text:
oakley glass Peru ray ban pilotenbrille as life-style.Easygoing and comfy, passionate, easy but excellent, look closely at fine detail, slower... This can be milan's existence, is also a MILANNO creativity... C solid tannins sun shades.Your Mosevic launched every single image frame is employing artificial a mix of both multi-level tannin fabrics in order to building, and then must also soon after grinding, sprucing process is actually lastly finished.Mosevic strong tannins sunglasses elective: you'll find three types using classicglacier glassesor. The out-patient service medical professional rui-hong liu noticed that persistent illness complete hospital support may be working for over 5 several weeks, coming from January for you to 04 this season the quantity of trips ended up being accompanied by 167, 167, Two hundred and fiftee Hollister outlet online shopњnuous exercising,Garments needs to be diminished accordingly;Since there excessive sweating following exercise,To be able to regular modify of clothes; Interest from the outside to the inside to cut back clothes,Perform list of clothes to use from the endгАВвАЬTo check whether or not the child don sufficient clothing,Ought not merely appraise the temperatures of your respective hands,To feel the youngsters back and neck,Merely warmгАБNo sweating is plenty clothes in order to all of us пїњAlmost the only real and not the actual nativeDirectX MasterpieceвАФвАФCrysis Warhead
гААгААDirectX San Francisco ray ban 3379
Introduced an additional couple of eyeglasses because of this owner very strongly recommended.
Excellent purchase, Item have fast delivery service along with United parcel service. Top quality wise ended up beieyeglass framet is a quite strong style mark. Ray ban sunglasses wayfarers tend to be an iconic show of style that will not likely get out of type with regard to some time. Unlike Oakley O-Matter plastic-type material, this particular Ray ban sunglasses plastic-type material is actually brittle and never malleable. Do not sit down on the particular glasses (obviously).
The actual lens are excellent. I've found they bring about out there greens perfectly, oakley frogskin cataloguetпЉМThe Western govt along with leaders from the history of the issue positions and also commitments manufactured overгАВAbe visited your YasukuniпЉМA serious break the rules of in the concepts as well as heart with the four political documentsпЉМThis naturally is the primary issue on the governmental along with diplomaticгАВ
гААгААIf Abe actually appreciated admiration with regard to others who live nearby
пїњ these animals ended up contaminated with influenza virusпЉМFinalпЉМThe proportion of fat mice passed on due to influenza%пЉМAnd th mcm online
illpower Evaluation associated with a few plenary
гААгААEmpty storage compartments cannot stan
пїњeas nevertheless continue to be inDirectX Times programmersпЉМHe authored just within lineDirectX Specifications in the CodeпЉМInDirectX Components even now could be absolute confidence with the rendering of fullгАВThis brings up an excellent mind upsetting outcomeвАФвАФDeve
lopers don't require newAPIDevelopment of the latest engineпЉМProgrammers don't need to study a new coding tactic and change their particular coding habitsпЉМThe total industry
пїњher groups all winгАВFor earningnys sunglasses Brand name Layout Glasses Large frame Toad wine glass HOT Trend 2015 Summer Coating Men's Antique Sun shades
Summer season Style Kitty Vision Sunglasse Women Eyewear
Glasse With Steel Shape Brandname Artist Aviator Sunglasse For males UV400 3057F
Summer season Fashion sunglasses adult men manufacturer custom Backyard Aviator Sun glasses for Men Girls Foshion Glasses Generating Wayfarer Sun's rays Goblet
2015 Summer season fashi ray ban 3357 reservationdren's palm, atart exercising . clothes, More prone to coldгАВThe most significant position will be,The age of must a youngster become Lengnuanzizhi large,Mom and dad may talk to the youngsters sensation past too far to alter garments
пїњeir academic dreamsпЉМWith their particular loveгАБPatientгАБProfessional expertise in order to form all of it... Detailed
гААгААLi Qing
гААгААNie Yanjun
гААгААCentury competitionпЉМTalent competitionпЉМCompetition is amongst the thorough ability and also qualityгАВCon


text:
Nematode effector proteins: an emerging paradigm of parasitism
Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
I. Introduction
Phytonematodes are unique among the plant pathogens because they are animals (microscopic roundworms) that develop an intimate and sustained obligate parasitic relationship with their host plants. When compared with the microbial-feeding nematode Caenorhabditis elegans, a premier biological and genetic model (Jones et al., 2011; Yook et al., 2012), the two most striking adaptations for parasitism in phytonematodes (Hussey, 1989; Baldwin et al., 2004; Davis et al., 2004) include the development of a stylet and elaborate specialized secretory gland cells within the nematode esophagus (Fig. 1a,b). The stylet is a protrusible hollow mouth spear in the nematode head that is used to pierce host plant cell walls to access host cell contents for ingestion. The stylet also serves as a structure to deliver the secreted effectors produced in the nematode esophageal gland cells (Hussey, 1989; Davis et al., 2008) to modify host cells for a permanent source of nutrients.
The definition of effectors offered by Hogenhout et al. (2009) as 'all pathogen proteins and small molecules that alter host-cell structure and function' provides a broad palette that is inclusive of all current investigations of phytonematode effectors. Research on avirulence (avr) genes of microbial pathogens has emphasized the role of secreted pathogen effectors (avr gene products) with respect to their functions in incompatible (resistance gene-mediated) host–microbe interactions (Desveaux et al., 2006). By contrast, most studies of phytonematode effectors have focused primarily on their roles in promoting compatible (susceptible) parasitic interactions with their hosts (Davis et al., 2008; Rosso et al., 2012). Emerging evidence from functional investigations of pathogen effectors (Deslandes & Rivas, 2012; Feng & Zhou, 2012; Rafiqi et al., 2012; Win et al., 2012), including effectors of phytonematodes (Gheysen & Mitchum, 2011; Haegeman et al., 2012; Rosso et al., 2012; Hewezi & Baum, 2013), suggests roles not only in conditioning the host defense response but also in the augmentation of multiple processes of host cells during the course of parasitism.
Parasitic strategies differ among species of phytonematodes, from those that only pierce and ingest cellular contents as they migrate among host cells to species that induce elaborate modifications in plant cells selected to serve as the permanent feeding site for sedentary life stages of the nematode (Hussey & Grundler, 1998). The feeding sites formed in host plant tissues (Fig. 2) by root-knot (Meloidogyne spp.), cyst (Heterodera and Globodera spp.), and reniform (Rotylenchulus reniformis) nematodes have received particular attention in research for the dramatic changes in host cell morphology (Jones, 1981) and gene expression (Escobar et al., 2011) that are induced and the resultant plant damage that culminates in billions of dollars of annual global crop losses (Chitwood, 2003). Plant response to nematode parasitism in either susceptible host genotypes or resistant crop cultivars occurs in the feeding sites that surround the nematode head (Jones, 1981; Williamson & Kumar, 2006), suggesting that effectors are secreted from where the stylet and amphid (labial sensory neuron) openings exist.
Decades of research demonstrating the differential secretory activity of phytonematode esophageal gland cells throughout plant parasitism have identified these glands as the primary source of secreted effectors (summarized in Hussey, 1989; Davis et al., 2008; Rosso et al., 2012). Analyses of esophageal gland cell transcription coupled with bioinformatics have identified numerous parasitism genes that encode different putative effector proteins in cyst and root-knot nematodes (Davis et al., 2000, 2008; Haegeman et al., 2012; Rosso et al., 2012; Hewezi & Baum, 2013). More recent genomic and transcriptional analyses have extended effector identification to other phytonematode species including some migratory plant parasites (Rosso et al., 2009, 2012; Abad & McCarter, 2011; Gheysen & Mitchum, 2011; Jacob & Mitreva, 2011). While exciting discoveries such as nematode cell wall-modifying enzymes of ancient microbial origin (via horizontal gene transfer; Smant et al., 1998; Yan et al., 1998; Scholl et al., 2003; Jones et al., 2005; Mitreva et al., 2009; Danchin et al., 2010; Kikuchi et al., 2011) and secreted nematode effectors that mimic endogenous plant signaling peptides (Wang et al., 2005, 2010a, 2011; Mitchum et al., 2008, 2012; Lu et al., 2009) have been described, a majority of putative phytonematode effectors encode novel proteins (Davis et al., 2008; Rosso et al., 2012). The advances in our knowledge of the identities and functions of phytonematode effector proteins have been extraordinary and are beginning to paint a complex picture of nematode regulation of the parasitic process (Gheysen & Mitchum, 2011; Haegeman et al., 2012; Rosso et al., 2012; Hewezi & Baum, 2013), yet we have just begun to scratch the surface in our understanding of these unique host–parasite interactions.
II. Nematode effector regulation and delivery into host cells
As already noted, the three large specialized secretory gland cells, one dorsal and two subventral, in the nematode's esophagus are the principal sources of effectors essential for phytonematodes to parasitize plants (Fig. 1a,b; Hussey, 1989). The complexity and secretory function of these unique unicellular glands are revealed in ultrastructural studies (Endo, 1984; Hussey & Mims, 1990). Each gland contains a large lobed nucleus with a prominent nucleolus and abundant Golgi complexes, rough endoplasmic reticulum, and other organelles typical of secretory cells. Effector proteins are synthesized in the nuclear region of the gland cells and N-terminal signal peptides direct them to the secretory pathway where they become packaged in membrane-enclosed spherical secretory granules (dense-core vesicles) formed by budding from the trans-Golgi network (Fig. 1c). The secretory granules are transported forward by a microtubule network in the glands' cytoplasmic extensions to distal elaborate valves where the effector proteins are released into the valve's end sac by exocytosis (Fig. 1d) and subsequently secreted into host tissues through the nematode's stylet. During the parasitic cycle of sedentary endoparasitic nematodes, developmental changes occur in the activity of the gland cells and the effector proteins they produce. The subventral glands function primarily but not exclusively in the penetration and migration phases and the dorsal gland, which enlarges after the onset of parasitism, has a principal role in the formation and maintenance of the feeding cells.
While recent advances in molecular biology have enabled significant progress to be made in the identification and functional characterization of nematode effector proteins, little is known about the actual process of effector secretion. Although effectors have been shown to be developmentally expressed, how they are packaged in the granule matrix or what regulates the synthesis and secretion of different effectors throughout the parasitic process remains elusive. Nonetheless, the effector proteins appear to be synthesized in response to specific signals, because different effectors are required to be produced in time and space during the parasitic process. The secretion of effectors appears to occur via a regulated exocytosis pathway in response to external stimuli (Miller & Moore, 1990; Burgoyne & Morgan, 2003; Kim et al., 2006). The observations of nerve processes and neurosecretory cells apposed to the gland cell cytoplasmic extensions and ampulla also indicate that effector secretion is probably regulated by the nematode's nervous system (Endo, 1984; Hussey & Mims, 1990).
