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Research ArticleResearch Article
cDNA-AFLP Reveals a Striking Overlap in Race-Specific Resistance and Wound Response Gene Expression Profiles
Wendy E. Durrant, Owen Rowland, Pedro Piedras, Kim E. Hammond-Kosack and Jonathan D. G. Jones
The Plant Cell June 2000, 12 (6) 963-977; DOI: https://doi.org/10.1105/tpc.12.6.963
Wendy E. Durrant
Sainsbury Laboratory, John Innes Center, Colney Lane, Norwich NR4 7UH, United Kingdom
Owen Rowland
Pedro Piedras
Kim E. Hammond-Kosack
For correspondence: jonathan.jones@bbsrc.ac.uk
© 2000 American Society of Plant Physiologists
The tomato Cf-9 gene confers resistance to races of the fungal pathogen Cladosporium fulvum expressing the Avr9 gene. cDNA amplified fragment length polymorphism analysis was used to display transcripts whose expression is rapidly altered during the Avr9- and Cf-9–mediated defense response in tobacco cell cultures. Diphenyleneiodonium was used to abolish the production of active oxygen species during gene induction. Of 30,000 fragments inspected, 290 showed altered abundance, of which 263 were induced independently of active oxygen species. cDNA clones were obtained for 13 ACRE (for Avr9/Cf-9 rapidly elicited) genes. ACRE gene induction occurred in the presence of cycloheximide. Avr9 induced ACRE gene expression in leaves. Surprisingly, ACRE genes were also rapidly but transiently induced in leaves in response to other stresses. The amino acid sequences of some ACRE proteins are homologous to sequences of known proteins such as ethylene response element binding protein transcription factors, the N resistance protein, a calcium binding protein, 13-lipoxygenase, and a RING-H2 zinc finger protein. Rapid induction of ACRE genes suggests that they play a pivotal role during plant defense responses.
Disease resistance in plants often involves recognition of invading pathogens followed by activation of a defense response. Such incompatible interactions are dependent on the presence of a resistance (R) gene in the host and an avirulence (Avr) gene in the pathogen (Flor, 1971; Keen, 1990). Many plant R genes have been identified. Their products have motifs consistent with potential roles in pathogen detection and subsequent signal transduction (Bent, 1996). However, the signal transduction activated by R gene products is still poorly understood.
Cladosporium fulvum is a biotrophic fungus that causes leaf mold disease of tomato. The tomato Cf-9 gene confers resistance to C. fulvum races expressing the corresponding Avr9 gene. The Avr9 protein is secreted by the fungus and is processed to a cystine knot peptide of 28 amino acids, which can be retrieved in intercellular washing fluid (IF) from infected leaves (De Wit and Spikman, 1982; Van den Ackerveken et al., 1993). Infiltration of Cf9 tomato or transgenic Cf9 tobacco with Avr9 leads to necrosis within 24 hr. This response is faster in tobacco than it is in tomato (Hammond-Kosack et al., 1998). Cell suspension cultures derived from Cf9 tobacco plants, when challenged with Avr9, rapidly produce active oxygen species (AOS) (Piedras et al., 1998) and activate two mitogen-activated protein (MAP) kinases (Romeis et al., 1999) and a calcium-dependent protein kinase (Romeis et al., 2000).
The mode of action of the Cf-9 protein is not known. Changes in gene expression are likely to be important for activation of defense mechanisms, and transcriptional changes have been reported in several plant–pathogen interaction systems (Rushton and Somssich, 1998). The nonhost resistance responses of parsley and tobacco cells to elicitors from cultures of Phytophthora spp have been particularly well studied (Somssich et al., 1989; Suty et al., 1996). However, analysis of transcriptional regulation has often focused on induction of pathogenesis-related (PR) proteins and enzymes of phenylpropanoid synthesis (Linthorst, 1991; Dixon and Paiva, 1995). Cf-9 gene–dependent induction of chitinase and 1,3-β-glucanase expression has been documented (Wubben et al., 1996), but these responses were 4 to 8 hr after elicitation. We sought to identify genes that exhibited rapid Cf-9–dependent induction by Avr9. We used Cf9 tobacco cell cultures, which provide an amenable experimental system in which to study rapid Avr9 responses. Synchronous delivery of ligand to cells can be achieved more reproducibly in cultures than in leaves, making them ideal for biochemical and pharmacological studies (Piedras et al., 1998).
The rapid production of AOS is one of the earliest defense responses (Lamb and Dixon, 1997). AOS may contribute directly to plant defense and also play a role in signaling, leading to the induction of defense genes (Jabs et al., 1997; Yang et al., 1997). Cf9 tobacco cell cultures provide a system in which we can distinguish between AOS-dependent and -independent events by preincubation with diphenylene-iodonium (DPI) to inhibit AOS production (Piedras et al., 1998). This study provides comprehensive expression profiling of rapid R gene–dependent, AOS-independent gene induction during the plant defense response. The RNA fingerprinting technique of cDNA amplified fragment length polymorphism (cDNA-AFLP) (Bachem et al., 1996, 1998) was used. Of ∼30,000 cDNA fragments inspected, 290 fragments showed altered abundance within 15 to 30 min after adding Avr9. Of these, 263 were induced by Avr9 even in the presence of DPI. Some showed homology to known genes, of which many may have roles in further signaling. We also observed the induction of Avr9/Cf-9 rapidly elicited (ACRE) genes in Cf9 tobacco leaves in response to Avr9 infiltration. We discuss the importance of this large set of induced genes in the defense response and in other stress responses.
Rapid Alterations in Cf9 Tobacco Transcript Levels in Response to Avr9
Cf9 tobacco cell cultures were treated with IF containing Avr9 (IF[Avr9+]) or lacking Avr9 (IF[Avr9–]) or pretreated with DPI for 5 min before adding IF(Avr9+). Cells were harvested after 30 min, and RNA was extracted and used for cDNA-AFLP. In experiments conducted simultaneously, Avr9-dependent AOS production was observed. This production could be inhibited by adding DPI, as shown by Piedras et al. (1998) (data not shown). Gene expression was surveyed in two independent sets of cells using 512 primer combinations for selective amplification. A section of a typical AFLP gel used in this analysis is shown in Figure 1A. The majority of bands showed no change in intensity between the three treatments. Changes in the intensity of individual bands did not affect others in the same lane, indicating that product accumulation was not affected by the concentration of individual substrates in the reaction. The AFLP fragments ranged in length from 50 to 400 bp; for each primer combination, 50 to 70 bands were observed, with ∼30,000 fragments being analyzed. Altered expression patterns were observed for 290 fragments (Figure 1B); of these, 273 were induced by Avr9. Only 4% of the changes in expression were inhibited by DPI, indicating that most rapid changes are independent of AOS.
Control experiments on 30 fragments confirmed that their differential expression required both Avr9 and Cf-9 (Figure 1C). No induction was observed in nontransformed tobacco cell cultures. Treatment with the chemically synthesized Avr9 peptide was sufficient to produce the differential expression observed after IF(Avr9+) treatment, confirming that Avr9 was responsible for the changes (Figure 1C). These controls demonstrate that the altered gene expression is Cf-9 and Avr9 dependent.
Compilation of Sequences from Induced and Repressed cDNA Fragments
The differentially expressed fragments were excised from the gels, reamplified by polymerase chain reaction (PCR), and sequenced. DNA sequences were obtained for 260 fragments. The other sequences were a mixture of PCR products and could not be directly sequenced. The sequences were compared with those in the GenBank database using the BLAST program (Altschul et al., 1997). Sequence similarity was found for 37 induced and five repressed sequences (Table 1). These sequences were similar to those of protein kinases, transcription factors, calcium binding proteins, a protein phosphatase, a 13-lipoxygenase, and other proteins. Their roles in signal transduction suggest that many Avr9-induced proteins have signaling functions. The sequences of the majority of the fragments showed no homology to known sequences. They may represent previously uncharacterized genes, or the AFLP fragments may have been too short to reveal significant homology. The sequence data for the fragments listed in Table 1 have been submitted to GenBank, and their accession numbers are listed in Table 1.
Time-Course and Inhibitor Studies of Selected cDNA Fragments
We focused our attention on 18 cDNA-AFLP fragments induced independently of AOS. These were selected based on their homology to known genes or because they were strongly induced. Their expression patterns were examined in Cf9 cells during a 4-hr time period after IF was added. All of the cDNA fragments were induced by IF(Avr9+) (Figure 2) but not by IF(Avr9–) (data not shown), but their patterns of expression varied in strength and timing. Fragments 1 and 111 were rapidly but transiently induced within 15 min, with maximum expression after 30 to 60 min; expression decreased after 2 to 4 hr. Fragment 4 showed very transient expression; it was induced 30 min after elicitation, but expression had decreased by 1 hr. The other 15 fragments were also induced rapidly, but their induction was more sustained and transcript levels remained high for at least 4 hr. These patterns were reproducible in at least two independent experiments.
Most of the genes were induced in the presence of DPI, suggesting that AOS are not intermediates in the pathway to gene induction and that an alternative pathway must be involved. The protein synthesis inhibitor cycloheximide (CHX) was used to test whether induction of these 18 genes required de novo protein synthesis. Cf9 cells were pretreated for 1 hr with 5 μg/mL CHX. IF was then added, and cells were harvested after another 30 min. Treatment with CHX did not inhibit Avr9-dependent gene induction; rather, it induced the expression of these genes in the absence of Avr9 (Figure 3). This suggests that new protein synthesis is not required to induce the 18 genes tested, reinforcing the idea that their induction is an early target of Cf-9. Induction by CHX alone is typical of many early-response genes and suggests the involvement of a short-lived repressor (Koshiba et al., 1995).
cDNA-AFLP Display of Avr9/Cf-9–Dependent Changes in Gene Expression.
