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Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.
Citrus greening, or Huanglongbing (HLB), is the most devastating disease of citrus. The disease is caused by the Gram-negative phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus, and Candidatus Liberibacter americanus. CLas is found in Southeast Asia, the Indian subcontinent, the Arabian Peninsula, the United States, Cuba, Mexico, West Indies, Honduras, and Brazil, and it is exclusively transmitted by the Asian citrus psyllid Diaphorina citri. Fruits from infected trees are green, misshapen, and bitter (Bove, 2006). Disease symptoms include blotchy mottled leaves (nonsymmetrical chlorosis or mottling), pale yellow leaves, yellow shoots, corky veins, stunting, and twig dieback. Roots are also affected, with dramatic decreases observed in the mass of fibrous roots in infected plants (Johnson et al., 2014). In leaves, another phenotype associated with HLB is the accumulation of callose inside the sieve plate pores of the infected plant's phloem. Accumulation of callose was demonstrated both by aniline blue staining and by immunogold labeling (Kim et al., 2009; Achor et al., 2010; Deng et al., 2019; Granato et al., 2019). This accumulation is an early response that begins at early stages of the disease and probably leads, at more advanced disease stages, to collapse of the cells. An associated third disease symptom is the accumulation of excessive amounts of starch in the leaves of infected plants (Kim et al., 2009; Achor et al., 2010; Granato et al., 2019). This phenotype is not observed in the roots (Kim et al., 2009; Etxeberria et al., 2009; Folimonova and Achor, 2010). It was shown that the accumulation of starch occurs only after the accumulation of callose in the sieve elements and after the collapse of phloem cells. Leaf chlorosis was related to the disruption of the cell inner grana structure and happened only in parts of the leaf where plugging of the phloem occurred (Achor et al., 2010). These phenotypes raised the hypothesis that the blotchy mottled leaf symptom, and the damage caused to the fruits, result from the plugging of the phloem cells, leading to decreased translocation of sugar and the accumulation of starch in the source tissues. Unplugging the phloem may therefore provide an attractive way to increase the productivity of affected plants. Association of callose with sieve areas and sieve plates in angiosperms has been widely studied (Behnke and Sjolund, 1990; Stone and Clarke, 1992). Induced phloem callose led to a decrease in the lateral movement of C14-assimilates and auxin, whereas treatments that stimulate breakdown of sieve plate callose led to increased movement of fluorescein through the sieve tubes (Webster and Currier, 1965; McNairn and Currier, 1968; Hollis and Tepper, 1971; McNairn, 1972; Aloni et al., 1991; Maeda et al., 2006). In citrus leaves, callose accumulation during CLas infection impaired symplastic dye movement into the vascular tissue and inhibited photoassimilate export in the infected leaves (Koh et al., 2012). On the other hand, a phloem-specific callose synthase (CALLOSE SYNTHASE7 [CalS7]) was recently identified, and its absence resulted in carbohydrate starvation (Barratt et al., 2011; Xie et al., 2011; Xie and Hong, 2011). These results suggest that the presence of basal levels of callose may actually be required for efficient carbohydrate transport in the phloem and pointed to a more complex relationship between sieve pore callose and phloem transport, where the balance is important and either too much or too little callose can have a negative effect.
An additional mechanism for phloem plugging that was also shown to occur in HLB-infected plants is the induction of phloem proteins (P-proteins). These gel-forming proteins were shown to undergo a rearrangement in the sieve elements after injury or irradiation (Knoblauch and van Bel, 1998; Knoblauch et al., 2001). These proteins are suggested to play a role in plugging of sieve plates to maintain turgor pressure within the sieve tube after injury and during pathogen and pest infection, but their exact role in these processes is still unclear (Knoblauch et al., 2014). In Cucurbita spp., two predominant P-proteins, the phloem filament protein or PHLOEM PROTEIN1 (PP1) and the phloem lectin or PP2, have been associated with the structural P-protein filaments. PP2 is a dimeric poly-GlcNAc-binding lectin that was shown to be covalently linked to P-protein filaments by disulfide bridges (Read and Northcote, 1983). In HLB-infected sweet orange plants, sieve elements were obstructed by filamentous protein material and it was shown that the plugging material contained PP2 by immunogold labeling (Achor et al., 2010). PP2 gene expression was also shown to be upregulated in leaves of HLB-infected sweet orange plants compared to healthy plants (Kim et al., 2009). Moreover, PP2 transcript levels were also upregulated in an HLB-susceptible citrus variety compared to that in a tolerant variety, suggesting that PP2 expression and phloem plugging may play a role in the onset of disease symptoms in susceptible varieties (Wang et al., 2016).
Whereas complete plugging of the phloem cells was clearly demonstrated, very little is currently known regarding sieve pore closure and the intra- and intercellular movement of CLas between sieve tubes. Here, we focused on the cellular processes that take place at the phloem pores. By examining the ultrastructure of the phloem pores and the movement of CLas in the infected sink citrus tissues of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi), we show that whereas sieve pores are plugged by callose in cells of young leaves, the callose is not induced in the fibrous roots and seed coats. In the unplugged sieve elements, we show that CLas can move between cells and that the bacterium's ability to change its morphology enables it to enter the pores. We also show that CLas can move into nucleated cells in the seed coat phloem tissue, whose identity is still unknown. Finally, we show that CLas is associated with the phloem pores via adhesion to the plasma membrane adjacent to them. This interaction may target CLas to the pores and play a role in the cell-to-cell movement of the bacteria.
