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- American Society of Plant Biologists
Pathogen-inducible plant promoters contain multiple cis-acting elements, only some of which may contribute to pathogen inducibility. Therefore, we made defined synthetic promoters containing tetramers of only a single type of element and present evidence that a range of cis-acting elements (boxes W1, W2, GCC, JERE, S, Gst1, and D) can mediate local gene expression in planta after pathogen attack. The expression patterns of the promoters were monitored during interactions with a number of pathogens, including compatible, incompatible, and nonhost interactions. Interestingly, there were major differences in the inducibilities of the various promoters with the pathogens tested as well as differences in the speed of induction and in the basal expression levels. We also show that defense signaling is largely conserved across species boundaries at the cis-acting element level. Many of these promoters also direct local wound-induced expression, and this provides evidence for the convergence of resistance gene, nonhost, and wound responses at the level of the promoter elements. We have used these cis-acting elements to construct improved synthetic promoters and show the effects of varying the number, order, and spacing of such elements. These promoters are valuable additions to the study of signaling and transcriptional activation during plant–pathogen interactions.
The availability of a range of defined synthetic plant promoters that direct controlled local gene expression in response to pathogens would be a major advance. These promoters could be used to help define signaling pathways, to isolate novel mutants using "targeted genetics" (Hooley, 1998), and to engineer plants with increased disease resistance. The control regions of plant genes are modular and contain a number of cis-acting elements, each of which may contribute to one or more aspects of a complex expression profile. One strategy to overcome this complexity is to produce synthetic promoters containing only defined individual elements, thereby reducing expression profile complexity (Salinas et al., 1992). However, although there are numerous reports of synthetic promoters being inducible by elicitors in transient expression systems (Rushton and Somssich, 1998), in most cases it is not known to what extent individual cis-acting elements retain their functionality in planta when removed from their native promoter context and whether we can use these individual "modules" to make synthetic promoters that direct a desired expression pattern.
Pathogen-inducible promoters represent an attractive system for the production of synthetic promoters. There are a large number of known pathogen-inducible genes (Rushton and Somssich, 1998), and their promoters are among the best studied in plants. Two groups of pathogen-inducible cis-acting elements, the GCC-like elements (Ohme-Tagaki et al., 2000) and the W boxes (Rushton et al., 1996; Eulgem et al., 2000), have been well studied. The GCC box (AGCCGCC) often is found in the promoter regions of defense genes (Ohme-Takagi and Shinshi, 1995). A similar element has been reported to direct jasmonate and elicitor-responsive expression (JERE; AGACCGCC) (Menke et al., 1999), and another (DRE; TACCGAC) directs cold-, salt stress–, and dehydration-responsive expression (Yamaguchi-Shinozaki and Shinozaki, 1994). Recently, another similar GCC-like element called box S (AGCCACC) has been identified that directs expression by fungal elicitors (Kirsch et al., 2000). It appears that minor variations in the core sequences impart responsiveness to different stimuli.
The W box [(T)TGAC(C/T)] is the binding site for members of the WRKY family of transcription factors (Rushton et al., 1996). There is increasing evidence that W boxes are a major class of cis-acting elements responsible for the pathogen inducibility of many plant genes (Raventós et al., 1995; Rushton et al., 1996; Wang et al., 1998). The importance of W boxes was illustrated recently by studies of the Arabidopsis transcriptome during systemic acquired resistance (Maleck et al., 2000; Petersen et al., 2000). In some cases, clustering of W boxes may be associated with inducibility by pathogens.
Given that GCC-like boxes and W boxes have been so well studied, it is surprising that there is almost no direct in planta evidence that they can mediate pathogen-inducible expression. Although W boxes have been shown to impart elicitor-inducible expression on a minimal promoter in transient expression systems (Raventós et al., 1995; Rushton et al., 1996; Eulgem et al., 1999), there is only one report suggesting that isolated W boxes can function alone in planta and direct pathogen-inducible expression (Kirsch et al., 2001). It was shown that the W box–containing promoter element E17 mediates gene expression at pathogen infection sites in transgenic Arabidopsis plants. Data concerning GCC-like boxes are equally scarce. A synthetic promoter containing four copies of a GCC box directs ethylene-inducible expression in tobacco (Ohme-Takagi and Shinshi, 1995), but pathogen inducibility has yet to be shown.
Both GCC-like elements (Suzuki et al., 1998) and WRKY transcription factors (Hara et al., 2000) have been implicated in gene expression in response to wounding. It was shown recently that wound- and pathogen-induced signaling consists of networks with some shared components (Romeis et al., 1999). It remains an open question, however, whether specific cis-acting elements can direct both pathogen- and wound-induced expression in planta or whether these two activities are characteristics of separate elements.
Here, we present a comprehensive study of pathogen-inducible synthetic plant promoters constructed from a range of both well-studied and novel cis-acting elements. At least seven different elements can alone direct local pathogen-inducible gene expression in transgenic Arabidopsis plants. Major differences are seen between many of the elements with regard to their background expression, their induction by different pathogens, and their speed of induction. Additionally, we demonstrate that several pathogen-inducible elements also direct local wound-inducible expression and therefore that components of pathogen- and wound-induced signaling are shared.
Elicitor-Inducible Synthetic Promoters
Our approach to making synthetic promoters that are induced locally by pathogens was first to test candidate elements in a parsley transient expression system for inducibility by a pathogen-derived peptide elicitor, pep25. Promising candidates then were introduced into Arabidopsis plants to evaluate their in planta expression patterns and inducibility by pathogens. Figure 1A shows how the synthetic promoters were constructed. Each element was inserted between the SpeI and XbaI restriction sites upstream of the −46 35S minimal promoter of Cauliflower mosaic virus.
Initially, synthetic promoters containing tetramers of candidate elements were constructed. The reason for the use of tetramers was the observation that promoters with multimers of elements are stronger than those with just one or two copies (see below). Because it was unknown whether any of the elements were functional in planta, we used tetramers in initial experiments to ensure that any expression would be strong enough to be detectable by β-glucuronidase (GUS) staining. The first elements tested were boxes W1 and W2 from the parsley PR1 genes (Rushton et al., 1996), box S from the parsley ELI7 genes (Kirsch et al., 2000), a novel element called box D from the parsley PR2 gene (P.J. Rushton and K. Hahlbrock, unpublished results), and an element we termed the Gst1 box from the potato gst1 gene (Strittmatter et al., 1996). The Gst1 box contains an S box and a W box separated by just 4 bp in a region of the promoter that mediates transcriptional activation in response to pathogens, during senescence, and in root apices (Figure 1B). All of the promoters showed inducibility by pep25 in transient expression studies (Figure 1C), although the strength and inducibility of the elements varied greatly. Tetramers of most elements showed inducibilities of five- to 30-fold, whereas four copies of box S had a remarkably high inducibility (>400-fold), which was attributable to an almost complete lack of expression in the absence of pep25.
Although box S is very similar in sequence to the GCC, JERE, and DRE boxes, they appear to direct different patterns of gene expression (Yamaguchi-Shinozaki and Shinozaki, 1994; Ohme-Takagi and Shinshi, 1995; Menke et al., 1999). To investigate these GCC-related elements, we constructed a series of synthetic promoters that are based on box S (Figure 2A) . To determine the effect of changes within the core sequence, the flanking sequences remained identical and we altered only the bases necessary to change one variant into another. A construct containing the GCC core sequence was slightly stronger than box S but showed greatly reduced inducibility by pep25 (Figure 2B). This reduction to ∼3% of the box S value is remarkable considering that this is the result of a single base pair change and is attributable primarily to an increase in the background level of the GCC box. Results with JERE were almost identical to those with box S, both in strength and fold induction. In contrast, DRE directed a lower level of expression and showed little elicitor inducibility. A nonfunctional version of the GCC box (GCC mut) (Figure 2A) (Ohme-Takagi and Shinshi, 1995) had very low activity but was slightly inducible by pep25. These results show that minor variations in the core sequences of GCC-like elements can have profound effects on both the strength and the elicitor inducibility of these elements.
Expression Patterns of the Synthetic Promoters in Planta and in Response to Wounding
We next addressed the question of whether the synthetic promoters with defined cis-acting elements are functional in planta. Promoters containing tetramers of the elements were introduced into Arabidopsis plants. We first determined the levels of background expression for each construct. The majority of the synthetic promoters had little background expression in leaves (Figure 3A and data not shown). By comparison, the background level of expression was sometimes higher in roots (data not shown), and in the case of 4 × W2, this level was very high. Only 4 × D had no appreciable background expression in any parts of the plant.
Many of the leaves shown in Figure 3A manifest local induction by wounding where the leaves have been excised. This response is very rapid, being induced during the time taken to harvest the plants. Therefore, we wounded leaves by cutting through one-half of a leaf and monitored changes in promoter activity. Except for box D, expression from all promoters was induced locally by cutting (Figure 3B and data not shown). Figure 3B also illustrates that this massive damage to the leaf sometimes led to a lower level increase in GUS expression over the entire leaf (cf. Figures 3A and 3B). However, when plants grown in greenhouse conditions exhibited local wound-induced expression, presumably because of insect damage, we never observed expression in other parts of the leaf (data not shown). This finding suggests that local wounding of Arabidopsis plants leads only to highly restricted expression of these promoters. Only when more extensive damage is done (e.g., severing of vascular tissue) is there a response throughout the leaf and subsequent nonlocal induction.
We observed two different patterns of local wound-induced expression after cutting (Figures 3C and 3D). Most common was expression in a layer of cells at the actual cut site (Figure 3C). However, with the same promoters, we sometimes also observed local expression in a ring of cells some distance away from the cut site (Figure 3D). The exact conditions necessary for the production of each pattern were not investigated further.
Synthetic Promoters That Direct Local Gene Expression in Response to Peronospora parasitica
Having determined the level of background expression with the synthetic promoters, we looked to answer the following question: Do they direct pathogen-inducible expression? To do so, we investigated their expression during interactions with a range of different pathogens. We inoculated with the biotrophic oomycete P. parasitica pv Cala2, which produces an incompatible interaction with Columbia (Col-0) plants (Holub et al., 1994). Local gene expression, limited to small areas around infection sites, was seen with 4 × W1, 4 × GCC, 4 × S, 4 × Gst1 (Figure 4) , 4 × W2, and JERE (data not shown). By contrast, no expression was detectable with 4 × D. Figure 4 demonstrates that although 4 × GCC is pathogen inducible, it has a much higher level of background expression in uninfected parts than does 4 × S, and these data accurately reflect the transient expression data described above (Figure 2). For all of the tested elements except box D, these results demonstrate that these elements alone can direct pathogen-inducible expression in planta when removed from their native promoter context.