(A) Cf9 tobacco cells were treated with IF(Avr9+) or IF(Avr9–) or pretreated for 5 min with DPI before addition of IF(Avr9+). Cells were harvested after 30 min. Lanes are in groups of three: each group was amplified with a different pair of selective AFLP primers. (+) indicates Avr9 or DPI added; (–) indicates Avr9 or DPI not added.
(B) Enlarged view of the boxed area in (A). The arrows indicate repressed (1) and induced fragments (2) and a fragment whose induction is inhibited by DPI (3). (+) indicates Avr9 or DPI added; (–) indicates Avr9 or DPI not added.
(C) Induction requires both Cf-9 and Avr9. Cell cultures of Cf9 and nontransformed tobacco (PG) were analyzed 30 min after elicitation with IF(Avr9+) (+), IF(Avr9–) (–), or chemically synthesized Avr9 (syn.) and compared with untreated Cf9 cells (0).
Isolation of cDNA Clones Corresponding to 13 cDNA-AFLP Fragments
Sixteen out of the 18 AFLP fragments described above were used to screen a cDNA library for full-length clones (Table 2). The cDNA library was made from Cf9 tobacco cells harvested 30 min after the addition of IF(Avr9+). Before using the fragments for library screening, their identity was verified by repeating the AFLP technique using three selective nucleotides (rather than two) on the MseI primer and sequencing the band excised from the gel. Repeating the AFLP is important because no part of the gel is free from other PCR products, and it is likely that more than one fragment will be excised from the gel. Increasing the number of selective nucleotides decreased the number of fragments being amplified, thereby reducing the likelihood of amplifying contaminating fragments. The sequences of these fragments matched those from the original AFLP screen for all fragments used for library screening (data not shown). The reamplified fragments were used as probes to screen the library.
The results of the library screening are summarized in Table 2. We isolated and sequenced single clones for 13 AFLP fragments. Nicotiana tabacum is an allotetraploid originating from two distinct diploid species (N. sylvestris and N. tomentosiformis). The clones hybridizing with AFLP fragments 31, 75, 111, 137, and 180 and the AFLP fragments themselves had mismatched nucleotides, suggesting that these clones derived from a homolog of the cDNA from which the AFLP fragment originated. Clones with a 100% match at the nucleotide level with the AFLP fragment were obtained for the remaining eight fragments. When AFLP fragment 111 was used as a probe, two hybridizing clones were isolated, ACRE-111A and ACRE-111B, with a 99 and 94% match at the nucleotide level with the AFLP fragment, respectively. In some cases, cDNAs of different lengths with different polyadenylation sites were obtained. The genes were named ACRE genes, followed by the number corresponding to the AFLP fragment used for library screening.
Homologies of Sequences of AFLP Fragments to Sequences in the Databases
Seven ACRE transcripts encoded proteins with similarity to sequences in the GenBank databases (percentage identity; amino acids in Table 2). The remainder showed no homology and represent previously unidentified gene products. ACRE-1, ACRE-111A, and ACRE-111B have amino acid sequences homologous to those of the ethylene response element binding protein (EREBP) transcription factors. ACRE-1 was similar to Arabidopsis ERF6 and tobacco EREBP4, which is induced by ethylene and wounding (Ohme-Takagi and Shinshi, 1995; Suzuki et al., 1998). The amino acid identity over the entire protein was low (49% for AtERF6) but much higher (78% for AtERF6) in the DNA binding domain, which is conserved throughout the EREBP family. ACRE-111A and ACRE-111B were most similar to Arabidopsis DREB1A (for dehydration-responsive element binding protein), which is induced by cold stress (Liu et al., 1998). Both ACRE-1 and ACRE-111A had small open reading frames (uORFs) upstream of the major ORF. ACRE-1 had three uORFs of 78, 9, and 18 bp, and ACRE-111A had two uORFs of 15 and 18 bp. uORFs have been described in other transcripts, and although present in <10% of eukaryotic mRNAs, they are more common in regulatory genes and have been shown to repress translation of the main ORF (Wang and Wessler, 1998).
ACRE-4 is 82% identical to the N resistance protein from tobacco. This homology extends throughout the N terminus of the protein but stops before the leucine-rich repeats. The predicted structure of ACRE-4 is similar to the truncated N polypeptide, Ntr, a product of alternative splicing (Whitham et al., 1994). No clone was identified encoding a full-length N-like protein.
ACRE-31 is 83% identical to CAST, a calcium binding protein of unknown function from potato (Gellatly and Lefebvre, 1993). It has three putative EF hand calcium binding domains.
ACRE-132 is homologous to a number of zinc finger proteins of the RING-H2 (for really interesting new gene) class E and is most homologous to the auxin-induced ATL2 gene product of Arabidopsis (Martínez-García et al., 1996). It has the conserved RING-H2 motif and a hydrophobic domain at the N terminus (predicted to be a membrane anchor), which are characteristic of this class of RING-H2 proteins (Saurin et al., 1996; Jensen et al., 1998).
ACRE-75, ACRE-137, ACRE-146, ACRE-169, and ACRE-231 are similar to hypothetical proteins from Arabidopsis identified during genome sequencing. The ACRE-231 sequence also has 22% amino acid identity to glycosyl transferases of bacteria involved in lipopolysaccharide biosynthesis. ACRE-65, ACRE-75, ACRE-169, ACRE-180, and ACRE-194 encode small peptides of unknown function of 97, 139, 124, 102, and 156 amino acids, respectively. The sequences of the ACRE cDNA clones have been submitted to GenBank as accession numbers AF211527 to AF211540.
Expression Patterns of 18 Induced Genes.
Cf9 tobacco cells were treated with IF(Avr9+) for the times indicated above the gels (in hours) before harvesting. Expression was examined by using cDNA-AFLP analysis. Each gel is labeled with the number of the AFLP fragment, and the corresponding band is labeled with an arrow.
RNA Gel Blot Analysis Verifies Gene Induction in Cell Cultures
To verify the expression patterns observed after cDNA-AFLP analysis, RNA gel blot analysis was performed using probes derived from four ACRE cDNAs. For ACRE-31, ACRE-146, and ACRE-169, the full-length cDNAs were used as probes. To avoid cross-hybridization with related EREBPs, we used a 600-bp fragment from the 5′ end of the ACRE-1 cDNA. This fragment was used because it did not include the region encoding the conserved DNA binding domain. The induction pattern we observed in cell cultures by using this method was the same as that seen when cDNA-AFLP analysis was conducted (Figures 4A and 4B).
CHX Treatment Does Not Block Avr9-Dependent Gene Induction, but It Induces Expression in the Absence of Avr9.
Cf9 tobacco cells were pretreated with 5 μg/mL CHX or ethanol for 1 hr, and then IF(Avr9+) or IF(Avr9–) was added; cells were harvested after another 30 min. Expression was examined by using cDNA-AFLP analysis. (+) indicates Avr9 or CHX added; (–) indicates Avr9 or CHX not added. Each gel is labeled with the number of the AFLP fragment, and the corresponding band is labeled with an arrow.
ACRE Gene Expression in Leaves: Transient Nonspecific Induction and Sustained Avr9-Dependent Induction
We investigated the expression patterns of ACRE-1, ACRE-31, ACRE-146, and ACRE-169 in leaves of nontransformed (data not shown) or Cf9 tobacco after injection with IF. Surprisingly, the ACRE genes were rapidly but transiently induced in leaves after 30 min, regardless of the genotype or elicitor used. The induction by Avr9 was stronger and/or more sustained (Figure 4C). Additional experiments were conducted in which the leaves were cut with a razor blade and either injected with water, MgCl2, or MgSO4 or not injected and harvested after 30 min. Induction of 18 AFLP fragments was investigated by using cDNA-AFLP analysis (Figure 5), and induction of ACRE-1, ACRE-31, ACRE-146, and ACRE-169 was also examined by RNA gel blot analysis (Figure 4D). Infiltration of leaves with any solution produced strong but transient (peaking after 30 min) induction of all AFLP fragments inspected. Cutting the leaves without infiltration induced all fragments, except for fragments 3, 65, 157, 169, and 192. Induction by cutting was in most cases weaker than that elicited by infiltration but provoked a similar response for fragments 1, 31, 44, 75, 111, and 194. These results are consistent with other data suggesting an overlap between the responses induced by different stresses, such as fungal elicitor, wounding, and flooding of the apoplast in plants (Romeis et al., 1999). Recent data from mammals also suggest that overlapping sets of genes are induced by the different signal pathways activated by growth factor receptors (Fambrough et al., 1999).
Summary of Clones Obtained from the Cf9 Tobacco cDNA Library
Hin1, Hsr203J, and EREBP1 Transcripts Also Accumulate in Response to Avr9
Using RNA gel blot analysis, we were able to determine whether other defense-induced genes were rapidly elicited in the Cf-9/Avr9 system. Hin1 (for harpin-induced) and Hsr203J (for hypersensitivity-related) are reported to be rapidly induced by bacterial harpins and Avr gene products during the hypersensitive response of tobacco (Pontier et al., 1994; Gopalan et al., 1996). The tomato homolog of Hsr203J is induced by Avr9 in Cf9 plants within 3 hr (Pontier et al., 1998).
