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- © 2012 American Society of Plant Biologists. All rights reserved.
Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.
MicroRNAs (miRNAs) are versatile regulators of gene expression in plants and animals. They are 21 to 24 nucleotides long and processed by a Dicer nuclease from long RNA precursors with base paired foldback structures (Baulcombe, 2004). The single-stranded form of the miRNA forms a ribonucleoprotein complex with Argonaute (AGO) that can bind by base pairing to a target RNA (Bartel, 2009; Voinnet, 2009). In plants, the successful targeting reaction requires complementarity of the miRNA at most of the residues (Mallory and Bouché, 2008).
The consequence of the targeting reaction depends on the nature of the targeted RNA and the extent of complementarity with the miRNA. The target RNA is cleaved and the level of the protein product is reduced if there is near complete complementarity, including positions 9 and 10 of the miRNA. Translational suppression without turnover of the target RNA is mediated by miRNAs with incomplete complementarity to their target (Brodersen et al., 2008; Lanet et al., 2009). In addition, there may be miRNA-mediated targeting of chromatin-associated RNAs that leads directly or indirectly to targeted epigenetic modification (Khraiwesh et al., 2010; Wu et al., 2010).
In some instances, miRNA-mediated gene silencing is a simple negative switch: whenever the miRNA gene is active the target mRNA is silent. However, these versatile RNA regulators may also participate in feedback loops and carry out more subtle roles in genetic regulation. They might dampen fluctuations in target gene expression, for example, or influence temporal changes (Voinnet, 2009). In some instances, the miRNAs or their precursors may move through plasmodesmata and different stages in the feedback system occur in adjacent cells or in separate roots and shoots (Bari et al., 2006; Pant et al., 2008).
miRNAs may also initiate regulatory cascades with multiple mRNA targets (MacLean et al., 2010). These cascades involve secondary small interfering RNAs (siRNAs) that associate with AGO proteins, similarly to miRNAs. The first step in these cascades requires an RNA-dependent RNA polymerase (RDR; RDR6 in Arabidopsis thaliana) and it takes place when the initiator miRNA duplex structure is asymmetrical (Manavella et al., 2012), if the initiator miRNA is 22 nucleotides rather than 21 nucleotides long (Chen et al., 2010; Cuperus et al., 2010), or if there are two target sites for 21-nucleotide RNAs (Axtell et al., 2006). The initiator miRNA stimulates the RDR to convert the targeted RNA into long, double-stranded RNA that is then processed by Dicer into secondary siRNAs. A high proportion of the secondary siRNAs are in a 21-nucleotide phased register in which the first position is the cleavage target of the initiator miRNA (Chen et al., 2007).
The best characterized secondary siRNAs are known as trans-acting siRNAs, and their targets include a pentatricopeptide (PPR) and auxin response factor (ARF) gene mRNAs (Axtell et al., 2006). These secondary siRNAs may move between cells and establish developmental gradients (Chitwood et al., 2009). Both PPR and ARF mRNAs are targeted separately by secondary siRNAs and by miR161 (Rhoades et al., 2002; Allen et al., 2005; Yoshikawa et al., 2005), miR160, or miR167 (Rhoades et al., 2002; Allen et al., 2005). Therefore, it is likely that the secondary siRNAs play a role in either reinforcing or coordinating the action of primary sRNAs or miRNAs.
The secondary siRNA loci, like miRNA genes, have evolved independently at different times (Cuperus et al., 2011). The TAS3-derived secondary siRNAs targeting ARF mRNAs, for example, are conserved in distantly related plants (Axtell and Bartel, 2005), and it is likely that the TAS3 loci evolved in a common ancestor of seed plants. Other secondary siRNA loci in rice (Oryza sativa) and Brachypodium are species specific and are likely to have arisen more recently (Johnson et al., 2009; Vogel et al., 2010). Similarly, the TAS1 secondary siRNAs that target PPR mRNAs are specific to Arabidopsis and they are also likely to be relatively modern.
Both ancient and modern secondary siRNA loci are targeted by miR2118. In Medicago (Zhai et al., 2011), the miR2118 family sequences initiate secondary siRNAs on the mRNAs for nucleotide binding site–Leu-rich repeat (NBS-LRR) proteins in a process that is likely to have originated in a common ancestor of monocots and dicots. The miR2118 family also initiates secondary siRNAs on uncharacterized mRNAs expressed in rice flowers (Song et al., 2012). However, these alternative secondary siRNAs are not found in other species and so their origin is likely to be in the rice evolutionary lineage and more recent than a monocot/dicot common ancestor.
Here, we characterize secondary siRNA loci and the initiator miRNAs in Solanum species. The loci correspond to the genes for NBS-LRR disease resistance proteins, and we demonstrate that at least one of the secondary siRNAs targets the mRNA that, in a related form, has been implicated in basal immunity in Arabidopsis. The initiators of secondary siRNAs on NBS-LRR mRNAs comprise an unusually diverse superfamily comprising miR482 and miR2118 in other species. The sequence of this miRNA superfamily is variable and a high proportion of the NBS-LRR mRNAs are targeted. The miR482-mediated silencing of NBS-LRR mRNAs is lost in virus- and bacteria-infected tissues most likely due to the action of pathogen-encoded proteins. Based on these data, we propose a model in which the plant exploits pathogen-derived suppressors of RNA silencing to achieve inducible expression of defense-related genes.
An Unusually Diverse miRNA Superfamily
The miR482 family is unusual in that the members are 22 nucleotides rather than 21 nucleotides long and they have more variable sequences than other miRNA families. In different plant species, the miR482 sequences vary at nine positions (Figures 1A to 1C) and there are at least 31 isoforms. Other miRNAs, even when they exist as families, have fewer differences and generally fewer isoforms (Felippes et al., 2008; Meyers et al., 2008; Cuperus et al., 2011).
The miR482 family could be extended to include miR472 in Arabidopsis by allowing an additional variation at position 2 (Figure 1B). In addition, if 22-nucleotide miRNAs with a first position that aligns with position 3 of miR482 are also allowed, a superfamily would form that includes the miR2118 subgroup (Zhai et al., 2011). The nomenclature of these miRNAs in the online miRBase (Griffiths-Jones et al., 2008) is inconsistent because there are miR2118-like sequences named as miR482, miR5300, or miR2809. In this article we refer to the 22-nucleotide miRNAs that align with position 1 of miR482 as "miR482 type" and those that align with position 3 or 4 of miR482 as "miR2118 type" members of the miR482/2118 superfamily (see Supplemental Figure 1 online).
Our sRNA data sets from tomato seedlings (Solanum lycopersicum cv M82) include six isoforms of mature miR482 with variation at seven of the nine variable sites (Figure 1A). Two other tomato sequences are of the miR2118 type (Mohorianu et al., 2011). One of these is similar to Medicago miR2118a and it is named as slmiR482 in miRBase. The second is named as miR5300. The miR2118 family in rice is even more diverse. There are 18 pre-miRNA variants corresponding to 16 different mature miRNAs (Johnson et al., 2009; Vogel et al., 2010).
If variation in the miR482/2118 superfamily had been generated in an ancient ancestor, we would expect different plants to share similar isoforms of miR482. However, most of the 31 isoforms of miR482 (Figure 1A) are species specific and a more likely scenario is that most of the variation was generated since the divergence of even closely related species. There are only a few instances of identical sequence variants in more than one plant; thus, stumiR482 (potato [Solanum tuberosum]) is identical to slmiR482b (tomato), and gmmiR482a (soybean [Glycine max]) is the same as pvmiR482 (Phaseolus) (Xie et al., 2011; Zhai et al., 2011). In these examples, the common form may have evolved before the divergence of the two species but not in an ancient ancestor.
There are only four examples of distantly related plant species with an identical isoform of miR482. These are tomato with slmiR482f and cotton (Gossypium hirsutum) with ghmiR482a; grape (Vitis vinifera) with vvmiR482 and Picea with pabmiR482c; and poplar (Populus spp) with miR482c and Picea with pabmiR482b. In addition, there is one variant that is shared by three distantly related species in the Pinaceae (Pinus-ptamiR482a/b and Picea-pabmiR482a) and the Salicaceae (Populus) (ptmiR482a/b).These exceptions to the species-specific pattern are probably due to independent evolution of identical isoforms in different plant lineages.
Diversification of the miR2118 subfamily is similarly species specific. The pre-miR2118 sequences from rice, for example, are all more similar to each other than they are to the corresponding sequences from other species.
The abundance of miR482/2118 superfamily RNAs varies greatly between species and families of plants. These RNAs are rare in monocots and infrequent in Caricaceae, Asteraceae, Cucurbitaceae, Lamiaceae, Brassicaceae, Caryophyllaceae, basal angiosperms, ferns, and mosses (Figure 2A). The miR482 members are abundant in cycads, gymnosperms, members of the Malvaceae and Vitaceae, and the Solanum and Capsicum genera in the Solanaceae. In seedling data sets of tomato and in its wild relative Solanum pennellii, the miR482 type are predominant with miR482f represented 100-fold more frequently than miR482a (see Supplemental Figure 2 online). The miR2118 family members, by contrast, are most abundant in the Rutaceae, Solanaceae, and, particularly, in the Fabaceae. The miR5300 that is assigned as miR2118 type is most abundant in the Solanaceae.
RNA gel blot analysis was consistent with Solanum species having unusually high levels of miR482: probes for isoforms miR482a and f detected abundant RNAs in S. lycopersicum (tomato), S. pennellii, and S. tuberosum but not in Nicotiana or non-Solanaceous species (Figure 2B; see Supplemental Table 1 online). A probe for Arabidopsis miR472 hybridized with an RNA in potato but only weakly with tomato miRNAs.
Together, these various analyses indicate that the miR482/2118 superfamily is unusually variable both in sequence and in expression level. The pattern of variability indicates that this superfamily is in constant evolutionary flux with different species and genera having specific isoforms or combinations of isoforms.
miR482/2118 Superfamily Members as Potential Regulators of NBS-LRR Disease Resistance Genes
The psRNATarget algorithm that predicts targets of plant miRNAs (Dai and Zhao, 2011) (see Supplemental Tables 2 and 3 online) identified the mRNAs of disease resistance proteins as having binding sites for miR482 species. These proteins have NBS and LRR domains (Meyers et al., 2003), and, of the 186 predicted NBS-LRR proteins encoded in the tomato genome, there were 58 with miR482 target motifs as assessed by the TAPIR protocol (Bonnet et al., 2010) (Figure 3A). It is highly likely that these are bona fide targets because they have a high degree of complementarity to the miR482 sequences with more than half having a TAPIR algorithm score of >8. The mRNAs of NBS-LRR proteins have also been identified recently as targets of the miR2118 family in Medicago truncatula (Zhai et al., 2011).
A minority of the miR482 isoforms had unique NBS-LRR targets in tomato (Figure 3B). However, most miR482 family members had multiple NBS-LRR targets (see Supplemental Table 4 online). This pattern arises because the target sequence encodes a variant of the P-loop or Walker A motif that is specific to R proteins (Figure 3C), and, of the seven variable positions in the tomato miR482 family (Figure 1A), there are six that are complementary to the wobble position of conserved amino acids in the NBS domain of R proteins or to amino acid position 5 in this motif (Meyers et al., 2003) (Figures 3C and 3D) that is not conserved. The miR2118 family shows a similar pattern of variation, and we conclude that the miR482/2118 superfamily has the potential to control expression of many NBS-LRR genes and to be a master regulator of disease resistance in tomato.
To find out whether the miR482 family targets a particular type of NBS-LRR protein, we aligned its sequences against the well annotated resistance (R) gene sequences in the Arabidopsis genome. Using the TAPIR rules for miRNA targeting, we established that mRNAs of coiled-coil NBS-LRR (CNL)-type proteins (Meyers et al., 2003), with a coiled-coiled domain at the N terminus rather than a Toll/Interleukin Repeat (TIR) motif, were the preferred targets of miR482 (Figure 4A). There was further differentiation because the CNL–D-type mRNAs are less represented in the targets than in the total CNL-type NBS-LRRs (Figure 4B).
The miR2118 family from M. truncatula also targets the P-loop of NBS-LRR proteins as revealed by alignment with sequences of M. truncatula (Zhai et al., 2011). The miR2118b variant, like miR482, targets CNL-NBS-LRR mRNAs (see Supplemental Figure 3 online). However, targets of Medicago miR2118a and c as well as tomato miR482 (a miR2118 type sequence) include both TIR- and CNL-NBS-LRR proteins. This targeting prediction is consistent with the high sequence homology of slmiR482 (slmiR2118) with Medicago miR2118a (see Supplemental Figure 1 online).