Local Expression during Powdery Mildew Challenge
We next investigated the inducibility of the promoters during the nonhost interaction with the barley powdery mildew Blumeria graminis f. sp. hordei. Only four synthetic promoters (4 × S [Figure 5A] , 2 × W2/2 × S/2 × D [Figure 5B], 4 × GST [data not shown], and 4 × W2/4 × S [data not shown]) directed significant local gene expression under the conditions tested. These promoters all contain box S, suggesting that box S may play a role during nonhost signaling. The germinating spores (sp) form appressorial germ tubes (agt), and although most do not penetrate the plant, local gene expression is found predominantly in the underlying mesophyll cells (Figure 5A) just beneath the attempted penetration site, suggesting that recognition events on the leaf surface trigger defense responses in the underlying cells. Importantly, the level of expression from these promoters and the number of expressing cells correlated directly with the extent of differentiation of the fungus. This is illustrated in Figure 5B, where at one infection site, haustorium initial formation has triggered cell death. Here, a larger number of cells show activation of the promoter, and levels of expression are higher than in the two other infection attempts, in which infection progressed only as far as papilla formation.
During the compatible interaction with the powdery mildew Erysiphe cichoracearum, all of the promoters except DRE directed local expression. After 7 to 10 days, areas of powdery mildew growth could be seen, and expression of the synthetic promoters was very high in these infected areas (Figure 5C). When viewed closely, intense GUS staining was apparent in these areas, and a wave of gene expression was apparent in advance of the growing hyphae (Figure 5D).
Local Gene Expression during Interactions with Pseudomonas syringae
We also performed infections with the necrotrophic bacterial pathogen P. syringae pv tomato using two different isolates: a Col-0–compatible isolate (DC3000) and a Col-0–incompatible isolate (DC3000 carrying the avrRpm1 gene) (Debener et al., 1991). Figure 6 shows the expression patterns obtained with some of the synthetic promoters. Expression of 4 × W2 was induced locally within 6 hr of infection with the incompatible isolate (Figure 6A). Expression also was apparent with the compatible isolate, although the level of expression was lower. Similar results were obtained with 4 × JERE (Figure 6B), 4 × W1, 4 × S, and 4 × Gst1 (data not shown).
Although untreated leaves from plants containing 4 × GCC showed some background expression, often this was increased greatly by mock infection with MgCl2 (Figure 6D); it was difficult, therefore, to determine whether induction by P. syringae was occurring. By contrast, 4 × GCC showed clear induction by P. parasitica (Figure 4B), even though background expression was apparent. This finding suggests that the process of injecting liquid into the leaf is itself a sufficient abiotic stress to induce high-level expression of 4 × GCC. Closer inspection of plants containing other synthetic promoters (most notably, 4 × W2, 4 × Gst1, and 4 × W1) showed that background expression levels also are increased sometimes after infiltration of MgCl2 (data not shown). This is similar to the results obtained when a cut was made through one-half of a leaf (Figure 3) and suggests that wounding/stressing a large portion of a leaf can lead to a response in the entire leaf.
Infection of 4 × D–containing plants led to qualitatively different results. No expression was detected in control or mock-infected leaves or with either isolate 6 hr after infection. By contrast, strong local gene expression was seen 24 hr after infection with both isolates (Figure 6C). Again, the level of expression was higher during the incompatible interaction.
Effect of Element Number on Strength and Inducibility
Although the synthetic promoters containing tetramers of elements direct local gene expression upon pathogen attack, many are not yet ideal for our purposes because they have more background expression than desired or they respond to a number of biotic and abiotic stresses. Therefore, we have started to construct improved "second-generation" synthetic promoters using these cis-acting elements as building blocks.
The first parameter investigated was the effect of the number of copies of an individual cis-acting element in a synthetic promoter. We constructed a series of promoters containing one, two, four, and eight copies of an element and tested these by transient assay. Figure 7A shows the results obtained with 1 × W2, 2 × W2, 4 × W2, and 8 × W2. Increasing the number of copies of W2 increased the strength of the promoter progressively. However, 2 × W2 had the best inducibility, because additional copies caused a proportionally greater increase in background expression. Similar results were obtained with boxes S and D (data not shown).
These findings suggest that promoters containing fewer copies of an element may be better suited to mediate pathogen-specific inducibility in planta. Accordingly, we introduced promoters with two copies of elements into Arabidopsis plants. Figures 7B and 7C show a comparison of 2 × S and 4 × S plants treated with P. syringae and demonstrate that the differences between the two promoters that were observed in the transient assay also are apparent in planta. Both promoters were inducible by pathogens, but 4 × S showed a higher background level and clear wound induction where the leaf was excised. By contrast, 2 × S, although clearly pathogen inducible, showed on average less background and in many cases no apparent wound induction.
Spacing Effects and Promoter Strength
Our data show that not all W box–containing synthetic promoters behave similarly. For example, both transient expression experiments (Figure 1) and results from transgenic plants show that box W2 is much stronger than box W1, even though both contain the same TTGACC core element. Therefore, we asked the question: What makes a W box strong or weak? To eliminate possible spacing effects, we made new versions of both box W1 (box W1 new) and box W2 (box W2 new) that are identical in length and have TTGACC core sequences in identical positions (Figure 8A) . The only difference between the new version of box W1 and the original is the addition of the preceding 7 bp from the PR1-1 promoter. To our surprise, both of the new elements were strong, with no detectable difference between 4 × W2, 4 × W2 new, and 4 × W1 new (Figure 8B). The seven additional bases in box W1 new have increased the strength either by adding a positive element or by altering the spacing between TTGACC core elements. Therefore, we made a version of 4 × W1 new in which an unrelated sequence was substituted for the original seven bases (Figure 8A). This construct (4 × W1 5 prime mut) directed a similar level of expression to 4 × W1 new (Figure 8B). Thus, the difference in strength between the two versions of box W1 was not sequence dependent, excluding the possibility that a new cis-acting element was generated. The pronounced difference in strength between 4 × W1 and 4 × W2, therefore, seems to be a spacing effect caused by different distances between core TTGACC elements.
To establish whether spacing from the TATA box had any effect on promoter strength or inducibility, we inserted elements 18 bp farther upstream into the SpeI site instead of between the SpeI and XbaI sites (Figure 1A). Figure 8C shows that this resulted in a small increase in strength but had little effect on inducibility. Placing the elements at other positions farther upstream had little effect (data not shown). Together, our results suggest that positioning an element closer to the TATA box can result in some change in promoter strength but that there is little effect on inducibility.
Box D and the Role of Flanking Sequences
Box D is of considerable interest because box D–containing synthetic promoters have no appreciable background expression or wound inducibility in planta, and they also show different kinetics of induction by pathogens. Box D was discovered as a DNase1 footprint from approximately −76 to −52 in the parsley PR2 promoter (P.J. Rushton and K. Hahlbrock, unpublished results). This region (box D short) (Figure 9A) had a high elicitor inducibility but was weak (Figure 9B). Because the exact extent of the element was unclear, a longer version (box D) containing the next six bases from the PR2 promoter at the 3′ end was constructed (Figure 9A). Box D was almost 30 times stronger than 4 × D short (Figure 9B), although inducibility was reduced as a result of increased background levels. We then constructed a version of box D in which the six additional bases were exchanged (Figure 9B, box D mut), and this was almost identical to box D short (Figure 9A), demonstrating that the considerable increase in strength is a sequence-specific effect that is dependent on the six bases GGAACC. Therefore, we believe that box D consists of at least two elements: an elicitor-responsive element and a positive-acting element. This positive element cannot function alone (Figure 9, 4 × D 3 prime and 4 × D 3 prime mut) but appears to be a coupling element that forms a functional unit with the elicitor-inducible element(s). Alternatively, the shortened version of box D simply may lack crucial 3′ nucleotides of a single element required for selective high-affinity binding of a specific transcription factor, thus allowing binding to other related factors, leading to the observed differences in inducibility.
Promoters with Combinations of Elements
Having taken a reductionist approach, reducing synthetic promoters to a single type of cis-acting element to demonstrate functionality, we next took the first steps toward making improved synthetic promoters that contain more than one type of defined cis-acting element. The question that we asked was the following: What effect does the insertion of a second element have? To answer this question, we inserted four copies of box S either upstream (4 × S/4 × W2) or downstream (4 × W2/4 × S) of 4 × W2. Figure 10A shows that this insertion had little effect on the strength of the promoters and a relatively minor effect on inducibility. Mixing up the elements to make 2 × S/2 × W2/2 × S/2 × W2 led to only a slight increase in promoter strength.
In contrast to the transient expression results, in planta studies produced encouraging results. We introduced two synthetic promoters (2 × W2/2 × S/2 × D and 4 × W2/4 × S) that contain combinations of different elements into Arabidopsis plants. Both showed good inducibility by a range of pathogens and were among the best promoters tested, combining high inducibility with low background (Figures 10B and 10C). It seems likely that all of the cis-acting elements contribute to the overall expression of the synthetic promoter and that promoters containing carefully selected combinations of elements may be among the best pathogen-inducible promoters.
Pathogen-Inducible Synthetic Promoters
In this report, we provide direct evidence that a range of pathogen-inducible cis-acting elements can alone mediate pathogen-inducible expression in planta. When taken out of their native promoter contexts, they retain pathogen inducibility as components of synthetic promoters, directing expression that is local and that correlates with the extent of growth of the pathogen. The cis-acting elements tested fall into three groups: W boxes, GCC-like boxes, and box D. Our observations suggest that binding sites for WRKY (W box) or AP2/ERF (GCC-like box) transcription factors can be sufficient to confer pathogen inducibility on a promoter. This is an important observation because these represent two of the three largest families of plant-specific transcription factors (Riechmann and Ratcliffe, 2000). It will be interesting to extend these studies to include other pathogen-inducible elements such as Myb Recognition Elements (MREs) and as-1–like elements, although evidence suggests that these elements might not be able to function alone (Rushton and Somssich, 1998).
Interestingly, although more than one type of cis-acting element is not required for pathogen inducibility, some pathogen-inducible promoters contain elements of more than one type. An example is the Gst1 box, which contains both a W box and an S box. This places the gst1 gene under the control of both WRKY and AP2/ERF transcription factors. The potato gst1 promoter has been well studied (Strittmatter et al., 1996), and our work provides the first clear evidence of how this gene is activated transcriptionally in response to pathogens. It may be common that signaling pathways operating via different transcription factors can target the same gene; another example is the parsley WRKY1 gene (a W box and a GCC box) (Eulgem et al., 1999).