RNA Gel Blot Analysis Confirms ACRE Gene Induction in Cf9 Cell Cultures.
ACRE genes are also induced in leaves by Avr9 and infiltration of fluids. Total RNA (5 to 10 μg) extracted from cell cultures or leaves was used for RNA gel blot analysis. Loading of gels is shown by ethidium bromide (EtBr) staining. The same blots were probed with four ACRE cDNAs.
(A) and (C) Cf9 cell cultures (A) or leaves (C) were treated with IF(Avr9+) or IF(Avr9–) for the times indicated (given above blots in hours).
(B) Cf9 or nontransformed (PG) cell cultures were analyzed 30 min after treatment with IF(Avr9+) (+), IF(Avr9–) (–), or chemically synthesized Avr9 (syn) and compared with untreated Cf9 cells (0).
(D) Tobacco leaves were cut with a razor blade and either injected with water (H20), MgCl2, or MgSO4 or not injected (Cut) and harvested after 30 min.
In Cf9 tobacco cells, Hin1 and Hsr203J were expressed at a low constitutive level and were strongly induced by Avr9 (Figure 6A). Maximal induction of Hsr203J was after 2 hr. Its induction was later than that of the ACRE genes. The Hin1 transcript accumulated to the same level 30 min after the addition of either IF(Avr9–) or IF(Avr9+); however, induction by IF(Avr9–) was transient, whereas induction by IF(Avr9+) continued for at least 4 hr. The late induction of Hsr203J and the accumulation of Hin1 at 30 min after either treatment explain why they were not detected in our AFLP screen for rapidly induced genes.
In Cf9 tobacco leaves, there was also some constitutive expression of both genes, and the pattern of induction by IF(Avr9+) was similar to that observed in cells. The Hin1 transcript accumulated after 15 min and Hsr203J after 2 hr (Figure 6B). Some induction by infiltration of IF(Avr9–) and water or buffer was also observed after 30 min (Figures 6B and 6C).
To test further the role of EREBPs in Cf-9–mediated defense, we examined mRNA accumulation of EREBP1, which is induced in response to many stresses, including ethylene, salicylic acid, and wounding (Ohme-Takagi and Shinshi, 1995; Horvath et al., 1998; Suzuki et al., 1998). It is also induced by Pseudomonas syringae pv tabaci expressing the avrPto avirulence gene in tobacco plants expressing the Pto resistance gene (Zhou et al., 1997). EREBP1 was expressed at a low level in untreated Cf9 cell cultures (Figure 6A). The transcript levels 30 min after treatment with IF(Avr9–) or IF(Avr9+) were the same, explaining why EREBP1 was not identified in the original AFLP screen, despite having suitable restriction sites. In leaves, EREBP1 expression was induced within 30 min after leaf infiltration, which is consistent with the results of Suzuki et al. (1998) (Figures 6B and 6C). However, induction by Avr9 was stronger and more prolonged than induction by wounding (Figure 6B).
To understand the Cf-9–mediated plant defense response, we undertook a comprehensive analysis of rapid Cf-9/Avr9–dependent changes in gene expression. We identified genes induced early in the defense response because these are most likely to have regulatory and signaling functions. In other studies, analysis of early gene induction has been performed using elicitation with fungal proteins that are involved in non-host interactions. These include the addition of the peptide elicitor Pep-25 from P. sojae to parsley cell cultures, a well-established model system for a non-host interaction, and cryptogein from P. cryptogea to tobacco cells (Somssich et al., 1989; Suty et al., 1996). A number of rapidly induced genes were observed; some had maximum expression 30 to 60 min after the elicitor was added. However, few have addressed whether the same genes are induced in a gene-for-gene system.
ACRE Genes Are Induced in Leaves by Avr9 and Infiltration of Fluids.
Tobacco leaves were cut with a razor blade and either injected with water (H20) or MgCl2 or not injected (Cut), harvested after 30 min and compared with untreated leaves (0). Expression was examined by using cDNA-AFLP analysis. Each gel is labeled with the number of the AFLP fragment, and the corresponding band is labeled with an arrow.
We used cDNA-AFLP analysis to detect immediate early gene expression during the Cf-9– and Avr9-dependent plant defense response in Cf9 tobacco cell cultures. The response of these cultures to Avr9 elicitation has been well characterized (Piedras et al., 1998; Romeis et al., 1999). Cell cultures provide a more reproducible system than do leaves for the analysis of induced responses. The rapid production of AOS is characteristic of plant defense responses, and it has been suggested that AOS play a role in signaling, leading to changes in gene expression (Jabs et al., 1997; Yang et al., 1997). The Avr9-elicited production of AOS is abolished by DPI (Piedras et al., 1998), which allowed us to investigate the contribution of AOS to Cf-9–dependent gene expression. Many genes are rapidly induced in Cf9 cell cultures by elicitation with Avr9. This induction is dependent on the presence of Cf-9 and Avr9 and for most induced genes does not require the presence of AOS. CHX did not inhibit this induction. On the contrary, it strongly induced ACRE gene expression even in the absence of Avr9. This is typical of immediate early response genes (Koshiba et al., 1995) and suggests regulation by a rapidly turned over repressor protein. In comparison to Hin1 and Hsr203J, which were previously described as rapidly induced genes, it became clear that ACRE gene induction is a very early response. This suggests that ACRE genes are involved in the initial development of the defense response.
Advantages of cDNA-AFLP Analysis
By using cDNA-AFLP analysis, we were able to survey transcriptional changes with no prior assumptions about which genes might be induced or repressed. cDNA-AFLP analysis gave reproducible results that were confirmed using RNA gel blot analysis (Bachem et al., 1996, 1998). Amplification of fragments from constitutively expressed genes provided internal control bands with every primer combination. cDNA-AFLP analysis has advantages over other RNA fingerprinting techniques and DNA chip-based approaches (McClelland et al., 1995; Baldwin et al., 1999). DNA chips can only be used to analyze the expression of cloned genes (Kehoe et al., 1999), whereas cDNA-AFLP analysis can reveal altered expression of any gene that carries suitable restriction sites. With the enzyme combination chosen, ∼30,000 cDNA fragments were inspected, of which 290 were differentially expressed; therefore, ∼1% of the transcripts rapidly alter in abundance after the addition of the Avr9 elicitor. To visualize a cDNA by using an AFLP, it must contain ApoI-MseI fragments of 50 to 400 bp. We inspected the sequence of 50 tobacco mRNAs and found that 76% contained suitable ApoI-MseI fragments. This means that 76% of genes expressed could potentially be visualized by using this enzyme combination. Also, tobacco is an allotetraploid between two polymorphic species; therefore, if one copy of a gene cannot be visualized, then there is still a chance of seeing another copy. Thus, it seems likely that a large proportion (>75%) of the transcripts were inspected for their response to the addition of Avr9.
A Limited Role for AOS in Rapid Defense Gene Induction
Most of the early Cf-9– and Avr9-dependent changes in gene expression were not inhibited by DPI and therefore are not dependent on AOS. Studies with other systems have concentrated mainly on genes with roles in phytoalexin production or cellular protection. In soybean, H2O2 induces expression of genes required for cellular protection but not those that encode phenylalanine ammonia lyase or chalcone synthase, which are involved in phytoalexin synthesis (Levine et al., 1994). In parsley cell cultures challenged with the peptide elicitor Pep-13 from P. sojae, treatment with DPI, but not catalase, inhibited both phytoalexin production and the induction of many defense genes, indicating a requirement for superoxide rather than H2O2 (Jabs et al., 1997). It is feasible that although the oxidative burst is not required for rapid induction of ACRE genes, it may play a role in the expression of genes that respond at later times when AOS levels are higher.
Hin1, Hsr203J, and EREBP-1 Expression Is Induced by Avr9/Cf-9 and Infiltration of Fluids into Leaves.
Total RNA (5 to 10 μg) extracted from cell cultures or leaves was used for RNA gel blot analysis. Loading of gels is shown by ethidium bromide staining.
(A) and (B) Cf9 cell cultures (A) and leaves (B) were treated with IF(Avr9+) or IF(Avr9–) for the times indicated (given above blots in hours).
(C) Tobacco leaves were cut with a razor blade and either injected with water (H2O), MgCl2, or MgSO4 or not injected (Cut) and harvested after 30 min.
ACRE Gene Products and Their Possible Roles in Signal Transduction
ACRE-65, ACRE-75, ACRE-169, ACRE-180, and ACRE-194 encode small peptides of <160 amino acids. A number of small peptides are induced during the defense response. Some of these have antimicrobial properties, for example, defensins and thionins (Broekaert et al., 1997). However, these small ACRE proteins are unlikely to be defensins or thionins because they are not cysteine rich and have no apparent signal peptide. ACRE-137, ACRE-146, and ACRE-231 are similar to hypothetical proteins predicted from the genome sequence of Arabidopsis. These ACRE proteins may play roles in signaling or other aspects of the defense response, but their function is unknown.
Some of the induced genes are candidates for components of signaling pathways, which may be responsible for induction of other defense responses. Alternatively, they may encode proteins involved in pathways leading to gene expression that are induced by a positive feedback pathway to replace the protein as it is degraded. Protein phosphorylation cascades play an important role in many signaling pathways. The sequence homology of seven AFLP fragments to sequences of protein kinases and one to a phosphatase (Table 1) is therefore not unexpected.