Therefore, this computational analysis indicates that the miR482 and miR2118 families constitute an unusually diverse miRNA superfamily with sequence variation that corresponds to the mRNAs of NBS-LRR proteins. The presence of the miR482/2118 superfamily would have the potential to suppress expression of NBS-LRR proteins to an extent that varies between plants. Those with abundant and diverse miR482/2118 would silence these disease resistance proteins more extensively than plants like Arabidopsis in which this superfamily is represented by a single nonabundant miRNA (miR472).
Secondary siRNAs Initiated by miR482 Family Members
A property of 22-nucleotide miRNAs is the ability to initiate the synthesis of secondary siRNAs from the 3′ side of the target RNA sequence (Chen et al., 2010; Cuperus et al., 2010). These secondary siRNAs align in a phased register with respect to the target site of the 22-nucleotide miRNA and, in some instances, they also have mRNA targets. In our tomato sRNA data sets, we identified 15 genomic RNA loci with phased siRNAs (Chen et al., 2007, 2010). Of these, three corresponded to the conserved TAS3 RNA that has been well characterized in Arabidopsis, one matched AGO1 mRNA that is also known in Arabidopsis to have secondary siRNAs initiated by miR168, five had unidentified miRNA initiators, and the remaining six were predicted targets of miR482 and encoded NBS-LRR proteins (see Supplemental Tables 5 and 6 online).
Figure 5 illustrates the distribution of phased siRNAs that target an NBS-LRR mRNA named LRR1 (Solyco02g036270.1.1). Most of the abundant siRNAs are in the phased register, and they align to a region close to the 3′ side of the miR482a target site. It is likely that the targeted mRNA would be cleaved by an AGO/miR482 nucleoprotein because there is complete miRNA/mRNA complementarity at positions 9 and 10 of the target site (Figure 5). This cleavage site defines the first position in the phased registers. Also consistent with this proposed cleavage site, we used 5′ rapid amplification of cDNA ends (RACE) analysis to detect cleavage of LRR1 mRNA (18/24 clones) between positions 10 and 11 in the predicted miR482a target site (Figure 5). This cleavage site matches well with a previous analysis of the tomato degradome (13 out of 22 clones; Li et al., 2012a). The degradome corresponds to mRNA fragments resulting from miRNA-directed cleavage.
In at least two of the NBS-LRR protein mRNAs (Figure 5; see Supplemental Figure 4 online), there is a second miR482 target site at the 3′ end of the region with phased siRNAs. This second target site has less complementary to the mRNA, and, as expected, 5′ RACE analysis did not identify cleaved products matching this region (Figure 5). However, the final position in the register aligns opposite to position 10 of miR482 and so the target site is in phase with the secondary siRNAs. These secondary siRNAs along with the accumulation of cleaved RNAs provide direct evidence that NBS-LRR protein mRNAs are targeted by miR482.
miR482 Silences NBS-LRR Protein mRNAs
The sequence of miR482 family members and the miR482-initiated secondary siRNAs aligned to NBS-LRR protein mRNAs suggest that miR482 is a suppressor of R protein biosynthesis. In line with this possibility, tomato CNL-NBS-LRRs (LRR1, Solyco02g036270.1.1; LRR2, Solyc04g005540.1.1) that are good predicted targets of miR482 members are less abundant than the Hero and Mi NBS-LRR mRNAs that are not miR482 targets (see Supplemental Figure 5 online). It is likely that this difference is due to miR482 expression because the wild tomato relative S. pennellii, in which miR482a is more abundant than M82, has lower expression of LRR1 and LRR2 than M82 but similar levels of Hero and Mi mRNAs. Similarly in F1 and F2 lines from an M82 × S. pennellii cross, the LRR1 and LRR2 mRNAs are less abundant than in M82 but expression in an introgression line (IL8-3; Eshed and Zamir, 1995) resembles that of M82 tomato.
We next used a transient assay system in Nicotiana benthamiana to test the contribution of miR482 and secondary siRNAs as mediators of NBS-LRR mRNA silencing. This species has only low levels of miR482 (Figures 2A and 2B), and we used plants that were either wild type or with reduced expression of RDR6 (RDR6i; Schwach et al., 2005). RDR6 is an essential factor in secondary siRNA production (Yoshikawa et al., 2005).
Tomato miR482a was expressed transiently using Agrobacterium infiltration, and we assayed the levels of a tobacco NBS-LRR mRNA-EU713768.1 (Figure 6A) that is conserved in N. benthamiana and that has a good target site for miR482a. The expression construct had the miR482a precursor sequence coupled to the cauliflower mosaic virus 35S promoter. We also expressed miR482aΔ that contains alterations to the mature miR482 sequence so that the complementarity with the NBS-LRR mRNA is lost.
Expression of the native miR482a but not miR482aΔ in this assay initiated the expression of secondary siRNA D1 (Figure 6B) and silencing of EU713768.1 mRNA by 10-fold or greater (Figure 6C). In N. benthamiana wild-type and RDR6i lines, transient expression of miR482a also targeted mRNA from an EU713768.1 mRNA (Figure 6C), but there was no secondary small RNA production at the D1 register in RDR6i lines (Figure 6B). Therefore, in this system, the secondary siRNAs were not required for suppression of the NBS-LRR mRNA; miR482 primary targeting was sufficient.
A second assay exploited the potential of plant viruses to produce suppressors of RNA silencing. We predicted that these suppressor proteins in virus-infected plants would interfere with either the production or targeting of miR482 so effectively that NBS-LRR mRNAs with target motifs would increase in abundance in virus-infected tissue. NBS-LRR proteins without the target motifs should not be affected in the same way.
The level of miR482 was reduced slightly in the inoculated leaves of tomato plants infected with Turnip crinkle virus (TCV), Cucumber mosaic virus (CMV), and Tobacco rattle virus (TRV), whereas miR168 was unaffected (Figures 7A and 7B). The LRR1 and LRR2 mRNAs with miR482 targets were both more abundant in plants infected with these three viruses, with the increase in both instances being most pronounced with TCV (Figure 7C). Neither of two NBS-LRR mRNAs (Mi and Hero) without miR482 targets was more abundant after virus infection (Figures 7C and 7D). TRV and CMV had no significant effect on Mi and Hero, whereas TCV led to a decrease in the level of these mRNAs.
In a similar experiment, we exploited the finding that the bacterial pathogen Pseudomonas syringae DC3000 encodes suppressors of RNA silencing (Navarro et al., 2008). These suppressors are transported into infected plant cells via a type III secretion system. We predicted that, as in virus-infected plants, the DC3000 infection of tomato would induce LRR1 and LRR2 mRNAs but not Mi and Hero mRNAs. We further predicted that any increase in LRR1 and LRR2 mRNAs would be reduced in a hrcC mutant of P. syringae DC3000 in which the type III secretion system would be less active. The results were fully consistent with these predictions (Figures 8A to 8C). There was a slight induction of LRR1, LRR2, Mi, and Hero mRNAs in hrcC mutants independent of miR482 levels, indicating that the observed effects are partially independent of the delivery of effectors.
From these data with viruses and bacteria, we conclude that miR482-mediated silencing of NBS-LRR mRNAs is relieved in pathogen-infected tomato plants. In principle, there could be a similar loss of miR2118 type silencing in other plants following infection with bacteria and viruses resulting in increased abundance of resistance proteins. However, the expression of miR2118 is specific to panicles in monocots (Song et al., 2012), whereas tomato miR482 is expressed at similar levels in every tissue tested (see Supplemental Figure 6 online). Hence, the induction of NBS-LRR mRNAs in infected plants may be specific to Solanum species and members of the Fabaceae (Figure 2A) in which the 482/2118 superfamily miRNAs are abundant.
Natural Variation in Secondary Silencing
The miR173 and miR390 initiate secondary siRNAs in Arabidopsis (Allen et al., 2005; Chen et al., 2007) that target mRNA for ARF or PPR proteins. To find out whether miR482 has siRNA-mediated secondary mRNA targets, we exploited a difference between tomato and S. pennellii in the profile of phased secondary siRNAs on the LRR1 mRNA. Tomato has abundant LRR1 secondary siRNAs in the D2 register (Figure 9A), whereas, in S. pennellii, this secondary siRNA is absent (Figures 9B and 9C). The introgression line IL2-1, in which the LRR1 locus is derived from S. pennellii, also lacks the 21-nucleotide siRNA from D2 (Figures 9F and 9G). The predicted target of the D2 siRNA is the mRNA for an unknown protein (Figure 9D). Consistent with this prediction, we detected mRNA cleavage products with a 5′ end that corresponds to position 10 of the D2 siRNA in extracts of tomato but not S. pennellii and that this mRNA is more abundant in S. pennellii and IL2-1 than in M82 (Figures 9E and 9H).
Variation in the miR482/2118 Superfamily
Defense against pathogens is one of the most potent drivers of evolutionary change in host organisms (Nilsson et al., 1997). It accounts for a high degree of variation both within and between species in host defense molecules and for diversifying selection in the corresponding gene sequences. In this analysis of miR482 in tomato and S. pennellii, we provide evidence for a different type of variation that would be associated with the pathogen-inducible expression of NBS-LRR mRNAs and possibly other mRNAs associated with defense.
The NBS-LRR proteins are normally associated with effector-triggered immunity against pathogens. From these and other data (Figures 5 to 8; Zhai et al., 2011), it is evident that their mRNAs are targeted by members of the miR482/2118 superfamily. Other mRNAs may also be linked to miR482 if they are secondary siRNA targets (Figure 9). In this study, for example, we identify the mRNA for an unidentified protein as a secondary target of miR482. This unidentified protein has similarity to PEN3 that is involved for basal immunity in Arabidopsis (Stein et al., 2006). In addition, a predicted target of the highly abundant D4-siRNA (normalized reads of ~200 in our data sets; Figure 9B) derived from LRR1 is a proteosome subunit (Les#S38989204/290-3111; TAPIR targeting score 4) that could also be involved in resistance gene function and disease resistance (Beers et al., 2000; Peart et al., 2002; Tör et al., 2003). We envision that miR482 regulates defense mechanisms in tomato via effects on NBS-LRR and other defense proteins.
A Role for NBS-LRR Proteins in Non-Race-Specific Immunity
The NBS-LRR proteins are normally associated with effector-triggered immunity in which there is a gene-for-gene relationship between the host and the pathogen (Jones and Dangl, 2006). The host gene encodes an NBS-LRR protein that mediates recognition, either direct or indirect, of a pathogenesis effector that is encoded by the pathogen. This effector-triggered immunity is normally specific for some but not all races or strains of a pathogen. However, if the NBS-LRR proteins are overexpressed (Bendahmane et al., 1999), defense can also be induced independently of protein-based recognition mechanisms. Based on the effect of NBS-LRR protein overexpression and on the analysis presented here, we propose a role of miRNA regulated NBS-LRR proteins in non-race-specific resistance against viral and bacterial pathogens that is linked to miRNAs and secondary siRNAs.
Our proposal involves an NBS-LRR induction mechanism that is mediated by suppressors of RNA silencing encoded by plant pathogenic viruses and bacteria (Brigneti et al., 1998; Voinnet et al., 1999; Navarro et al., 2008). In some instances, these proteins block the biogenesis of siRNA or miRNA, and in others, they target the RNA silencing effector complexes (Voinnet, 2005). Once the pathogen has become established, the suppressor would be produced in or transported into the infected plant cell and, as observed (Figures 7 and 8), the miRNA-mediated silencing of NBS-LRR proteins would be relieved. Presumably there would be the same release of secondary siRNA silencing resulting in increased levels of targets of NBS-LRR–derived siRNAs (Figure 9).
The release of silencing described here results in a two- to threefold increase of individual NBS-LRR mRNAs in infected plants (Figures 7 and 8). However, as the induction may involve many tens of mRNAs with miR482 targets (Figures 3 and 4), the net effect on the cell would be equivalent to induced expression of any one NBS-LRR mRNA by 100-fold or more. Thus, the release of miR482/2118 silencing in infected cells would have the same effect as overexpressing an individual NBS-LRR protein and the level of immunity in the plant would be enhanced. An effect on defense would be reinforced through loss of secondary siRNAs affecting other proteins with roles in defense (Figure 9).
The increased NBS-LRR proteins in infected cells would also potentiate effector-mediated immunity due to secondary infection. With an elevated level of NBS-LRR proteins, there would be accelerated activation of the defense pathways against secondary pathogens and the plants would be protected against double infection.
This proposal that expression of defense genes is coupled to the RNA silencing system adds another layer to the defense and counterdefense interactions of pathogens and their plant hosts. In the context of viruses, the RNA silencing system is a first layer of defense. The viral suppressors are a counterdefense system (Voinnet, 2005), and miR482 is then a counter-counterdefense system that is dependent on the counterdefense system.
There is also an involvement of miRNA-mediated silencing in the first layer of defense against bacterial pathogens of plants. Correspondingly, several of the bacterial effectors of disease are suppressor of RNA silencing (Navarro et al., 2008). This finding explains how there could also be induction of miR482 targets in plants infected with bacteria (Figure 8) and, possibly, with other pathogens if they encode RNA silencing suppressors.