Our results suggest that defense signaling is largely conserved across species boundaries, because an element from a potato gene (the Gst1 box) is active in a parsley transient expression system and in Arabidopsis plants. In fact, none of the elements tested originate from Arabidopsis promoters, yet all retain their functionality. These results also validate our use of a parsley transient expression system for the initial characterization of elements, because we have successfully identified and characterized numerous pathogen-inducible elements and synthetic promoters in a way that would not have been possible in planta. In almost all cases, elicitor inducibility in the transient expression system is accompanied by pathogen inducibility in planta.
Signaling in Different Types of Plant–Pathogen Interactions
Table 1 summarizes the responses of the synthetic promoters to pathogens under the conditions tested. Importantly, not all of the elements respond in the same way to pathogens and wounding. Even elements that are bound by the same family of transcription factors (e.g., GCC, JERE, S, and DRE) show different expression patterns. This is consistent with reports that these elements may respond to different hormones, for example, salicylic acid (W boxes) (Yang et al., 1999), jasmonate (JERE) (Menke et al., 1999), and ethylene (GCC) (Ohme-Takagi and Shinshi, 1995). Therefore, we have generated a spectrum of synthetic promoters useful for studying plant–pathogen interactions at the molecular level.
During compatible interactions, the synthetic promoters accurately report the in vivo situation in which gene activation is slower and perhaps weaker (Lo et al., 1999). In this context, the observed high-level expression of the synthetic promoters during the compatible interaction with a powdery mildew (E. cichoracearum) illustrates their potential usefulness during plant–pathogen interactions of commercial importance.
Under the conditions tested, only promoters containing box S consistently showed local expression during the nonhost interaction with barley powdery mildew. Box S, therefore, could be a useful tool for studying nonhost responses and for engineering plants with broad-spectrum resistance. These results are remarkable considering that there is only a single basepair difference between box S and the GCC box (Figure 2). This single alteration results in box S having much lower background levels both in the transient expression system and in planta. Given that both elements probably are bound by AP2/ERF transcription factors, experiments designed to identify family members with high affinities for box S could yield useful data concerning signaling during plant defense.
Box D is an extremely interesting novel element, unlike any of the others tested, because it combines an apparent lack of background expression or induction by wounding with strong induction during some, but not all, plant–pathogen interactions (Table 1). Box D has been found to be responsive to P. syringae, E. cichoracearum, the bacterially derived peptide elicitor flg22 (data not shown), and the oomycete-derived peptide elicitor pep25 in protoplasts. Box D also responds with different kinetics than other elements, being induced later than the other elements (Figure 6). The molecular characterization of box D and the identification of its cognate transcription factors would provide new insights into defense gene activation during plant–pathogen interactions.
Pathogen and Wound Signaling through the Same cis-Acting Elements
Recently, work on plant defense signaling has demonstrated the convergence of resistance gene, elicitor, wound, and salicylate responses at the level of mitogen-activated protein kinase (MAPK) activation (Romeis et al., 1999). It is not clear at present whether these signaling pathways merge at the MAPKs or upstream thereof or whether the same MAPK can mediate disparate responses by interacting with other proteins (Bent, 2001). Our results showing that tetramers of a range of pathogen-inducible cis-acting elements also direct local wound-induced gene expression demonstrate the convergence of resistance gene, nonhost, and wound responses at the level of promoter elements. This observation is in agreement with recent data demonstrating an extensive overlap in the transcriptional response of plants to race-specific elicitors and mechanical stress (Durrant et al., 2000). Studies of 290 Avr9/Cf-9 rapidly elicited genes demonstrated that many of these also are induced by wounding (e.g., by cutting or infusion of liquids).
The results presented here extend these observations from the level of the entire promoter to that of individual cis-acting elements. In addition, the identities of the elements suggest that pathways involving salicylic acid, jasmonate, and ethylene may converge at this level. One key question that remains is whether the same or different members of transcription factor families are responsible for both pathogen and wound responses. In other words, whether convergence occurs at the level of transcription factors as well as at the level of cis-acting elements and promoter architecture. Most likely, these signaling networks have shared components, with proteins at many nodes being capable of receiving inputs from multiple pathways (Bent, 2001). Many pathogen- and wound-inducible cis-acting elements could represent such nodes.
Improving Synthetic Promoters
We used synthetic promoters containing tetramers of elements to ensure that the promoters were strong enough to detect activity by GUS staining. These promoters, however, are not optimal for all purposes; therefore, we set about making improved second-generation promoters by varying several parameters. Most important was the number of copies of an individual element in a promoter. Both the strength and the inducibility of a promoter can be modulated by varying the number of copies of an element. Importantly, this also can have the effect of reducing/eliminating some background expression because pathogen inducibility appears stronger than basal or wound-induced expression (Figure 7B). Spacing between individual cis-acting elements and/or between these elements and the preinitiation complex also can have a profound effect (Figure 8), but spacing is difficult to predict (Wray, 1998), and the optimal spacing, like the optimal number of elements, needs to be determined experimentally.
Promoters with combinations of different elements may be among the best pathogen-inducible promoters (Figures 10B and 10C), because they often combine good inducibility with low background. Together, our results suggest that the optimal pathogen-inducible synthetic promoters may consist of combinations of one or two copies of defined cis-acting elements. Perhaps the best synthetic promoters, although made up of carefully chosen components, will not be that dissimilar to natural promoters after all.
Biological Importance and Applications
The pathogen-inducible synthetic promoters could have major applications, first, as molecular markers, and second, in engineering crops with increased disease resistance. As molecular markers, the promoters are attractive because they consist of one cis-acting element and therefore one defined end point of signaling pathways. This could have major advantages over the full-length promoters that are used commonly. Through introduction into mutant backgrounds, the synthetic promoters can be used to better characterize these mutants, and the use of defined synthetic promoters for this purpose could become a standard practice. Differences in responses during different plant–pathogen interactions also can be investigated at the molecular level. Synthetic promoters can be used as reporters for mutant screens using targeted genetics (Hooley, 1998), and this may prove a powerful approach.
The spread of plant pathogens and insect pests is increasing worldwide (Moffat, 2001). Researchers have identified numerous plant and pathogen genes that can be used to increase crop resistance toward invading pathogens. These strategies involve interfering with the replication of viruses in the plant, expression of gene products toxic to certain pathogens, and enhancement of the plant's own natural resistance mechanisms. Such introduced genes usually are placed under the control of strong promoters, yielding constitutive expression of the gene product in all tissues of the plant. This can have detrimental effects on plant growth, development, and crop yield. Use of the synthetic promoters presented here and future improvements thereof may prove valuable in engineering plants with increased resistance, because the use of such defined regulatory sequences may allow highly restricted expression of the desired gene product exclusively at the sites of attempted pathogen invasion. Thus, expression of the gene product is limited to cells surrounding an infection site and is not found in healthy parts of the plant. Moreover, expression via such promoters can be triggered by a range of different pathogens, including during compatible interactions with pathogens of commercial importance, such as powdery mildew. This expression may be sufficient to abort the progression of the invader even in such compatible interactions.
Construction of Synthetic Promoters
Promoter constructs were produced by annealing phosphorylated upper and lower strand oligonucleotides to create elements containing a SpeI restriction site at the 5′ end and an XbaI restriction site at the 3′ end. These were introduced into pBT10–β-glucuronidase (GUS) (Sprenger-Haussels and Weisshaar, 2000) between the SpeI and XbaI sites (Figure 1). Promoters containing multiple copies of elements or combinations of elements in any desired order were obtained by digesting the constructs with either SpeI or XbaI together with SacI, which cuts the plasmid at a site outside of the synthetic promoter. Ligation of two such fragments recreates the plasmid with an increased number of elements. This can be repeated as the 5′ SpeI and the 3′ XbaI sites are recreated, but internal SpeI-XbaI ligations result in the loss of these restriction sites. For analysis in Arabidopsis thaliana, the entire synthetic promoter was excised as a HindIII-SacI fragment and ligated into the binary vector pGPTV-GUS-KAN (Becker et al., 1992).
Transient Expression
Transient expression analysis was performed as described previously (van de Löcht et al., 1990) using 20 μg of ScaI-linearized DNA per assay. Protoplasts were harvested 8 hr after transfection. All results represent a minimum of seven independent experiments. Normalization control experiments were performed after cotransfection with a constitutively expressed Petroselinum crispum UBI4/2::luciferase construct (Sprenger-Haussels and Weisshaar, 2000).
Transgenic Arabidopsis Lines
Agrobacterium tumefaciens–mediated plant transformation was performed as described (Bechtold et al., 1993). Approximately 10 independent lines were isolated for each synthetic promoter-GUS reporter transgene, and between two and four representative lines (typically between T2 and T5) were subjected to detailed analysis. Tested lines varied between T2 and T5, depending on the promoter tested. Histochemical staining for GUS activity was performed as described (Jefferson, 1987).
Wounding of Plants
Leaves were cut with scissors and harvested 1 hr thereafter. Control leaves were excised and immediately stained for GUS activity.
Infection of Plants with Pathogens
Plants were infected with Pseudomonas syringae pv tomato strain DC3000 without an avr gene (compatible) or with the avrRpm1 gene (incompatible) or with Peronospora parasitica pv Cala2 as described (Kirsch et al., 2001) and stained for GUS activity. Four- to 6-week-old Arabidopsis plants were infected with Erysiphe cichoracearum UCSC1 according to the method of Vogel and Somerville (2000) and with Blumeria graminis f. sp. hordei K1 according to Peterhänsel et al. (1997).
Microscopic Analyses
Clearing of leaves and staining of fungal structures were performed according to Peterhänsel et al. (1997).
We thank all of those who supported and encouraged this work, especially Günther Strittmatter, Dietmar Stahl, Jonathan Phillips, Ursula Cordier, Silke Fontein, and Luca Santi. We also thank Maret Kalda for photography and Paul Schulze-Lefert and Klaus Hahlbrock for comments on the manuscript.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010412.
- Received September 24, 2001.
- Accepted January 11, 2002.
- Published March 22, 2002.