In the Flush, Sieve Pore Size Is Reduced by Callose
It was previously shown that in HLB-susceptible sweet orange and grapefruit, CLas infection leads to dramatic phloem phenotypes such as the swelling of the middle lamella between cells surrounding sieve elements and the complete plugging of phloem sieve tubes by a P-protein–like material (Folimonova and Achor, 2010; Supplemental Fig. S1, A–C). In this study, our focus was to analyze the sieve pore characteristics in the cells that are still functional (not completely plugged or collapsed) and to determine whether CLas can move between these cells. Because CLas accumulates in the phloem, we hypothesized that CLas moves mainly with the phloem flow and, thus, will accumulate in higher numbers in the sink tissues. We, therefore, focused on three sink tissues: the young leaves (flush), the emerging fibrous roots, and the seed coats of developing seeds. We used transmission electron microscopy (TEM) to further analyze the phloem at the ultrastructural level in Madame Vinous sweet orange and Duncan grapefruit. We first examined phloem cells from the young developing leaves (flush). The TEM images clearly revealed the ultrastructure of the pores, including the presence of callose and filamentous protein inside the pores (Fig. 1A). In TEM sections of uninfected sieve pores, we observed a basal level of callose, which partially reduced their opening, but clearly left available space for movement between cells (Fig. 1B). The average opening of the pores in uninfected plants flush was 265 ± 19 nm. Such an opening is similar to the described average width of CLas (Hilf et al., 2013). The callose we observed may be, in part, a result of our tissue preparation procedure. In sections collected from HLB-infected flush tissues, results were dramatically different. In many sieve plates, callose accumulated to a much higher level and almost completely filled the space of the pores, leaving no available space for movement (Fig. 1, C–F). Callose levels in the cells were variable. In some cells, we could detect a combination of open and occluded pores (Fig. 1C), whereas in the other cells callose completely filled the cell and plugged the whole sieve area (Fig. 1D). On average, opening diameters were about half of what was found in uninfected plants, with no difference between asymptomatic and symptomatic plants: The average pore opening was 130 ± 10 nm for infected asymptomatic and 105 ± 9 nm for infected symptomatic cells (Fig. 1G). Similar results were obtained from grapefruit (275 ± 30nm for healthy and 174 ± 20 nm for infected plants). Remarkably, we saw very few CLas bacterial cells in the images generated from the flush (Fig. 1).
In Roots, Pores Are Plugged by an Alternative Mechanism
Next, we examined the sieve pores in young fibrous root tissues in Madame Vinous and Valencia sweet orange and in Duncan grapefruit. In TEM sections from uninfected roots, callose could be seen in the pores of the phloem (Fig. 2, A, B, E, and F). As was previously shown, thickening and collapse was observed in infected root sieve elements, similar to that in the flush (Aritua et al., 2013; Supplemental Fig. S1D). However, in the noncollapsed cells of HLB-infected root tissues, very little callose was seen. Instead, we consistently detected the accumulation of dark extracellular material along the phloem pores (Fig. 2, C, D, G, and H). This accumulated material differed in its consistency and color from the healthy tissues callose (Fig. 2), and was deposited extracellularly between the plasma membrane and the cell wall (Fig. 2, G and H), which is different from the filamentous P-protein we observed inside the pores of the flush (Fig. 1A) and from the material that was previously shown to bind with PP2 antibody (Achor et al., 2010). These deposits accumulated at the openings of the phloem pores and inside the pores (Fig. 2, H–J). The dark extracellular material was detected in grapefruit and sweet orange (both Valencia and Madame Vinous), but we never detected it in phloem from infected flush or from roots of healthy plants that were identically stained (Figs. 1 and 2). In addition, we could rule out the possibility that this dark material was a callose-staining artifact, because we could observe it at the pores in addition to the brighter and smoother thin callose collar (Fig. 2, I–K). Unlike the flush, in the roots we could find the CLas bacteria associated with the phloem, but the number of the bacterial cells was still relatively low (Fig. 2, G and H). In some cases, we could detect CLas attaching to the cell membrane, with the extracellular deposits accumulating adjacent to CLas attachment place (Fig. 2H).