Given the large number of different types of effector proteins that are produced in both types of esophageal gland cell, how the effectors are packaged in the secretory granules is unclear (Fig. 3). As effector production is developmentally regulated, the gland cells must contain secretory granules comprising a mixture of different effector proteins and/or a mixture of secretory granules containing individually packaged effector proteins, at any particular time. Co-immunolocalization studies to localize multiple effectors simultaneously within the esophageal gland secretory granules will aid in our understanding of effector packaging.
How and what triggers the secretion of specific effectors in different host tissues and cells at critical time-points in the parasitic process remains a mystery. For example, some effectors (e.g. endoglucanases (ENGs)) are clearly secreted into the apoplast during nematode intra- or intercellular migration in the root (Wang et al., 1999), whereas other effectors, shown to directly interact with specific cytoplasmic proteins or targeted to the nucleus, must enter the host cell to function in parasitism (Hewezi & Baum, 2013). Intuitively, the stylet must have evolved for the purpose of penetrating plant cell walls so the parasite could deliver effectors directly into the host cell cytoplasm as well as for ingesting nutrients from the cell during feeding. Indeed, ultrastructural studies of nematodes feeding on host cells reveal that a small pore is formed in the plasma membrane at the stylet orifice, which would be consistent with the parasite being able to secrete effectors directly into and feed from the cytoplasm of host cells (Figs 4, 5; Rebois, 1980; Hussey et al., 1992a). And new molecular evidence is emerging that supports the notion that effector proteins are secreted directly into the cytoplasm of the parasitized host cell. For example, a nematode effector protein that functions as a peptide mimic of plant CLAVATA3 (CLV3)/endosperm surrounding region (ESR) (CLE)-like peptides, which normally function in the apoplast by binding to extracellular receptors, is first delivered to the cytoplasm of host cells. The nematode protein then functions as a ligand mimic in the apoplast after being retargeted by a trafficking domain embedded within the secreted effector protein (Wang et al., 2010a,b). Additional support for functioning of nematode effectors inside plant cells comes from numerous studies that identify intracellular host proteins that directly interact with nematode-secreted effectors and the subcellular compartments targeted by different effectors (Huang et al., 2006a; Elling et al., 2007a; Patel et al., 2010; Lee et al., 2011; Jaouannet et al., 2012). Furthermore, feeding tubes formed within the cytoplasm of feeding cells (Fig. 5; Rebois, 1980; Hussey & Mims, 1991) probably develop from nematode stylet secretions produced in the dorsal gland cell. Development of feeding tubes occurs quickly at the basal end near the stylet following its insertion into a host cell (Wyss & Zunke, 1986). In fact, accumulation of a crystalline secretory component similar in ultrastructure to the feeding tube wall was detected in an open dorsal gland valve in a female root-knot nematode (Hussey & Mims, 1990). Identifying the composition of feeding tubes presents an exciting challenge for the future.
Although the preponderance of the evidence supports the delivery of effectors directly into the cytoplasm, some effector proteins, including those that modify cell walls, have been found to accumulate in host extracellular spaces during feeding cell development and maintenance. Immunolocalization studies have shown that effectors from the amphids and dorsal gland cells of sedentary stages of root-knot nematodes accumulate at the wall of a feeding cell (Vieira et al., 2011). While the possibility exists that effectors could be deposited externally in the apoplast by the nematode and then translocated via the plasma membrane into the host cell cytoplasm, as determined for oomycete effector proteins (Birch et al., 2008), no signature translocation motifs have been identified in nematode effector proteins (Hewezi & Baum, 2013).
Clearly we have only a rudimentary understanding of effector synthesis and secretion by phytonematodes. In the future, determining the environmental triggers for the synthesis and secretion of different effectors throughout the parasitic cycle and where they function in recipient host cells will be required for a complete understanding of nematode parasitism of plants. If the plant signals are first perceived by the amphids to regulate secretion, silencing of known amphid chemosensory genes or mutants in the plant signaling molecules may provide clues to the triggers of differential secretion in phytonematodes.
III. Nematode effectors as probes of plant cell biology
Phytonematodes that gain entry into plant tissues vary in their migration patterns. Some nematodes, such as root-knot nematode, are more stealthy as they migrate intercellularly. By contrast, the forceful intracellular migratory pattern of most endoparasites triggers a path of destruction that elicits a robust plant response. Recent studies have begun to document the changes and differences in gene expression in host roots in response to these different infection strategies (Kyndt et al., 2012). Presumably, the plant's first line of defense against the invading nematode is triggered in response to damage-associated molecular patterns (DAMPs) or conserved pathogen-associated molecular patterns (PAMPS; Win et al., 2012), although these have yet to be identified for phytonematodes. Thus, many of the effectors secreted during the migratory phase of infection facilitate nematode penetration and migration by degrading components of the plant cell wall, as well as enable nematodes to dampen down the plant's immune system (Smant & Jones, 2011).
Sedentary endoparasites eventually settle down to feed near the vasculature. Successful establishment and maintenance of feeding cells most certainly requires sustained suppression of plant immune responses. Transcriptional profiles of feeding cells has identified downregulation of defense gene and hormone signaling as a common theme underlying feeding cell formation by sedentary endoparasites (Barcala et al., 2010; Damiani et al., 2012). Thus, effector proteins secreted during the sedentary phase of parasitism probably coordinately regulate plant defense suppression with cellular reprogramming to form a metabolically highly active feeding cell upon which it will depend for weeks to sustain its growth and development; that is, unless the plant carries genes conferring resistance to the nematode, in which case the interaction culminates in HR-like cell death of the feeding cell and ultimately death of the nematode (Williamson & Kumar, 2006; Kaloshian et al., 2011).
Histological studies provided the first glimpse into these unique feeding cells and detail the distinct morphological features characteristic of each plant–nematode interaction during compatible and incompatible interactions (Endo, 1965; Jones, 1981; Wyss, 1997). The feeding cells induced by root-knot (giant-cell) and cyst (syncytium) nematodes have received the most attention (Gheysen & Mitchum, 2011). Giant cells can be 100 times the size of a normal root cell and undergo repeated acytokinetic mitosis to become multinucleate (Fig. 2b). By contrast, a syncytium is a complex of hundreds of cells coalesced from adjacent wall dissolution to form a multinucleate feeding site (Fig. 2c). Some of the cellular changes leading to feeding cell establishment include alterations to the nuclei, cytoskeleton, hormone status, and metabolism of the selected cell. This is paralleled by extensive changes in cell wall architecture and may involve differing degrees of wall loosening and expansion, dissolution, and ultimately biogenesis, the latter leading to the formation of cell wall ingrowths to facilitate solute uptake typical of transfer cells. Conversely, these processes appear to be disrupted during incompatible interactions. More recently, transcriptome and metabolome analyses of feeding cells have provided insight into the massive changes in gene expression to reveal a complex network of molecular events leading to their formation in susceptible host plants (Ithal et al., 2007; Szakasits et al., 2009; Barcala et al., 2010; Hofmann et al., 2010) or their degeneration in plants resistant to the nematode (Kandoth et al., 2011).
While mechanisms underlying the development or demise of feeding sites for the most part remain a mystery, the end result is unquestionably under the direct influence of nematode stylet-secreted effectors. How do phytonematodes get away with such elaborate modifications of host cells? As we describe below ('An update on nematode effectors by genus'), some of these effectors are common among the various phytonematodes, whereas others appear to be lifestyle-specific (summarized in Haegeman et al., 2012). The varied host responses and different ontogenies of feeding cells among the various types of sedentary nematodes appear to be driven by the unique combination of effectors delivered to host cells. Consequently, nematode effectors should prove useful as molecular probes to study plant cellular processes such as the cell cycle, the cytoskeleton, cell wall architecture, and cellular metabolism, among other processes.
1. Cell cycle and cytoskeleton
Cell cycle activation is required for feeding cell formation (de Almeida Engler et al., 1999). The enlarged nuclei of giant cells and syncytia are the consequence of aberrant cell cycles leading to genome amplification through endoreduplication, and several endocycle activators involved in this process have been identified (de Almeida Engler et al., 2012; de Almeida Engler & Gheysen, 2013). Both endoreduplication and mitotic activity are required for feeding cells to progress to maturity. Cell cycle aberrations are paralleled by dynamic cytoskeletal rearrangements in syncytia and giant cells (de Almeida Engler et al., 2004). Multiple points of disruption to the cytoskeleton have been described (Caillaud et al., 2008; Clement et al., 2009) that could be influenced by the nematode. While nematode effectors directly targeting these cellular processes have not yet been identified, the apparent hijacking of components of the cell cycle and cytoskeleton is materializing as a point of convergence for effectors from a wide range of pathogens (Wildermuth, 2010; de Almeida Engler et al., 2012) and is expected to come into view in future studies of effector function.
2. Cell wall architecture
A host response pivotal to feeding cell formation is controlled cell wall modifications. The recent use of glycan-specific antibodies and fluorescence imaging is beginning to reveal the distinct molecular architecture of feeding cell walls (Davies et al., 2012). Contributing to these changes are numerous classes of plant proteins involved in cell wall modification and biogenesis, such as expansins, ENGs, extensins, cellulose synthases and UDP-glucose dehydrogenases (Goellner et al., 2001; Wieczorek et al., 2006; Sobczak et al., 2011; Siddique et al., 2012). However, how these are coordinately regulated in time and space remains unclear. These modifications appear, in part, to be mediated cooperatively with nematode effectors, such as secreted cellulose-binding proteins (CBPs) found to interact with plant pectinmethylesterase (Hewezi et al., 2008a). Hence, the nematode may have more direct control over feeding cell wall modifications than originally anticipated.
3. Metabolism
As feeding cells develop, the large central vacuole is reduced to several small vacuoles and is replaced by dense cytoplasm reflecting one of the most impressive features of nematode feeding cells – increased metabolism. The abundant changes in plant genes involved in primary metabolism are well documented from transcriptome studies (Ithal et al., 2007; Szakasits et al., 2009; Barcala et al., 2010), and these are now being coupled with metabolic profiling to reveal the complex metabolic reprogramming within feeding cells (Hofmann et al., 2010). Certainly, this altered metabolism is under the direct influence of the nematode, but how much of this is being driven by nematode secretions? Changes to the phytohormone status of selected cells is crucial to feeding cell establishment (Goverse et al., 2000; Wubben et al., 2001; Grunewald et al., 2009a,b), and evidence is accumulating that nematode effectors may indeed drive these processes by directly targeting hormone transport proteins (Lee et al., 2011). Another effector, chorismate mutase, identified from a wide variety of phytonematodes to date (Lambert et al., 1999; Gao et al., 2003; Jones et al., 2003; Lu et al., 2008; Vanholme et al., 2009a; Haegeman et al., 2011; Yu et al., 2011) and more recently the smut fungal pathogen (Djamei et al., 2011), appears to be a key regulator of metabolic priming to promote pathogenesis by altering the plant's shikimic acid pathway, although the exact mechanism of action remains to be elucidated. The shikimic acid pathway produces aromatic amino acids providing precursors for a variety of metabolites that may influence the plant–nematode interaction, including auxin, salicylic acid, and a variety of other phenolic compounds.