AFLP fragment 44 is similar to cDNAs encoding 13-lipoxygenases, which are enzymes that catalyze an early step in the synthesis of jasmonic acid, which accumulates after wounding and herbivore attack (Creelman and Mullet, 1997). Jasmonic acid has been shown also to be important for resistance to fungal pathogens. For example, the Arabidopsis mutant coi1 (for coronatine insensitive), which is blocked in its response to jasmonic acid, shows enhanced susceptibility to Alternaria brassicicola and Botrytis cinerea (Thomma et al., 1998). The incompatible interaction between tobacco plants and P. parasitica var nicotianae is abolished in plants expressing antisense lipoxygenase (Rancé et al., 1998).
Calcium channel blockers, such as lanthanum, interfere with Cf-9 signaling, suggesting that calcium influx is required for the oxidative burst (Piedras et al., 1998). ACRE-31, a putative calcium binding protein with three EF hands, may be involved in signaling cascades, and this suggests that calcium is also involved in other responses.
ACRE-132 is a putative zinc finger protein of the RING-H2 subfamily. The cellular functions of RING proteins are diverse, but they are often involved in formation of multiprotein complexes (Saurin et al., 1996; Jensen et al., 1998). ACRE-132 has a hydrophobic domain at the N terminus that may function as a membrane anchor, so it might be part of a multiprotein complex anchored in the plasma membrane.
The sequence of ACRE-4 is 82% identical to Ntr, the product of the differentially spliced transcript N Lfrom the N resistance gene. The induction of an N gene homolog during Cf-9–mediated resistance is particularly intriguing. Both ACRE-4 and N contain a putative nucleotide binding site and a domain with homology to Toll and the interleukin-1 receptor (Whitham et al., 1994). The ratio of the alternatively spliced N transcripts changes after inoculation with tobacco mosaic virus (TMV) and plays a critical role in the TMV resistance response. The NS transcript, which encodes the full-length N protein, is more prevalent before and during the first 3 hr of infection with TMV. The NL transcript, which encodes the truncated N protein (Ntr), is more prevalent 4 to 8 hr after infection with TMV (Dinesh-Kumar and Baker, 2000). The induction of an N homolog by way of a Cf-9–dependent pathway suggests that the two classes of resistance gene may have some downstream signaling components in common and that there could be interaction between components of pathways activated by different classes of resistance protein.
Importance of EREBP Transcription Factors in Defense
The amino acid sequences of ACRE-1 and ACRE-111A are 41 and 66% identical to those of EREBP4 and DREB1A, respectively. These are members of a large family of transcription factors thought to control expression of many genes during the defense responses induced by a number of biotic and abiotic stresses (Riechmann and Meyerowitz, 1998). Tobacco EREBP1 to EREBP4 bind GCC boxes in PR gene promoters, and their transcript levels are induced by C2H4 and wounding (Ohme-Takagi and Shinshi, 1995; Suzuki et al., 1998). DREB1A, DREB1B, and DREB1C bind the CRT/DRE sequence found in promoters of genes regulated by cold and drought (Liu et al., 1998). The rapid but transient induction of ACRE-1 and ACRE-111A by Avr9 suggests that they are required for induction of other defense response genes. ACRE-1 and ACRE-111A are likely to bind different promoter elements, the GCC box and CRT/DRE box respectively, based on their homology to known proteins. This suggests that they may regulate different sets of response genes required for plant defense. It has been suggested that EREBPs regulate the expression of the genes encoding them via interaction with GCC boxes in their promoters (Solano et al., 1998). Three tomato EREBPs, Pti4, Pti5, and Pti6, were identified as proteins that interact with the Pto resistance protein (Zhou et al., 1997). Pto is a protein kinase, which suggests that EREBP activity could be controlled by phosphorylation as well as by changes in the level of transcription. C2H4 plays an important role in pathogen resistance. For example, tobacco transformed with etr1-1 (for ethylene resistant) from Arabidopsis, conferring dominant C2H4 insensitivity, shows reduced basic PR gene expression and loses non-host resistance to soil-borne fungi (Knoester et al., 1998). C2H4 alone may not be sufficient for resistance; for A. brassicicola resistance in Arabidopsis, the C2H4 and jasmonic acid pathways must both be triggered (Penninckx et al., 1998). The translated sequence of another induced AFLP fragment is homologous to 1-aminocyclopropane-1-carboxylase (ACC) synthase, a key enzyme in C2H4 synthesis. C2H4 accumulates in Cf9 tomato 9 to 12 hr after injection with IF(Avr9+) (Hammond-Kosack et al., 1996). Increases in C2H4 also induce the expression of EREBP genes; therefore, this may amplify or prolong the Cf-9–mediated defense response by inducing the expression of the same or additional EREBP genes.
Model for the Role of ACRE Genes in Plant Stress Responses.
Upward pointing arrows indicate elevated protein levels. The double-headed arrow indicates a possible interaction between the Cf-9 and Avr9 proteins.
Other examples of rapid induction of transcription factors during the defense response have been reported. Treatment of parsley cells with the fungal elicitor Pep25 induces a rapid but transient increase in WRKY1 (named after the conserved amino acid sequence WRKY) and WRKY3 expression (Rushton et al., 1996). These bind elicitor-responsive W boxes in the parsley PR-1 promoter, suggesting that they play a key role in signal transduction. One AFLP fragment with homology to WRKY genes was identified during our survey of Avr9-induced genes, suggesting that WRKYs also play a role in Cf-9–mediated defense responses. Basic leucine zipper (bZIP) transcription factors are also involved in the defense response. Recently, several bZIP proteins were identified that interact with NPR1 (for nonexpressor of PR genes) and bind the PR-1 promoter, providing a direct link from a key signaling intermediate downstream of many R genes to a transcription factor (Zhang et al., 1999). We did not identify any bZIP transcription factors. However, the soybean bZIP, G/HBF-1, which binds the chalcone synthase promoter, is activated by phosphorylation and not by changes in expression (Dröge-Laser et al., 1997).
Functional Significance of Expression Profiling Data
Knowledge of the expression patterns of genes can provide important information about their function. The large sets of genome-wide expression data from DNA microarrays are often studied by using cluster analysis (Bassett et al., 1999). Cluster analysis uses statistical algorithms to group sets of genes with similar patterns of expression across a number of treatments or tissue types. In a clustering analysis of 2467 yeast genes of known function, many genes that share similar functions clustered together over several treatments (Eisen et al., 1998). When the analysis was performed using all of the ∼6200 yeast genes, the genes of known function clustered in the same pattern, but these clusters were expanded by the addition of uncharacterized genes. It has been inferred from such studies that the genes of unknown function that react in the same way as genes of known function have similar roles in the response to a particular stimulus. Furthermore, cluster analysis allows the grouping of different stimuli that elicit similar patterns of gene expression. This provides insight into the biological mechanisms of the responses to different stimuli but also raises new questions about how an organism decodes this to give a unique response.
In this study, we have performed a "poor man's cluster analysis." A large set of genes was identified whose members respond to a gene-for-gene stimulus in cell cultures. These could be grouped according to whether they were induced or repressed by Avr9 and whether this change in expression required AOS. The induction of ACRE genes by Avr9 was verified in leaves; during these experiments, it was observed that ACRE gene expression was also transiently induced by mechanical stress. The induction of 18 AFLP fragments by mechanical stress was therefore examined by cDNA-AFLP analysis. Of these 18 genes that were induced with gene-for-gene specificity in cell cultures, all were induced by mechanical stress in leaves. This indicates that even these very early R gene–dependent transcriptional responses are not specific to R gene function but are shared by responses to other stresses. This observation is important for the interpretation of gene induction data from other plant pathogen systems and in response to abiotic stress.
Significance of Common Responses to Different Stimuli
The ACRE genes were induced by cutting leaves with a razor blade and infiltrating liquid into the apoplast and by using Avr9 (Figure 5), suggesting a surprising degree of overlap between the responses elicited by Cf-9/Avr9 and other stresses. Other evidence for overlap in responses comes from studies on the activation of MAP kinases. Two MAP kinases, wound-induced protein kinase and salicylic acid–induced protein kinase, are rapidly induced in Cf9 tobacco by Avr9 (Romeis et al., 1999). These are also induced by a number of other stresses, including wounding, salicylate, and TMV infection (Scheel, 1998; Zhang and Klessig, 1998; Romeis et al., 1999). In mammalian cells, the numerous signal pathways activated by growth factor receptors induce many of the same genes, and the transcriptional responses to different growth factors overlap (Fambrough et al., 1999; Pawson and Saxton, 1999). The final responses to Avr9 and to mechanical stress are different. Both MAP kinase and ACRE gene induction are more transient after mechanical stress than after elicitation with Avr9, suggesting that the differences in final outcome may be achieved by differences in the duration and magnitude of the activation of the same pathway components and gene inductions.
A model for the role of ACRE genes during the defense response is proposed in Figure 7. Recognition of Avr9 in Cf9 plants results in the rapid production of AOS and induction of ACRE genes. These appear to be mediated by two different pathways because induction of most ACRE genes does not require AOS. The pathway leading to ACRE gene induction is also activated by wounding. ACRE proteins are candidates for components of signaling pathways involved in the activation of later defense responses.
This comprehensive survey of Cf-9–dependent gene induction has identified a number of genes that are induced during the plant defense response independently of AOS. There are two routes for further work. First, ACRE genes need to be studied to determine their function in the plant defense response by using reverse genetics. Second, the transcriptional regulation of the ACRE genes needs to be investigated. What transcription factors regulate ACRE gene expression, and how is Cf-9–dependent activation of these transcription factors achieved? The answers to these questions will provide further insight into inducible cellular defense mechanisms.