To provide supporting evidence for the differential plugging dynamics observed between the shoots and the roots in the microscopy images, we also conducted gene expression analyses for the phloem-localized PP2 and CalS7 genes. Samples were collected from the flush, bark, mature-leaf midribs, and roots of both healthy and infected plants, and the gene expression levels in HLB-infected tissues were compared to those in the same tissues of healthy plants (Fig. 3A). As expected, the expression levels of both CalS7 and PP2 were upregulated in almost all the CLas-infected shoot tissues, the only exception being PP2 expression in midribs. CalS7 expression levels were significantly upregulated in HLB-infected bark and midribs, and for PP2, there was a significant upregulation in the bark tissues. In sharp contrast to the shoot tissues, the expression levels of both CalS7 and PP2 were significantly downregulated in HLB-infected roots compared to healthy roots. For PP2, there was a 5-fold downregulation in the roots, and for CalS7 there was a strong 100-fold downregulation in the roots of infected plants compared to the roots of healthy plants. To verify that CalS and PP2 genes were expressed in grapefruit vasculature, and to further explore if there are additional phloem-related grapefruit orthologs of these families, we isolated RNA from the lateral veins of healthy and infected blotchy mottled leaves and compared gene expression levels of the different gene family members. CalS3, CalS7, CalS8, CalS9, CalS11, and CalS12 were all expressed in the lateral leaf vein, but there was no expression of CalS2, CalS5, and CalS10 detected, indicating these three family members are not phloem-related in grapefruit (Fig. 3B). For PP2, we could not detect any expression for orange1.1g041394m, orange1.1g045187m, orange1.1g042480m, and the orange1.1g039003m PP2 homologous transcripts. The only primer pairs that detected gene expression in the veins were designed for orange1.1g024966m and orange1.1g024822m (Fig. 3B). These two sweet orange transcripts share 90% identity and may represent a single PP2 family member.
In Seed Coats, Open Pores Enable Widespread Cell-to-Cell Movement of CLas
TEM sections from the phloem tissue in the chalazal end of young seed coats (Hilf et al., 2013) showed a dramatically different situation than what was seen in both the plant roots and shoots. In Valencia sweet orange seed coats, cells were almost free of callose, and phloem pores remained open, with an average available space of 332 ± 14 nm for cell-to-cell movement (Fig. 4, A and B). Inside these cells, bacteria accumulated to very high numbers, sometimes filling all the available space inside the cells, and were clearly dividing and propagating (Fig. 4). Bacteria appeared as both round and elongated bacilliform-like shapes, characteristic of CLas (Hilf et al., 2013). Similar results were observed in grapefruit seed coats, where we detected only moderate levels of callose that was comparable to healthy seed coats, indicating that only normal callose levels are present in infected tissue without further accumulation (Supplemental Fig. S2). In the infected Duncan grapefruit seed coats, we could detect high levels of CLas bacterial cells as well, some of which were entering or exiting the sieve pores (Supplemental Fig. S2). Remarkably, in both sweet orange and grapefruit seed coat tissues, we could clearly detect CLas in living nucleated cells (nonsieve element cells; Fig. 4C; Supplemental Fig. S2). The nature of these nonsieve elements cells and the possible bacteria movement mechanisms into them are still unclear.
The open sieve pores enabled CLas bacteria to move between cells (Fig. 5, A–C). The size of these open pores (average 332 ± 14 nm) were shown to fit perfectly with the diameter of the elongated form of CLas (Fig. 5C). Remarkably, this elongated form usually reached the pores in the right orientation for movement (perpendicularly to the plasma membrane; Fig. 5, B and C), suggesting an unknown targeting mechanism may be involved. Once at the sieve pores, the elongated bacteria could move, cross the sieve pores, and enter the adjacent cell (Fig. 5C).
The round forms of CLas, reaching a diameter of up to 1 μm, were bigger than the diameter of the open pores and, therefore, could not move cell-to-cell while in this form. However, the bacterium had the ability to change its form (Fig. 5D). When a circular bacterium reached the proximity of the phloem pores, sometimes it changed from a circular to an elongated form (Fig. 5E). The elongated part of the bacterium was then able to enter the pores (Fig. 5F).
CLas Bacteria Adhere to Plasma Membrane at the Sieve Plate
The targeting and cell-to-cell movement described here should require some active aspect for CLas movement to target the pores at the right orientation and to enable the translocation. This could be either a bacterial or a host activity, or both. We were therefore looking for a possible cellular mechanism that would enable this. We could detect CLas adhesion to the plasma membrane of the plant cells next to the pores (Fig. 6). CLas binding to the host plasma membrane took place mainly around the phloem pores, and less so at other areas of the cell periphery (outside the sieve plate; Fig. 6A). In some cases, a filamentous-looking link that connected some bacteria with the plasma membrane was seen (Fig. 6, B and C). The identity of this structure and whether it is of bacterial or plant origin is unknown. In other cases, we could detect a clear anchor that connected the bacteria to the plasma membrane (Fig. 6, D–F). These adhesion sites were observed in all the sink tissues we examined (flush, roots, and seed coats) and may represent a general mechanism for phloem pore targeting in citrus.
The phloem, a major pathway for long distance systemic movement in plants, is involved in the trafficking of photoassimilates, small signaling molecules, and larger macromolecules such as proteins and nucleic acids. The phloem also provides the highway for virus systemic spread in the plant. However, little is yet known about systemic movement inside the phloem, and even less is known about the systemic movement of bacteria. In this work, we documented the passage of the Gram-negative phloem-limited CLas bacteria between phloem sieve-element cells and some of the responses that occur in the phloem pores after bacterial infection of the plants.