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subset: commoncrawl


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When I was a kid a read a Popular Science article about how photos of a Soviet spacewalk seemed to be faked. Since then photography experts have uncovered problems with the US Moon landing in 1969. It has to do with multiple light sources and too much detail in 'silhouettes.' Other experts question how the nearly paper-thin skin of the Command Module protected astronauts from the Van Allen radiation belt.
Month – September 2015
How the Supremes Perverted the "Equal Protection" Clause
New Common Core Lawsuit Asserts Parents' Rights
Using Comedy to Expose the Iran Deal
How Bernie Sanders used the Bible at Liberty University
Students Punished for Saying 'Illegal Alien'
Hospital Medically Kidnaps Homebirthed Newborn
Graham Grandson Leaps into New Ministry Job After Scandal
The Christian world was shocked over the inappropriate relationship that prompted Billy Graham's grandson to resign from the pastorate of a megachurch. But it only took another Florida congregation less than three months to give Tullian Tchividjian another gig. He is the author of a book that many consider to be promoting hyper-grace.


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Event details
- April 2, 2028
- 9:00 am
- 31575 Hilltop Blvd, Running Springs, CA 92382
FIRST Service 9am CLICK HERE TO VIEW THE LIVE STREAM
We will be putting chairs out in the parking lot 6ft apart for people who feel comfortable to come sit and watch the service from their chair. Drive in parking will still be available for those who are high risk and feel safer continuing to watch from their cars with the windows down or windows up and tune in on the radio to listen.
Child care/classes for ages 0-18
* We will be allowing people to use the church restrooms.


text:
Here's how to order:
Look around Jamberry.com for any extra must's
Click HERE to contact Melissa to submit your order
Then check your email for an invoice securely pay from
Credit, Debit or PayPal are all accepted!
What else to expect:
Your order will ship directly from Jamberry
Order can take up to 2 weeks to arrive - so plan accordingly if it's a gift!
Melissa will send you a Thank You gift after your 1st order
You'll still receive the great customer service you've come to love
Thank you again for your understanding during this minor change!


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Footage showing alleged police brutality at a memorial event for Ian Tomlinson led to the suspension last night of a second police officer.
Shot the day after the main 1 April demonstrations, it shows an incident during which frontline police were attempting to contain protesters who had gathered at the Bank of England for the vigil.
The 47-year-old newspaper vendor had collapsed and died nearby the previous night, and demonstrators had come dressed in black to lay flowers and demand answers about his death. At the time protesters were not aware of footage showing Tomlinson being attacked from behind by an officer before he collapsed.
The film begins showing hundreds of protesters gathered opposite the Royal Exchange. They are surrounded by lines of police who appeared to have formed a cordon, or "kettle", to contain them.
A man holding a newspaper attempts to leave the police cordon. He is seen remonstrating with police, who refuse to let him out. The crowd reacts angrily when minor scuffles break out between the man and police.
It is at this point that a short woman standing outside the cordon reacts angrily toward police. Her identity is not known. Photographs taken at precisely the same time show her clutching a digital camera and carton of orange juice. Swearing, she directs her anger at one particular officer.
The officer, who has concealed his badge numbers, appears to shove her back, saying: "Move back." She replies: "[I'm a] fucking woman."
The officer then appears to slap her across the face with the back of his hand, saying: "Go away." The footage captures what appears to be an angry reaction from protesters who witnessed the strike.
The same officer then removes an extendable baton from his left pocket and strikes the woman on her legs. The footage is not clear, but the woman appears to fall to the ground. The crowd again erupts angrily with chants of "shame on you".
The footage was shot by Tristan Woodwards, a 25-year-old administrator from Basingstoke, placed on YouTube and subsequently handed to the Guardian. Woodwards said last night he felt the officer had "abused his power".
"If someone was chucking a bottle or hitting a police officer I could have understood their behaviour," he said. "She [the woman] wasn't doing anything out of order. He's pretty tall and pretty big. Hitting a small woman isn't the most acceptable thing he could do."
The film is one of a number showing alleged police brutalities obtained by the Guardian since the footage showing Tomlinson being attacked by an officer.
Referring to the latest footage, a spokeswoman for the Independent Police Complaints Commission said: "The IPCC has been made aware that the Metropolitan police service will be referring an incident to us following footage which has come to light this afternoon. As soon as we get the referral, we will look into it and decide the best way to progress an investigation into the actions of the officer involved."


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I heard Rush Limbaugh going on about Tiger last week, he was all like "isn't it amazing how Tiger has earned a second chance after he's been so disgraced". That got me thinking about the start of this thread - I just can't figure out how Tiger cheating on his wife (who is now a multimillionaire) with trashy white women was so horribly disgraceful. He didn't roofee anyone, he didn't masturbate in front of unwilling women, he didn't jokingly pretend to grab a woman's tits by putting his hands nowhere near a flack jacket, he didn't even beat his wife. How did his infidelities affect any of us?pinback wrote: ↑Tue Mar 12, 2019 7:35 amAthletes are more about, are you still going to root for them and cheer them on. I've found recently that an athlete I've been a huge fan of for decades did something, not illegal, but distasteful enough to me that I find myself not caring about what he does anymore, or even actively rooting against him.
Screw you, Tiger.
He was known as a golf prodigy, and yeah for his endorsement contracts he tried acting like a role model. Him turning out to have a fetish for trashy women makes me feel like he's a normal sports or celebrity guy, not some sort of magical guy who is perfect at everything. In fact, his wife coming after him with a golf club and the fact that he barely even bought the women fast food makes the whole story hilarious to me.
Pinner, how can you possibly let Tiger cheating on his wife obscure the fact that he's busted his ass for years to rehabilitate and play with an improved swing that doesn't destroy his body as much. He's paid his dues and worked his way back which is a great role model.
I thought we liked him for golf, not what he does with his penis.