IV. Functional characterization of effectors
We currently only have a rudimentary understanding of the regulatory networks controlling feeding cell formation. A clearer picture is expected to emerge as the functions of more nematode effectors come into view. Functional analyses of the effector proteins encoded by phytonematode parasitism genes remain a challenge because these nematodes are obligate parasites with limitations in forward genetic analyses and a current lack of a genetic transformation system (Davis et al., 2008; Rosso et al., 2012). However, the gene silencing assays and protein interaction studies highlighted below have proved invaluable to assess nematode effector function (Rosso et al., 2009, 2012; Hewezi & Baum, 2013).
1. Silencing
The discovery and development of target gene silencing in C. elegans (Fire et al., 1998) using RNA interference (RNAi) has provided a technology to specifically and transiently silence target phytonematode genes, including the parasitism genes that encode secreted effector proteins (Gheysen & Vanholme, 2007; Davis et al., 2008; Rosso et al., 2009; Dalzell et al., 2011; Lilley et al., 2012). Ingestion of double-stranded RNA (dsRNA) complementary to the gene to be silenced is critical to RNAi efficacy for most target genes in nematodes (Timmons & Fire, 1998; Bakhetia et al., 2007; Rosso et al., 2009) and was first successfully demonstrated in phytonematodes by soaking infective cyst nematode juveniles in a dsRNA solution with a feeding stimulant (Urwin et al., 2002). Subsequent studies have demonstrated decreased plant infectivity, reduced target transcript abundance, and altered developmental potential of phytonematodes in RNAi soaking experiments that have targeted parasitism genes (summarized in Lilley et al., 2012; Rosso et al., 2009, 2012). Plant transformation vectors that express target gene dsRNA within transgenic plants have been utilized for host-derived delivery of dsRNA for ingestion by nematodes and subsequent silencing of target nematode genes expressed during the parasitic process (Gheysen & Vanholme, 2007; Davis et al., 2008; Rosso et al., 2009; Lilley et al., 2012). When an apparently essential phytonematode parasitism gene is silenced by plant host-derived RNAi technology, a relatively drastic reduction in nematode parasitic success has been observed (Huang et al., 2006b; Hamamouch et al., 2012; Chronis et al., 2013; Jaouannet et al., 2013; Xue et al., 2013) – results that suggest the functional significance of the encoded effector protein in the parasitic process. Moderate effects on parasitism of RNAi silencing of other phytonematode parasitism genes have also been observed (summarized in Gheysen & Vanholme, 2007; Rosso et al., 2009, 2012; Lilley et al., 2012), suggesting potential functional redundancies among some nematode effector proteins or potential technical difficulties in host expression, delivery, or processing of the dsRNA construct. The design of more effective RNAi constructs and enlistment of microRNA (miRNA) expressed in nematode feeding sites (Hewezi et al., 2008b) as tools for efficient host-derived gene silencing remain as key strategies for functional analyses of phytonematode effector proteins and the production of transgenic plants for future nematode control strategies.
2. Host target identification
Methods employed to identify the host targets of phytonematode effectors have proved successful to begin to elucidate the network of interactions and functions of effector proteins within recipient plant host cells. A wide range of plant subcellular compartments, including the apoplasm, cell wall, cytoplasm, and nucleus, are targeted, which is indicative of a wide range of physiological functions in parasitism. Targets of nematode effectors suggest differential roles in host cell transcriptional, developmental, and metabolic reprogramming, protein degradation, and phytohormone transport and accumulation (Gheysen & Mitchum, 2011). These reported effector functions are consistent with the molecular and morphological responses observed in the complex feeding cells induced by sedentary species of phytonematodes (Jones, 1981; Escobar et al., 2011). Host proteins targeted by nematode effectors identified to date include putative SCARECROW-like (SCL) transcription factors (Huang et al., 2006a), a pectinmethylesterase (Hewezi et al., 2008a), a coiled-coiled nucleotide binding leucine-rich repeat (CC-NB-LRR) protein (Rehman et al., 2009a), an oxidoreductase (Patel et al., 2010), a spermidine synthase (Hewezi et al., 2010), an auxin influx transporter (Lee et al., 2011), several leucine-rich repeat receptor-like proteins (LRR-RLPs; Guo et al., 2011), a papain-like cysteine protease (Lozano-Torres et al., 2012), a β-1,3-endoglucanase (Hamamouch et al., 2012), and an aquaporin tonoplast intrinsic protein (Xue et al., 2013). In all cases, except for the LRR-RLPs, yeast two-hybrid analysis was the method of choice to identify the host-interacting proteins. In the majority of cases, these interactions were confirmed using either in vitro pull-down assays or in planta bimolecular fluorescence complementation (also known as BiFC) by bombardment into onion epidermal cells. In the case of nematode-secreted CLE effectors, radiolabeled peptides were tested for direct interaction with selected plant receptor proteins transiently expressed in Nicotiana benthamiana leaves (Guo et al., 2010, 2011).
The list of host cell targets and processes will greatly expand as existing and new putative phytonematode effectors are characterized.
V. An update on nematode effectors by genus
1. Heterodera
Cyst nematode species of the genus Heterodera are well known for their distinct host ranges. In susceptible hosts, these nematodes use a stylet to penetrate the cell wall of a selected root cell and secrete effector proteins to induce the formation of a syncytium. While the mechanisms underlying and driving the differences in host range among species are unknown, a role for nematode effectors in this process is anticipated. To date, effector discovery in Heterodera has focused on two species, Heterodera schachtii and Heterodera glycines. Unlike other cyst nematodes, the sugar beet (Beta vulgaris) cyst nematode H. schachtii infects Arabidopsis thaliana, and was adopted as a model system for studies of plant–nematode interactions nearly 20 yr ago (Sijmons et al., 1991; Gheysen & Fenoll, 2011). Heterodera glycines, the soybean (Glycine max) cyst nematode, has demonstrated utility as a genetic model system with which to study parasitism (Opperman & Bird, 1998; Niblack et al., 2006), but functional genomic approaches targeting identification of stylet-secreted effector proteins have accelerated the process. The earliest approaches focused on affinity purification of proteins using monoclonal antibodies to antigens in nematode stylet secretions (De Boer et al., 1996). This approach was successful in identifying the first nematode parasitism genes, encoding β-1-4-endoglucanases, in H. glycines and Globodera rostochiensis (Smant et al., 1998). The search for a higher throughput approach resulted in the identification of over 50 candidate parasitism genes encoding putative secreted effectors of H. glycines from cDNA libraries constructed from RNA isolated from cytoplasm microaspirated from esophageal gland cells of parasitic life stages (Gao et al., 2001a, 2003; Wang et al., 2001). A microarray-based expression atlas of the parasitism genes across H. glycines life stages followed (Elling et al., 2007b, 2009). The identification of orthologous H. glycines parasitism genes in the closely related H. schachtii has facilitated studies to acquire insight into the biological function of several nematode effectors using A. thaliana as a model system (Wang et al., 2005, 2010a; Hewezi et al., 2008a, 2010; Patel et al., 2010; Lee et al., 2011; Hamamouch et al., 2012). Additionally, approaches such as direct sequencing of in vitro-produced esophageal gland secretions and mining of expressed sequence tags (ESTs) generated from infective juveniles have identified a number of putative secreted effector proteins from H. schachtii (De Meutter et al., 2001; Vanholme et al., 2006).
To date, 70% of all effectors identified from Heterodera are novel proteins, which presents a considerable challenge in ascribing function. Among those identified from H. glycines and H. schachtii with sequence similarity to known proteins is an elaborate suite of cell wall-modifying proteins (CWMPs), including ENGs (Smant et al., 1998; De Meutter et al., 2001; Gao et al., 2002a, 2004a; Vanholme et al., 2006), CBPs (Gao et al., 2004b; Vanholme et al., 2006), pectate lyases (PELs; Vanholme et al., 2007), and an arabinogalactan endo-1,4-β-galactosidase (Vanholme et al., 2009b). These nematodes also produce secreted effector proteins with homology to chitinase (CHI; Gao et al., 2002b), chorismate mutase (CM; Bekal et al., 2003; Gao et al., 2003), ubiquitin (Gao et al., 2003; Tytgat et al., 2004), and venom allergen proteins (VAPs; Gao et al., 2001b, 2003), although their functions in nematode parasitism remain unknown. The first nematode-secreted peptide hormones sharing homology with plant CLE peptides were identified from H. glycines (Wang et al., 2001; Gao et al., 2003; Olsen & Shriver, 2003; Mitchum et al., 2012), and more recently from H. schachtii (Patel et al., 2008; Wang et al., 2011). These peptides were shown to functionally mimic the effects of plant CLE peptides in exogenous peptide assays and through complementation of the A. thaliana clavata3 mutant (Wang et al., 2005, 2010a, 2011). Although the biological function of these peptide mimics in feeding cell formation is unclear at present, they were recently shown to signal through A. thaliana LRR-RLPs CLV1, CLV2, and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2; Replogle et al., 2011, 2013), probably triggering developmental changes necessary for syncytium formation. Another nematode-secreted effector identified from H. glycines and H. schachtii that may mimic endogenous host proteins is annexin. Annexins are calcium- and membrane-binding proteins with wide-ranging cellular functions. Nematode annexin was able to complement the reduced germination phenotype of a plant annexin mutant under high salt stress and was found to interact with an A. thaliana oxidoreductase, possibly promoting parasitism by modulating host stress and defense responses (Patel et al., 2010). Despite the challenges, tremendous progress has been made to elucidate the function of several novel effector proteins, including 10A06, 19C07, and 30C02, identified from both H. glycines (Gao et al., 2003) and H. schachtii (Hewezi et al., 2010; Lee et al., 2011; Hamamouch et al., 2012). The 10A06 and 30C02 effector proteins were found to interact with spermidine synthase (Hewezi et al., 2010) and β-1,3-endoglucanase (Hamamouch et al., 2012), respectively, and functional studies have suggested a role for these proteins in modulating host defense responses. The 19C07 effector protein was found to interact with an auxin influx transporter, LAX3, presumably to regulate auxin balance during the early stages of feeding cell formation (Lee et al., 2011).