CLas infection is known to induce a reorganization of the citrus phloem. This reorganization includes the swelling of the middle lamina, callose deposition, P-protein accumulation, and phloem hyperplasia (Etxeberria et al., 2009; Achor et al., 2010; Folimonova and Achor, 2010; Deng et al., 2019). Here, we investigated different citrus sink tissues to gain further knowledge about the specific cellular reactions that are taking place in the sieve element. TEM images revealed that the responses varied dramatically between different tissues. We observed an increase in the callose levels in the flush, but not in roots or seed coats. Increased callose synthesis and deposition is a general response to plant pathogens (De Storme and Geelen, 2014; Ellinger and Voigt, 2014). It is hypothesized that its role is to isolate the damaged sieve elements and to confine the invading pathogen. However, too much callose may be a "double-edged sword," and its overproduction may be responsible for the mass-flow impairment and blockage of photoassimilates that were described for HLB. Koh et al. (2012) also showed that the phloem sieve pores' apertures were reduced by callose, and that photoassimilate export was delayed in CLas-infected plants. They suggested that the massive callose occlusion seen in sieve pores in previous publications might have been a tissue preparation artifact and did not represent the actual in vivo levels (Koh et al., 2012). Here we show that in the flush tissue there is a basal level of callose in healthy controls (that could result in part from tissue preparation), but also that the pore openings are significantly reduced by callose accumulation in the infected plants. When we examined other tissues (such as roots and seed coats) using the same fixation techniques, we found little or no callose at all. This indicates that wound-induced callose resulting from TEM techniques probably only represents a minor part of the plugs. Gene expression analysis of lateral veins from infected grapefruit leaves showed that CalS2, CalS5, and CalS10 were not expressed in the veins, indicating these are not responsible for phloem callose synthesis in grapefruit in response to CLas. CalS3, CalS7, CalS8, CalS9, CalS11, and CalS12 were expressed in the lateral veins. CalS3, CalS7, CalS8, and CalS12 are known to be involved in plasmodesmata/sieve pore callose formation and bacterial infection (Ellinger and Voigt, 2014; Cui and Lee, 2016). CalS9 and CalS11 are known to be mainly involved in callose biosynthesis during pollen development and cell division, but may play an unknown role in the phloem HLB response as well. In both this study and a previous study (Granato et al., 2019), CLas seems to cause a very moderate upregulation of many CalS genes rather than the strong upregulation of a single family member. This may indicate that callose synthesis is regulated at more than the gene-expression level. During abiotic stresses, stress-induced P-protein pore sealing is an almost instant response and callose synthesis rapidly occurs within minutes after abiotic stimulation (Furch et al., 2007; Zavaliev et al., 2011). This rapid response suggests a regulation mechanism at the protein level or the relocalization of P-proteins and CalS complexes which are already present in the sieve elements (Zavaliev et al., 2011). It is possible that an abiotic stress, in addition to the biotic one, is involved in the HLB-related occlusion. Overall, our results support a scenario in which disease symptoms in the flush result from "overproduction" of the occlusion mechanism.
Whereas callose plugging was the main response observed in the young leaves, in the root pores there was very little callose plugging. In the pores, we observed the appearance of an extracellular dark material between the plasma membrane and cell walls. In previous transcriptomic analyses that compared the shoots and roots, it was shown that there are dramatic differences in the symptoms and the transcriptional responses between citrus stems and roots to CLas infection (Aritua et al., 2013; Zhong et al., 2015). They also showed that CalS7 was downregulated in CLas-infected roots (Zhong et al., 2015). Here, we measured the response of CalS7 and PP2 genes to HLB infection in both the shoots and the roots. We found that CLas infection caused a dramatic 100-fold reduction in the expression of phloem-specific CalS7 in the roots, whereas in the shoot, CalS7 was upregulated in the presence of CLas. These results indicate there is a potential mechanism to avoid massive plugging of the root cells, or that a different unknown mechanism (that may be related to the dark deposits we observed) could be taking place. Johnson et al. (2014) showed that CLas accumulation caused severe dieback in the roots. Our results here may be related to cell death.
The seed coats were found to exist at the extreme opposite end of the phloem occlusion gradient. In these tissues, there was no clear sealing of the sieve pores, by either callose or P-protein accumulation. In some cells, sieve pores remained open with no callose at all. In others, callose was present, but only at levels similar to that in healthy controls, without further occlusion. Because there is no direct vascular connection between the seed coat and the developing embryo or endosperm, the area of phloem we studied in the seed coat is probably a trap with no outlet for CLas. As reported in Hilf et al. (2013), CLas multiplied and accumulated in these cells to high numbers, completely filling up the cells, supporting the assumption that sieve pores closure is a plant defense response that limits bacteria spread. Moreover, in this tissue, we could also detect CLas in nucleated cells, and their very high number suggest they are replicating in these cells as well. Phytoplasmas have been detected in phloem parenchyma cells close to the sieve elements (Siller et al., 1987). The identity of these CLas-containing nucleated cells (whether they are developing sieve elements, companion cells, or phloem parenchyma, all of which would have nuclei) and the mechanistic understanding of the bacterial movement into these cells are both still unknown.