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Solutions for solar cells in lab
h.a.l.m. offers ready-to-go high-end photovoltaic performance testers for lab use. The standard solutions cetisPV-Celltest3 and cetisPV-Moduletest3 include mechanical housings optimized for sun simulating. They are adaptable to many tasks. h.a.l.m. systems offer a wide scope of detailed analysis and measuring methods especially for the non-time-critical lab situation.
The cetisPV-Celltest3 is a complete class AAA rated high-precision manual tester system for the IV measurement of solar cells. It is designed for high R&D, laboratory, and start-up production use.
High precision measuring
The h.a.l.m. IV curve tracer cetisPV-CTL1 is a 3-channel 16-bit AD converter with an active electronic 3-quadrant load for synchronous measurement of current, voltage, and irradiance. Above that, it tracks different temperatures via RTD or contact-free IR sensors for temperature correction according to IEC 60891 Ed. 2.0.
Extendend flash performance
The high-speed sun simulator allows flash durations of up to 200 ms. h.a.l.m. offers unique features like single-flash hysteresis for capacitive cells and multi-level flashes, which allow to record IV curves at different intensities within one flash.
Flexible cell contacting
The contacting system cetisPV-Contact1 allows the user to accurately measure different cell layouts. With little hex key adaptions, the system can be switched from a two- to a three-busbar layout. The double IV contacting probes are easy to maintain. cetisPV-Contact1 can also be used in semi-automatic cell testers or in small production environments. Equipped with an optional pneumatic lift (two hand security push buttons), a complete handling cycle of loading, measuring, and unloading of a cell specimen can be performed within approximately 6 seconds.
Optimized dark chamber construction
The measurement dark chamber provides mounting facilities for the flasher light box and the cell-contacting station. The side walls and the front doors are coated with anti-reflective tissue. For easy handling, two light tubes illuminate the chamber only when the doors are opened. While closed, the operator is protected against the flash light and a proper dark measurement situation is provided. Since this type of measurement is extremely sensitive to residual light, true dark IV measurements including the evaluation of very stable RserDf and RshuntDf values can be carried out. The new chamber design incorporates mounting space for additional electro-optical measurement systems (optional): thermo-graphical camera for hotspot analysis and electroluminescence camera for micro-crack detection.
Advanced measuring and analysis methods
For lab use, h.a.l.m. devices allow further measuring methods like sectional measurement or double-flash hysteresis (or even the combination of both). The sectional measurement collects IV curve data over several flashes. Therefore, the total load sweep can be segmented over several flashes to reduce dynamical effects. Above that, two measurements (series) with inverted load sweep direction can be combined to reduce sweep directional influences.
Many hardware add-ons and retrofits available
Thanks to the modular principle, the h.a.l.m. systems in general can be easily extended and updated to the newest state-of-the-art. There are several hardware options available, which can be integrated and retrofitted. The cetisPV-GR (grid resistance), for example, analyses the cell surface printing quality and finger resistances on front and rear side. Additionally, it checks the contact resistance of the pins to ensure a good and reliable contact is made to the cell.
Visual inspection add-ons available
Also available for the cetisPV-Celltest3 are thermography and electroluminescence options, which are smoothly integrated into the system. This also allows the unique long-term thermography feature.


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From book fairs and beer festivals through to Weihnachten and Wurst, Germany provides everything you'll need for a fantastic visit. Fly to Germany in Alitalia's renowned 'Italian Style' and explore some of the country's greatest cities.
Berlin is a city that emanates culture and sophistication, particularly as you stroll along the famous boulevard Unter Den Linden. Try also the 'Berlinale,' the world's largest film festival with over 400 projections and 300,000 tickets sold. Düsseldorf is known as a center for business and fashion shows, as well as being home to some of Germany's best museums and galleries. In the city of Frankfurt you'll find one of the biggest exhibition halls in the world and a global reputation for science museums and great dining. The southern city of Munich is inescapably associated with its beer festival. For some contrast, visit its many castles and nearby breathtaking mountain scenery. While Stuttgart's ballet charms aficionados, devotees of other arts will be impressed by the collections in the State Art Gallery and City Art Museum.
The buses and trains are cheap and reliable. When you travel to Germany, book your internal travel tickets in advance to save money. Visit in December and be entranced by Germany's 'Christmas Wonderland' markets.


text: I would like to take this opportunity to thank you, Rob, and the Hammond/Osborne firm for the considerate and professional treatment I received while attending to my brothers affairs. As I told Rob during our discussions, my brother was adamant that all business concerning his affairs was to be conducted through your firm and I totally understand why. It is an arduous task at best to look after someone's affairs and having someone to clarify the issues and sort through the pitfalls for you is invaluable.


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Regular price
Flirty Full Apron - orange and white striped fabric, with navy blue Detroit Tigers accent and navy skirt trim. Features side pocket with accent fabric on the right side. (Solid blue) waist ties can be tied in front or back.
When I saw this Detroit Tigers fabric in a remnant pile at Joann's, I KNEW it would make a fab apron. There wasn't very much of the fabric, so I had to be strategic in how I placed it.
When I found this thrifted orange and white striped material in my stash, it was a match made in baseball heaven! I love how bold this pairing is. And a little bit referee-ish, yes? LOVE. I'm more of a football girl, but LOVE how this baseball apron turned out!
Shown on size S model - fits size S-L
Pattern is Luscious Layers by Modern Vintage Designs


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Board Meeting will be conducted at 7:00pm in the Old Town Brandon HD Training Room.
All Chapter Members are welcome.
Arrive 30 minutes prior to KSU with a full tank.
This allows you time to check in with the Road Captain, receive a ride sheet, and attend the important pre-ride meeting.


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Libya's weapons must be safeguarded, Baird says
Canada must help Libya make sure its weapons of mass destruction don't get into the wrong hands, Foreign Affairs Minister John Baird said Friday.
Speaking to CBC News from Paris the day after an international meeting on Libya's future, Baird said there are a number of fronts Canada can help with.
"There's significant stockpiles of mustard gas and other chemical weapons that have been secure for a number of years, but we want to make sure they don't fall into the wrong hands," Baird said. "So there's another area where we can help demilitarize a country so hopefully it'll have a peaceful future."
Canada is willing to work with the new Libyan government to promote democracy and the rule of law, write a constitution and train civil society members and police, as well as build capacity, Baird said.
But it's important for the UN to lift its sanctions so the new government can access Libyan assets, he said.
"We pressed hard for that yesterday. Obviously, the new government in Libya has to pay the salaries of police officers. Some of them in Benghazi [the rebel stronghold] haven't been paid in as many as six months," Baird said.
"The school year is starting so they'll have to pay teachers. They'll have to pay for the electricity, for the water systems, all the other public services that are essential to a stable and free society. So we're urging them to do it as expeditiously as possible and putting our weight behind others who are making similar calls."
Canada's military mission is due to end in Libya on Sept. 27. This week, Baird and Prime Minister Stephen Harper hinted it could go on longer, saying the goal of keeping civilians safe isn't done yet and Canada will continue as long as NATO is there.


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Voice of Iranian Kurdistan - Clandestine
*sign-on // parallel frequency
3970.04 kHz. Via Salah Al-Din, Northern Iraq, *0226-0335 UTC, Nov 17, 19, 21, 27 and 28. Trumpet interval signla to Kurdish ID as: " Era dengi Kurdistani Irana." Announcement, political talks about Iran and songs. Very clear as long as the Iranian jammer stayed on 3975 or 3980, SINPO 45333. Heard // 4870 or 4890, but 3970 was 12 seconds delayed! At at 0330 the jammer arrived to 3970 resulting in SINPO 43332. My logging on Nov 11 in DX-Window No. 415 with the clear ID (recorded): "Era dengi Kurdistani Iraq" seems to be a mis-announcement by the speaker in the early morning! (Petersen). At 1300 on Nov 19 I could hear it a short time without jammer, because they were late returning from 3980. The signal was very weak, but I think there was first a frequency announcement giving at least three frequencies and then lady IDs: "In Sedaye Kurdistani Iran" in Farsi. I could not confirm, if this was // with 4880, but probably not. They also signed off much earlier than 4880, on Nov 19, maybe 1330. (Ritola)
(DX Window # 416 via Anker Petersen)
(DX Window # 416 via Anker Petersen)


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We are planning to create a project in Jira to have all Test cases from all projects. That would be the centralized repository for test cases. We need to link this test case to a user story in the other project.
Hi iarvizu,
Thanks for your inquiry, but before we reply, could you clarify if you're using Zephyr for Jira (native to Jira) or Zephyr Standalone?
Hi Fernando,
We use Zephyr plugin for Jira
Hi iarvizu,
We dont have this option for Zephyr for Jira currently, but we are working on a feature called TEST REPOSITORY which should make this possible.


