification.
According to him, trump “has repeatedly voiced the truth about these murderous regimes.” “Mr. Putin may think that he wins in this new type of war… he May have thought that our free nation is weak and does not it will react, he said. But he’s wrong. Russian aggression only strengthens our resolve and confidence.”


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per group (up to 6)
North Side of Lake Union, Near Gas Works Park, 1341 N. Northlake Way, Seattle, WA, 98103 When looking at the front door for the Divers Institute, go to the left side of the building and down into the parking lot. Please arrive 15-20 minutes early as we leave right on time
Admission Ticket Free
Space Needle (Pass By)
Gas Works Park (Pass By)
Saint Mark's Episcopal Cathedral (Pass By)
You can cancel up to 24 hours in advance of the experience for a full refund.


In northern Iraq, where soldiers are battling the self-declared Islamic State, or ISIS, one fiery singer is on a mission to stir up patriotic sentiment.
Crimson-haired Iraqi performer Helly Luv has a recording contract in the U.S., but she didn’t want to shoot her latest video in Hollywood.
Her track, called “Revolution,” is a tribute to the ethnically Kurdish soldiers known as peshmerga who are fighting against ISIS. So she took a video team to a front-line village, as bullets flew and battles raged.
“Yeah!” she tells me. “We shot the video right there, and it was so crazy.”
The video features a bejeweled Helly Luv — herself Kurdish — dancing in a traditional peshmerga outfit, and in a tank firing a shell.
“Rise up, ‘cause we’re so much stronger as one,” she sings in English. “Breaking the silence as loud as a gun. Brothers and sisters, we all come from one. Different religions, we share the same blood.”
In Iraq, A Kurdish Warrior-Diva Sings Against ISIS, Despite Threats


The ready – just like the speaking, fortunately – is sort of over. The buildup to the Rugby League World Cup has been dominated by unfavorable headlines, ill-judged feedback and countless controversies. However now, with every week to go till England kick off the boys’s event in opposition to Samoa in Newcastle, it feels as if we are able to lastly give attention to the rugby in the end.
World Cup warm-ups don’t essentially act as a precursor for what’s going to observe within the event. England slipped up on this very floor in opposition to Italy 9 years in the past, earlier than occurring to make the semi-finals. However on a moist, depressing evening in Salford, this was a efficiency which is able to provide nice encouragement for a showdown in opposition to a Samoa group which are being tipped to trigger an upset in a recreation which is able to in impact determine who wins Group A.
Shaun Wane definitely has meals for thought, not least after an eye catching worldwide debut from Dom Younger. The Wakefield-born winger has been a sensation within the NRL with Newcastle, and after turning down Jamaica to function for England, Younger was topic to feedback from the broadcaster Eddie Hemmings, who mentioned he wasn’t “English within the strict sense of the phrase”. It led to a swift apology however Younger definitely let his rugby do the speaking right here.
He marked his worldwide debut with a try to had a hand in a number of others as England cantered to victory in opposition to Fiji, who’ve reached the semi-finals of the final three World Cups. Granted, this was not their strongest aspect, with a number of key gamers nonetheless making their approach to England for the event. However as Wane mentioned: “That was very spectacular given the situations. I assumed we did very well. A number of the talent was very spectacular.”
Wane was not incorrect. England led 28-0 at half-time and by no means appeared again, with Younger the star of the present in that first 40 minutes. He broke the impasse with a well-taken attempt within the nook earlier than turning supplier to help tries for George Williams and Kallum Watkins. That, mixed with one other debut attempt, this time for Salford’s hooker Andy Ackers, who raced over from close-range, and one for Joe Batchelor, who seized upon an error from Isaac Lumelume, put England in management on the break.
It was a show which left the opposition’s coach impressed, too. “We didn’t get a lot proper however England had been superb,” Brian McDermott, the previous Leeds coach who helps Fiji at this yr’s event, mentioned. “We’ll have a look at it but it surely was a disappointing efficiency from us.” McDermott, as brutal as ever along with his evaluation, was proper. Fiji didn’t deal with the abysmal situations fairly in addition to England, and the hosts took full benefit.
Younger was not the one star on present right here, however with Tommy Makinson and Ryan Corridor his competitors for a spot within the aspect subsequent Saturday at St James’ Park, this was an honest assertion from the 21-year-old. Has he finished sufficient to drive his manner into the 17 for Samoa? “We’ll see,” Wane mentioned. “I used to be very impressed although. I’ll take a look after a overview however there’s an excellent probability he’ll be in.”
The hours main as much as kick-off had been dominated by controversy once more, because the RFL confirmed their chief govt, Ralph Rimmer, wouldn’t face disciplinary motion for disparaging feedback made concerning the Fijians final week. However World Cup organisers will hope that’s now firmly put to mattress, and England did their half in serving to shift the main focus in direction of what ought to be a beautiful and unpredictable event.
They took their foot off the gasoline after half-time. However main 28-0, and with an enormous conflict subsequent week to sit up for, that’s maybe comprehensible. Wane launched a litany of interchanges from the bench and 6 minutes after the restart, Luke Thompson was subsequent to attain after grounding a well-weighted kick from Marc Sneyd, one other who caught the attention on debut.
There have been additionally additional tries from Mike McMeeken, Mike Cooper and Corridor within the ultimate quarter as England shifted via the gears. There can be loads of twists and turns within the weeks forward however when it comes to laying a marker down, this was about as satisfying an evening’s work as Wane might have hoped for.
England Welsby; Younger, Watkins, Farnworth, Corridor; Williams, Sneyd; Hill, Ackers, Cooper, Whitehead, McMeeken, Radley Tries Younger, Ackers, Watkins, Williams, Batchelor, Thompson, McMeeken, Cooper, Corridor Targets Sneyd 7 Interchange Marshall, Powell, Thompson, Burgess, McIlorum, Batchelor, Smith, Wardle, Pearce-Paul
Fiji Naiqama; Qareqare, Ravalawa, Valemei, Vulikijapani; Moceidreke, Wakeham; Vuniyayawa, Tagiuima, Kamikamica, Miller, Seci, Liolevave Interchange Joseph Ratuvakacereivalu, Nakubuwai, Sadrugu, Masima, Wong, Lumelume, Taira