An additional potential layer of regulation in this system could involve numerous truncated genes for NBS-LRR proteins in plant genomes (Meyers et al., 2003). Their transcripts could sequester miR482/2118 and thereby fine-tune the suppression of NBS-LRR mRNAs. There are precedents for regulation of miRNAs by target mimics in both plant (Franco-Zorrilla et al., 2007) and animal systems (Cesana et al., 2011; Karreth et al., 2011; Sumazin et al., 2011; Tay et al., 2011).
The Costs and Benefits of the miR482/2118 Superfamily
Disease resistance proteins may have a cost to the plant (Tian et al., 2003). The miR482/2118 system described here would minimize this cost because the NBS-LRR would be at a low level in the absence of a pathogen. In this connection, it is striking that NBS-LRR genes are unusually numerous (Pan et al., 2000; Xu et al., 2011; Zhai et al., 2011) in the members of Solanaceae and Fabaceae, in which the NBS-LRR mRNAs are regulated by miR482/2118 (Zhai et al., 2011). Perhaps the low-level expression of NBS-LRR protein mRNAs in these plants reduces any cost associated with individual gene family members and creates a background in which there can be diversification of the NBS-LRR gene family without there being a penalty for the plant. We envision that plants would benefit from low levels of NBS-LRR proteins due to miR482/2118 if infection pressure is low or if they have other layers of defense against pathogens.
However, for plants under high infection pressure or without alternatives to the NBS-LRR defense system, the benefit of low expression of NBS-LRR proteins could be offset by the other costs. For example, it could be that low level of NBS-LRR proteins results in a delay to effector-triggered immunity that would be damaging for plants that are frequently attacked by pathogens. We speculate that the variation in the sequences and expression level of the miR482/2118 superfamily (Figures 1 and 2) is likely to reflect the shift in the balance of the costs and benefits in different plants depending on the environment and other defense systems.
In this report, we focus on the link of the miR482/2118 superfamily and disease resistance. Further testing of the hypotheses and speculation based on our data will require Medicago and tomato lines in which RDR6 and other RNA silencing proteins are knocked down or knocked out. Unfortunately, such lines are not yet available. There is also an outstanding question about the possible alternative roles of the miR482/2118 superfamily. In rice, for example, TIR-NBS-LRR proteins are absent (Zhou et al., 2004), but the miR2118 subfamily that targets their mRNAs is particularly diverse. We would therefore expect that there are other, as yet unidentified, targets of these miRNAs. While this article was under revision, 2118 family miRNAs targeting the mRNA for a tobacco TIR-NBS-LRR (N) were described (Li et al., 2012b). When these miRNAs were transiently expressed in N. benthamiana, they interfered with N-mediated resistance to Tobacco mosaic virus. Li et al. proposed that shutdown of miRNAs and siRNAs upon pathogen infection could enhance R gene activity.
Plant Growth
Tomato (Solanum lycopersicum) plants were raised from seed in compost (Levington M3) and maintained in a growth room at 23°C with 16-h-light and 8-h-dark periods with 60% relative humidity and at light intensity of 300 μmol/m2/s. Two-week-old seedlings taken for RNA analysis generally had two simple leaves and one compound leaf and a leaf bud.
RNA Analysis and Cloning
RNA gel blot analysis was performed as described previously (Shivaprasad et al., 2012). Briefly, total RNA was extracted from 100 mg plant tissue (2-week-old seedlings) and ground into fine powder in liquid nitrogen using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. About 12 or 15 μg total RNA was resuspended in 10 μL loading buffer (0.10% bromophenol blue and 0.10% xylene cyanol in 100% de-ionized formamide), heated at 95°C for 1 min, and loaded on 15% polyacrylamide denaturing gel (a 19:1 ratio of acrylamide to bisacrylamide, 8 M urea). The gel was run using electrophoresis apparatus (Bio-Rad) at 110 V for 2 h, and then RNAs were transferred to a Hybond N+ membrane by electroblotting in 1× tris-boric acid EDTA buffer at 10 V overnight. The hybridization was performed at 35°C for 18 to 24 h in UltraHyb-oligo buffer (Ambion) using short DNA oligos as a probe (see Supplemental Table 1 online) end-labeled with 32P by polynucleotide kinase (New England Biolabs) and purified through MicroSpin G-25 columns (GE Healthcare) according to the manufacturers' recommendations. The blot was washed twice with 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.5% SDS for 30 min at 35°C. The signal was detected after exposure to a phosphor imager screen using a molecular imager (Bio-Rad).
Small RNA cloning was performed using an Illumina sRNA cloning kit. About 20 μg of total RNA was separated on a 15% 1× tris-boric acid EDTA polyacrylamide gel, and a small RNA region between 15 and 40 nucleotides was excised and purified. This small RNA fraction was used for ligation of adapters and amplification of fragments. Sequencing was performed at Cancer Research UK (Cambridge, UK).
Reference DNA Sequence
The small RNA libraries were aligned to the tomato genome sequence (Mueller et al., 2005, 2009) downloaded from The International Tomato Genome Sequencing Consortium (http://solgenomics.net/), release bacs v340, as well as the EST collection from solgenomics.net website. For identifying tomato NBS-LRRs, the latest version (SL2.40) of the genome as well as entries in the National Center for Biotechnology Information were used to collect sequences.
The small RNA high-throughput sequencing libraries were aligned to the reference sequence using the PatMaN (Prüfer et al., 2008) alignment program. Only reads with 100% match to the genome were used in further analysis.
Identifying miR482 Diversity, Sequence Alignments, and Its Targets
The UEA small RNA analysis toolkit (Moxon et al., 2008) was used to identify members of a given miRNA family (miRProf and miRCat). Sequences of miR482 members were obtained from miRBase release 18 (Kozomara and Griffiths-Jones, 2011) and aligned using ClustalW and ClustalX2 (Larkin et al., 2007). Protein sequence logos were generated using seqLogos (http://imed.med.ucm.es/Tools/seqlogo.html). Targets of miRNA were identified using two different algorithms, namely, psRNATarget algorithm (Dai and Zhao, 2011) and TAPIR algorithm (Bonnet et al., 2010). To find targets of miR482 family in the Arabidopsis thaliana genome, target NBS-LRR sequences were taken from the NIBLRRS Project website (http://niblrrs.ucdavis.edu/index.php) that was supported by the National Science Foundation Plant Genome Program Award 9975971. Images of small RNAs mapped to NBS-LRRs were generated using the UEA small RNA toolkit function SiLoMa (Moxon et al., 2008).
Analysis of abundance of miR482/2118 superfamily was performed through miRProf analysis of published large-scale data sets derived from various plant species available through the Gene Expression Omnibus (GEO) platform (Edgar et al., 2002). These libraries have been described previously (Pilcher et al., 2007; Havecker et al., 2010; Ma et al., 2010; Molnar et al., 2010; Wang et al., 2011; Zhai et al., 2011; Shivaprasad et al., 2012; Song et al., 2012). miRNA sequences were checked to compensate for the misannotation of miR482 type and miR2118-type sequences in miRBase.
Phasing Analysis of Small RNAs
The identification of phased segments in the genomes was performed by calculating the log P value from the hypergeometric distribution adapting the method used previously (Chen et al., 2007, 2010).
Quantitative PCR
cDNA synthesis was performed according to the manufacturer's protocol using random hexamers or oligo(dT) (Superscript II or III; Invitrogen). Quantitative PCR was performed using the CFX96 real-time system (Bio-Rad) using SYBR Green JumpStartTaq ReadyMix (Sigma-Aldrich). Data were analyzed as described (Schmittgen and Livak, 2008). Primers used for amplification are as follows: LRR1 (forward 5′-TGATGTGGTCCGAGACGTGGCT-3′ and reverse 5′-TCCCAGCAACTCGGTCAGCAC-3′: 814 bp), LRR2 (forward 5′-TGGTAGGCACAGAGAAACAGAGGT-3′ and reverse 5′-TCCACACATCATCCAGGGCAACA-3′; 298 bp), Mi (forward 5′-GCTGGAGTCATTGCTGGGAGGG-3′ and reverse 5′-GCACCCACGGACAGCACTCG-3′; 857 bp), Hero (forward 5′-TCCAAGGACGCATGGTAGCCGA-3′ and reverse 5′-AGCCGGTGACTTGTGCCACG-3′; 633 bp), Target of D2+ mRNA (forward 5′-TGCAGATGTGGACTTGGAGCAATCG-3′ and reverse 5′-GCCGTTGCGGAAAATGCCCC-3′; 325 bp); and Nicotiana tabacum NBS-LRR (forward 5′-GGGCATGGGCGGTGTAGGGA-3′ and reverse 5′-ACGCAGCAAAGAACCCCACACTT-3′; 526 bp). GAPDH (forward 5′-GGAGGAGGGAACAACAAGAGG-3′ and reverse 5′-AGATGCCGTCAGTGCCGA-3′; 238 bp) was used as internal control.
Validation of miRNA Cleavage
miRNA target validation was performed using the GeneRacer kit (Invitrogen) as per the manufacturer's instructions. Briefly, total RNA derived from 2-week-old seedlings was used for RNA extraction using Trizol (Invitrogen). RNA was ligated with an adapter and reverse transcribed using gene-specific primers that annealed 150 to 300 nucleotides downstream of the predicted cleavage site within the corresponding target mRNAs. The 5′ RACE amplification products were gel purified (Qiagen), cloned into TOPO TA cloning vector (Invitrogen), and sequenced.
Construct Design and Mobilization to Agrobacterium tumefaciens
miR482 was PCR amplified from tomato genomic DNA. The following primer combinations were used to amplify 35Spro:miR482: 482_35spro_BamHI_For (5′-CCGGggatccTCTACACTTTTCCACCATATCC-3′) and 482_wtpro/35spro_BamHI_Rev (5′-GGCCggatccTCGTGTCATGCAATTTTAAATTCTTGCC-3′). To introduce mismatches into the miR482 sequence (miR482Δ), a PCR ligation method (Trinks et al., 2005) was used. For 35Spro:miR482Δ, the following primer combinations were used for PCR #1: 482_35spro_BamHI_For and 482_Δ_Rev (5′-GCATTTATGGCATGTTCTTTGTATTCAAGACCACTCTTGCG-3′); PCR#2: 482_Δ_For (5′-AGTGGTCTTGAATACAAAGAACATGCCATAAATGCAGAGG-3′) and 482_wtpro/35spro_BamHI_Rev. PCR ligation for 35Spro:miR482Δ was performed with 482_35spro_BamHI_For and 482_wtpro/35spro_BamHI_Rev. pBIN61 (Bendahmane et al., 2002) was used as binary vector. For 35Spro:miR482 and 35Spro:miR482Δ, pBIN61 was linearized with BamHI and miR482pro:miR482 and miR482pro:miR482Δ were cloned downstream of the 35S promoter. Constructs were confirmed with standard sequencing, and correct constructs were transformed into Agrobacterium strain C58C1 (Sciaky et al., 1978) by electroporation using the Gene Pulser Xcell Electroporation System (Bio-Rad).
Bacterial and Viral Inoculation
Pseudomonas syringae DC3000 and hrcC was inoculated into 4-week-old tomato leaves as described (Sohn et al., 2007). TCV and CMV infections were performed as described (Harvey et al., 2011). TRV was a gift from S.P. Dinesh-Kumar (Liu et al., 2002). Tomato plants were infected with sap from Nicotiana benthamiana infected with TRV and CMV, while sap from uninfected N. benthamiana was used as control.
Accession Numbers
RNA sequencing data are available from Gene Expression Omnibus GSE23562.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Sequence Alignment of the miR482/2118 Superfamily.
Supplemental Figure 2. Abundance of miR482 Family Members in Total Extracts of Tomato (M82) and S. pennellii.
Supplemental Figure 3. NBS-LRR Targets of Medicago miR2118 Family Members and slmiR482 in Arabidopsis.
Supplemental Figure 4. Secondary siRNAs Derived from the LRR2 NBS-LRR mRNA.
Supplemental Figure 5. Abundance of miR482a and Two Target mRNAs in Tomato (M82) and S. pennellii.
Supplemental Figure 6. Accumulation of miR482 in Different Tissues of M82 and S. pennellii.
Supplemental Table 1. Oligonucleotides Used for RNA Gel Blot Analysis.
Supplemental Table 2. Targets of miR482 among Tomato Unigenes.
Supplemental Table 3. Targets of miR482 among Tomato Genomic Sequences.
Supplemental Table 4. Targeting Abilities for 33 Tomato NBS-LRRs by miR482 Isoforms.
Supplemental Table 5. Phased sRNA Loci in Tomato Matching Genomic Sequences.
Supplemental Table 6. Phased sRNA Loci in Tomato Matching Unigenes.
We thank Dani Zamir and the Tomato Genetics Resources Center at the University of California, Davis, for providing various tomato lines. We thank the Genomics and Bioinformatics Core Facilities (Cancer Research UK Cambridge Research Institute) for Illumina sequencing and Jonathan Jones for Pst DC3000 and hrcC strains. We thank Sol Genomics Network and the International Tomato Genome Sequencing Consortium for sequences. We also thank Krystyna Kelly and Risha Narayan for helping with accessing GEO data sets. We thank Gail Preston, Natasha Elina, and D.C.B. laboratory members for fruitful discussions. We thank Shuoya Tang for plant care and James Barlow for media preparations. This work was supported by the Biotechnology and Biological Science Research Council (Grant BB/E006981/2), the Gatsby Charitable Foundation, and European Research Council Advanced Investigator Grant 233325 REVOLUTION. H.-M.C.'s visit to the Baulcombe lab was supported by NSC 97-2311-B-001-003-MY3. D.C.B. is a Royal Society Research Professor.
P.V.S. and D.C.B. designed the study and drafted the article. P.V.S. carried out the bioinformatic, RNA, and phenotypic analyses. K.P. and D.M.B. made constructs. H.-M.C. and B.A.C.M.S. carried out phasing analysis.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: David C. Baulcombe (dcb40{at}cam.ac.uk).
↵1 Current address: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan.
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] Online version contains Web-only data.
- Received December 29, 2011.
- Revised February 16, 2012.
- Accepted February 22, 2012.
- Published March 9, 2012.
Open Access articles can be viewed online without a subscription.