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NAVATKADU (605) in Navatkadu division, Akkarai pattu south, Puttalam Pattu division, Puttalam District. Given in the Censuses for 1881 and 1891 as Navakkadu. v . MARIPO.
Derivation.- Casie Chitty derives the name (Nawa Kadoo) from Nava, nine, and kadoo, sword. "It is remarkable for the pompous visits which the Kings of Kandy were formerly accustomed to make to it, as soon as they were crowned, to assume the sword of State after bathing in the sea in the neighbourhood."
Situation.- "Situated directly opposite to Puttalam. about 15 miles south-west of Calpentyn" (Ceylon Gazetteer 170-3). It is not so ; but to the south-west of Puttalam. Valentyn (Ceylon. p.27) says that on the "Island" of Calpentyn "are two villages,to wit, Maripo almost on the south-west point and Tellewari, about the middle also situated on the west side ; there is also a town (Vlek) , Navacar, on the east side, about 2 miles from Maripo, &c." "Nevacar" is ,of course, Navakkadu (Ceylon Literacy Register, N.S.,Vol.IV.,p157). From Puttalam it would be about 8 miles, 3 miles down the Chilaw road, turn to right, 5 miles thence. 10 miles from Puttalam on the Akkarai pattu road.
Inhabitants.- "It contain about 200 inhabitants (1834), the majority of whom are the descendants of the ancient Mookwa chieftains."
Roman Catholic Church.- "There is a small church for the use of the Roman Catholics."
Planting and Agriculture.- " There are several coconut and areca nut plantations, which are generally interspersed with small patches of paddy fields." Also tobacco galas.
Water.- "The Water at this place is the best in the district, and the inhabitants obtain it by sinking wells to a little depth." (Ceylon Gazetteer, pp. 170-2.)
Struck by Lightning.- Three men were struck by lightning in this village in March, 1891 ; one died, and two were stunned only.(Diary of Assistant Government Agent, Puttalam, 1891.)
Government Building.- A Receiving Office.
Mission School.- Roman Catholic.
Salt.- Fish- curing yards and stores.
School.- Government.
Tavern.- Licensed.
Population.- 1881 : 77 houses ; 175 males, 146 females, 321 persons. 1891 : 92 houses ; 170 males, 159 females, 318 persons.
Race.- 3 Low-country Sinhalese ; 309 Tamils ; 6 Moors.
Religion.- 312 Christians ; 10 Hindus ; 6 Muhammadans.
Literacy.- 87 males, 17 female literates ; 1 male English literate.
Age.- 0-5 30 males, 23 females ; 5-15 42 males, 34 females ; 15-30 51 males, 59 females ; 30-55 46 males, 26 females ; over 55 4 males, 3 females.
Occupation.- 4 manufacture ; 85 pasture and agriculture ; 6 traders ; 1 professional ; 2 labourers ; 2 others ; 218 dependents.
P.C. – Puttalam
(GAZETTEER of the PUTTALAM DISTRICT of the NORTH WESTERN PROVINCE of CEYLON – FRANK MODDER F.R.G.S. – 1908)


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This section contains 475 words
(approx. 2 pages at 400 words per page)
Twenty men; now twenty-five. Circling the circling cars. Ninety miles from the nearest O for operator and ninety from the nearest badge." Ruby, Page 13
"There were no whites (moral or malevolent) around to agitate or incense them, make them ugly-up on the oven and defy the adults. In fact, local citizens were prospering, had been on a roll for more than a decade: food dollars for beef, for wheat, gas rights sold, oil fueling purchases and backing speculation." Seneca, Page 102
"The man whose hand was healing from a chopping block mistake would not get to the second clean bandage before a fresh cord was finished and stacked. Having been refused by the world in 1890 on their journey to Oklahoma, Haven residents refused each other nothing, were vigilant to any need or shortage." Seneca, Page 108
"Whatever else, thought Anna, the Convent women had saved the day. Nothing like other...
This section contains 475 words
(approx. 2 pages at 400 words per page)


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Read Daily Updated Light Novel, Web Novel, Chinese Novel, Japanese And Korean Novel Online.
This chapter is updated by NovelFree.tk
Continuing the trial of the joint conference, the Dense Fog Forest would most likely not be destroyed, but there would be bloodshed. In addition, a decrease in their evaluation was a possible punishment, which would be a big loss in benefits!
Needless to say, the "seriously injured" Leylin was here too!
Since there was so much hatred, the only way to end this would be through complete destruction, was something that the other party could not tolerate.
Thus, they would take advantage of Leylin being in the midst of recovery by gathering all their manpower, and destroy the Nature's Alliance!
This was what Leylin also hoped for.
"I had specially given him a few days to rally his men. I hope he doesn't let me down!" Leylin sneered.
Shortly after, he turned and looked towards the canyon's exit.
At this moment, Magi wearing black Chinese-styled gowns stood in a circle outside the Canyon, silently crowding around several figures in the middle.
The leader of the group, a middle-aged man with a face worthy of veneration, was the escaped guild leader of the Dense Fog Forest.
"Tsk Tsk! Gus, you really should be embarrassed, being reduced to this miserable state after getting injured from an honorary professor in a small-scaled guild!"
So 'Gus' was his real name, and the previously used name, 'Xerxes,' was fake!
Another man who stood tall beside Gus, was an elderly man in his 50s or 60s. He had wrinkles all over his face. With his slanted nose, sharp, narrow pairs of eyes, it made him look even more sinister than Gus.
Even as he mocked Gus, no anger was seen in the eyes of the surrounding Magi from the Dense Fog Forest, as if they never heard what was said.
The reason for their change in attitude was that this old man was radiating huge waves of energy.
Among the emptiness in the middle of the circle formed by the old men, radiations of spiritual force could be observed. Impressively, it was the strength of a powerful peak rank one Magus!
"If you had been me, the result would not have been any better!"
Gus sighed in the face of mockery, but deep down, was still fearful of Leylin's strength.
Having profound knowledge of Gus' strength, the old man turned solemn too; "If Leylin has already recovered from his injuries, then I want twice the compensation!"
"If it's doubled, let it be doubled!" Gus promised immediately with clenched teeth.
"I have actually used an ancient 'light protector' rune. That's the reason why I could recover fast in such a short period of time. The other party could not possibly have such a thing…"
This was the confidence Gus had, as he hailed from a medium-scaled guild. To him, as a small-scaled guild, it was impossible for Nature's Alliance to have such an asset.
"Je Je! You're right. Besides, they are at most at the same level with you, how can they stop us two on their own?"
The old man chuckled.
"My Lord guild leader! The broken signal has been repaired!" At this moment, a semi-converted elemental Magus walks over and spoke to Gus
"Great! Attack immediately! I want the Nature's Alliance guild removed entirely from the Twilight Zone!"
A malevolent look swept across Gus' face, and he suddenly raised his hand.
More than a dozen of different coloured bright lights fluttered out of the Canyon. Unexpectedly, each one was a rank one Magus!
As a medium-scaled guild, it was not a big problem if the Dense Fog Forest nurtured or recruited official Magi. considering the long, accumulated history of the more than 30 Magi.
This time, Gus planned to use a large number forces from his guild.
More than a dozen brilliant lights rushed to the entrance of the canyon, but were immediately stopped by a membrane formed from a defensive layer of light.
"It's just defensive formation made by an insignificant small-scaled guild! Destroy it!"
From the layer of bright light, a screeching sound was heard and black light converged, forming the shape of a large axe. The head of the axe even had strong black iron chains coiled around it which were threads of bright, cold and threatening light.
*Ka Cha!*
The giant hatchet ruthlessly struck!
In the wake of the loud strike, the entire membrane of light fell apart, disintegrating and dispersing into small specks of light that gradually darkened.
"Enemy Attacking!"
As an official Magus, Celine was the first to react. Her face turned deathly pale and a gush of cold wind sent her to the entrance of the experiment lab within the icy mountain.
*Shaaa!* The black light flashed past, a mole no smaller than the size of a young boy crawled out, his two long, grizzly beards dragging across the floor.
"This Celine! Causing trouble this time! If I am still alive later, you'll have to send me 10 times as many berries as before!"
The mole stroked his beard, with an unwilling look on his face.
"Damn it! If it wasn't for the deal that I made with that stupid old man at that time, I wouldn't have to be stuck here defending his place!"
"All of the acolytes, meet me here now!"
Celine did not even have the time to force a smile, let alone talk to the mole. She just communicated directly with the rest of the acolytes in the guild.
She was extremely sure that the attack this time around was from the Dense Fog Forest's men!
In fact, during a war between the official Magi, acolytes were just cannon fodder! By sending them out was only a disgrace, and even causing needless casualties.
Currently, they had a slim chance of survival!
Celine's eyes flashed with a tinge of hope, and started shouting towards the laboratory, "My Lord Leylin! It is the Magi of the Dense Fog Forest! They have violated the rules of the joint conference and launched an attack on our guild!"
But what took with Celine by surprise even more than the sudden attack was the fact that there was no reply from inside.
Celine repeated the message thrice, and it was only after she could not wait any longer and broke the layer of ice, that Leylin's voice responded. "I'm still in the midst of recovering! If this gets interrupted, I'm afraid that there will be immediate repercussions…"
"How much longer do you need?" Celine clasped onto her handkerchief tightly.
"Ten minutes!" After some silence, Leylin's voice was heard again.
"Alright! I will fight for you during this period of time!" Celine took a deep breath towards the acolyte that was previously there and cleared her throat.
"As the guild leader, I command you to protect this place at all costs! It might be hard to you, but with the help of spell formations, I need you to persevere just for ten minutes!
"Yes, my Lord! We will fight to the end!"
After being distracted for a moment, the acolytes below started an uproar one after another, Obo and Ilya were among those acolytes.
It was obvious and they clearly understood what the future had in store for them if their guild was defeated today.
"That's great! Let's activate it! Grandpa Cedric!!!"
Celine took a deep breath.
"Okay, alright! I can't stand youngsters like you all!" the mole shook his head and suddenly plopped to the ground!
In some instant, black rays of light spread out, forming a big spell formation runes on the ground.
A barrier of light rose and combined with the original spell formation, engulfing Leylin and the experiment lab within.
*Boom! Blast!*
At this moment, a group of enemy Magi arrived at the scene, and the acolytes that were not able to get into the barrier in time were severely injured.
A small boy was cut in half by a blade of light. His upper torso was still wailing as he tried to breathe.
Another female acolyte, who was decimated into a pool of blood by a green sphere that fell from the sky, did not even had time make a sound.
More and more acolytes were instantly buried to death, by huge broken structure of the building that was shattered by magic spells, also without being able to make any sound.
As Celine looked at the devastating scene in front of her, she was unable to control herself and broke down into tears. They were the future potential Magi of Nature's Alliance, where her sweat and blood were poured in.
"I found it! It's here!"
This new barrier of light, which was visibly thicker and stronger than the ones before, caught the attention of the enemy Magi quickly. Very soon, more than a dozen rays of light enveloped the entire place, creating faint shadows in the middle.
*Clang!* Celine threw a communication device on the ground.
"The signal is disrupted! Hold on!"
She placed her palm on the barrier, as a huge wave of spiritual force and magical power was poured forth and injected into the barrier.
The rest of the acolytes followed suit, though their spiritual powers were very weak. With a mere touch, most of the acolytes' faces turned pale, as though they had been beaten up by a large hammer.
Both sides did not exchange many words, and more than a dozen colourful energy beams bombarded the spell formation.
*Bang!* Numerous acolytes lay paralyzed on the floor, most of them having fainted. Celine could barely even stand up straight and was on the verge of collapsing.
Upon seeing two shadows behind the numerous Magi, Celine's expression was filled with despair.
At this time, it has not even been half the time that Leylin had requested. Fighting against the two peak rank one Magi had made Celine lose all hope and strength to fight back.
In the laboratory behind Celine, Leylin quietly waited, a cold and detached expression on his face…
"It should be roughly about now, right?" He muttered.
In fact, Leylin could have fought back, but he had waited until the Nature's Alliance had racked up a disastrous amount of casualty…
As for the dead acolytes, they did not have much importance to Leylin, and their deaths weighed lighter than specks of dust in his heart.
As long as the opponent left irrefutable evidence of an attack, what were the deaths of a few acolytes? Also, these acolytes were under Celine's guidance. If he really wanted to gain complete control of the Nature's Alliance, more blood would have to be shed!
Now, however, it was time.
Gus and the rest of them had done so much damage to the Nature's Alliance that even if Leylin were to kill them all, it could be reasoned as self-defense.
This excuse may not be very good, but it was definitely better than having none! The acolytes that have died and the data that have been perished
"Magus Gus! Why? Just why?"
Celine retracted her hands and looked at the barrier which was damaged by the combined attacks of the enemy. The guardian animal too was severely injured as it spat out blood, smiling bitterly.
"Benefits! Naturally, everything is for our own benefits!"
Gus' voice was ice cold, as if not having any of the emotions that a normal person would have. He had also turned a blind eye to Celine's charms.
"You hindered my path, and I just want to settle the dispute, that's all!"
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text:
Detect and recognize license plates, if the license plate is ocluded, the vehicles are detected and reported.
Dont miss any event!
Real-time video analysis for vehicles at speeds of 200 km / h. we can recognize from 96% to 98% accuracy, count cars and create complete reports.
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© 2018 Ebenezer Technologies