Plant and Cell Culture Conditions and Treatments
Plants (Nicotiana tabacum cv Petite Havana and transgenic Cf-9– expressing tobacco) were grown as described by Romeis et al. (1999). Intercellular washing fluid (IF) was obtained from cultivar Petite Havana plants expressing Avr9 (IF[Avr9+]) or nontransformed (IF[Avr9–]) (De Wit and Spikman, 1982; Hammond-Kosack et al., 1994). Leaves were infiltrated with IF from a syringe barrel through small razor blade cuts. At given times, two leaf panels were cut from the injected area and frozen in liquid nitrogen.
Tobacco cell cultures were maintained as described by Piedras et al. (1998). Before use, cells were washed, and aliquots of 2 g of cells in a 40-mL assay buffer (5 mM MES, pH 6.0, 175 mM mannitol, 0.5 mM CaCl2, and 0.5 mM K2SO4) were dispensed into single flasks and left to equilibrate for 3 hr at 22°C. To each flask, 150 μL of IF(Avr9+) or IF(Avr9–) was added, and at times given in the figures, two 2-g aliquots of cells were harvested by filtration and frozen in liquid nitrogen. The synthetic Avr9 peptide was used at a final concentration of 10 nM. To study the effects of inhibitors, we pretreated cells before elicitation for 5 min with 1 μM diphenyleneiodonium (DPI) or for 1 hr with 5 μg/mL (17.7 μM) cycloheximide (CHX). Compounds were from Sigma.
Measurement of Reactive Oxygen Species
Aliquots (0.2 mL) of all cell samples were tested for active oxygen species production using the ferricyanide-catalyzed oxidation of luminol (Schwacke and Hager, 1992).
Total RNA was extracted according to Logemann et al. (1987). Poly(A) RNA was isolated from 150 μg of total RNA using oligo(dT) coupled to paramagnetic beads (Dynal A.S., Oslo, Norway). cDNA was synthesized using Expand reverse transcriptase (Boehringer Mannheim), RNase H, and DNA polymerase I.
Template Preparation, Amplified Fragment Length Polymorphism Reactions, and PAGE Analysis of Products
The template for cDNA-AFLP was prepared according to Vos et al. (1995) and Bachem et al. (1996), using ApoI and MseI. Sequences of primers and adapters used for amplified fragment length polymorphism (AFLP) reactions are as follows (N can be any nucleotide): ApoI adapter top strand, 5′-CTCGTAGACTGCGTACC-3′; ApoI adapter bottom strand, 5′-AATTGGTACGCAGTCTAC-3′; MseI adapter top strand, 5′-GACGATGAGTCCTGAG-3′; MseI adapter bottom strand, 5′-TACTCAGGACTCAT-3′; ApoI preamplification primer, 5′-CTCGTAGACTGCGTACCAATT-3′; ApoI selective amplification primers, 5′-GACTGCGTACCAATT(C/T)NN-3′; MseI preamplification primer, 5′-GACGATGAGTCCTGAGTAA-3′; and MseI selective amplification primers, 5′-GATGAGTCCTGAGTAANN-3′. AFLP reactions were performed according to Bachem et al. (1996), except that a mixture of Taq and Pfu DNA polymerases (160:1 units) was used for polymerase chain reaction (PCR) in a final volume of 10 μL. Selective amplification products were separated on a 5% polyacrylamide gel run at 100 W until the bromophenol blue reached the bottom. Gels were dried onto 3MM Whatman paper (Whatman, Maidstone, UK) and positionally marked before exposing to Kodak Biomax film (Sigma) for 2 days.
Isolation and Sequencing of Fragments
Films were aligned with markings on the gels. The bands of interest were marked, cut out with a razor blade, and incubated in 150 μL of TE (10 mM Tris, pH 7.5, and 1 mM EDTA, pH 8.0) overnight at 37°C. AFLP fragments were recovered by PCR under the same conditions as used for the preamplification. The dideoxy termination method (Sanger et al., 1977) was used for sequencing, and sequences were determined using an automated sequencer. Nucleotide sequences and translated sequences were compared with nucleotide and protein sequences of the GenBank nonredundant databases and sequences of the expressed sequence tag databases by using the BLAST sequence alignment program (Altschul et al., 1997).
Construction and Screening of cDNA Library
RNA was extracted from Cf9 tobacco cells 30 min after adding IF(Avr9+). Poly(A) RNA was isolated from 1.25 mg on oligo(dT) cellulose columns (Pharmacia). cDNA was synthesized and inserted into the Uni-ZAP XR λ insertion vector (Stratagene, La Jolla, CA). After packaging (Gigapack III Gold; Stratagene), the library was plated at a density of 50,000 plaques per 20 × 20-cm plate. Plaque lifts were screened with the reamplified AFLP fragments, which were radiolabeled with 32P-dCTP using an oligolabeling kit (Pharmacia). Hybridization was performed following the protocol of Church and Gilbert (1984). Filters were washed four times for 15 min with 100 mL of 1% SDS and 1 × SSC (0.15 M NaCl and 0.015 M sodium citrate) at 65°C.
RNA Gel Blot Analysis
Aliquots (5 to 10 μg) of total RNA were separated by electrophoresis on a denaturing 1.4% agarose gel (100 mL of denaturing gel contained 1.4 g of agarose and 5.4 mL of 12.3 M formaldehyde [37% w/w]) followed by blotting onto nylon membrane (Hybond-N; Amersham, UK) (Sambrook et al., 1989). The RNA was cross-linked to the membrane using a UV Stratalinker (model 2400; Stratagene). Probe labeling and hybridization were performed as given above. The membranes were washed with 0.1% SDS and 2 × SSC twice for 10 min and subsequently with 0.1% SDS and 0.5 × SSC twice for 30 min at 65°C.
Isolation of Hin1, Hsr203J, and EREBP1 cDNAs from Tobacco
The Hin1, Hsr203J, and EREBP1 cDNAs were amplified from tobacco cDNA by PCR using specific primers. PCR products were ligated into the pGEM-T EASY vector (Promega, Southampton, UK), and the identity of the clones was verified by sequencing. Clone inserts were used as probes on RNA gel blots. For the EREBP1 probe, a 239-bp C-terminal fragment was used. This fragment did not contain the DNA binding domain.
We thank Kate Harrison and Matthew Smoker for propagation of cell cultures, Sara Perkins, Simon Daplyn, and Justine Campling for horticultural assistance, and Patrick Bovill and David Baker for assistance with sequencing. We are grateful to Martin Parniske forcritical reading of the manuscript. This research was supported by the Gatsby Charitable Foundation. O.R. is supported by a Human Frontiers Science Program fellowship.
1 Current address: Departemento Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Córdoba, 14071-Córdoba, Spain.
2 Current address: Monsanto Europe S.A., Maris Lane, Trumpington, Cambridge CB2 2LQ, UK.
Received January 10, 2000.
Published June 1, 2000.
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Loss of cabin pressure
Bodrum, Turkey
London Gatwick, United Kingdom
TC-IZD
A Pegasus Airlines Boeing 737-800, registration TC-IZD performing flight PC-5121 from Bodrum (Turkey) to London Gatwick,EN (UK) with 49 passengers, was enroute at FL400 about 30nm north of Thessaloniki (Greece) when the crew initiated an emergency descent to FL110 (average rate of descent 3600fpm) due to the loss of cabin pressure, the passenger oxygen masks were not released. The aircraft subsequently turned around and diverted to Izmir (Turkey) for a safe landing about 65 minutes after leaving FL400.
A replacement Boeing 737-800 registration TC-IZI departed Izmir about one hour after landing and reached London with a delay of 3 hours.
Pegasus A20N at Bucharest on Mar 30th 2018, touched down before runway threshold
A Pegasus Airbus A320-200N, registration TC-NBF performing flight PC-361 from Istanbul Sabiha Gokcen (Turkey) to Bucharest Otopeni (Romania), was on…
Pegasus B738 at Istanbul on Jan 7th 2020, runway excursion on landing
A Pegasus Airlines Boeing 737-800, registration TC-CCK performing flight PC-747 from Sharjah (United Arab Emirates) to Istanbul Sabiha Gokcen…
Pegasus A20N at Basel on Jan 26th 2021, nose gear tilted 90 degrees on landing
A Pegasus Airlines Airbus A320-200N, registration TC-NBH performing flight PC-939 from Istanbul Sabiha Gokcen (Turkey) to Basel/Mulhouse…
Pegasus B738 at Istanbul on Feb 5th 2020, overran runway, impacted wall, broke up
A Pegasus Boeing 737-800, registration TC-IZK performing flight PC-2193 from Izmir to Istanbul Sabiha Gokcen (Turkey) with 177 passengers and 6 crew,…
Pegasus B738 at Istanbul on Feb 11th 2020, captain incapacitated
A Pegasus Airlines Boeing 737-800, registration TC-CPI performing flight PC-902 from Vienna (Austria) to Istanbul Sabiha Gokcen (Turkey), was…

redpajama_set_name: RedPajamaCommonCrawl


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Spirit expands manufacturing operations in Malaysia
Construction is set to begin in October with a target for operations to begin in mid-2018.
Spirit AeroSystems is in the architectural and engineering planning stages to add a 50,000-square-foot manufacturing facility adjacent to other buildings on its campus in the Malaysia International Aerospace Centre near Kuala Lumpur.