text: DUSTY HEARTwith BBGUN and JOE GEORGE Thursday, September 29, 2016 at 7:30pm$8.00 advance$10.00door21+Venue: Turf Club https://www.facebook.com/events/173329993097250/Info TabsDetails PerformersPerformer: DUSTY HEART WEBSITEFACEBOOKTWITTERINSTAGRAMRich vocal harmonies merge with intimate layers of guitar, fiddle and banjo to define the lush sound of Dusty Heart. Barbara Jean and Molly Dean are two songwriters recognized in their own right for their poetic lyrics and distinct vocal styles that when combined create a powerful and haunting sound that takes on a life of its own. Read more about DUSTY HEART Special GuestsSpecial Guests: BBGUN WEBSITEFACEBOOKTWITTERSOUNDCLOUDBBGUN began in a sunny living room, over coffee, on a Friday morning that quickly turned into every Friday morning. Musicians and songwriters Al Church and Neal Perbix's distinct styles, matured over years of Minneapolis gigs, wove to form lyrics that are both contemplative and playful and harmonies both sophisticated and singable. Complemented by Jeremy Hanson's expert, easy drumming, their rock 'n' roll tunes sound best blasted from the speakers of a pickup truck. BBGUN's solid debut album documents the anxiety and surprising joys of trading in late-night cigarettes for a kind of settling down. Read more about BBGUN JOE GEORGE WEBSITEFACEBOOKTWITTERINSTAGRAMJoe George is a guy who loves songs. He loves writing them, loves singing them, and loves and sharing them. He has always felt at home while creating music. Read more about JOE GEORGE