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More than a third of employees believe that flexible working is key to competitiveness in the current digital landscape, according to a new White Paper called Beyond Digital, which has been published by computer software company Infomentum.
The study asked more than 1,000 UK office workers about the current and future state of business, and 40% revealed that they would like to be able to embrace more flexible working hours outside of the traditional office. However, the majority said that they are still not allowed to do so, with 57% admitting that their employer believes that they cannot be trusted to perform their roles if they are outside the office.
There appears to be a gender gap when it comes to attitudes about flexible working, as those surveyed said that men were 12% more likely than women to be given the chance to work from home and 8% more likely to be offered working hours on a flexible basis.
The report noted that there is now a “worrying disconnect between current management and the expectations of the Gen C [digital native] workforce,” which has been caused by disparate company cultures, budget restraints and a lack of clear direction throughout the enterprise.
Looking ahead to 2020 and beyond, 37% of workers said that their organisations would not be competitive in the future if they did not embrace flexible working practices, while 50% think that enterprises need to invest in more innovative technology to keep ahead of the curve. A further 60% believe that the rise of digital and the Internet of Things (IoT) will make their own professions more automated by the end of the decade. Overall, 91% said that their businesses would no longer be competitive if they did not embrace change.
“As we move towards 2020, this constant change is no longer something that businesses must prepare for, but rather a reality that all organisations must embrace,” Infomentum chief commercial officer Vikram Setia said.
He added: “Businesses, employees and even governments will all be affected. Attempting to fight against this torrent of change is to try and fight the future itself. Improvements in technology, automation and efficiency are coming, the only thing we as businesses can do is embrace this fact and attempt to lead the change.”
The report also found that workers are now experiencing an increase in their workloads due to the emergence of flexible working technologies. Almost two-thirds use a tablet, while 56% make video calls and use VOIP on a daily basis.

domain: sociology


Few locations can rival Reading’s rail, road and bus service connectivity
Reading Bridge House is conveniently located just a short walk from Reading station. From there, frequent direct trains into London Paddington take just 23 minutes. Reading is also a major rail hub with services across the Thames Valley and other parts of the country.
The Heathrow RailAir link stops outside the station providing easy access to and from the airport. This runs every 20 minutes and every 30 mins at weekends.
The Elizabeth Line rail service between Reading and London serves a total of 40 stations along the entire route, passengers can easily travel into, and beyond Central London.
Reading also has an award-winning bus service. Its river walkways and cycle paths make green travel a pleasure.
Reading benefits from excellent road connections, served by three motorway junctions (M4 J10, 11 & 12).