Spirit AeroSystems
Spirit AeroSystems (Wichita, Kan.) will expand its manufacturing operations in Subang, Malaysia to accommodate increasing volumes of assembly work on aerostructure components for commercial airliners. The company is in the architectural and engineering planning stages to add a 50,000-square-foot manufacturing facility adjacent to other buildings on its campus in the Malaysia International Aerospace Centre near Kuala Lumpur. Construction is set to begin in October with a target for operations to begin in mid-2018. Spirit expects to eventually add about 300 workers to the current site population of 700.
"We are excited to see our Malaysia site in a strong growth mode as we begin the process of adding capabilities that will allow us to better support the needs of our customers," says Scott McLarty, Spirit AeroSystems vice president and general manager, U.K. and Malaysia.
Advanced materials for aircraft interiors
Applications aren't as demanding as airframe composites, but requirements are still exacting — passenger safety is key.
Composites 101: Fibers and resins
Compared to legacy materials like steel, aluminum, iron and titanium, composites are still coming of age, and only just now are being better understood by design and manufacturing engineers. However, composites' physical properties — combined with unbeatable light weight — make them undeniably attractive. This month, CAMX Connection introduces to composites novices the fibers and resin systems commonly used in composites manufacturing.
Composite parts are formed in molds, also known as tools. Tools can be made from virtually any material. The material type, shape and complexity depend upon the part and length of production run. Here's a short summary of the issues involved in electing and making tools.


How To Write An Introduction Of Newsletter?
Newsletter formats differ from one another in their design, functionality and use. In today's post we will give you a brief review the three majorly used newsletter formats and their advantages and disadvantages along with examples of sample newsletter formats.
HTML based newsletters give you more flexibility than any other format. You can play around with fonts, change background colors, add headers etc. These features give your newsletter a lively feel and make them more engaging. Vast options of customization allow you to design your newsletters in many ways.
HTML format allows extensive use of graphics. If you plan to promote a product through your newsletter, use of graphics and product pictures can really help you get more customers. Moreover, graphics and pictures make your newsletter more appealing and attractive.
You can use hyperlinks with HTML format unlike plain text where the links appear broken while in the case of a PDF newsletter, they render browser security issues.
The main problem people face with this format is that their HTML knowledge is not enough for them to develop their own newsletters. It is possible to acquire sample newsletter formats and tweak them according to your needs, but you might not find one good enough to your liking. If you are serious towards using this format, you can hire a coder who can do the work for you.
One of the key disadvantages of this format is its compatibility issue with email clients. Not all email clients support this format. Therefore, if your subscriber is using an email client that does not support this format, they will not receive your newsletter.
The best advantage of using plain text format is the ease with which they are created. No technical knowledge is required as well. No special software is required as well since a simple word processor can do the job.
Newsletters written in plain text format will always be delivered to the subscriber since email clients have no issues with this kind of format.
A simple looking newsletter may or not be a problem depending how you look at it. If you appreciate trendiness, graphics and images, you might say that a simple newsletter is lacking majorly.
On the other hand, some readers might get distracted with flashy looks and color contrasts. Your content is what matters the most in this case. less options with creativity might be a hindrance for you to choose this format.
Since plain text does not allow hyperlinks, rather longer links in your newsletter will appear broken due to email client restrictions. This situation gets very out of hand with affiliate links since they all look like a URL spread over two, three lines.


Michael Menchaca earned his MFA from the prestigious Rhode Island School of Design in 2015 with a concentration in printmaking. A San Antonio native, Menchaca is known to juxtapose images from popular culture with Mesoamerican iconography. Vignettes from San Antonio is a new series of work that is inspired by the artist's hometown. Large collage works speak to the cultural landscape of San Antonio and correspond with the city's celebration of its tricentennial anniversary. Highlighting such iconic San Antonio scenes as Easter weekend at Brackenridge Park, the campus at San Antonio College, and the Battle of Flowers Parade, Menchaca expands his personal codex to include San Antonio specific iconography.
The artwork of Michael Menchaca can be found in the collections of the McNay Art Museum, San Antonio, TX; Mexic-Arte Museum, Austin, TX; Museum of Latin American Art, Long Beach, CA; National Museum of Mexican Art, Chicago, IL; Rhode Island School of Design Museum, Providence, RI; San Antonio Museum of Art, San Antonio, TX; Austin Museum of Art, Austin, TX; Crystal Bridges Museum of American Art, Bentonville, AR, and Spencer Museum of Art, Lawrence, KS.


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HomeWORLDWIDEARTS & CULTUREFilm, TV & Books"Netizens" Preview – Powerful and Very Important
"Netizens" Preview – Powerful and Very Important
September 16, 2019 Barbara Keer Film, TV & Books 0
Carrie with Client
Recently a very powerful and important feature documentary, Netizens has come to my attention. Directed by Cynthia Lowen, it is about revenge porn. Lowen is an Emmy-nominated filmmaker and writer. This timely 96-minute documentary features prominent Brooklyn attorney Carrie Goldberg and author of Nobody's Victim; Anita Sarkeesian, founder of Feminist Frequency and target of the 'Gamergate' attacks; and Tina Reine, a businesswoman whose career is destroyed by reputation-harming websites. With Soraya Chemaly, Danielle Citron, Mary Anne Franks and Jamia Wilson.
The film premiered at the 2018 Tribeca Film Festival and its international premiere at Toronto's Hot Docs, and opening soon in selected theaters around the U. S. beginning September 23rd, 2019.
Netizens
Cynthia is also the producer and writer of BULLY, a feature documentary film following five kids and families through "a year in the life" of America's bullying crisis, which she developed in partnership with Emmy- and Sundance-award winning director Lee Hirsch. Filmed over the course of the 2009/2010 school year, BULLY opens a window onto the pained and often endangered lives of bullied kids, revealing a problem that transcends geographic, racial, ethnic and economic borders.
Lauded by reviewers, BULLY was awarded a prestigious Alfred I. DuPont-Columbia Award for excellence in journalism, as well as the 2013 Stanley Kramer Award from the Producers Guild of America, the True Life Fund Award, the Cinema Eye Audience Award, the Emery Award, the Bergen Film Festival Audience Award, and more. Drawing on BULLY's success, the filmmakers created The BULLY Project Social Action Campaign, a collaborative effort in partnership with multiple organizations, foundations, brands and corporate sponsors, sharing a commitment to ending bullying and transforming society.
The film and campaign have garnered support from such prominent voices as Anderson Cooper, Ellen DeGeneres, Kelly Ripa, Meryl Streep, Katie Couric, Justin Bieber, and many others. Featured at several summits on education and school climate, in April of 2012 BULLY was screened at the White House. The impact of this documentary and the associated social action campaign was recently explored in Anderson Cooper's 1-hour special, The BULLY Effect, premiering on CNN.
Cynthia Lowen, Photo: Joseph Michael Lopez
In addition, Cynthia is an award-winning poet and winner of the 2012 National Poetry Series for her collection The Cloud That Contained the Lightning , published by University of Georgia Press. Using the character of J. Robert Oppenheimer, known as the "father of the atomic bomb," as a jumping-off point, the collection explores the enduring legacy of nuclear weapons. Of these poems, Pulitzer Prize-winning author Richard Rhodes writes, "No biographer in 600 pages has come closer to understanding him [Oppenheimer]–and the bomb–than does Cynthia Lowen in these subtle, resonant poems." Cynthia is the recipient of the Hedgebrook Women Authoring Change Fellowship sponsored by William Morris Entertainment, the Discovery Prize, residencies to the MacDowell Colony, Yaddo, The Fine Arts Work Center in Provincetown, among other honors.
Cynthia Lowen generously agreed to an interview with Chicago Splash Magazine as follows:
I have been aware of children being bullied on the internet but not at all aware of the impact of internet bad behavior impacting women as the movie describes. How did this topic come to your attention?
In the fall of 2014, there were several coordinated cyber-attacks targeting women in the video game industry which came to be known as "Gamergate." These attacks were so violent that many women who were targeted had to leave their homes and there was little recourse from law enforcement or tech companies. As a filmmaker, it seemed to me like that there was a huge problem here that needed to be unpacked. One of the women targeted was Anita Sarkeesian who subsequently became one of the three women featured in Netizens.
Anita at Computer
I am very impressed with the workbook that you have available for schools and teachers in regard to your prize-winning movie, BULLY. Has the workbook been impactful? Do you plan a workbook for Netizens?
We had over five million students in America watch BULLY with their educators, and use our materials free-of-cost. As a result, we were effective in shifting bullying from being seen as a "normal" right of passage, to being something all schools and communities now know they must respond to. My hope for Netizens is that we can similarly shift the needle from online harassment and digital abuse being a "normal" part of the internet's wild west, to being something we need not accept in our digital society. We have are developing tools are resources that audiences can engage with, including six discussion guides available specific to various groups including Employers, Law Enforcement and Policymakers, University Students and Educators, Violence Prevention Advocates and Women and Girls. These are available on our website www.NetizensFilm.com
The movie, BULLY influenced legislation. Are you hopeful that Netizens will have the same impact? What changes would you wish to see?
I hope that as a result of Netizens audiences will feel empowered to take an active role in shaping their digital communities and will hold tech companies and policy-makers accountable for upholding our digital privacy and safeties. One great development is that Facebook is actively reducing "revenge porn" by creating tools that stop images at the source from being spread across all Facebook platforms (Facebook, Instagram, Messenger) as soon as an image has been identified as revenge porn.
I note that as Netizens opens across the country, there are also going to be Q and A sessions. Can you describe how that will work?