Despite the considerable progress in nematode effector discovery in Heterodera species, we are just breaking the surface in our understanding of effector function.
2. Globodera
Species of nematodes belonging to the genus Globodera feed on plants of the Solanaceae family. The majority of effector studies to date have focused on the potato (Solanum tuberosum) cyst nematodes (PCNs; G. rostochiensis and Globodera pallida), internationally recognized quarantine pests that infect a number of important agricultural crops such as potato, tomato (Solanum lycopersicum), and eggplant (Solanum melongena). Like Heterodera species, these sedentary endoparasitic nematodes secrete a battery of CWMPs during their invasion of plant roots. Genes encoding ENGs, PELs, CBPs, and EXPs (expansins) that were found to be expressed within the subventral esophageal gland cells have been cloned from both PCN species (Smant et al., 1998; Popeijus et al., 2000; Qin et al., 2004; Kudla et al., 2007; Jones et al., 2009; Rehman et al., 2009b). Knocking down of Gr-eng genes by RNAi reduced nematode infectivity, probably as a result of impaired root penetration at the onset of parasitism (Chen et al., 2005; Rehman et al., 2009b). Gr-Exp genes were demonstrated to have cell-wall-extension activity in planta (Qin et al., 2004; Kudla et al., 2005). Predicted structural differences between GrPEL1 and GrPEL2 suggested that these enzymes may target different pectic polysaccharides (Kudla et al., 2007). These nematode-secreted CWMPs appear to work in concert to weaken cell walls to facilitate nematode penetration and intracellular migration through the root tissue.
The two largest families of effector proteins identified from PCNs include the CLE-like proteins and secreted SP1a and Ryanodine receptor (SPRY) domain (SPRYSEC) proteins produced within the nematode's dorsal esophageal gland cell (Jones et al., 2009; Lu et al., 2009; Rehman et al., 2009a). Unlike A. thaliana and Heterodera CLE genes, which encode proteins with a single C-terminal CLE motif, many Globodera CLE genes encode proteins with multiple CLE motifs that vary in their sequence. Ectopic overexpression of the multidomain GrCLE proteins in A. thaliana produces a wide range of plant phenotypes (Lu et al., 2009). The three identical CLE motifs of GrCLE1 were shown to be processed into an active 12-amino acid CLE peptide by plant proteases (Guo et al., 2011). These studies suggest that, once secreted in planta, the multidomain Globodera CLEs may give rise to an assortment of functional CLE peptides potentially acting in different plant CLE signaling pathways to facilitate nematode parasitism. The search for receptors in potato that perceive Globodera CLE peptides is actively being pursued. Multiple SPRYSEC genes have been cloned from PCNs and the genome of G. pallida is predicted to contain > 200 different SPRYSEC genes (Jones et al., 2009; Rehman et al., 2009a; Postma et al., 2012). The SPRYSEC family may target many different host proteins through the hypervariable regions residing in the SPRY domain (Rehman et al., 2009a) and were recently found to play roles in activation and suppression of effector-triggered immunity (ETI). Coexpression of GpRBP1 (RBP: Ran-binding protein), a member of the SPRYSEC family from G. pallida, with the Gpa2 (Globodera pallida 2) resistance protein in potato induced programmed cell death in N. benthamiana leaves, indicating that GpRBP1 triggers Gpa2-mediated nematode resistance (Sacco et al., 2009). The effector SPRYSEC-19 of G. rostochiensis was demonstrated to bind directly to SW5-F, a CC-NB-LRR type of disease resistance protein, in planta (Postma et al., 2012), but this association did not activate ETI in host plants. Instead, a role for GrSPRYSEC19 in suppressing ETI and disease resistance was recently demonstrated (Postma et al., 2012).
Ubiquitin carboxyl extension proteins (UBCEPs), VAPs, and CMs are also expressed in the two PCN species (Jones et al., 2003, 2009; Lu et al., 2008; Lozano-Torres et al., 2012; Chronis et al., 2013). GrUBCEP12, which is expressed exclusively within the dorsal gland cell of G. rostochiensis, was found to be processed into free ubiquitin and a 12-amino acid peptide (GrCEP12) in planta and the released GrCEP12 peptide could suppress plant immune responses. Once secreted into plant cells, GrUBCEP12 becomes two functional peptides that may act in different host cellular pathways to promote nematode parasitism (Chronis et al., 2013). GrVAP1, expressed within the subventral gland cells of G. rostochiensis, was recently shown to specifically interact with the papain-like cysteine protease Rcr3pim (Rcr3 Solanum pimpinellifolium) in tomato, and this interaction could perturb the protein active site, resulting in increased plant susceptibility to the nematode (Lozano-Torres et al., 2012). Interestingly, GrVAP1 could also trigger Cladosporium fulvum 2 (Cf-2)-mediated nematode resistance, probably through perturbation of Rcr3pim (Lozano-Torres et al., 2012). A unique finding for Globodera CM genes is that they undergo alternative splicing (Lu et al., 2008; Yu et al., 2011). Alternative splicing may be an important mechanism regulating CM activity and other nematode effector functions during nematode parasitism.
Putative novel effectors, such as transthyretin-like proteins and proteases, as well as homologs of a large number of novel effectors from Heterodera species, were identified from both PCN species through mining of nematode EST sequences (Jones et al., 2009; X. Wang et al., unpublished). The genome sequences of the two PCN species will become available soon, which will reveal a complete catalog of effectors used by Globodera nematodes in plant parasitism.
3. Meloidogyne
Among all phytonematodes discussed here, root-knot nematodes (Meloidogyne spp.) clearly hold an elevated status. Most Meloidogyne spp. have a remarkably wide host range and it is safe to say that there is no higher plant that is not host to at least one root-knot nematode species. Why are Meloidogyne spp. able to infect such an enormous variety of plants, whereas cyst nematodes have very narrow host ranges? The secret probably lies in Meloidogyne stylet-secreted effectors. Apart from cell wall-degrading and -modifying enzymes such as ENGs (Rosso et al., 1999), PELs (Doyle & Lambert, 2002) and CBPs (Ding et al., 1998), or enzymes that target the plant shikimate pathway (i.e. CMs; Lambert et al., 1999), there is little overlap between the battery of effectors we currently know in root-knot and cyst nematodes. This is especially true for those effector genes that are considered novel and that do not share similarity with known genes in other organisms (Huang et al., 2003, 2004; Davis et al., 2004; Bellafiore et al., 2008). Even 16D10, a 13-amino acid Meloidogyne peptide effector that has some homology to the C-terminal CLE motif of plant CLE peptides, functions differently from CLE-like effectors in cyst nematodes. Whereas the cyst nematode CLE-like effector HgCLE was able to rescue clv3 mutant phenotypes in A. thaliana (Wang et al., 2005, 2010a), 16D10 failed to do so (Huang et al., 2006a). Interestingly, 16D10 interacts with plant SCL transcription factors SCL6 and SCL21 (Huang et al., 2006a), which suggests a high-level upstream regulatory role for this effector. Even though both cyst and root-knot nematodes probably target similar plant processes to initiate and maintain feeding sites, they clearly have different molecular means to accomplish their goals by using distinct sets of effectors.
Recent studies are beginning to untangle some of the complex molecular interactions between effectors of Meloidogyne spp. and their host plants. For example, the amphid-secreted Meloidogyne amphid protein 1 (MAP-1) effector, which was found by screening near-isogenic nematode populations that were avirulent or virulent in plants carrying the Meloidogyne incognita resistance gene Mi, seems to be involved in ETI (Semblat et al., 2001). Recent analyses indicate that highly variable tandem repeats in the MAP-1 protein correlate with nematode (a)virulence and that map-1 is part of a gene family (Castagnone-Sereno et al., 2009; Tomalova et al., 2012). In addition to a classic role in ETI, it is conceivable that Meloidogyne effectors are responsible for defense suppression and the massive modifications of the host plant's physiology to induce and maintain giant cells. Secreted CMs exist in several variants in a number of Meloidogyne spp. and seem to play an important role in the molecular interaction with the host plant (Doyle & Lambert, 2003; Huang et al., 2005; Long et al., 2006). Meloidogyne CM genes are highly upregulated 2 d after infection, which suggests a role in early events related to parasitism (Painter & Lambert, 2003). Overexpression of MjCM-1 in planta lowered the concentration of indole-3-acetic acid, presumably by competing for chorismate and thereby altering chorismate-derived metabolism, including salicylic acid-mediated plant defense reactions (Doyle & Lambert, 2003). Mi-CRT (CRT: calreticulin) from M. incognita was demonstrated to suppress PAMP-triggered immunity (PTI; Jaouannet et al., 2013). Transgenic A. thaliana overexpressing Mi-CRT showed increased susceptibility to M. incognita and the induction of defense-related genes and callose deposition after treatment with the PAMP elf18 (N-terminal 18 amino acids of Elongation factor Tu). Similarly, overexpression of effectors MJ-NULG1a (Meloidogyne javanica nuclear-localized gene 1a) (Lin et al., 2013) and 7E12 (Souza et al., 2011) in planta enhanced Meloidogyne infection, but the exact role of these effectors remains elusive. Interestingly, 7E12 overexpression also accelerated feeding site formation and vacuole size increase in giant cells (Souza et al., 2011). Meloidogyne giant cells are metabolic sinks and are speculated to function as highly specialized phloem transfer cells. The M. incognita effector Mi8D05 interacts with plant aquaporin tonoplast intrinsic protein 2 (TIP2), which supports this idea and suggests that effectors regulate solute and water transport in giant cells. Interestingly, Mi8D05 also induced accelerated shoot growth and early flowering when overexpressed in A. thaliana, but had no discernible phenotypic effect in roots (Xue et al., 2013).
Even though the current state of our knowledge of how exactly Meloidogyne effectors enable plant infection still resembles an incomplete jigsaw puzzle with an unknown number of pieces, promising progress in recent years suggests that we are beginning to put the pieces together and will eventually be able to fully decipher the molecular dialog between plant and nematode.