text:
The ultimate showdown of North versus South!
One of our staple street food here in the Philippines is Isaw or pig and chicken intestines. These are then cleaned, skewered and grilled topped with barbecue sauce then dipped in vinegar. There are many street food vendors selling isaw in the late afternoon but none are more popular than Mang Raul's and Mang Larry's.
Mang Larry's started in 1983 as the first one who sold isaw in the UP Campus, two decades later he is still doing it. Known for his isaw manok and cheap prices. Now he is armed with a staff and several branches around the UP Diliman campus and the UP Village area. He even inspired other street vendors to also sell around the campus.
In contrast, Mang Raul's started as a small barbecue stall inside a village in Las Pinas City. He started selling in the 1970's and while his business grew with loyal customers coming back for his signature 4 sauces and grilled meats. The local neighborhood eventually was able to upgrade all the surrounding stalls all because they sold rice to his loyal customers. This area is now known as Happy Place.
Round 1: Taste
Let's start with Mang Larry:
The first thing I noticed in his isaw and other barbecued meats is that they are small in size, it's around 4 inches long on the barbecue stick. The taste of their isaw is a lightly brined and seasoned with salt and pepper. They cook their isaw well done and torch a little so the outside skin layer will be crispy. When you dip it into the spicy vinegar with a hint of the sweet sauce, the flavors come together in unison.
On the other hand, Mang Raul's:
The isaw is also around 5 inches long, although his pork barbecue is around 8 inches. The taste is very sweet, you can tell that their isaw is well coated with their barbecue sauce, it is also a little charred on the outside, and the sauce is really sticking to the meat. Now when you want to use the dipping sauce, there are 4 choices, what I usually do is first to dip the isaw in the sweet barbecue sauce and then to the spicy vinegar so I have a bit of a kick at the end.
If you compare the two, they're really polar opposites. While Mang Larry is very simple in his sauce and marinade, Mang Raul uses his rich barbecue sauce to cover his isaw and his many dips. Although it's nice to note that both of them have clean tasting Isaw. Round 1 goes to Mang Raul.
Round 2: Dipping Sauce
Mang Larry uses the classic sweet sauce similar to those in fish ball food carts and a spicy vinegar as dipping sauces for his simple isaw. As oppose to the many choices, of Mang Raul, he provides you with 4 flavors of the sauce "Tamis Anghang", "Sweet", "Mild Vinegar", and "Spicy Vinegar".
For variety, Mang Raul wins this round.
Round 3: Price
In terms of price, there are very small differences here, as well as, menu choices. Both are similar.
Round 3 goes to Mang Larry.
This battle is going to come down to the taste.
While the isaw in both places are exceptional. I have to go with Mang Raul's based on:
- Taste: meat is perfectly brined that oozes out succulent flavor with every bite
- Dipping sauce: the ratio of spice, vinegar is perfect, variety of sauces available
- Price: although there's a 2-3php difference, the size per serving is bigger.
The char taste together with that sweet tangy sauce with a hint of spice is just addicting. The meat is perfectly marinated, flavorful with every bite. I recommend you order a side of hot steaming rice.
So if you are one of those foodie warriors in search for the beast isaw. Head down to Las Pinas and taste this isaw sorcery.
Mang Larry's Isawan
- Address: Magiting, Diliman, Lungsod Quezon, Kalakhang Maynila, Philippines
- Facebook: https://www.facebook.com/Mang-Larrys-Isawan-The-Famous-Isawan-in-UP-155375931160899/
- Operating Hours: 3pm- 9pm
Mang Rauls BBQ Haus
- Address: CRM Avenue, BF Homes, Almanza, Las Pinas City
- Facebook: https://www.facebook.com/MangRaulsBBQHaus/
- Operating Hours: 2pm – 9:30 pm
Do you want to collaborate? or recommend other places? Comment on the line below or send us an e-mail over at email@example.com
Make sure to check out our other Where to Eat Series and follow us on our Instagram and Facebook page for real time updates.


text: Focus groups of black voters put together by a Democratic pollster found that Hillary Clinton is underperforming among black voters. The focus groups were organized by Cornell Belcher of Brilliant Corner Research & Strategy. Belcher previously worked as a pollster for the Democratic National Committee. He found that Hillary is underperforming with black voters in the key states of Ohio and Florida and that they didn't feel compelled to vote for her out of disdain for Donald Trump. Black America is starting to slowly figure out Hillary Clinton, and starting to realize how much she despises black Americans. When in 1996 Hillary stated that young blacks are super predators and we have to bring them to heel. Yet some black Americans are still blind to what she said back then, but it's only a matter of time before black America realizes in mass that Hillary is the racist candidate. Hillary and the left have been pushing Trump as a racist, because she doesn't want Latino or Black America to find out she's only talking about herself. We haven't even had a major Black Lives Matter protest for almost two weeks now, so maybe even they are finding out all the lies they have been told by the Democratic party all these years, since the Democratic plantation forced most of black America on to the identity politics train in the 1960's. The Democratic party birthed the KKK, Robert Byrd, and the Jim Crow laws, maybe after all these years of lies to Americas black communities maybe there is hope they will still wake up. Remember also it was Hillary's husband Bill who as president made prison terms longer for black people in the 1994 crime bill.