Netizens will launch a national community tour starting September 23rdin New York City, Chicago, Washington, D.C., San Francisco and Miami and each screening will be accompanied by a Talk-Back and Q&A, with such participants as myself (the director), people from the film and experts. We welcome any groups or communities that wish to book a screening and they can visit www.NetizensFilm.com for tickets and more information.
What was your inspiration for this very important project?
To shine a light on forms of violence that are ubiquitous yet normalized in our communities and to depict these stories in such a way as to transform our attitudes that this violence is inevitable or something we have to accept.
Thank you to Cynthia Lowen for the insightful answers. We encourage all of our readers to see this extremely important film.
Photos: Courtesy of Netizens
Note: Check Netizens Film to find out what cities the film is playing and where to buy tickets that the Netizens documentary will be available on Amazon, Apple, X-Box and Google Play. Netizens trailer
Cynthia Lowen
Freaky Friday's Full Moon Wine Fest review – Wine, Trees and Water
'To Dad With Love", a Tribute to Buddy Ebsen – Beautifully Done
"How to Build A Girl" is Must-See Film
June 19, 2020 Michele Caprario Film, TV & Books 3
Spread the loveUS – Caitlin Moran's film adaptation of her 2014 novel How to Build a Girl is daring, funny, sweet, and never for a moment predictable. First in a trilogy of refreshingly female coming-of-age […]
A Vegan Holiday Season – Ask Elysabeth Alfano
November 7, 2019 Barbara Keer Food, Drinks, & Sweets 0
Spread the love I recently learned that November is World Vegan Month which surprised me, and also learned that 2019 marks 75 years since the foundation of The Vegan Society, which I found even more surprising. […]
Cirque du Soleil – The Sun Rises Giveaway
July 10, 2021 Stephan Martin Entertainment 0
Spread the loveThis summer, the sun rises. Experience the excitement of Cirque du Soleil in Las Vegas with tickets starting at $75! Intermission is Over. Buy tickets now at cirquedusoleil.com/las-vegas To help celebrate the return of Cirque du Soleil […]


We want you to have a voice and be aware of public hearings, development projects and bid opportunities. This page will be continually updated to include city public notices.
The City of Marion is looking to construct a post-and-frame addition of 4,800 square feet to an existing post-and-frame maintenance building at Lowe Park. The addition is fire separated from the existing building.
Public hearing regarding the proposed Housing Plan of Inspections and the Housing Fee Schedule.
Small Wireless Facility Design and Review Guideline Policy - provides aesthetic requirements and other specifications and reasonable conditions that small wireless facilities installed within the public ROW must meet prior to installation and following installation within the City.
GovDeals is the place to bid on government surplus and unclaimed property including heavy equipment, cars, trucks, office equipment and so much more. Each participating agency has its own auction rules and regulations and may be subject to government ordinances. Find items the City of Marion is looking to dispose of via www.govdeals.com/CityofMarionIA.


Sponsored by the Consulate General of India and co-presented by Indian Americans of Lexington and NetSAP. Directed by Rohena Gera US premiere | India, France | 2018 | 99 min. | Hindi, English, Marathi with subtitles In order to escape the pitfalls of the old-fashioned village that condemned her as a young widow, a woman moves to Mumbai to become a servant for a privileged businessman, with dreams of becoming a fashion designer. Winner of the Gan Foundation Award for Distribution at the Cannes Film Festival. Preceded by an optional dinner reception from 6-7 PM at the theater featuring Indian cuisine catered by Spice Delight in Belmont. Tickets to the dinner must be purchased by Monday, April 22, no exceptions. Movie tickets are not included in the price of the dinner and must be purchased separately.
Food: $30/person for optional Indian dinner catered by Spice Delight in Belmont.


STAR: Student Action for Refugees at Nottingham is 1 of 42 student lead groups at Universities across the UK. All STAR groups are affiliated members of the charity STAR, 'The National network of student groups working to improve the lives of Refugees in the UK'. This national charity is governed by a board of trustees elected annually by the student committee members themselves.
Student Action for Refugees at Nottingham run the weekly 'Tuesday Night Project', with our partners at the Red Cross. This English conversation class provides refugees and asylum seekers living in Nottingham with an invaluable opportunity to practice their English in a safe and supportive environment. Each class is equipped with English worksheets of varying difficulty to support the Student teachers and Refugee students. Through money raised by STAR Nottingham and the opportunities provided by this class, this summer 5 Refugee students passed their IELTS exams (an English language proficiency test) which means they can now start applying for university.
Your STAR Nottingham society also runs various campaigns, fundraising and social events across the academic year; providing students with an opportunity to help raise money and awareness for an incredibly important cause, while socialising and meeting likeminded people.
We are always looking for more volunteers to support our Tuesday Night Project. We have been so excited by how many new volunteers have joined us this year, and if the numbers keep going up we will consider starting some new regular volunteering opportunities to support our refugee and asylum seeker community in Nottingham. Aside from teaching at the conversation class, if you are keen to get involved your role could be anything from helping out at a bake sale and making placards to raising awareness or joining the committee itself.
There is a membership cost for joining societies to be paid when you sign up online.


Outslayer 80-Pound Punching Bag # 801 – Fighters Shop !
– 4ft tall 14" diameter.
– The USA heavy duty vinyl used is extremely tough and holds over 200lbs.
YOU WILL ABSOLUTELY LOVE THIS BAG OR YOUR MONEY BACK GUARANTEED!
Color : Red , but you can choose any other color from the drop down box on the bottom of this page. If you dont choose any color we will send you the same color bag as on the picture.


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August 31, 2016	/ Rouf Dar
Azaadi is the Sound of July in Kashmir
A protest rally had just begun from the village. It was organised by the committee, formed to supervise future programmes and tackle forthcoming problems in the village. Youths mounted bikes, cars and load carriers with protest songs on their lips. Women were still glued to the windows and waited in anticipation for everyone to return safely. 'Leftover' people resigned to their homes to relax and refill their bellies. Some elderly men chatted at a shopfront. An occasional motorcycle plied on the empty road.
Sharp slogans broke the silence. Slogans of Azaadi and India Go Back were heard. Two boys, both minor, appeared from behind one curve of the road. One had a handkerchief worn around his face. Another walked by his side. "Hum kya chahte?", shouted the former. "Azaadi", came the reply. The series of slogans went on as they passed through the village square with all eyes fixed at them. They seemed unfazed by the attention. It was just them and Azaadi.
"I will protest in the summer of Kashmir" has become a cliche. This summer too has Azaadi written all over it. Everything seems to utter Azaadi. Every sound rhymes with Azaadi. Maa ki mamtaa. Behno ki ismat. Water flowing steadily in a nearby river. Tyres of vehicles hitting the newly macadamized road hard. Ticking of the clock at night. Chirping of birds every morning. Fan rotating at full speed as if spinning the yarn of freedom. Small children playing games of Azaadi, aping adults and inculcating resistance.
Kashmir is "on the boil" for the umpteenth time ever since India wrested control over the Valley from the Dogras in 1947. A recurring phenomenon, that revisits after irregular periods, the season of Azaadi has once again engulfed Kashmir. Life has come to a halt, in a sense. In other terms, the Kashmiri life is searching again for what it has been longing for the last seven decades or so. The failure of India to win the hearts and minds of the people has resurfaced yet again. The philosophy of 'drag them by their balls' has been tested yet again. India has piled up such strong memories that forgetting is a luxury, unaffordable.
To dismiss the fresh spate of killings as a once-in-a-blue-moon incident is the gravest misreading that the Indian State and its policymakers have been preoccupied with for the last decade. In 2008, more than 80 youths succumbed to the state force over the Amarnath land row. In 2010, it swelled upto 120. 2011 had 56 recorded civilian deaths. 2016 has already more than 70 youth dead, around 8000 injured with more than 200 cases of visual impairment. It has been nearly two months into the protests now. And the cycle of protests appears unstoppable. Hence, kisi ek aadmi ke marne se taareekh khatam nahin hoti.
These years of "unrest" cannot be rubbed aside as occasional instances of "alienation" or "unemployment". These are results of the suppression of the common will of a people who have been tyrannized by a military occupation. These are currents of hurricanes that have built up violent speeds and threaten to bury Indian rule in Kashmir forever. The tide is certainly not in India's favour especially during such periods of anger and outburst. The silence between these periods is consumed by, and misconstrued as, "peace" enforced under the barrels of guns and in the concertina wires of curfew.
Burhan Wani's martyrdom was an impetus to the eruption of an inextinguishable sentiment in Kashmir. The sentiment stays dormant for a moment but it only signifies a lull before the storm. This has more to do with Azaadi and less with personality cult. Burhan is not Hailey's Comet but a revolution. People mourn Burhan because he was the manifestation of the most Azaad person in the valley. He was (comparatively) the closest to the stand he died for. And this proximity with Burhan has ignited the fire this time round. Mourning for a militant cannot stretch for a month. This is the rumination of a wish deeply embedded in the physiology of political Kashmir.
Contrariwise, the Indian State hasn't changed much from what we saw in 2010. After all what difference does a government-transition make in an occupied land. Congress is not any organisation in Kashmir. BJP is not a party in the valley. We see UPA and NDA as coalitions that share a common agenda — the forcible occupation of Kashmir. Leftists are a recent, welcome addition. Modern states, which are built upon dictated contracts, are sustained by force, military and legal. This force multiplies while extending the boundaries of a state to areas which yearn for secession. The might of the Indian state, is thus on display in the Valley every moment these days.