Overall, in our study, phloem plugging seemed to restrict CLas levels: There was almost no bacterial accumulation in the tissues where a strong callose production and plugging were observed, whereas bacteria accumulated to high numbers in the areas with little callose plugging. This may reflect differences in plant responses to keep the balance between the need to defend against CLas and the need to maintain plant growth and reproduction. The plant may plug and sacrifice new flush to block the bacteria, but may be more careful with extensive plugging in the roots, which would reduce water and nutrient acquisition. The same could be suggested of the seed coats, because plugging the phloem there would put the developing seed embryo at risk. CLas is not seed-transmittable and does not seem to enter the endosperm and embryo (Tatineni et al., 2008; Hartung et al., 2010; Hilf et al., 2013). The high accumulation of CLas in the seed coats provided a unique opportunity to look at the cell-to-cell movement of CLas. In the nonplugged seed coat phloem, CLas seems to move rather easily between cells. The sieve plate pores that do not contain any callose fit perfectly with the width of the elongated form of the CLas cells. In this conformation, CLas appears to pass through the pores with little or no interference as long as it reaches the pores in the correct orientation (perpendicularly to the membrane). In our images, CLas reached the pores at this exact orientation most of the time, suggesting that the bacterium is using a mechanism for targeting the pores in this orientation. Unlike the elongated form, the circular form of CLas was too large to pass between cells even in the nonplugged seed coats sieve pores, but, as we show here, the bacterial morphological plasticity allows it to change to the narrow form to move.
We further show that CLas cells adhere to the plasma membrane exclusively at the sieve plate, adjacent to the sieve pores. Attachment sites were also described for phytoplasmas (Buxa et al., 2015; Musetti et al., 2016). Plasma membrane attachment next to the pores may provide the necessary targeting mechanism for CLas to reach the pores. Recent studies on plant viruses showed that attachment to plasma membrane proteins serves to target viruses to the plasmodesmata (Raffaele et al., 2009; Levy et al., 2015). It is possible that a similar targeting mechanism is taking place in the sieve pores. The membrane attachment may provide the necessary help to carry the bacteria to the pore in the right orientation, and may also provide a driving force, in addition to the phloem flow, to enable the CLas bacteria to squeeze and pass through the plugged sieve pores. Membrane surface proteins carrying an adhesion motif were described for Onion (Allium cepa) yellows phytoplasmas and Spiroplasma citri (Neriya et al., 2014), but there is no known adhesion protein for CLas that has an additional outer membrane which is not present in phytoplasmas and spiroplasmas.
Our results show that CLas triggers different mechanisms that affect the structure of the phloem sieve elements at different sink tissues of trees, and that these mechanisms can limit the accumulation of the bacterium. Our study supports the hypothesis that foliar symptoms result from plant responses rather than the degree of CLas accumulation or activity, because even in the symptomatic flush tissue we could hardly find any CLas cells. We also show that CLas adheres to the plasma membrane at the sieve plate pore, and this mechanism may guide the bacterial cells to pass through the pores, with the help of CLas' pleomorphic nature. Our results provide visualization of intercellular movement by Gram-negative bacteria, and indicate that important crosstalk is taking place between CLas and the plant at the cellular level, which results in changes to both the bacteria and the host plant.
Plant Material
Young Leaf Flush
HLB-infected Madam Vinous sweet orange (Citrus sinensis 'Osbeck') and Duncan grapefruit (Citrus paradisi 'Macompare' with 'Duncan') were generated as described in Folimonova and Achor (2010). In short, HLB inoculum was collected from symptomatic field trees located in a grove in Highlands County, Florida and was further propagated by grafting into Madam Vinous sweet orange. Each variety was graft-inoculated with three pieces of budwood from PCR-positive HLB source trees and propagated in the greenhouse. Two weeks after grafting, the plants were trimmed back to stimulate the development of new growth. Visual observation of symptoms along with PCR assays were performed.
Seed Coats
Developing immature seeds (half to two-thirds mature size) from field-infected Valencia sweet orange and Duncan grapefruit showing visible symptoms of infection (small, lopsided growth) were collected in early summer when the sweet orange fruits were 3.7 cm in diameter and grapefruit fruit were only ∼6 cm in diameter, from the Teaching Grove at the Citrus Research and Education Center (University of Florida). Healthy Duncan grapefruit seeds of the same age were collected from plants grown in citrus under protective screen. Phloem tissue was imaged from the chalazal end of these seeds, as described in Hilf et al. (2013).
Fibrous root tissue was taken from Duncan grapefruit and Madam Vinous sweet orangeseedlings, and from Valencia grown on Volk (Citrus volkamericana) rootstock. Plants were graft-inoculated as above and green-house–propagated 5–7 months before sample collection.
Electron Microscopy
Electron microscopy analysis was performed as described in Folimonova and Achor (2010), using a standard fixation procedure as follows. Samples were fixed with 3% (v/v) glutaraldehyde in 0.1 m of potassium phosphate buffer at pH 7.2 for 4 h at room temperature, washed in phosphate buffer, then postfixed in 2% osmium tetroxide (w/v) in the same buffer for 4 h at room temperature. The samples were further washed in the phosphate buffer, dehydrated in a 10% acetone (v/v) series (10 min per step), and infiltrated and embedded in Spurr's resin over 3 d. Sections (100-nm) were mounted on 200-mesh formvar-coated copper grids, stained with 2% aq uranyl acetate (w/v) and Reynolds lead citrate, and examined with a Morgagni 268 transmission electron microscope (FEI).