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I have been thinking recently about software and product quality. There is a software quality conference this fall here in the Pacific Northwest and I recently read an article on the top ten software blunders of the last decade. As we rush products to market, are we compromising quality? What negative effect does that have on our product? Is it worth it? Is it acceptable? Is it the price we pay for doing business in a hypercompetitive world?
Continuous Exploits
In late April, it was discovered that there was yet another hole in Internet Explorer that allowed hackers to exploit vulnerabilities and plant malicious code on individual computers via infected websites. This is just one example of applications and operating systems with bugs waiting to be exploited. My question is this—are product developers and quality assurance teams releasing inferior, not-yet-ready–for-prime-time products, or are the products so complicated that developers do not understand all of the implications until after they have been tested by consumers? If it is the former, then the answer is to wait until all of the bugs are detected and corrected to release a superior product. If the answer is the latter, then that means that you and I are paying for the privilege of being product testers. Personally, I can think of better things to do with my time and money.
A Simplistic View
I will admit that I may be taking a simplistic view. My experience runs towards hardware products and support, although there are still quality products in that arena as well. According to Microsoft, Windows XP, which was released in 2001 and recently became unsupported, was compiled from forty-five million lines of code. Thirteen years later we have Windows 8.1. How many lines of code are in this operating system? Is the complexity sustainable or are we building products that we cannot manage? With this increasing complexity, have we resigned ourselves to a certain number of acceptable bugs? What is our tolerance level? One percent of nonfunctioning or potentially compromising code? Is that acceptable?
Nineteenth-century writer Oliver Wendell Holmes once said "I would not give a fig for the simplicity on this side of complexity, but I would give my life for the simplicity on the other side of complexity." I believe that we are stuck in the middle of that complexity right now. While our products are sophisticated, they lack that elegance on the other side of complexity. We have learned to write incredibly complex code, which is understood in part by individual coders but in entirety by no one. This is the very thing that makes that code vulnerable to exploits and security breaches. If we could somehow find that simplicity or elegance on the other side of complexity, then we could enjoy robust, secure, and usable products.
Do you have or use a product or application that you think has broken through that complexity curtain? Share your find with me.
About Kelly Brown
Kelly Brown is an IT professional, adjunct faculty for the University of Oregon, and academic director of the UO Applied Information Management Master's Degree Program. He writes about IT and business topics that keep him up at night.


text: Escape Game: Lake House is another point and click escape game developed by 5ngames. Someone is trapped inside the lake house. May be the person might have been kidnapped. No one is around to help the person. So you must do something about it. Help the person escape from the lake house with your puzzle solving skills. Relate objects and use clues to solve puzzles. Have fun!


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On March 12th 2014 the members with the secretary and the president of the JCU Theater Society performed their annual first show in Aula Magna: "Some…Noises Off", an adaptation of the acclaimed comedy written by Michael Frayn, "Noises Off".
The show was completely organized by the members of the club, who were all passionate and committed to the world of theater.
The script of "Noises Off" was adapted by the secretary Federica Brizzi, while the costumes, settings and props were managed by Menna Mohammed, the treasurer of the society. "Some…Noises Off" was directed by Sofia Martuscelli, the president of JCU Theater Society. The cast was composed of degree-seeking students of different majors: Samantha Abear, Federica Brizzi, Antonia Di Nucci, Matteo Franzese, Sofia Martuscelli, Mateo Proietti and Corinna Rombi.
The show was a great success, the public laughed and enjoyed the performance! Definitely a great start for the JCU Theater Society!


text:
Date: 13 November 2020, repeated May 07 2021
Venue: Online via Zoom
Cost: BAISIS Member £125 / BSA Member £150 /Non-member £170
Audience: Humanities teachers and Heads of Humanities
Practical Strategies for teaching Humanities subjects to international pupils in the mainstream classroom
Why should you attend?
If you teach a Humanities subject to international pupils in a mainstream school, this seminar will help you learn, refresh or build on your classroom strategies and teaching techniques to meet the needs of those pupils. This is a practical course which will help you to source suitable materials and consider how to support your pupils with both language acquisition and concept development. We consider the principles of language learning as they relate to subject specific teaching and learning, and relate this to CLIL (Content and Language Integrated Learning).
To give Heads of Humanities subjects and humanities teachers a better understanding of how concept development and language learning are related, so as to be able to support the needs of international pupils in the mainstream subject classroom.
• The principles of collaborative learning and the immersive context of EAL in Humanities
• Universal strategies for teaching and engaging international pupils in the Humanities.
• Strategies for reading, writing and vocabulary development in the Humanities.
Wider context and/or application to the Boarding sector:
Gone are the days when subject teachers could place the responsibility for international pupils in the hands of the EAL teacher. Now, the ISI says subject teachers should equip themselves with the skills to meet the language needs of the international pupils in their classroom.
Outcomes and resources:
Participants will develop empathy for international pupils, identify and examine potential barriers to their learning in Humanities lessons and explore the use of lesson planning, resources and teaching techniques to maximise outcomes for international pupils in the Humanities.
Speaker: Catharine Driver
Catharine is a consultant and trainer for EAL and Academic Literacy. She has wide experience of teaching and training in multilingual schools. She has been Head of EAL and Literacy and a school improvement consultant. Catharine was senior adviser for the British Council's EAL Nexus project and is now secondary school adviser at the National Literacy Trust. In 2018, she co-wrote "100 Ideas for Secondary Teachers supporting EAL" (Bloomsbury).
For further information contact firstname.lastname@example.com


text:
Health Insurance
The PEBA Website is the Perfect Source for Annual Enrollment Info
Some enhancements have been made to the Insurance Benefits website, http://www.peba.sc.gov/ to make annual enrollment easier for you.
One of the buttons on the bottom of the homepage has changed to Enrollment Info. This links to a general information page about annual enrollment. The page includes links to the Insurance Advantage (both editions), the annual enrollment video, MyBenefits and enrollment documentation information.
It is strongly encouraged for you to visit the Insurance Benefits website to learn more about annual enrollment and the changes they can make.
Helpful Benefits Tools
Enrollment and Coordination of Benefits:
- Within 31 days of hire or retirement date.
- PEBA Updates
Annual Enrollment:
- Change health plans
- Enroll or re-enroll in MoneyPlu$
- Enroll or drop State Vision Plan
- Enroll in or increase Optional Life with medical evidence
- Decrease or cancel Optional Life
- Make other changes as announced
Open Enrollment Period:
- October of odd-numbered years
- Enroll in or drop State Dental and/or Dental Plus
- Add or drop dependents from health or dental coverage
- My Benefits - This helpful video will show you how to get your BIN (Benefits Identification Number), how to Register in My Benefits, how to reset your password, how to sign in and how to make a change.
- 2019 Premiums
Dental Plus, Life Insurance Premiums also Increasing - Dental implants and related services will be covered under the State Dental and Dental Plus Plans. Services will be covered at 50% of the allowed amounts. The current benefit period maximums for all covered dental servicess will not change.
State Dental Plan - $1000 per benefit period
Dental Plus - combined $2000 per benefit period
- MoneyPlu$ Worksheets
- Blue Cross/Blue Shield - This website has a very helpful video demo to see how you can create an account, check your doctor visits, view health and dental claim summaries, health & dental eligibility benfits and a personal health assessment.
- Express Scripts - Prescription drugs website
- EyeMed - Vision care
- USC Benefits website
- How to Order Replacement ID Cards
- 2019 Insurance Summary
Dependent Certification
The Employee Insurance Program (EIP) is conducting an extensive audit of dependent eligibility. EIP is the Division of the State Budget and Control Board that provides the insurance coverages for State Employee and their dependents. Blue Cross/Blue Shield currently has the contract for the management of these benefits but EIP is the actual provider. If you see an envelope from the SC Budget and Control Board or EIP - Employee Insurance Program do not disregard.


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Avert your eyes? Nadya Suleman, AKA the Octomom, has just made her stripping debut.
The mother of 14 danced to Rihanna at the The Playhouse Gentlemen's Club in Miami as she stripped for about 400 customers.
According to the Daily Mail, the Octomom started her set wearing a tight black PVC ensemble of mask with thigh-high boots. Suleman also dressed in a school girl's outfit, drank from an over-sized baby bottle, and swung around the stripper's pole during her strip club debut.
Club owner Greg Louis said that the club saw about twice as many patrons than a regular night.
Louis said:
"The phone's been ringing off the hook. Several people asked me if it was really Octomom or just an impersonator."
Suleman has recently been turning to the adult industry to pay her bills. She recently released a sex tape and this weekend she made her stripping debut for $5000 for two 12-minute sets. Suleman also made tips and stayed after the show to sign autographs for $10-a-piece, take photos for $20-a-piece, and gave private dances for $200.
Some unfortunate Daily Mail photographer was on hand to take some pictures of the Octomom's debut. (You can see photos here.)
Kimmel isn't the only one mocking the Octomom. Even some of Suleman's fellow strippers have criticized her dance moves.
One of the girls told ABC:
"She's really stiff… Oh my God, she can't dance. But it's good for the rest of us girls because she made it a busy Friday night."
Meanwhile #OctoMom in Fort Lauderdale last Saturday… twitter.com/MrBrickell/sta…
— Mr. Brickell (@MrBrickell) July 16, 2012
The 18 Best Faces Watching Octomom Strip bit.ly/MDMPf9 twitter.com/MastersProject…
— Masters Projects (@MastersProjects) July 17, 2012