4. Rotylenchulus
The genus Rotylenchulus contains 10 known species that thrive in the tropical and subtropical parts of the world. All species are sedentary semi-endoparasites, which means that the posterior portion of their body is exposed on the root surface. Upon maturation, the adult female attains the characteristic reniform (i.e. kidney) shape. Rotylenchulus reniformis, the reniform nematode, is the most damaging species, having a broad host range that includes both dicot and monocot plants from 77 families (Robinson et al., 1997). Economically important hosts for R. reniformis include upland cotton (Gossypium hirsutum), soybean, pineapple (Ananas comosus), and sweet potato (Ipomoea batatas) (Robinson et al., 1997). The infective life stage (young adult female) of the reniform nematode establishes a feeding site within the root similar to the syncytium formed by Heterodera and Globodera spp. Rotylenchulus reniformis syncytia usually initiate from an endodermal cell and expand via partial cell wall dissolution (Rebois et al., 1975). A preliminary assessment of ESTs derived from R. reniformis sedentary females identified a number of sequences homologous to cyst nematode effectors including cellulase, SEC-2, G22C12, G16B09 and one EST that shared similarity with the HgCLE peptide (Wubben et al., 2010). Dorsal esophageal gland-specific expression of the R. reniformis CLE homolog has been demonstrated by in situ hybridization (L. Gavilano et al., unpublished). A much larger transcriptome survey of sedentary parasitic R. reniformis females using next-generation sequencing has identified additional potential effector homologs, including homologs of H. glycines CM, annexin, UBCEP, VAP, PEL, and CHI (K. Showmaker et al., unpublished). While homology-based approaches to effector discovery can quickly identify candidates for further study, effectors that are either specific to R. reniformis or sufficiently divergent from other species will go unnoticed. Thus, employing approaches for comprehensive effector gene discovery (Maier et al., 2013) is warranted.
VI. New approaches for effector identification
Despite concerted efforts for more than two decades, we can safely say that we probably have not discovered the full effector spectrum of any plant or animal parasitic nematode. The fact that effectors appear for the large part synthesized in the esophageal gland cells has provided a clearly defined target for effector identification and led to the intense focus of research efforts on the esophageal glands.
The identification of effectors in phytonematodes undoubtedly has been aided by the availability of genome sequences of root-knot nematodes (Abad et al., 2008; Opperman et al., 2008). Bioinformatic genome mining identified large panels of effector candidates with certain protein similarities and the presence of N-terminal signal peptides. Similarly, the impending release of genome sequences of other nematodes, notably potato cyst nematodes, clearly will aid effector identification in additional taxa. But even so, genome sequences alone will not allow the identification of complete effector repertoires and will miss effector types not fitting a preconceived notion of what effectors should look like. Rather, combinations of genome mining with other avenues of inquiry will be required. In particular, transcriptomic investigations of the esophageal glands combined with genome studies stand a grand chance of truly expanding our understanding of effector repertoires and their variability among genera, species and pathotypes.
Focus on the esophageal gland cells as the immediate target opens up the possibility of unravelling a complete effector spectrum of an individual nematode species. Over the years, different methods to target this cell type have been used, and most recently culminated in a method that allows the actual direct isolation of these gland cells (Maier et al., 2013). This new gland isolation technique will allow the identification of effectors from nematodes with different modes of parasitism. So far the research community has generated a catalog of cyst nematode and root-knot nematode effectors and found surprisingly few that overlap. But what about other sedentary nematodes, such as R. reniformis or Tylenchulus semipenetrans, or even Cactodera or Meloidodera isolates? But that's not all – what about migratory nematodes and then, of course, those nematodes that are in between – such as ring nematodes (Criconematid spp.), which retain their ability to move about between feedings but have prolonged their feeding sessions considerably and have adopted the ability to profoundly alter their host feeding cells (Hussey et al., 1992b; Westcott & Hussey, 1992)? Exploring the effector spectra in these taxa should prove to be fascinating. As a consequence of such studies, evolutionary relationships may have to be rewritten, the origin of certain effectors may become clear, and exciting revelations about the mechanisms that govern host range or virulence will be made.
In addition to merely searching for protein sequence similarities, more in-depth future analyses will identify other criteria that define effectors. For example, it is very possible that certain small domains of conserved amino acids or conserved protein structures will be identified as hallmarks of nematode effector groups, and such discoveries will allow additional queries into available genomes and transcriptomes.
One of the challenges of identifying the complete effector repertoire of a single nematode species lies in the fact that not all effectors are released through the stylet. Clearly, nematodes are most likely, or already have been shown, to release bioactive proteins from their amphids and their hypodermis along the body. Furthermore, release of compounds from the excretory/secretory system or the cloaca is a possible avenue of effector delivery. While stylet delivery has all the hallmarks of being the route through which the feeding cells are manipulated, the other avenues could function in suppression of defenses during migration as well as during the disruptive growth of the sedentary adult female stages as they expand. Similarly, not all effectors are released from the nematode through a signal peptide-dependent secretory pathway. Several effector candidates released from nematodes without a signal peptide have been reported (Bellafiore et al., 2008), and it remains to be seen how many additional such proteins will be uncovered in the future. In addition to the transcriptomic and genome sequence mining of nematodes, proteomic analyses of plant tissues or cells that have been in contact with parasitizing nematodes provide a potential means to identify proteins that are released by nematodes in planta. While sensitivity limitations currently exist for available technologies, it is expected that technical advances will open up new opportunities.
The picture becomes considerably more daunting when taking into account that, while the research community has focused on proteins as effectors, it is hard to imagine that the successful group of phytonematodes would have limited itself to only one class of effector molecules. Instead, nonproteinaceous molecules, potentially derived from diverse secondary metabolites, may also play important roles in parasitism. Indeed, there are literature reports that support this notion. De Meutter et al. (2003) described plant hormones being released from phytonematodes. Also, considering that rhizobial bacteria use carbohydrates as nodulation (NOD) factors and that McCarter et al. (2003) have reported nod gene homologs in the RKN genome, carbohydrate effectors may not be that unexpected. Interestingly, Weerasinghe et al. (2005) also observed root hair curling in advance of infective root-knot nematode juveniles, supporting a NOD factor-like function of an unknown nematode secretion.
VII. A global perspective of nematode effector function
In addition to the identification of all effectors, the characterization of their individual and integrative functions dwarfs all other challenges for a complete understanding of plant–nematode interactions. The current approach of overexpression of effector genes in plants, target gene silencing via RNAi, and identification of target host proteins, while successful (Hewezi et al., 2008a, 2010; Lee et al., 2011; Hamamouch et al., 2012), allows analyses of only a single effector at a time. Future investigations must consider how multiple nematode effectors function in concert during the parasitic process – as they do very successfully in nature (often to the detriment of agriculture). How many effectors (and which types) are required to form a feeding cell (or trigger host defense)? A fundamental change in approach is needed, or better, a bigger picture view. Interdisciplinary approaches, capitalizing on potential synergisms between experts in different fields, appear to be the most promising way to make comprehensive progress. What are some of the options that present themselves?
High-throughput assays clearly are urgently needed. For example, high-throughput assays for the identification of defense suppression activities of effectors are available and should be applied to nematode effectors. Similarly, high-throughput in planta effector expression approaches such as viral expression systems (Zhang et al., 2010) will allow the phenotypic analyses of a large number of effectors when introduced into plants. Moreover, the use of other pathogen effector delivery systems, most notably bacterial type III secretion systems (Sohn et al., 2007), to introduce nematode effectors into plants has significant promise for exploring effector traits in planta.
Approaches that allow high-throughput interactor screens, such as robotic yeast two-hybrid analyses, are particularly useful, and comprehensive interactome maps are realizable (Arabidopsis Interactome Mapping Consortium, 2011; Mukhtar et al., 2011; Van Leene et al., 2011; Hu, 2013; Mongiovi & Sharan, 2013). The prospect of identifying effectors interacting with other effectors also exists. While effector–effector interaction studies with yeast two-hybrid analyses may reveal such interactions, methods to identify multivalent protein interactions, such as techniques based on affinity purification of protein complexes followed by proteomics analyses, are highly desirable. Some effectors probably function in concert while interacting with host factors. For example, assembly of the feeding tube (Hussey & Mims, 1991) might involve a multi-effector/plant protein interaction, as the current mining of effector candidates has not yet revealed a clear candidate for an auto-assembling single effector feeding tube protein.
The localization of some nematode effectors to the plant host cell nucleus (Elling et al., 2007a; Jaouannet et al., 2012) and the demonstration of an interaction with plant transcription factors (Huang et al., 2006a) suggest potential regulatory roles at the transcriptional level. A related type of interaction is the direct binding of pathogen effectors to nucleic acids. Although discovered in other pathosystems (Römer et al., 2007; Boch et al., 2009), DNA-binding activity and direct transcriptional regulatory function of nematode effectors have not yet been reported, but have the potential to exist. In that case, high-throughput yeast one-hybrid analyses to identify DNA-binding activities of effectors will be very desirable.
Computational approaches to reveal integrative effector functions should play a prime role in future strategies. Prediction of protein interaction sites or protein structures with known functions undoubtedly will become available for analyses of novel nematode effector proteins. While analyses of plant response to single effectors has utility, a contrasting approach may be to remove (silence) a single nematode effector and monitor the plant response (i.e. like using transcriptomics or, histology) in its absence upon host infection. Systems biology approaches (Lucas et al., 2011; Collakova et al., 2012; Benfey & Scheres, 2013) to elucidating global effector functions, that is, the integration of several 'omics' data (such as metabolomics, proteomics, and transcriptomics) will ultimately play a key role. By focusing on the plant response at the infection site with high resolution coupled with similar assays of effector function, systems biology holds tremendous promise in separating true effects from experimental noise. Data from the types of analyses presented above can provide the raw materials for integrative approaches to model the collective host cell pathways and hubs targeted by nematode effectors to orchestrate these fascinating parasitic interactions. Undoubtedly, the prospects are exciting and bright for more fully revealing the role of nematode effector proteins in this new emerging paradigm of parasitism.
The authors gratefully acknowledge the members of their laboratories and collaborators over the years for their contributions to this work. We thank Richard Howard and Nancy Rizzo, DuPont Pioneer, for providing the pictures showing localization of nematode CLE effector proteins in the secretory granules of the dorsal gland cell. Research support for the authors has been awarded by the United States Department of Agriculture (USDA)-National Research Initiative Competitive Grants Program, USDA-Agriculture Research Service, United Soybean Board, Washington State Department of Agriculture, Washington State Potato Commission, Idaho Potato Commission, Washington Grain Commission, Washington Red Raspberry Commission, Pioneer Hi-Bred, Experiment Stations of University of Missouri, University of Georgia, North Carolina State University, and Iowa State University, and Agricultural Research Center at Washington State University.

subset: commoncrawl


text:
Post Content
Posted by Tracy Evans, MPH with help from Lindsay Tallon
Tracy is a Program Coordinator at the MA Department of Public Health
Once upon a time I used to be a very different person. I was the girl who would leave work at the end of a long day with both the freedom and energy to go for a run. I was motivated. I was a follower of Nike's Ad Campaign, "Just Do It." Before becoming a mom, making time for some form of exercise on a weekly basis was a top priority for me. But I don't know where that girl is anymore. I think she finally ran off to take a nap.
A few years into being a mom, I found myself slipping away into sedentary land. And I continued to live happily ever after wearing bigger pants and taking the elevator way too often.