Workers and shoppers made dozens of frantic calls to police in the moments after a gunman opened fire in the Garden State Plaza Mall. Here are some of those 911 calls, obtained by NBC 4 New York. Brian Thompson reports. Watch Part 2 here.
Published at 7:12 PM EST on Nov 6, 2013 | Updated at 6:17 AM EST on Nov 7, 2013


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Russia's MTS acquires stake in parent's bank
MOSCOW (Reuters) - Russia's top mobile phone operator MTS said on Friday it has acquired a 25.1 percent stake in MTS Bank for 5.1 billion roubles ($164 million) by buying additional shares issued by the bank.
The deal was concluded in accordance with the terms of an indicative offer between MTS, MTS Bank and their majority shareholder Sistema that were announced in October 2012.
MTS now owns around 27 percent of MTS Bank, Sistema has a 65.3 percent stake, while the balance of shares is held by other minority shareholders, MTS said in a statement.
($1 = 31.0844 Russian roubles)
(Reporting by Maria Kiselyova; Editing by Douglas Busvine)
- Thai PM calls snap election, protesters want power now |
- North Korea says Kim's powerful uncle dismissed for 'criminal acts'
- Protesters fell Lenin statue, tell Ukraine's president 'you're next'
- Storm pushes up U.S. East Coast after deep-freeze in the South
- Venezuela's Maduro to raise pressure on business after local vote
Nelson Mandela: 1918 - 2013
Reuters looks at the life and times of Nelson Mandela, an icon of peace and reconciliation who came to embody the struggle for justice around the world. Video


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Comet Gain album news
We’re very excited to say A Little Orchestra feature on Paperback Ghosts, the latest album from Comet Gain, released on Fortuna POP! records. We play violin and cello on four songs on the record.
The album was recorded at Soup Studios with producer Simon Trought at the helm, and was inspired by the psycho-geography of walks in North London woods and the forgotten grey hinterland of the city’s back streets. Paperback Ghosts is the seventh album from Comet Gain, and comes soaked in autumn melancholy. Tender-hearted but not miserable, defiant but not angry, it maintains the delicate balance that has always been Comet Gain’s strength.
To buy the album, visit the Fortuna POP! website.

domain: entertainment


Hanover Twp. bar burglarized
HANOVER TWP. - Police are trying to find out who burglarized Shmoopy's Bar & Grill overnight on Saturday.
The owner of the Main Street business in the Buttonwood section arrived at 12:40 p.m. Saturday to find someone had entered the bar through a basement window.
Inside, the cash register was open and money stolen, the jukebox was broken open and coins removed from it, and the kitchen had stock knocked off the shelving units. Police said the owner is taking inventory of the stolen items. Anyone with information can call Hanover Township police at 570-825-1254 or 911.


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The Alliance of International Football Coaches is an open forum providing dedicated resources to individuals working in the sport of Football around the Asia Pacific Region and throughout the rest of the world. A wide spread platform for professional development, support, and high-level educational resources for the betterment of the sport in Hong Kong and Asia, AI Football Coaches are leaders in the Football field.
CCW is able to provide comprehensive insurance support to members of the Alliance of International Football Coaches. Please click AI Football Insurance Options for further details of this insurance program.
More information about the Alliance can be found by visiting their website. Please click Alliance of International Football Coaches.