Reverse Transcription Quantitative PCR
To determine differences in CalS7 and PP2 genes in citrus (Citrus macrophylla) tissues, we utilized reverse transcription quantitative PCR (RT-qPCR) to quantify the relative abundance of CalS7 (GeneID: 102612996) and PP2 (GeneID: 102625021) transcripts in different tissues between noninfected and HLB-infected citrus plants (three plants each). Flush (young leaves), bark (internode between flush and first fully expanded leaves), midrib of mature leaves (from two to four fully expanded leaves from flush), and roots (young fibrous roots) tissues were excised from three plants each of HLB-infected and noninfected plants for RNA extractions. Total RNA was extracted using TRIzol (Life Technologies) followed by DNase I (Life Technologies) treatment and LiCl precipitation to purge DNA contaminants. The SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen; https://www.thermofisher.com/order/catalog/product/18080051#/18080051) was used to synthesize complementary DNA (cDNA) using 250 ng of total RNA extract per sample, alongside 0.2 μm of gene-specific primers (CalS7, TGGGCAGACGAAGATTTGGTA/GACATGAAGCCAAGGAATAGGA; PP2, CGGCATACGGATGGGAAGTAC/TCGCCAACAGGGATCTCTATC; ActB, GTTGCCATTGGTTGGTATTTGATAC/CGTCGACTGCCATTCCAGAT; GAPDH, TGGCGACCAAAGGCTACTC/TTGCCGCACCAGTTGATG reference genes; Harper et al., 2014) in 20-μL reactions. The TaqMan Universal PCR Master Mix (Applied Biosystems) was used for qPCRs as follows. Each reaction (10-μL volumes) was set up in triplicates consisting of: 2 μL of cDNA template, 0.4 μM of forward and reverse primers, and 50 nm of gene-specific 6-FAM/BHQ-1 labeled TaqMan probe (CalS7, 6-FAM-TCAGCTCATGTTCAGGATTCTCAAAGCA-BHQ1; PP2, 6-FAM-CAGTGAGCCTAAGACTCCTCTTACCA-BHQ1; ActB, 6-FAM-TGGTCGATGATTTGTCCGATTCACA-BHQ1; and GAPDH, 6-FAM-TGCTAGCCACCGTGACCTCAGG-BHQ1). Cycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min. Raw qPCR data were analyzed with the program LinRegPCR v2013.0 (http://linregpcr.nl) to correct for differences in reaction efficiency. The Ct values were averaged for each target, then used to calculate the relative quantification (RQ) values by the method described by Livak and Schmittgen (2001) against the geometric mean of the Ct values of GAPDH/ActB reference genes. RQ values were Log10-transformed before multiple Student's t tests (Holm–Sidak method, α = 0.05) to compare tissue means with the software GraphPad (https://www.graphpad.com/scientific-software/prism/).
To determine gene expression in lateral veins of grapefruit, leaf samples (three plants each) were collected from healthy and infected grapefruit trees. RNA was extracted from 100 mg of lateral leaf vein tissue with the RNeasy Plant Mini Kit (Qiagen). Using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), cDNA was prepared from the RNA. RT-qPCR was performed with the PowerUp SYBR Green Master Mix (Applied Biosystems). Each cDNA sample was diluted to a standardized concentration of 40 ng/μL. Separate qPCR reaction mixtures for each sample contained 160 ng of cDNA and primers for each gene at a concentration of 10 mM, with a total volume of 10 μL.
Thermocycling was performed using a model no. 7500 Fast Real-Time PCR system (Applied Biosystems) with the following settings: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 35 s and 60°C for 30 s. For CalS genes, we employed the primer sequences that were designed by Granato et al. (2019), based on the C. sinensis genome that contains homologs for the Arabidopsis (Arabidopsis thaliana) CalS2, CalS3, CalS5, CalS7, CalS8, CalS9, CalS10, CalS11, and CalS12 genes. For citrus PP2 gene expression, primers were designed to target citrus homologous regions of Arabidopsis PP2-B10 and PP2-B15 genes (Table 1). Gene expression was compared to citrus ActB reference gene, and analysis was performed using the 2−ΔΔCt method (Livak and Schmittgen, 2001).
Image and Statistical Analyses
Pores openings were measured using the software ImageJ (https://imagej.nih.gov/ij/). Statistical analysis was performed using the software JMP 14 (https://www.jmp.com/en_us/home.html). Means were compared using Tukey's honestly significant difference test and Student's t test.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: PP2s: XM_006489708 (orange1.1g024966m), XM_006489711 (orange1.1g024822m), XM_006420794 (orange1.1g041394m), XM_025092274 (orange1.1g045187m), XM_006420801 (orange1.1g042480m), and XM_006481644 (orange1.1g039003m). CalSs: XM_025101248 (orange1.1g001004m; CalS2), XM_006492601 (orange1.1g000171m; CalS3), XM_025101748 (orange1.1g045737m; CalS5), XM_006484824 (orange1.1g000389m; CalS7), XM_006477876 (orange1.1g000165m; CalS8), XM_006492604 (orange1.1g000179m; CalsS9), XM_025098709 (orange1.1g000180m, CalS10), XM_006467737 (orange1.1g000258m; CalS11), and XM_025092689 (orange1.1g000259m; CalS12).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. TEM images of completely plugged phloem cells from HLB-infected sweet orange flush and grapefruit roots.
Supplemental Figure S2. TEM images of grapefruit seed coats.
We thank Dr. Vladimir Orbovic, Dr. Arnold Scumann, and Laura Waldo (University of Florida) for providing us healthy Duncan Grapefruit seeds, and Dr. Robert Turgeon (Cornell University) for helpful suggestions during the preparation of this article.