But wait – this fairy tale has a better (and healthier) ending. Fast forward to one day last month when I saw a woman entering my office building after commuting to work on her bike. Wow, I thought, I have to meet her and steal some of her motivation. Okay, not steal, maybe borrow, or beg her to share some secrets! I just knew I had to meet her and get RE-inspired.
So I met with Lindsay Tallon, and want to share with you what I learned from this working mom with two children who rides her bike to work on a regular basis.
What motivated you to ride your bike to work?
Other DPH employees that ride their bikes to work and my husband, who does the same, inspired me. DPH also makes it super easy and worry free to store my bike with inside bike racks. Being motivated by a little competition, the DPH Green Streets Initiative where you pledge to go car-free the last Friday of the month at http://www.gogreenstreets.org was another incentive. Signing up gets you registered for DPH, provides discounts at local businesses, and calculates the calories burned and carbon offsets from your green commute.
Describe your healthy commute:
It's about 5 miles each way, and as a busy mom I consider the ride as treasured "me" time where I get to decompress before work or after a long day. Multi-tasking is an essential skill that any mom does on a daily basis and biking to work is just that–I get to work and exercise at the same time.
Anything you would like to say to other moms?
Take it one step at a time and just get started. Find ways to sneak fitness into your daily routine and treasure the time to take care of yourself. You deserve it!
What did I learn from Lindsay? I am a different person now, and I need a different plan. Time is different when you are a parent. I discovered getting up early is the most reliable time for me to work out while my son and husband are sleeping.
We all know where to find credible health information, but actually finding the motivation and inspiration and time is challenging. I want to encourage other moms to Just Do "Something". Find the time. Borrow the motivation. Look around at your family members or friends or neighbors or coworkers and see if someone has recently started a good eating plan or has found ways to increase physical activity/exercise and take the time to ask them about it; you may leave with more inspiration and tips, and please share them with me too.
Join us tomorrow, September 28, for the Walk/Ride Challenge by visiting http://www.gogreenstreets.org.
Tags: bicycle, bike, green streets, health, healthy, Mass in Motion, moms, walk/ride day, women
Recent Posts
Some Workers Face Higher Risk of Injury – But Employers Can Make a Difference posted on Jul 25
In the six years from 2008 through 2013, 356 workers died on the job in Massachusetts. This is about one worker every five days! The MDPH CFOI and MA FACE projects developed this infographic to illustrate these tragic worker deaths and to underscore the human costs …Continue Reading Some Workers Face Higher Risk of Injury – But Employers Can Make a Difference
Making Your Summer POP! posted on Jul 19
Few things are better than an ice-cold popsicle on a hot summer day! I have vivid memories from my childhood running around through the sprinkler and various neighbors' yards enjoying the summer sun. At that age, having a messy red ring around your mouth from …Continue Reading Making Your Summer POP!
Massachusetts is Rewarding Small Businesses for Healthy Workplaces posted on Jul 18
Wellness programs are good for business. They can lead to better performing workers, lower absenteeism and decreased healthcare and insurance costs. Now, small businesses can be rewarded for these efforts! The Massachusetts Wellness Tax Credit Incentive gives small businesses in Massachusetts a state tax credit …Continue Reading Massachusetts is Rewarding Small Businesses for Healthy Workplaces


text:
Content Tagged with "boobs"
Random Photos 433
Oh no, wet paint!! I don't know how anyone could concentrate on golf playing with these two. Yeah, that's not suspect at all.. And finally, now that's what I call good service!
Pappupassq's Best 100 Pics on S&F Battle!
This battle includes the 100 best pics on S&F as chosen by user Pappupassq for your viewing/voting pleasure. This is gonna be a monumental task, good luck!
Random Photos 432
Nice bikinis ladies! In other news the Pokemon Go craze is showing no signs of slowing down.. Looks like it's quite nipply out. Dani shows off her boobs in the car. And finally, nice scenery!
Honk The Horn for Dani's Boobs!
The truck driver finally obliges after much deliberation. And by deliberation I mean checking out Dani Daniel's awesome rack.
Random Photos 431
See through shirt for the win! Speaking of winning, she has won the genetic lottery! Jeny shows off impressive skills. Are you obsessed with PokemonGo yet? And finally, nude gaming!
Maintaining Eye Contact Challenge
We tracked down the video that this Random Photos gif came from and it turns out it's full of awesome cleavage. These guys are better men than I.
Hilary Flashes in the Snow
Apparently the cold doesn't really bother Hilary as she flashes various body parts for the camera while in a snow covered field.
Top 5 Nude Scenes From 80's Comedies
Possibly one of the finest top lists Mr Skin has put together yet. Full of classic scenes, they don't make them like this anymore..
Topless Beer Pong Gets Crazy at a College Party
These girls have their tits out before the game even starts. They're not great at beer pong but they are half-naked.
Random Photos 430
A brand new edition of everyone's favorite collection of sexy and funny photos! A little heavy on the Independence Day pics as you may imagine, but also some funnies! And finally, wow!
A Topless Jeny Asks Random People Sexy Questions
It looks like our Jeny has taken a leaf out of this Jenny's book. A very sexy leaf. She's a natural!
Suzanna's Naked at the Pool
Suzanna brought a big, pink float over for some fun in the pool. She didn't bring anything to wear, which is great.
Jemma's Home from Work Early
Jemma took the afternoon off and it looks like she has something special in mind. The purple thong was a nice choice.
Random Photos 429
This guy deserves an award of some kind. This blonde is having a bad day, while this brunette is enjoying her drive. Way to go Iceland! And finally, the elusive triple flash!
Janessa Brazil Strips for Us. WOW!!
This could just be the hottest fan video ever. Janessa (Penthouse model, Miss Howard TV and Random Photos star) has really outdone herself in this video for us. Go watch it, now!


text: Angela Carella: Quest on to quiet the roar of money Back to Article Comments Need to Register? X Remember Me Forgot your password? Today's Deals iView Digital Converter Box w/ Media Player for $20 + free... Cap Barbell 12-Piece Puzzle Equipment Mat for $6 + pickup at... Ozark Trail 10x10-Foot Gazebo Top for $25 + pickup at Walmart Stanley 1-Gallon Wet Dry Vacuum for $20 + pickup at Walmart Powered by


text:
I just bought a 2001 Mitsubishi montero and there is a vibration when you are between the speed of 40 to 45 miles per hour any suggestions my mechanic can't figure it out
4 Answers
could be the wheel needs balancing or a broken belt in the tire,, look for budging on the tire,, you have snow where you are? you could have some snow or ice packed inside of your rim
btw if you bought this car from a dealer they should be taking care of the problem for you
There is a service bulletin about that particular issue, mine was more of a shutter than a vibration.
1 out of 1 people think this is helpful.
Had a 96 ford explorer which had the same kind of problem. it was due to some water in the tire, i am guessing from a compressor not being drained regularly.Odd as it sounds ,never would have thought to look for that. Good luck.
Your Answer
Looking for a Used Montero in your area?
CarGurus has 152 nationwide Montero listings starting at $2,500.
Search Mitsubishi Montero Questions
Mitsubishi Montero Experts
Daniel Foster
Reputation 150
Reputation 90
Reputation 70
Related Models For Sale
Used Mitsubishi Montero Sport"
3 Great Deals out of 395 listings starting at $1,950
Used Toyota Land Cruiser"
15 Great Deals out of 1,046 listings starting at $3,995
Used Toyota 4Runner"
252 Great Deals out of 15,018 listings starting at $1,995
Used Nissan Pathfinder"
376 Great Deals out of 21,628 listings starting at $500
Used Jeep Grand Cherokee"
1,145 Great Deals out of 72,901 listings starting at $1,200
Used Ford Explorer"
976 Great Deals out of 63,578 listings starting at $995
Used Toyota RAV4"
885 Great Deals out of 45,736 listings starting at $1,350
Used BMW X5"
364 Great Deals out of 12,186 listings starting at $2,500
Used Honda CR-V"
1,151 Great Deals out of 82,911 listings starting at $1,750
Used Jeep Wrangler"
264 Great Deals out of 13,649 listings starting at $2,795
Used Toyota Sequoia"
128 Great Deals out of 4,854 listings starting at $2,500
Used Toyota Tacoma"
528 Great Deals out of 25,749 listings starting at $2,499
Used Acura MDX"
303 Great Deals out of 14,916 listings starting at $2,500
Used Cars For Sale
2006 Mitsubishi Montero Limited 4WD For Sale
8 listings starting at $3,500
2005 Mitsubishi Montero Limited 4WD For Sale
11 listings starting at $4,995
2004 Mitsubishi Montero Limited 4WD For Sale
8 listings starting at $5,500
2003 Mitsubishi Montero Limited 4WD For Sale
19 listings starting at $4,950
2002 Mitsubishi Montero Limited 4WD For Sale
33 listings starting at $2,500


text:
'Get in early. Stay late, and don't complain': From battling jealous male co-hosts to having to wear a swimsuit on air, Barbara Walters reveals how she overcame rampant sexism
- While working on The Today Show in the 1970s, her male co-host refused to do joint interviews with her unless he could ask the first three questions
- Ms Walters advises women to 'just do your job' and 'fight the big fights'
Veteran broadcaster Barbara Walters has revealed that she battled rampant sexism early in her career by not getting angry and staying focused on her job.
The 83-year-old ABC News anchor and co-creator of The View has interviewed every president since Richard Nixon, but in the 1970s her male co-host - who was paid twice her salary - refused to do joint interviews with her unless he could ask the first three questions.
'I felt I had to just do my job. And I have said this to other women: "Just do your job. Get in early. Stay late, and don't complain. Fight the big fights",' she tells Bloomberg Businessweek.
Blazing a trail: Veteran broadcaster Barbara Walters has lifted the lid on how she battled sexism early in her career by keeping her head down and working hard
Ms Walters began her broadcasting career in 1962 doing women's issues for The Today Show, which she calls 'tea pouring interviews', and admits that 'it was a very different time.'
She once even had to wear a swimsuit on air after a model failed to appear.
'Who knew from sexism?' Ms Walters told the magazine.
'Now if somebody says "I like your sweater" you can go to human
resources, and the person could get fired.'
She had a great relationship with Hugh Downs, but when he left in 1971 his replacement Frank McGee was much less welcoming.
'He didn't want me to participate at all,' she reveals. 'The compromise was that after the third question, I could come in.'
Getting answers: Ms Walters says that her friendly manner when interviewing guests like Senator Hilary Clinton has helped her ask more probing questions
Range of guests: Throughout her career Ms Walters has interviewed everyone from Dr Jack Kevorkian (left, pictured in 1993) to Sesame Street's Elmo (right)
She adds: 'I worked very hard on that fourth question.'