subset: commoncrawl


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Andy Serkis
Jump to navigation Jump to search
Andrew Clement "Andy" Serkis (born 20 April 1964) is an English movie, television, stage, voice actor, comedian, movie producer, and writer who is known for his roles in The Lord of the Rings movie series, King Kong, Rise of the Planet of the Apes, and in The Adventures of Tintin. He has won two Saturn Award in 2002 and in 2011 and a Screen Actors Guild Award in 2003.
Serkis is set to play Alfred Pennyworth in the DCEU 2021 movie The Batman.[1]
Serkis was born on 20 April 1964 in Ruislip, Middlesex, England[2] and studied at St Benedict's School, Ealing, Lancaster University, and in The County College. Serkis has been married to Lorraine Ashbourne since 2002. They have three children.
References[change | change source]
- ↑ Aguilar, Matthew (November 13, 2019). "Batman Director Confirms Andy Serkis as Alfred". Comicbook.com. Retrieved November 13, 2019.
- ↑ "Mr Andrew Clement Serkis | Level". Levelbusiness.com. Retrieved 21 August 2011.
Other websites[change | change source]
Media related to Andy Serkis at Wikimedia Commons
- 1964 births
- Living people
- Actors from London
- Actors from Middlesex
- English movie actors
- English stage actors
- English television actors
- English voice actors
- Movie producers from London
- People from Hillingdon (London borough)
- Saturn Award winners
- Screen Actors Guild Award winners
- Writers from London
- Writers from Middlesex