domain: sports


The Judiciary Committee voted in support of the nomination of Solicitor General Elena Kagan to the U.S. Supreme Court seat being vacated by Justice John Paul Stevens, sending the nomination to the full Senate for consideration. On a vote of 13-6, with Sen. Lindsey Graham (R-S.C.) as the only Republican voting yes, the Senate Judiciary Committee took the action after each senator was given time to explain his or her vote.
As with the hearings, much of the Republican opposition circles around Kagan’s dealings with military recruitment at Harvard Law School as it related to the “Don’t Ask, Don’t Tell” policy and her potential views on same-sex marriage.
“I’m concerned with [her ability] to defend the Defense of Marriage Act [as solicitor general]” Sen. Chuck Grassley (R-Iowa) said, noting the Justice Department’s brief defending DOMA included a statement that the Obama administration opposed DOMA as a policy. “How do we believe that she’ll uphold a law as a Supreme Court justice when she disagrees with that law?”
Sen. Jon Kyl (R-Ariz.) criticized the decision not to appeal the Ninth Circuit’s Witt decision, calling her reasoning “hollow.” He also criticized the distinction between Kagan’s response to Sen. John Cornyn (R-Texas) during her solicitor general confirmation hearings about whether there is a constitutional right to same-sex marriage and her answer to similar questioning at this hearing. At that time, she had replied that there was no right to same-sex marriage, which she explained at this hearing as referencing the established law. Now, however, she refused to discuss the matter because, she said, a case on the topic is likely to come before the Supreme Court.
Sen. Lindsey Graham (R-S.C.), the sole Repeublican expected to support the nomination, said on that topic, “If you think same-sex marriage is prohibited by the Constitution, I doubt this is the place you’re going to be saying it, because you’re going to have to be deciding it” eventually.
Graham circles around the military recruitment issue, concluding, “I don’t believe that” Kagan has “animosity” toward the military, calling her a “loyal American.”
He then begins to explain his expected vote for Kagan, saying at one point, “I view my role … in part, protecting the independence of the judiciary. “After the hearing, it was not a hard decision to make” to support Kagan, adding that Obama chose “wisely.”


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Discover Shakespeare Country
Budget full day sightseeing tour to Oxford and Stratford-Upon-Avon including lunch in the Cotswolds including a free hotel pickup!
Oxford- We will enjoy a gentle walking tour through Oxford with our guide through the narrow alleys and ancient squares where time stands still. We will see the Bodleian Library and the beautiful college courtyards.
The Cotswolds- An area steeped in history, rich in heritage and awash with natural beauty, the Cotswolds is a charming blend of bustling market towns, sleepy villages and breathtaking views and natural scenery. An optional lunch will be taken in one of the Cotswold villages
Stratford Upon Avon- A delightful market town located at the edge of the Forest of Arden that was the birthplace of the world-famous playwright, William Shakespeare. We see the half timbered house where he was born in the year 1564, followed by a photo stop at the picture postcard thatched cottage which was the home of his wife Anne Hathaway.
In Stratford Upon Avon you will have a choice of one of the following options:
Option 1* - Entry to William Shakespeare's Birthplace.
* Entrance fee is not included but can be pre-purchased before departing on the tour
Option 2 - Walking tour and explore the historic market town at your leisure.
This tour returns to London at around 6.30pm

domain: library


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WASHINGTON — The Senate Judiciary Committee's Subcommittee on the Constitution, Civil Rights and Human Rights is now just the Subcommittee on the Constitution.
A spokeswoman for Sen. John Cornyn, the number two Senate Republican and chairman of the subcommittee, defended the name change.
"The Constitution covers our most basic rights including civil and human rights," said Megan Mitchell, Cornyn's spokeswoman, in an email to BuzzFeed News. "We will focus on these rights along with other issues that fall under the broader umbrella of the Constitution."
Now that Republicans control the Senate, senators who have ascended to the role of chair on the various committees are free to alter the names if they choose.
A spokesman for Sen. Richard Durbin, the ranking member on the subcommittee, said that despite the name change the committee would still work on civil and human rights.
"The name of a subcommittee speaks to its priorities," Ben Marter, a spokesman for Durbin, said in an email to BuzzFeed News. "Senator Durbin will make sure that civil rights and human rights aren't dropped from Congress' agenda."
A watchdog group, the Leadership Conference on Civil and Human Rights, lambasted Cornyn for truncating the name.
"The new Senate Republican Majority's decision to expunge civil rights and human rights from this subcommittee's name is a discouraging sign given the growing diversity of our nation and the complex civil and human rights challenges we face," said Nancy Zirkin, the executive vice president of the Leadership Conference on Civil and Human Rights. "Names matter. This, after all, is a subcommittee with jurisdiction over the implementation and enforcement of many of our most important civil rights laws."