But instead of
getting angry, Mr McGee's studio rules drove Ms Walters to go out into
the field and do her own interviews. This earned her, she says, a
reputation as a 'pushy cookie'.
McGee died in 1974 - which she says no one expected - she became the network's first female co-host.
A few years later she also became the first female to host the ABC Evening News.
Career highlights: Ms Walter's interview with Monica Lewinsky in 1999 was viewed by 74 million people and remains her highest-rated interview of all time
Battling sexism: Ms Walters (pictured with Harry Reasoner in 1976) has battled sexism during her a 50-year career by staying focused on the job
Whether it's a head of state or celebrity, Ms Walters says that the secret to doing a great interview is doing your homework.
'It used to really annoy me when someone would come to interview me and say, "I'm sorry. I've never seen you on the air",' she admits.
She also discovered that that low-key friendly approach worked to her advantage during interviews.
'If you say "What's the biggest misconception about you?", that's easier than saying "Tell me why you married your sister".' she explains.
'It was a very different time. Now if somebody says "I like your sweater" they could get fired'
But she says that some guests, like former president Richard Nixon, have still been painfully awkward.
'Nixon wanted you to like him. It was almost pathetic,' she says.
'He would tell off-color jokes to the camera crew. He was such an uncomfortable person that he didn't know how to do it.'
This isn't the first time that Ms Walters has expressed contrarian views on feminism.
She made headlines in 2012 for taking former Republican presidential candidate Rick Santorum's side after blamed 'radical feminism' for convincing women that working was the key to happiness.
Through it all, Ms Walters says that she is proud of the fact that she has affected the way that women are regarded.
'If I have done stories and interviews that have in the past been done by men, and I opened the door a little bit, and now it's taken for granted, that would be a legacy I could be proud of.'
Most watched News videos
- 'You Christians, you kill us': Nun reveals words of ISIS...
- Special delivery! Democrats procure flags for convention...
- 'Our Mom and Dad would have been immensely proud of you,...
- 'Merkel is RUINING our country!' Germans revolt over four...
- 'I may become the first woman president, but one of you is...
- 'My best friend Hillary': Bill Clinton relives a 'lifetime...
- Gang of Muslims storm nudist pool in Germany yelling 'Allahu...
- Mommy's little helper! Chelsea Clinton heads off to the DNC...
- Kate and Wills fly to France for holiday on £8million...
- 'When I saw them I thought that's it. It's over': Nun who...
- 'Russia, if you're listening, I hope you can find Hillary's...
- 'Hillary Clinton isn't afraid to say Black Lives Matter':...


text:
Crazy Substitute Teachers
Enjoy, NG4.03 / 5.00 2,605 Views
Vacuum Salesman
This dude and his Megazord vacuum cleaner4.02 / 5.00 2,879 Views
Markiplier Animated | Mark Of The Survey Corps
This took far too long to make...3.96 / 5.00 7,327 Views
very cute
And ver funny. It's always cool when cc movies capture the zeitgeist.
Not too bad, but I'd expect better
Not too bad, but I'd expect better form somone with an A+ batting average. Still, the fact it was about a minute long and had a Gorge-Bush Clock in it made it better. Hope to see you doing better work in the future.
this was more of a joke submission.


text:
Crazy Substitute Teachers
Enjoy, NG4.06 / 5.00 892 Views
Vacuum Salesman
This dude and his Megazord vacuum cleaner3.98 / 5.00 2,231 Views
Markiplier Animated | Mark Of The Survey Corps
This took far too long to make...3.95 / 5.00 2,363 Views
At The End...
I'd like to call that the "MIC-SPLOSION!" :P
Holy moly, these graphics were intense! The paint like special effects mixed with explosions, rainbows, and glowing things everywhere made this pure action eyecandy! It was the best stick fight I have ever seen. I have seen quite a few, hoping to see something a bit different from the others, and finally I found THIS.
Get ready to be on the top for a bit... this flash deserves it!
Whoa kay.
Well that was pretty amazing. Definitely going on my favorites.
Great job.
~ Jay
i saw a double rainbow all the way across the sky at the end.
this is your whole flash summed up in just one word.(or two?)
best thing EVAR
hehe thanks!
I'm ace ing
I've seen tons of stickmen animations before, but this is surely the best one.
Music is amazing as well.


text:
Chair in Integrated Sensor Technology
- Edinburgh, City of Edinburgh
The University of Edinburgh is one of the world's top 20 institutions of higher education.....
- Recruiter: The University of Edinburgh
Principal Electrical Engineer - Power
- Bristol, England / Cumbria, Barrow-In-Furness, England
- Negotiable
Principal Electrical Engineer - Power Join our Electrical Power team and help design the self-contained generation and distribution system for the Successor submarine - a new generation of submarine designed to carry the UK's independent nuclear deterrent
- Recruiter: BAE Systems
Operations Supervisor (Mechanical/Electrical/Instrumentation)
- England, Cambridgeshire
- £33000 - £39000 per annum
Operations Supervisor - (Mechanical/Electrical/Instrumentation) Salary: Circa £33k - 39k dependant on experience + vehicle and great additional benefits (share scheme, pension, potential bonus).Location: Wisbech - Cambridgeshire We currently have an excit
- Recruiter: National Grid
Lead NDT Trainer
- England, Lancashire
- Competitive package
Would you like to be involved with training UK and international teams in Non Destructive Inspection (NDI) to support the in service fleet (Typhoon Tornado, and Hawk)?
- Recruiter: BAE Systems
Systems Design - Emerging Portfolio
- Bristol
- Competitive Salary & Benefits
What?s the opportunity? There are fantastic opportunities in Systems Design for engineers to work within Future Systems. These are highly visible, fast paced roles, in...
- Recruiter: MBDA
Metering Engineer
- Teddington, United Kingdom
- £24,109 - £27,961 plus EO Electronics PE of £8,090.00
We are now looking for a Metering Engineer to deliver RD's In-Service Testing (IST) scheme for gas and electricity meters.
- Recruiter: Department for Business, Innovation and Skills
Head of Operational Estates
Premium job
- Shrewsbury, Shropshire
- £46,625 to £57,640 per annum
As an experienced Estates Manager, you will play a key role in helping to shape the future of the Estates service.
- Recruiter: The Shrewsbury and Telford Hospital NHS Trust
Assistant Professor (Tenure Track) of Smart Building Solutions
Premium job
- Zurich, Canton of Zürich (CH)
The successful candidate is expected to develop a strong and visible research programme in the area of control and diagnostics of building systems
- Recruiter: ETH Zurich
Process Controls Leader
Premium job
- Humber Refinery, South Killingholme, North Lincolnshire DN40 3DW
- £60k - 75k plus extensive Compensation and benefits package, dependent upon experience
Experienced Process Control Leader providing leadership and technical support for Oil Refinery. Extensive Compensation and benefits package.
- Recruiter: Phillips 66
Regional Technical Support Manager
Premium job
- Warwick, Warwickshire
You will be required to lead the regional Customer Services strategy and resources to maximise Customer satisfaction.
- Recruiter: Siemens
Apple co-founder outlines 'human' computer vision
Apple co-founder Steve Wozniak speaking at the European Business Network's annual conference
A human-like computer of the future could give every child a personal tutor, according to Apple co-founder Steve Wozniak.
The former teacher says he envisages a day when information technology allowed one-on-one teaching and said it was important young people "wrote the book" rather than followed tried and tested ideas.
The entrepreneur was speaking today at Londonderry's Millennium Forum for the European Business Network's annual conference.
"We are moving closer to where a computer is like a person and we can have normal conversations with it," he said. "We will be able to have one teacher per student and let students go on in their own direction."
He added: "A computer is an awful cheap teacher, it has to get more human in its characteristics; anything another human being can understand is what I want my phones to understand."
The entrepreneur predicted mobile devices would become more like real humans over the next couple of decades.
He said that process has been on-going for years, with people "lifting" information into virtual rubbish bins on desktops and using mouses designed to mimic the two-dimensional human experience. Dropdown menus are another example of applying human thinking to computers.
Wozniak said for many years foldable LED displays had been the goal and added he wanted to see a glowing globe to zoom into for Google Earth-type applications.
He claimed people were being more and more attracted by Google glasses and wearable technology like watches or devices worn on the head.
"I would love to get low-cost plasters to push buttons and change the colour of my car, wearable technology is something we are hearing so much about at the moment," he said.
He described his philosophy on work simply.
"Our meaning in life is to follow the urges that we are born with," he said, adding that sometimes students with the least money were the most likely to succeed because they were forced to innovate and were not constrained by the need to earn a wage or follow accepted practice.
He said he invented the floppy drive because he wanted to see Las Vegas and the company was having a meeting there and he needed to earn his inclusion on the delegation, also recalling his work on programming scientific calculators at Hewlett Packard, which he said was simply about making life simpler by thinking differently.
Wozniak said when he first teamed up with Steve Jobs he managed to get Intel chips from a salesman for free and added they founded their company with $300 each.
At one point, opening garage doors at the push of a button was the frontier of technology and computers could not handle chess games, but now they are becoming more like humans in their ability to handle complex problems, he said.
"We did not invent the internet to be a brain, we stumbled on it by accident," he added.
Latest Issue
"As the dust settles after the referendum result, we consider what happens next. We also look forward to an international summer of sport."
Most viewed
- HMS Ambush submarine crashes into ship, again
- Tesla's 'Master Plan' future for self-driving cars and solar power
- Chip and pin compromised by hackers 'within a year'
- Flight MH370 search to be suspended, relatives informed
- MH370 search team may have looked in the wrong location for two years
- Lithuania launches campaign to lure away UK's car-makers


text:
Workout Buff Christmas Ornaments
Workout ornaments make great gifts for personal trainers or anyone who could use a little encouragement for changing their lifestyle and adding some physical fitness to their regimen. He loves to work out and sculpt his body, hoping to create six-pack abs from his time spent at the gym. The weight lifting ornaments, runner ornaments, swimming ornaments and exercise equipment ornaments are great gifts for a guy who is ripped or give as a gift to celebrate that someone on your shopping list who has beaten the battle of the bulge and has successfully reached their weight loss goals.
- View All
- ‹ Previous
- Page
- 1
- 2
- Next ›
- Running Price: $13.95
- Triathlon Price: $13.95
- Weightlifter Male Price: $13.95
- Weightlifter Male holding a Barbell Price: $13.95
- View All
- ‹ Previous
- Page
- 1
- 2
- Next ›
Read what our customers say about our Personalized Christmas Ornaments and their shopping experience!Hover over testimonials to stop scrolling
12/7/2015 - "Very nice selection. Arlene S."
8/12/2015 - "I have purchased ornaments from this website for years. I keep coming back because you always have what I have in mind for the perfect gift! Debbie M."