A.L., S.Y.F., D.A., M.D., and S.G. designed the experiments; D.A., S.W., C.W., and S.B.-M. collected the data; A.L., S.W., and S.B.-M. analyzed the data; A.L. conceived the project and wrote the article with contributions of all the authors.
↵1 This work was supported by the University of Florida, Institute of Food and Agricultural Sciences Institute of Food and Agricultural Sciences Early Career Seed Grant (No. 00127818 to A.L.).
↵2 Present address: Department of Entomology, University of California, Davis, California 96616.
↵[OPEN] Articles can be viewed without a subscription.
- Received November 7, 2019.
- Accepted November 23, 2019.
- Published December 9, 2019.


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Despite playing inside one of the NBA's most acidic environments, the Washington Wizards' Otto Porter is silently having a breakout year. He's surged beyond competence, thriving within his role as the exclamation point at the end of a good chunk of sentences set in motion by John Wall and Bradley Beal.
In his fourth NBA season, Porter, a former third overall pick out of Georgetown, has made a much-anticipated leap. The 23-year-old (who doesn't turn 24 until June) is posting career-high per-36-minute numbers across the board, with the league's ninth-best effective field goal percentage and ninth-lowest turnover rate. He's shooting over 41 percent behind the three-point line and over 53 percent from the floor, with as many Win Shares as Draymond Green. There are 123 players who attempt at least three shots in the restricted area per game, and only five are more accurate than Porter.
Porter's game is a tender eye of round roast—it's all lean, direct, and efficient. According to Synergy Sports, only two players who've used at least 205 of their team's possessions are more efficient: Kevin Durant and Steph Curry. Real Plus-Minus currently ranks Porter as a top-20 player and the Washington Wizards get slaughtered when he's off the floor.
And yet, it hardly matters. Porter's rise can't overcome one of the worst benches in basketball. The Wizards are a depressing 7-13; they rank 21st in net rating, and are 3.5 games out of a playoff spot despite a fairly easy schedule and no major injuries besides the newly acquired 30-year-old defensive center Ian Mahinmi. FiveThirtyEight gives them a 28 percent chance to make the playoffs.
The fact that a rapidly improving, home-grown building block who plays one of the game's most important positions isn't inspiring much optimism for Washington's present or future is both strange and sad.
Porter is a free agent this summer, and thanks to a salary cap that's estimated to increase by another $10 million, he's about to make "franchise player" money (and probably more than Giannis Antetokounmpo). But while Porter is improving, he most certainly isn't a franchise player yet, and the Wizards already have clear-cut 1 and 1A scoring options in Wall and Beal. There's a real possibility that he stalls if he stays in Washington. Sure, he can bulk up a bit, improve on the defensive end, maybe become a better passer and work on his outside shot, but the way this Wizards roster was constructed limits how much more Porter's role—and his game—can expand. He currently has Washington's seventh-highest usage rate and literally all his threes have been assisted.
Porter is an energetic wing who lingers on the perimeter while Wall and Beal draw the defense's attention. (Roughly half his field goal attempts come after a pass from those two.) He regularly punishes his man for ball watching, and knocks down open outside shots. He has mastered when and where to cut.
He does an incredible job running the floor and knows how to finish on the move. Porter is also one of seven players 6-foot-8 or shorter to grab at least 13 percent of available rebounds when they're on the court. He keeps possessions alive and is pretty great at making something out of nothing.
But the play types we commonly associate with All-Stars may not be in his DNA. Porter doesn't isolate, post-up, embark on pick-and-rolls, or come off screens very often. He's starting to throw the occasional pocket pass, but rarely sets teammates up when given a ball screen. He lacks an explosive first step and wiggle as a ball-handler, and the game hasn't slowed down for him as a playmaker just yet.
There are subtle ways to change this, though. Washington's reserves are a wet bag of trash, and it might be worth Wizards coach Scott Brooks' time to extend Porter's leash while Beal and Wall rest.
According to NBAWowy, the 98 minutes Porter has played without that duo have yielded mixed results. His usage rate spikes by ten points and the team's offense functions at a high level. But Porter's individual scoring takes a dramatic blow, and it's hard for Brooks to rationalize reducing minutes for his starting five, a group that's absolutely crushing people.
Porter also doesn't get to enjoy life as a small-ball four, where he has thrived on offense, all that often. One of Washington's most successful lineups pits Porter at the four and Kelly Oubre Jr. at the three, but it has played just 15 minutes so far this season.
There's also a question of whether Porter can hold up defensively in that role, despite having an ideal body for switch-happy schemes. On paper, Porter is versatile. In reality, smaller guards treat him like wrapping paper. Porter is long, but not particularly quick moving laterally.
Some stars, like the San Antonio Spurs' Kawhi Leonard, have been able to evolve without switching teams because their growth happened to align with organic roster changes. Leonard, for example, was always far more impressive on the defensive end and his improvement with the ball was never a given, but it coincided with the decline of San Antonio's veteran icons. Porter's situation isn't like that at all, and his duties are unlikely to change so long as Beal and Wall are around.
It's a dilemma similar to the one Harrison Barnes and the Golden State Warriors faced last season, and, to a lesser extent, what happened a few years ago between the Oklahoma City Thunder and James Harden. Most scoffed at the Dallas Mavericks when they maxed out Barnes, and he has clearly exceeded expectations. Same with Harden.