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The online course is predominately videos with the scripted text written below. The online course is comprised of 5 chapters and a total of 23 lessons. Material will take novice students about 8-10 hours to complete.
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Scott Ian is the official SciFi.com blogger for the final season of Battlestar Galactica.
Who is Scott Ian?
Let me rephrase. Frakkin' Scott Ian, the man who lent headbanger cred to the shaved-head-and-long-goatee look so popular in the '90s, is blogging about BSG.
Who is Scott Ian?
Frak you, you don't know who Scott Ian is. Don't you know he's the man? He's so bad, he should be in detention.
For more than two decades, Ian's been a thrash guitarist for the metal band Anthrax, an outfit that by its very composition, represented the bridge between hardcore punk and its hair-metal brethren.
And if expanding the genre that far wasn't enough, Anthrax were the advanced guard of the rap core movement, releasing "I'm the Man," their answer to the Beastie Boys and then collaborating with Public Enemy on "Bring the Noise." Eat that, Deftones.
On top of that, Ian won last year's "VH1 Rock 'n Roll Celebrity Poker Tournament."
I first encountered Anthrax on, of all things, Married with Children. The whole band guest-starred, and eventually died (I think) from eating some sort of green sludge that had been in the fridge so long it had evolved into a radioactive life form.
"If it was in the refrigerator, why is it warm?"
That line, if I'm remembering correctly (probably not) was issued by Ian himself and it's haunted me for nearly 20 years. I don't know why. It just sends a shiver down my back.
A couple years later, as I entered my teens, I found myself craving hard music, hard guitars, hard drums, speed and screaming; Ricky Rachtman became my new best MTV friend. I wanted to one-up my friend Matt, who had discovered Metallica before I had. So, I got into Anthrax. I even bought a T-shirt that my parents wouldn't allow me to wear to school.
After graduating from junior high, I forgot about Anthrax. What can I say? Korn hit the scene. I only thought about them again in 2001, when the news was all ga-ga over the suspected anthrax packets being mailed to US Congressmen. After all those years of listening to "Belly of the Beast" I realized I'd never actually known what "anthrax" actually was.
I digress. As it turns out, Ian's not only a veteran of the metal revolution, but he's a sci-fi fan-boy and a pretty decent writer. He's blogging each episode (except Friday's will be late because he's in Costa Rica, lucky bastard) and should feature guests who know more about what's going on behind the scenes. And with posts titled "Drop your dicks and grab your sticks!!" it's pretty clear that they've given him a long leash.
Ian's blog replaces executive producer and creator Ron Moore's blog, which hasn't been updated since last summer. To tell you the truth, I actually prefer to hear from someone who doesn't know what's going on behind the scenes; it means he makes predictions and interpretations I don't always agree with. Now if only they would activate comments, so I can tell him as much.
But if they do, I'll have to resist gushing. I just realized that I'm nearly as much of a Scott Ian fanboy as I am a Battlestar geek. Frak me.


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06 Sep 2019
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At The Festival
Visit the Seedlip stand at Taste of London to discover their two unique products:
SEEDLIP Spice 94 – an aromatic & complex blend of all spice berry, cardamom, bark & citrus peel distillates.
SEEDLIP Garden 108 – a herbal & floral blend of hand-picked peas & homegrown hay with traditional plant distillates in celebration of the English countryside.
Both served with tonic for an adult & sophisticated alternative.


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Monday, October 19, 2009
Dinosaur Research Institute's annual fundraising dinner
The Dinosaur Research Institute's annual fundraising dinner will be held on November 7, 2009 at 6PM at the Earl Grey Golf Club in Calgary. Tickets are $150 per person with tax receipts available for a large portion of the ticket cost. The funds raised go towards dinosaur research based in Alberta or for researchers from Alberta to conduct research outside of the province (such as visiting distant museums, etc.).
The Dinosaur Research Institute also maintains the Rene Vandervelde grant, which provides funds for Alberta-based dinosaur researchers to present at conferences as well as the Darren Tanke Neoceratopsian Scholarship which provides funds for ceratopsian workers to visit distant museums, present at conferences, etc.
During the dinner, several presentations from past recipients will occur and a paleontology student from Alberta will be present at each table. A silent auction will be held after the dinner with numerous interesting items available.
For more information, visit www.dinosaurresearch.com. Hope to see you there!
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This Girl Can Personalised positive Gift for Girls
This Girl Can Personalised positive Gift for Girls
Here's to strong girls everywhere! May we know them and may we raise them.
Encourage the girls you know to be who they want to be with this roomy accessory bag.
Perfect as a wash bag, a makeup bag, a pencil case - it has so many uses. It measures approx. H21 x W24 x D2 cm.
We personalise each bag with the name of your choice using pink glitter letters in our fierce font and lightning bolt motif. Don't forget to leave your personalisation in the Notes section of your order.
You can also add a clip-on strap in pink or rainbow webbing.


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Active CBD Pen by Savage CBD
Savage CBD presents our premiere CBD Active. This pen is infused with Maui Wowie and Caffeine – perfect for day time or just any time throughout the day you want to grab a relaxing jump start. Simply grab one of these, use as needed, and enjoy!
Ingredients: Full Spectrum Hemp Extract, Caffeine & Terpenes.
Brand Description:
We are one of the largest CBD brands in the Vape, Eliquid, and Ejuice industry. There are many CBD disposable pens on the market, but none of them quite stacked up to the Savage CBD disposable CBD Pens. Savage CBD's motto is full transparency, high-quality CBD products. We ensure a low cost to end-users so that everyone can enjoy CBD. Savage CBD keeps innovating to make sure they stay one step ahead of the competition in the CBD & Ejuice industry.
Full Description:
Savage CBD took a hard look at the CBD industry and saw where a lot of companies were falling short. It seems like everyone has a CBD disposable pen on the market but the flavors weren't very great and very few are rechargeable. What Savage did is they hired on a team of mixologists and chemists to nearly perfect the CBD mixing process. Meaning that you can almost always count on getting a good flavor and clear liquid with every CBD disposable pen. We found the best rechargeable pens so you can enjoy our product to the very end. Since Savage CBDs parent company, Savage Enterprises, is a dominator in the Ejuice industry it's no secret they already know how to craft near-perfect flavors. Taking the best Maui Wowie terpenes and full-spectrum hemp extract we crafted the perfect flavor for disposable CBD pens.
Due to Washington state laws & regulations, we are unable to process or sell any CBD vape products to the residents of the state of Washington.


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You've hidden your smile for the last time! You are finally ready to improve the appearance of your teeth, and you have some ideas about how you'd like your smile to look. You have some consultations scheduled and you're ready to learn more about cosmetic dentistry. Here are some important questions to discuss with your prospective dentists about cosmetic dentistry in Irvine, CA.
What are Your Ideas and Recommendations for My Smile?
As you visit with different dentists, you will find that they all have different perspectives and treatment philosophies. This is a good thing! You may learn that you have multiple treatment options to choose from as they present different plans to you. There may be possibilities you hadn't even thought of. Be sure to work with the dentist that will be most effective in helping you achieve your goals for your smile.
Can I See Before and After Pictures of Cases Similar to Mine?
Cosmetic dentistry is truly an art. Creating symmetry and harmony within a smile requires more than just excellent technical skills. You want a dentist who combines both skills and artistry, and there's no better way to determine this than by looking at pictures of their work.
What Will My Smile Look Like when It's Completed?
Most dentists don't have a crystal ball, but some like Dr. Jeffrey C. Robertson use a diagnostic wax-up for the same purpose. A diagnostic wax-up is a wax model created of your teeth and designed to show you what your finished smile will look like. It's very exciting and much easier to make final decisions about your treatment plan when you can see the finished product right in front of you!
Dr. Robertson would love to help you achieve your dream smile, and with over 30 years of experience, he has the skills to do it. Call us today to schedule your consultation and learn more about what's possible for your smile.