6/30/2016 - "I thought they were beautiful ornaments and I can't wait for my daughter to open it at Christmas. Suzmarie S."
7/14/2016 - "So cute! I love how it is personalized and I got it so quickly. Arlene C."
11/8/2015 - "Received my ornament today, super fast service and the ornament is beautiful. Love it...Betty G."
6/30/2016 - "This is my second year ordering ornaments! I am very happy with the number of selections! Glenda C."
12/30/2015 - "These ornaments are wonderful including the personalization! Ann L."
8/31/2015 - "Hello. I Got my ornament back today with the changes I asked for. Absolutely love it!!! Thank you! You are one awesome company. Excellent ornaments!! Will buy again next year!! Thanks again so much!! Pam"
7/27/2016 - "Amazing choices - personalization additional date options were allowed on all items. Well Clarified in all process and coupon free shipment appreciated with discount. Thomas H."
8/30/2015 - "I am very pleased with OrnamentShop. About a year ago I purchased a "First Christmas" baby ornament, for a child in my family. I made a mistake on the year he was born. I confirmed it and everything. These people went above and beyond to find the order and correct it for me. It may have even been personalized by the time I realized my mistake. With no extra charge or complaints,it was fixed and shipped. I will always go to OrnamentShop first when I am looking for something unique and special. Beverly P."


text:
Follow us
From the Mary Celester to missing pilots, history is chock full of stories about unexplained disappearances. Join Matt and Ben as they take a look at some of history's top unexplained disappearances - and solve a few others.
UP NEXTMysterious Disappearances
UP NEXT4 Unexplained UFO Sightings
UP NEXTPolybius: The Facts and Fiction
UP NEXTWhat is Jade Helm 15?
UP NEXT3 Facts About Modern Slavery
UP NEXTWhat is a conspiracy theory?
UP NEXTWhat is apocrypha?
UP NEXTInfiltration: Corporations
UP NEXTThe Pineal Gland: Mysticism and Neuroscience
UP NEXTWhat's inside the catacombs?
UP NEXT4 Serial Killers on the Loose
UP NEXTWeather as a Weapon: Digging Deeper
UP NEXTExploiting Your Reptilian Brain for Profit
Stuff They Don't Want You to Know: Is public WiFi dangerous?
Public wifi can be an enormously convenient thing -- it helps people dodge data charges in coffee shops, airports and other places around the globe. But how safe is it? Tune in to learn more.
Stuff They Don't Want You to Know: Zombies: Part 1 - CLASSIC
Nowadays, zombies are big business. But outside of the horror films and video games, is there any truth to the zombie legends? Tune in to find out.
Stuff They Don't Want You to Know: What would happen if the world's debt disappeared?
It's often said that money makes the world go round – so what would happen if there was no money left? Would it be a paradise or pandemonium?
Stuff They Don't Want You to Know: Banks, Cartels and Money Laundering
How do criminals turn dirty cash into clean money? Who helps them? And more importantly, what happens when the launderers are caught?
Stuff They Don't Want You to Know: Drones, Part 1: Target Killing | CLASSIC
Unmanned Aerial Vehicles, or UAVs, have fundamentally changed the nature of warfare. But who controls them? What are they doing, and why?
Stuff They Don't Want You to Know: Religion and Crime: The Narco Saints
Criminals, from cartels to thieves and more, have adopted the practice of praying to entities like Santa Muerte for forgiveness, protection and aid. But why?
Stuff They Don't Want You to Know: What is the 'Red Market?'
You've heard of the Black Market, but what about the Red Market? Watch to learn about the secret world of the illegal human organ trade.
Stuff They Don't Want You to Know: 5 Things You Didn't Know About Satan
Where did the idea of Satan originate? What's his true name? What does he really look like?
Stuff They Don't Want You to Know: Real-life Super Powers
While it might sound like something out the X-Men, a few people really do have what could be described as genuine superpowers. Tune in to learn more about human echolocation, super-strength, eidetic memory and more.
Stuff They Don't Want You To Know: The Mafia: Fact and Fiction
For millions of people, the word 'mafia' conjures images of "The Godfather" and "The Sopranos". But what is 'the Mafia', exactly? Where does the term come from, and how has the definition changed over time?
Stuff They Don't Want You to Know: 4 Weird Things about the Washington Monument
The Washington monument is one of the strangest structures in DC. Join the guys as they explore the weird history of this iconic obelisk.
Stuff They Don't Want You to Know: Drones, Part 2: Domestic Use | CLASSIC
Unmanned aerial vehicles (UAVs) have changed the nature of war. But where are they flying, and what are they doing? The answers might surprise you.
Stuff They Don't Want You to Know: 5 Secret Space Programs
The path to the stars isn't all altruism and noble scientific advancement -- in fact, human activities in space have often been shrouded in secrecy, and the practice continues today.
Stuff They Don't Want You to Know: Berenstein or Berenstain? The Mandela Effect
How permanent is the past? Could facts, times, and specifics of events somehow change after the fact? If so, why? Learn more about the tricky nature of memory and allegations of the multiverse in today's episode.
Stuff They Don't Want You to Know: Targeted Online Advertising
Have you ever searched for something online, only to find an eerily related series of ads popping up at each website you visit afterward? If so, you've encountered the Brave New World of targeted online advertising.
Stuff They Don't Want You to Know: Chemtrails: Experimenting on the Public
You've probably heard of chemtrails theories before -- they're often derided in the mainstream. But has something like the chemtrail theory ever been proven? Have governments really poisoned unsuspecting civilians with airborne chemicals?
Stuff They Don't Want You to Know: Ghosthunters: The Warrens
For decades Ed and Lorraine Warren claimed to investigate paranormal phenomena, confronting the forces of darkness with the power of faith. They've inspired numerous films and become a part of American folklore -- so how much of their story is true?
Stuff They Don't Want You to Know: Who was the Original Night Stalker?
From 1979 to 1986, an unknown serial killer assaulted and murdered at least 10 people. He remains uncaught to this day. So who was he? Is it possible to find the Original Night Stalker?
Stuff They Don't Want You to Know: 5 Things You Should Know About Primary Elections
To outside observers, the US primary elections can seem pretty confusing. So how do political parties use this system to select candidates - and why do critics feel the process is so corrupt?


text: Ad Decapitator Stalks LondonHamilton Nolan6/05/08 12:45pmFiled to: things we actually likeDecapitatorGood IdeasTrendsAdvertisingArtPop Culture33EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalinkCall it what you will—street art, culture jamming, or protest. I'll call it some guy who's been going around London and graphically hacking the heads off of models in all types of ads. With fantastic attention to detail. Pictured: Carrie Bradshaw, improved. You know all the cool kid brands are just dying of impatience waiting for him to hack up one of their ads. Lovely. Two more pics of the mystery chopper's graphic, allegorical work, after the jump. Advertisement [Flickr via Environmental Graffiti]Reply33 repliesLeave a reply


text:
Used Lincoln Town Car for Sale in San Diego, CA
New Search
Filter Results
|- Loading...
|- Loading...
Dealer Promotions
Days on Market
|- Loading...
Accidents Reported
Text Search
Add Similar Cars
Choose Other Models to Add
Listings for these models will be added to the listings you are currently viewing.
CarGurus Quick Poll
|by kayeed44
"Great for what we use it, road trips and chauffering relatives."
Comfort and surprising MPG=22 combined. Mostly 55 mph highway driving. We use this car for traveling, chauffering relatives and going to parties/celebrations. No major mechanical problems, but did have heater blower motor (burn out), power seat (quit on trip to Florida) and hub caps (plastic retainers broke off) problems. Acceleration, brakin...
1989 Lincoln Town Car Base Used Cars in El Cajon, CA 92021
Price: $1,995
Mileage: 95,300
Location: El Cajon, CA 92021
2001 Lincoln Town Car Signature Used Cars in La Mesa, CA 91942
Price: $4,199
Mileage: 136,754
Location: La Mesa, CA 91942
2000 Lincoln Town Car Executive Used Cars in San Diego, CA 92111
Price: $4,999
Mileage: 94,493
Location: San Diego, CA 92111
2001 Lincoln Town Car Cartier Used Cars in Spring Valley, CA 91978
Price: $5,995
Mileage: 112,413
Location: Spring Valley, CA 91978
2002 Lincoln Town Car Executive Used Cars in San Diego, CA 92120
Price: $5,997
Mileage: 61,954
Location: San Diego, CA 92120
2005 Lincoln Town Car Executive L Used Cars in La Mesa, CA 91942
Price: $6,999
Mileage: 219,866
Location: La Mesa, CA 91942
2000 Lincoln Town Car Signature Used Cars in San Diego, CA 92108
Price: $7,999
Mileage: 27,123
Location: San Diego, CA 92108
2007 Lincoln Town Car Signature Used Cars in Spring Valley, 91977
Price: $3,150
Mileage: 220,000
Location: Spring Valley, 91977
Filter your search criteria!
A large number of listings matched your criteria. You are seeing the top 2,000 listings.
Used Lincoln Town Car By Year
|Starting Price
|Total Available
|2011 Lincoln Town Car
|3 Great Deals
|62 listings
|2010 Lincoln Town Car
|6 Great Deals
|51 listings
|2009 Lincoln Town Car
|6 Great Deals
|55 listings
|2008 Lincoln Town Car
|2 Great Deals
|77 listings
|2007 Lincoln Town Car
|7 Great Deals
|182 listings
|2006 Lincoln Town Car
|5 Great Deals
|215 listings
|2005 Lincoln Town Car
|11 Great Deals
|202 listings
|2004 Lincoln Town Car
|5 Great Deals
|222 listings
|2003 Lincoln Town Car
|6 Great Deals
|281 listings
|2002 Lincoln Town Car
|1 Great Deal
|132 listings
Similar Cars
Mercury Grand Marquis
70 Great Deals out of 2,549 listings starting at $1,599
Cadillac DTS
82 Great Deals out of 1,872 listings starting at $2,900
Cadillac DeVille
2,360 listings starting at $1,999
Used Lincoln Town Car By City
Lincoln Town Car in Bakersfield CA
4 listings starting at $5,399
Lincoln Town Car in Fresno CA
4 listings starting at $8,999
Lincoln Town Car in Indio CA
5 listings starting at $3,995
Lincoln Town Car in Lake Havasu City AZ
2 listings starting at $11,996
Lincoln Town Car in Las Vegas NV
6 listings starting at $5,999
Lincoln Town Car in Los Angeles CA
43 listings starting at $3,495
Lincoln Town Car in Phoenix AZ
15 listings starting at $4,950
Lincoln Town Car in Santa Barbara CA
10 listings starting at $3,495
Lincoln Town Car in Yuma AZ
1 listing