By JOHN T. WARD
After a post-election lull, Red Bank Democrats plan to bring in a parking consultant to offer guidance on how to fix parking issues downtown, Councilman Erik Yngstrom said Wednesday.
With the Democrats back in command of the borough agenda with a 4-2 majority, Yngstrom, starting his second year on the council., was appointed on January 1 to head the powerful three-person parking committee. He’s backstopped by fellow council Democrats Kathy Horgan and Ed Zipprich, who is also head of the local party operations.
Sidelined in the transition was Councilman Mike Whelan. He had advanced a possible parking garage and ancillary development on the site of the 2.3-acre White Street municipal lot onto the agenda to a degree not seen in more than a decade. But concept plans submitted by developers met resistance because of their size and scope, while opponents also argued that a fresh and comprehensive parking study was overdue.
It appear they’ll now get it. At Wednesday’s council meeting, Yngstrom said the parking committee had held its first meeting a night earlier, when “great ideas about how to improve parking” were discussed, he said.
“However, the first thing that we do want to do is release an RFQ [Request for Proposals] for a parking consultant to come in and evaluate the downtown business district from the train station to Riverview [Medical Center], and provide us with ideas and solutions for our parking problems,” Yngstrom said in a brief update.
“We as council members are not parking experts up here, and we need this data in order to make such a big decision for our town moving forward.”
Yngstrom said the committee will work with the police department, the planning department tand downtown promotion agency Red Bank RiverCenter “to get this right.”
“We’ve been pushing for this study for a year, and believe that once we have this data and a recommendation from an expert, then we can have a game plan to move forward and then fix our parking problems.”
Yngstrom said the committee hopes to have the RFQ ready for introduction at the next semimonthly meeting, scheduled for January 24.
Yngstrom made no mention of a plan to designate a redeveloper for the White Street lot. Two builders, BNE Realty and borough-based Yellow Brook Property, owned by Roger Mumford, were the finalists in a process that began last April with five contenders for the project.


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Task 4 Research
Principal Investigator(s): Shad Nelson
- Develop water conservation methodologies for South Texas citrus production using flood, micro-spray and drip irrigation systems and comparisons. Results for the 1st crop to be available February 2003.
- Prepare N and P fertilization treatments in combination with irrigating on citrus to determine fertility recommendations for citrus production under varying irrigation management. Results to be available February 2003.
- Develop crop coefficients for citrus under varying irrigation methods based on soil moisture monitoring.
- Develop drip irrigation and soil solarization studies on young citrus groves to minimize soil-borne pests (like citrus nematode) while enhancing irrigation efficiency. Results available by Jan. 2003. Publication target in conjunction with previously collected data by Dr. Meni Skaria from the TAMUK Citrus Center of May 2003.
- Use K fertigation through drip irrigation lines to improve phyto-chemical production in citrus fruit while minimizing water loss. Results to be combined and compared to preliminary work obtained by Dr. Bihmu Patil from TAMUK Citrus Center for presentation at professional meetings in 2003.

domain: biology


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At Open Country we value and embrace Equality, Diversity & Inclusion (EDI) and the wellbeing of everyone involved with the charity. We believe in creating a diverse and inclusive culture which supports people to reach their full potential. We have a long history of supporting and campaigning for a disability-inclusive society and have always welcomed people from all walks of life.
In 2022 we commissioned an external body to undertake an ‘Equality, Diversity & Inclusion Gap Analysis’. During this process they consulted with our disabled members, volunteers, staff and trustees through a number of interactive workshops.
As a team we welcomed the findings of the report, as they encouraged us to continue to examine our procedures, services and work practises to identify areas where improvements can be made. We are committed to listening, learning, and improving, so please get in touch with any feedback – positive or negative!
For more information please see our Equality, Diversity & Inclusion Policy.
For the past 25 years that I have been leading Open Country, I have been committed to ensuring that equality, diversity and inclusion has been at the core of everything that we do and that approach has enabled us to embed ourselves into the heart of the communities we serve.
Get In Touch
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