As I edit this, Kashmir is shut for the 53rd consecutive day — the writing started on the 40th day but with no internet access, completion dragged on further. The state government has failed to evolve methods to tackle a population that idolised Burhan. Within moments of this news on the third day of Eid, mobile networks were scrapped in South Kashmir and later in the whole valley. Heavy deployment of forces was started to thwart possible outrage. The outrage seemed uncontrollable though. Police stations and army camps became primary targets with many of them put to flames. Lakhs thronged Tral for the funeral which included many active militants, if reports are to be believed.
To prevent the world from getting information about its crimes in Kashmir, a perfect media blackout has been ensured. Mobile networks are always the primary victims. Television and radio reports have never portrayed a 'fairer' picture. The physicality of Kashmir as a military prison is compounded by the social imprisonment, by preventing communication. There is no contact between localities, villages, towns and regions. There is no news of death being sent across to the world that is busy with ding-dong elections in USA. There are no candle marches or protest rallies happening anywhere. India is not being shamed. India is the US' nuclear partner, France's jet partner, Iran's gas partner and Saudi Arabia's oil partner. And in politics, vested interests take precedence over everything else, even political and human rights.
More than 70 youth chose to follow Burhan to the grave for reasons incomprehensible for the state. The Indian state considered Burhan to be a thorn in the supposedly prospective "peace" in Kashmir. With his death, they would have anticipated peace to ensue — job done. But ask any passerby. He will testify that times ahead are more troubling for he has inspired an entire generation to desert fear and hug martyrdom. He is being imitated by little children who play with wooden rifles. He is being mourned by every kid. Tera bhai mera bhai — Burhan bhai Burhan bhai reverberates in the streets.
"The best jihad is to speak a word of truth to the oppressor", reads a Hadith. But words don't affect India anymore. Laaton ki boot baaton se nahin maante. Thus goes the story. The first day after Burhan was martyred, people stormed army camps, police stations and other signs of occupation in the whole valley. The brutal response of forces caused more than half of the current death toll within the first three or four days of the backlash. A police station was torched in Kulgam district and people decamped with rifles, the exact number unknown. How long can such people be held under subjugation? How on earth can their hearts and minds be occupied?
The armed forces have been employing more subtle tactics of muting the brimming population other than killing, torturing and shelling. Clerics who attend public gatherings are being picked up. In many areas, water and electricity supplies are cut off to culminate a perfect seige on the people. In an instance, they even poisoned the water filtration plant but in vain. They turn off electricity before raiding villages and localities. Darkness brings a sense of fear. Night means nocturnal raids in Kashmir. Dark uniforms and dark weapons in complete darkness turn into a dreaded combination. And Kashmiris have more reasons to be wary of the enemy. There is no time to rest. Aaraam haraam hai.
There seems no predestined, immediate result of this round of shutdowns, especially when this whole game of death — the death of around 70 youths — is termed as Pakistani mudakhlat by India. It fails to acknowledge, as it has historically done, that Kashmiris have an independent existence of their own, as human and political beings. That we have the right to choose a befitting future for ourselves. That our country is not a bob in the pendulum that necessarily has to swing between India and Pakistan and cannot balance itself independently in the middle. India attaching Pakistan's name to every indigenous uprising in Kashmir rubs salts on the wounds inflicted on us. The movement however goes on, unabated.
It is astonishing how far Kashmiris have grown up as a people who have kept the resistance coming. Yet it is pretty understandable that this generation has learnt the ABCs of resistance in the placental sheaths of mothers, who were witnesses to the torment of 1990s. So, fearlessness is but an inherent trait. Now youths don't run away from encounter sites but towards them. Now people are not beaten in crackdowns but it requires draconian laws like AFSPA and PSA to pick up youths and torture them in untraceable locations. Now, as mosques announce the arrival of the army, people grab axes, spades, rods, sticks and assemble. The army is chased away.
It is said that death is the jurisdiction of Izraaeel, the angel. The Izraaeel in Kashmir is India. For decades India has been piling up numbers of dead, raped, tortured, disappeared, widowed, jailed Kashmiris. This generation of Kashmiris has come a long way, upholding ancestral tradition, and in no way fears death now. We don't embrace death because better lives have been denied to us by India. Our youth don't take up arms because, as some analysts would say, they are disappointed and alienated by the failure of Indian democratic processes in the valley. This all is the natural corollary of an unfulfilled demand of freedom.
In a valley that has been under multiple regimes of occupation since 1586, the stubbornness of India, subsequent attempts to stifle popular consciousness and withhold genuine demands has drawn failure every time. The public will has been evident as ever. The demands have been as vocal as ever. Band-aiding the problem has never worked. Economic packages and political promises, knowingly fake, haven't improved the situation. Interlocutors have unsuccessfully tried to bargain and buy our souls. Relief operations haven't worked either. The wound has found no clot till date. Kashmir continues to bleed.
The bleeding will continue until our political aspirations are met according to our wishes. For it to happen, both India and Pakistan will have to shed egoistic inhibitions and reconcile their ruptured baahmi taaluqaat. Even if that doesn't happen, the struggle will sustain itself. It is, and will get, tougher by the day but stronger along the same proportions. The rallying cry will persist — Hum Kya Chahte?
TagsAzadi Kashmir Uprising
Written by:Rouf Dar
Storyteller from Kashmir read his work at https://roufdar.wordpress.com/a


Software Advice, an online resource for HR software buyers, recently posted their 2011 Market Trends Report for Human Resources Information Systems (HRIS). There is an expected employment growth rate of about 8% over 2011 according to a survey from Manpower's Outlook. However, that growth will not drive demand for core HRIS systems. Software Advice suggests that firms will better leverage existing systems by implementing new applications.
While HRIS demand will remain steady, it is likely that demand for strategic HR software will increase as firms up their hiring efforts and aim for a higher-performing workforce. Leading vendors will enhance their app portfolios through acquisitions, and we can expect to see some new blood in the market as the barriers to entry have lowered considerably in the past few years thanks to new deployment options.
You can read about each of these trends individually on the Software Advice blog, but we will dig into one in particular right now: Strategic HR.
The report predicts that this area will be the most active in terms of consolidation activity and will experience noticeable growth over 2011, despite the lowered demand during the recession. Talent management and performance management overlap quite a bit, sharing features such as recruitment automation, applicant tracking, and the onboarding and assessment of employees. Because of these apps, HR software vendors have been able to extend their reach, deploying hiring managers across the entire organizations. There have been several leaders in this area, but there is a micro-trend of core HR vendors entering this space as well. It will be interesting to watch this particular area develop over the year.
You can read more about these trends on the Software Advice blog, and be sure to take their survey at the bottom of the article that asks, "When do you think your organization is going to bite the bullet on a new or upgraded HR system?" They will post the results in the coming weeks.


Joint Base San Antonio - Fort Sam Houston, Texas (March 13, 2017) -- Thirteen dental warriors from various dental activities across Dental Health Command - Central (DHC-C) convened at Camp Bullis recently to compete in the DHC-C regional Best Warrior Competition.
The grueling competition is designed to test the competitor's mental and physical toughness along with their Soldier and leadership skills. Leading the way in the year's competition were Sgt. Juan Fernandez from Fort Bliss, Texas who took the NCO Best Warrior title and Spc. Anthony Lee from Fort Carson, Colorado who earned the top spot as Soldier Best Warrior. These two dental warriors will represent DHC-C at Regional Health Command- Central's (RHC-C) Best Warrior competition later this year.
The punishing 5-day competition included the; Army Physical Fitness Test (APFT), obstacle course, day and night land navigation, individual marksmanship, Army warrior task lanes, written essay, written test, oral board, 12-mile ruck march and mystery event.
During his remarks at the award ceremony, Command Sgt. Maj. Roderick Batiste, Brooke Army Medical Center Troop Command, praised all the competitors for their warrior spirit and reflected upon the Soldier's creed. He also took the opportunity to lift the spirits of those competitors who did not win. Batiste said, "Come back again next year, compete and when you feel like giving up, remember our warrior ethos - I will not accept defeat and I will not quit."
Col. Michael Roberts, DHC-C Commander, also commended the competitors for their hard work and perseverance. "We might be health care providers but we are Soldiers first and this kind of competition gives us an opportunity to test our Soldier skills," said Roberts.


pulled pork with a spicy chili rub.
Wash pork shoulder and pat dry. Apply chili powder rub (combine ingrediencts listed), place in slow cooker, pour in marinate and set for 8 hours. Once pork shoulder is cooked (tender and pulls apart), remove from slow cooker and shred disgarding any fat and marinate. Combine red sauce ingredients in a sauce pan and bring to a low boil. Turn temp to medium low and simmer for 5 minutes. Pour over shredded pork and serve on a gluten-free bun. You can alway use BBQ sauce in place of marinate and red sauce.


@Jennifer.Garner is gonna be the most beautiful person in the school drop-off line.
Here she is! I revealed People magazine's Beautiful Issue 2019 cover star... and I couldn't agree more. Don't miss any of it, tomorrow.
I had to wait to park this morning, but it was worth it. #repost @theellenshop ・・・ @thekalenallen knows a fashion statement when he sees one.
When all else fails, do what @brielarson does and start naming members of One Direction.
You won't believe what Portia put in my Easter basket.
If you have a tattoo of @BrieLarson or Scarlett Johansson, chances are I showed it on the air today.
Don't miss this dance party with Captain Marvel and Black Widow tomorrow.
There's some method to my madness.
@Pink's not gonna let anyone mess with her kids.