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Defining Resonance Structures
Moderators: Chem_Mod, Chem_Admin
4 posts • Page 1 of 1
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Defining Resonance Structures
Are resonance structures only the Lewis structures by which you can move the double triple bonds? Or do they include all the structures that you can draw for a molecule? For example, with N2O, you can draw different structures with single, double, or triple bonds, so would all those structures be considered resonance? Or is it only when you move the bonds are they then referred to as resonance structures?
- Posts: 103
- Joined: Sat Aug 17, 2019 12:17 am
Re: Defining Resonance Structures
Resonance is when there are multiple correct ways to draw the Lewis Structure for a molecule because the bonds are movable and stable in either position, either from rotating the molecule or just simply changing the bonds around for usually every other bond pair. An example of this can be the ion Nitrate NO3- which has multiple ways of drawing its Lewis Structure but it simply moves the double bond pair to different locations
Re: Defining Resonance Structures
Resonance structures I believe only apply to when there are multiple ways of drawing the exact same structure. For example, moving the location of a double bond would make another resonance structure.
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Re: Defining Resonance Structures
I think that formal charge also comes into play.
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Adapt Forward looking for 100 Percent Remote Senior Software Engineer / Full Stack Developer
Job Description
Adapt Forward is a cybersecurity solutions provider for some of the nation's most valuable information systems. Leveraging advanced threat assessment technology and experience in building high-level information security infrastructure, we develop adaptive solutions uniquely tailored to our customers' business objectives to protect sensitive data against sophisticated threats in an increasingly complex security environment.
Job Summary
We are a technology-driven Cybersecurity Service Provider (CSSP). Our mission is to provide remote asset protection for our customers at distributed sites around the world. The development team provides an application used for triaging active cybersecurity incidents and facilitating rapid response for risk mitigation. The successful candidate will be developing application features for the web-based application that we develop and support in a highly fluid team environment.
Key Responsibilities
- Advanced software-to-database integration
- Developing efficient software features to deliver dynamic content to Jinja2 templates
- Working together with front-end developers as well as server-side developers to ensure both sides understand one another.
- Ensuring that the developing codebase is conforming to best practices in regard to coding standards, form validation (both on front-end and server-side), placement and flow of business logic, etc.
- Limited after hours availability required
Basic Requirements
- Eligibility for a DoD Secret Clearance. Candidates with a current/active DoD clearance will be given preference. ** Adapt Forward will sponsor a clearance for those that do not currently possess one.
- Bachelor of Science degree in Computer Science, Software development or equivalent technical major.
- Knowledge of relevant state-of-the-art technologies and solutions.
- DoD approved 8570 baseline certification(s): IAT Level II Certification.
Professional Qualifications
- Expert level skills (8+ years) development in a LAMP stack environment
- Minimum of 10 years' experience developing innovative web applications
- Expert level (10+ years) experience with Object-Oriented design principles
- Expert level skills (8+ years) in Python (PHP considered), JavaScript, HTML5, CSS3, and SQL
- Experience (5+ years) with Bootstrap, jQuery, and AJAX
- 8+ years' experience developing in a Linux environment
- Strong experience using version control systems; SVN, GIT, or other equivalent experience
- Strong documentation and communication skills
- Ability to handle multiple projects with flexible requirements
Desired Skills and Experience
- Cybersecurity Experience
- Experience with Bootstrap, jQuery, and AJAX
- Experience developing on the Flask python framework
- Experience with Angular/Node
- Experience with RESTful design principles
- Threading/multiprogramming experience
- Full-stack development experience
- Experience with Oracle
- Test-Driven Development experience as well as Unit Testing
- Agile development methodology
- Docker and Kubernetes / containerization
- CI/CD pipeline
- Cloud development / implementation
- Master's degree in Computer Science, Software development or related field
- Current portfolio
Summary of Benefits
- Comprehensive Physical Wellness Package, including Medical, dental, vision care, plus flexible spending accounts for health- and dependent-care are included in our standard benefits plan.
- 401k Retirement Plan with Matching Contribution is immediately available and vested
- Annual Training Budget to be used for conference attendance, school enrollment, certification programs, and associated travel expenses.
- Ten Federal Holidays, plus three weeks of PTO/vacation/sick leave that accrues at a rate of ten hours per month.
- Employee Assistance Program: Counseling/legal assistance and other employee well-being programs are also offered.
Adapt Forward is an equal opportunity employer that values diversity in the workplace and does not discriminate or allow discrimination on the basis of race, religion, age, gender, sexuality, national origin, citizenship, disability, veteran status or any other classification protected by federal, state, or local law. Adapt Forward promotes affirmative action for minorities, women, disabled persons, and veterans
Click below link to apply for this job:
Apply Here


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Holden has cemented the foundations of its future SUV family with confirmation it will add the American-sourced Acadia mid-sized soft roader from 2018.
The Australian brand announced the seven-seat Acadia during an event at its Port Melbourne headquarters where it outlined some of its immediate plans in preparation for the closure of its local manufacturing operations by the end of 2017 and the transition to becoming a full import brand.
While the Acadia, which is badged as a GMC product in the US, won't arrive in local showrooms for another 18 months at least, Holden is preparing to roll out five new models in the next five months in a bid to strengthen the supporting cast in its range before the popular locally-built Commodore is replaced by the next-generation Opel Insignia.
The five new vehicles include the updated Colorado pick-up that was launched a fortnight ago and the Trailblazer wagon that uses the same underpinnings which arrives in showrooms in October. The all-new Astra small car and updated Barina city car will be added in November and followed by a heavily revised version of the Trax baby SUV in January.
"This level of launch activity hasn't happened at this company for nearly 15 years and signals our intent loud and clear," said Holden Chairman and Managing Director, Mark Bernhard.
The Acadia is the newest addition to Holden's lineup and slots in below the rugged Trailblazer with a more urban focus as a rival to the likes of the Toyota Kluger, Mazda CX-9, Hyundai Santa Fe and Kia Sorento. It will also face-off against Ford's similarly US-sourced Edge, which is set to replace the locally made Territory at around the same time.
While Holden has yet to reveal full details or give any indication on pricing, the Acadia is available in the US in both front and all-wheel drive variants and in five-, six- or seven-seat configurations across seven different model grades.
It is powered by a choice of either a 2.5-litre four cylinder that produces 144kW and 254Nm (available only in front-wheel drive) or a 3.6-litre V6 that generates 231kW and 367Nm. Both powertrain options are offered with a six-speed automatic only.
And it has the latest suite of connectivity functions and safety features, including automated emergency braking, lane keeping assist, blind spot warning and a 360-degree camera.
While the Acadia will bolster Holden's SUV portfolio, it won't be the only new soft roader in its showrooms with a replacement for the existing Captiva due to be revealed within the next six months and on-sale at around the same time as the Acadia in 2018.
Bernhard told Drive the company is exploring every option within General Motors' portfolio to build on its existing SUV line-up but said the likelihood of anything larger than the Acadia, such as Chevrolet Subarban/GMC Yukon, coming from America is marginal.
Instead, Drive understands Holden will capitalise on its conventional SUV range - from Trax through Captiva, Acadia and Trailblazer - with niche high-riding variants of the Astra and Commodore wagons that follow in the wheeltracks of the Subaru Outback.
Beyond the new models, Holden also announced a new marketing campaign that is underpinned by a yet-to-be-released track from local electronic artist Flume, a streamlined logo redesign and a renewed focus on customer initiatives, including a 24-hour test drive program.
Read all the latest Holden news and reviews here


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Our clients benefit from the knowledge that Medirest is the largest provider of support and food services to hospitals and the senior living industry, operating in every State and Territory of Australia including regional areas. Medirest is a valued partner because of our unique understanding of the key success factors facing healthcare service providers and we develop and deliver innovative solutions to meet those challenges.
Our aim is to excel in the key areas of patient and resident satisfaction, quality and nutrition, with cost effective strategies that reinforce our commitment to our clients.
Medirest is the market leader because:
For more information on how Medirest can help you with your catering and support services needs, please contact us via the details below:
National Office
Ph: +61 2 9797 4900


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30 Lonsdale St Braddon, ACT 2612
No Place Like Home is a pop up exhibition by ceramic artist Gemma Bonshek Kane that is supported by the Assemblage Project as part of the Australian Ceramics Triennale in Canberra from the 9th to the 11th of July 2015. Bonshek Kane's "No Place Like - Series 2" captures the intrinsic connection between people, place, and memory within our own physiology. Bonshek Kane questions how places we identify with become part of us, the outside existing within, sometimes long after those places are gone. Bonshek Kane's work draws inspiration from her immigrant childhood, the loss of her Polish heritage, and her many moves between Australian cities as both a child and an adult. "No Place Like – Series 2" speaks to the connection between people, homeland's and generations that lasts much longer than any built form.
Image: Gemma Bonshek Kane, work title - "No Place Like - Series 2"


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This tool makes it easier for you to understand EXOS stacking and hardware compatibility. With so many hardware configurations to choose from, we understand it can be challenging to know which ones can work together.
With the EXOS Stacking Tool v2.0, users can:
- Build a stack by selecting various EXOS models so the tool can verify compatibility
- See which stacking technologies (VIM and stacking modules) are compatible and recommended
- See the minimum and maximum EXOS versions supported by the entire stack
- See the theoretical table size limits for the entire stack based on model selections
- See the suggested stack master and backup
- Access product information and knowledge articles linked from within the tool
See this article for more information: Where Can I Find the EXOS Stacking Tool?
Ask questions or provide feedback by commenting below. We want this tool to benefit you, so your feedback is welcomed and encouraged!
Extreme Networks Services Team


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Quilt Documentation Being Held at:
Lake Havasu City, AZ 86403
(First Documenting Day was held on July 14, 2016 -- 2:00pm)
Want to do your own??? If your quilt is "yours" and you know everything about it, then yes, complete the paperwork, Release Form and the Overall Quilt Description Form (7pgs) and send 3 photographs (1-Overall, 1-Upclose, 1-Back including the label) of your quilt to us we can get you into the database - send to Havasu Stitchers, PO Box 2875, Lake Havasu City, AZ 86404.
|Quilt Documentation Forms
|[Click on each of the items below to downlaod file]


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5Dimes is a US-focused bookmaker covering a wide range of sports, including cricket, with a simple and straightforward online betting site. 5Dimes was established in 1998 and markets itself as the betting site for seasoned gamblers. Regulated in Costa Rica, 5Dimes is open to customers from all over the world, including US clients.
⇒ ⇒ Click here to visit 5Dimes.com
How to Bet on Cricket at 5Dimes
It is not possible to view the markets on offer with 5Dimes prior to opening an account, which is a straightforward process. All accounts are held in US dollars. Once logged in account-holders can access the Sportsbook page. From this page a menu on the left-hand side lists the various types of bet available – straight bets, reverses, parlays, and so on. Selecting one of these reveals the range of sports for which that type of bet is available in the centre of the page. Select the preferred sport using the tick box menu and click the continue tab to open all the available markets if that type of bet and for that particular sport.
Odds can be displayed for each market in decimal or American format, and the bet is be selected by entering a stake.
5Dimes Cricket Betting Markets
The site has a limited range of cricket markets, usually involving current matches and mostly with outright result markets. There are generally no prop bets, futures, handicap or live betting options for cricket markets.
Depositing and Withdrawing Funds
For non-US clients, funds can be deposited in a 5Dimes account through a variety of methods, including debit/credit card, bank transfer, Western Union transfer, cheque, or through an online payment service (Neteller, Moneybookers, Click2Pay). The minimum deposit amount is US$50 regardless of the deposit method used. Withdrawing funds for non-US clients is straightforward. Payments can be made via bank transfer, cheque or through an online payment service. There are fees for some types of withdrawal method, and availability depends on the country of residence.
For US clients, the options for depositing and withdrawing funds are more limited due to the various laws relating to online gambling. 5Dimes allows US clients to deposit funds using MoneyLineWallet as an intermediary. Funds are first deposited into a MoneyLineWallet account through bank transfer or eCheck, and then transferred into a 5Dimes betting account. Money Gram and Western Union can also be used to deposit cash into a 5Dimes account. Withdrawals are made by bank transfer onto a savings or checking account.
Features of the 5Dimes Site
The 5Dimes site is simple and uncluttered, and provides a wide variety of bets aimed at the experienced gambler. This allows clients to combine different types of combination bet across various sports, selecting a result market from a cricket match and a result market from a different sport.


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Last Updated: May 4, 2017
How can you generate enough content ideas to keep your website fresh and visitors coming back? Coming up with topics to write about is easier than you think. Here are 10 tips that can help you generate new content ideas for your website on an ongoing basis.
You already have enough traffic, right? (Cue the laughter.) Regardless of how big you get, you never have enough traffic. Traffic leads to profit and the more profit, the better, right?
And, because the rules of content marketing are constantly changing, you can't read enough articles or try enough new tactics. If you find the right ideas for your audience, that's when the fun really starts.
The buzzword (or phrase) that every Internet entrepreneur knows is content marketing—using content like articles, video, podcasts, and images, to draw people to your site where you will hopefully monetize them.
But content marketing is a crowded field. Everybody is doing it now. Standing out and getting the eyes and ears of your readers is exceedingly difficult but what could set you apart from others is the quality of your ideas. Your readers will only read so many of the same tired, rehashed topics and even the cutest pictures and videos of puppies get old before too long.
Unless you're one of the very few people that can create the most viral of ideas by yourself, you're going to need help. Everybody has different experiences and those experiences will allow them to think of things you couldn't. Grab a pizza, a whole lot of paper and technology, and start brainstorming as a team. If you help one blog owner, he or she will help you.
Buzzsumo watches social media platforms and will alert you when something is about to go viral. If you have it alert you after a modest amount of likes and shares, you will know when something goes viral before the rest of the Internet does.
If you know what's trending, you can produce content to go with it. Don't copy anybody's work. Come up with a new angle to present a topic people are already reading. Go to buzzsumo.com to get started. The platform offers a free plan.
Buzzfeed—the site that people love to hate. Think what you want but Buzzfeed has its fingers on the pulse of digital content trends. In fact, the company plays a large part in creating the trends. Check out its recent articles and see which ones people really liked. Create content around those subjects.
You may not fancy yourself a videographer but the data doesn't lie. People want video. Text and images still work but not much beats a video—at least not in 2015. Want proof? Check out your content consumption habits. Check out your kids' too. Likely, you stop and watch those videos that Facebook throws in your newsfeed. Watching a video is much more mindless than reading—at least for most people. Sometimes a video where you talk about your blog post is enough to catch readers' eyes.
RELATED: How to Grab Your Audience's Attention with Video
If you're a personal finance writer, keep a list of issues you tackle in your personal life and write about them. Did you have to deal with your neighborhood HOA over an issue? That's an article. Also ask friends, family, and professional acquaintances about things they've experienced and use those as ideas. It's so easy to forget the most obvious of sources of inspiration, isn't it?
If you have a blog with a reasonable amount of traffic or an e-mail list, ask them what they would like to see. Make your readers partners in your content versus seeing them only as customers.
All of the major social media sites have a list of what's trending right now. Some things will come and go rapidly but others are big enough to have staying power. Think of ways to jump on trends even if it's a fun way to insert your business into an area it wouldn't normally belong. Animal Plant created the Puppy Bowl to bring in animal lovers on the day of the Super Bowl.
Find an expert and interview them on a topic relevant to your readers. Even better, ask your readers to submit questions for the interview.
Guest posts work well when the guest is an authority in his or her field. Don't take offers from people who want to pay you or clearly aren't looking to add value to your readers. When they have the right attitude—and the credentials to back it up, guest posting saves you time while putting something new in front of your readers.
RELATED: How to Do Business Blogging the Right Way
Go to YouTube, find a great video, write an amazing headline (or two or three), include some backstory to make an article out of it, and post it on your site. That's how sites like Buzzfeed and ViralNova got their start. When you don't have a giant writing staff, let other people create the content. As long as you're embedding the video from the original source and writing your own original text, you aren't violating any laws.
Of course, always be careful with content curation. Stick with embeddable content that you know is suitable for sharing.
Bottom Line
You're not alone. Unless you have a team of creatives sitting around generating content each day, the struggle for high-quality engaging content is a reality for most blog owners. Always try something new. Don't be afraid to fail, and don't continually fall back on the same ideas. You can fail multiple times but one piece of content that gets giant traffic is enough to make up for all of the duds.
© 2015 Attard Communications, Inc. All Rights Reserved. May not be reproduced, reprinted or redistributed without written permission from Attard Communications, Inc.


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Time for giving
A massive increase in homelessness in Croydon has seen 100% increase in demands made on The Wellington, formerly the YMCA, a homeless hostel based in the centre of Croydon. And so, the Axis Croydon Office decided to help the homeless in Christmas 2018.
Elaine Adamson, Senior Administrator, had started to collect some key essentials for homeless people and the whole Croydon Team soon got involved. A few volunteer hours and a drive to make a difference produced 75 gift bags comprising hats, gloves, socks, toothpaste and brush, deodorant, hand warmers, toilet rolls and blank Christmas cards and a pen so that residents of the hostel can contact family or friends. Axis people collected, franked and posted the envelopes.
Croydon Council held a bedding drive collecting over 30 duvets and pillows for Wellington residents – all of which were delivered just in time for Christmas when Ian Rhodes, Divisional Manager and Sadie Hack, Resident Liaison Officer also took the gift bags to the Wellington.
There they talked with Lee who is homeless.
Ian explained, "Lee has been homeless for five years due to losing his job with a local removal company. He was happy to help us unload the vans. We presented his gift bag. He was over the moon stating he hadn't had a bar of soap to himself for a very long time. He was also very excited to be given a brand-new duvet and two pillows.
"I cannot describe the feeling and emotions that came over me when visiting this place – what we consider as everyday items are considered as luxury and beyond grasp by many of the residents. . It goes to show that every little does and can help. I'm proud to say that Team Croydon have undertaken this great work with compassion, empathy and most importantly respect. This has certainly been a leveller for my team and me!"
Just the job
In another initiative, also in December the Axis Croydon Gas Team entertained the residents of Freeman Court sheltered housing scheme with a cup of tea, a slice of cake and the great gift of practical help and advice. Rebecca Lock, Axis Contracts Manager, with colleagues Carol Thompson, Alison Macey, Georgia Halliday and Jackie Avis, assisted residents in reporting any repairs that needed attending to, and a handyman was available to help with any minor jobs that could be completed there and then.
Dinner in a bag
Axis Repairs & Maintenance Croydon Team and Lidl organised 'Christmas dinner in a bag' for 47 local residents. Bags contained a chicken joint/nutroast, Yorkshire puddings, stuffing, gravy, carrots, parsnips, Brussels sprouts, potatoes, sparkling non alcoholic drink and Christmas pudding. Our team gave away 19 bags to residents in the north of the borough identified as suffering from severe deprivation and 28 bags to residents currently on probation and identified by the London Probation service as vunerable or in severe need.


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Introducing Vuttaline - Handwritten Font
Vuttaline is a Script Font with Handwritten Style. The Vuttaline font made with digital brush pen strokes that making this font look authentic. This font is perfect for fashion brand, wedding invitation, business card, logo brand, signature, and then calligraphy.
This Font contains Elegant, Authentic, Luxury and has a Unique Concept.
- Ligatures
- Uppercase and Lowercase letters
- Numbering and Punctuations
- Multilingual Support
- Works on PC or Mac
- Simple Installation
- Support Adobe Illustrator, Adobe Photoshop, Adobe InDesign, also works on Microsoft Word
Hope you Like it.
Introducing Design School – our jam packed platform with its own mini search engine! Design School was made to help you learn all there is to know about fonts, graphics, templates, designs, file types, different software programs, Frequently Asked Questions and much much more.


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