Is Porter ready to take on more responsibility, or is his development and recent stellar play more reliant on Wall and Beal doing a majority of the leg work? More to the point, is Porter worth maximum money?
It's impossible to say for sure, but some team will likely offer it, whether it's a growing franchise with cap space that's thin on the wing (Philadelphia 76ers, Brooklyn Nets, New Orleans Pelicans, Denver Nuggets, Phoenix Suns), or—and this is highly unlikely—a team that has given up on finding a fully realized superstar and wants the next best thing: an All-Star in the making who has yet to become one (the Boston Celtics and Miami Heat).
Two front office execs in last 24 hours predicted to me he's getting a max. He's been really good. — Zach Lowe (@ZachLowe_NBA)December 6, 2016
Porter is restricted, which means Washington can match any offer presented by another team. If it's a max contract, the Wizards could get dangerously close to the luxury tax by keeping him on board; precisely zero owners are happy to step over that line with a team that isn't a lock to make the postseason. Even if the Wizards say "thanks but no thanks," they still won't have enough cap space to afford anybody who can come close to replacing Porter's production. If they let him walk, praying for improvement out of Oubre Jr. is the only option. As was the case with Beal last summer, Washington has all but assured itself of paying a market rate price or above for a player—a hallmark of general manager Ernie Grunfeld's 13-year tenure with the Wizards.
For now, few players are dominating in their roles like Washington's third-leading scorer. Whether that role lowers the ceiling on Porter's game—and whether it could be higher elsewhere in the league—is something teams will need to decide.
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From the classic fairytale, Goldilocks and the Three Bears, this nursery rhyme-inspired costume will have your Book Week or Fairytale Dress-Up worries all sorted. It's not too big, not too small, it's just right! But do be careful not to spill porridge down the front of it....
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Jobe Sofia Shorty Short 2mm Wetsuit Women
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You'll stay warm and protected from wind and rain in this value-priced soft shell. Welding details on the pockets and at the elbows give this jacket a sharp technical look. 100% polyester woven shell bonded to a water-resistant film insert and a 100% polyester microfleece lining 1000MM fabric waterproof rating 1000G/M2 fabric breathability rating Reverse coil zippers Full-length interior storm flap Zip-through cadet collar with chin guard Welded chest pocket Front zippered pockets Open cuffs Open hem with drawcord and toggles for adjustability
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Commit statistics for a0223952a6d7ca01cca465730bb150050967920d Sep 15 - Jan 10Excluding merge commits. Limited to 2,000 commits.
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text: Originating in France, baguettes are made from basic lean dough. It is recognizable by its length and crisp crust. The supposed history of baguettes is an interesting one. A law in the 1920's was put in place to keep bakers from working before 4 am, this didn't give them time to make proper rounded loaves. Making slender baguettes was a simple solution to this problem since they bake more rapidly.


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Theodore Roosevelt Moore, Jr., 89, of Bryan, Texas went to be with the Lord on the 24th of June 2020. He fought death as valiantly as he did defending this country through two foreign wars, finally succumbing to the exceptional wear and tear that he subjected himself in order to be the best husband, father, soldier, and Christian.
Ted only knew how to give 100 percent, never doing anything half-way. He would always give his time freely to help anyone in need, never thinking twice about his own sacrifices. He was preceded in death by his son Theodore Kurt Moore, and four sisters.
He is survived by his wife of 58 years, Mary Moore; and two sons Norbert Alexiou and Thomas Vaden Moore; and two daughters, Jamie Wright and Jennifer Moore; along with seven grandchildren and five great-grandchildren.
To send flowers to the family or plant a tree in memory of Theodore Roosevelt Moore, Jr., please visit our floral store.


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WALKERTON – Brockton council has filled the vacant chair around the horseshoe.
At a special council meeting on Tuesday morning at the Bruce County administration building, Tim Elphick was selected as the newest member of Brockton council.
Elphick was selected from seven candidates who applied for the position.
"It's certainly an honour and a privilege to be Brockton's next councillor," said Elphick after being sworn in on Tuesday morning. "I am certainly excited to get to work for the citizens of Brockton and look forward to advancing the interests of our community."
The newest member of council was selected after council heard from the seven candidates – Elphick, former Brockton mayor Charles Bagnato, Philip Englishman, John Fruin, Carl Kuhnke, Gregory McLean and Henry Simpson (Paul Crysler withdrew his name for consideration on Jan. 20) – and two rounds of voting.
Getting to those two rounds of voting, however, were not without some excitement.
At the beginning of the meeting, two members of council – Mayor Chris Peabody and Councillor Dean Leifso – declared a conflict of interest. For Peabody, the conflict was that one candidate (McLean) is his brother-in-law, and for Leifso, he cited professional reasons for why he could not participate in the selection process.
Both Peabody and Leifso observed the proceedings, with Peabody being able to return and participate if McLean was no longer in consideration.
The first candidate to speak was Englishman, who immediately asked that Deputy Mayor Dan Gieruszak declare a conflict of interest due to a dispute between the two in relation to issues at the Saugeen Municipal Airport.
After a brief recess to allow staff to review procedure, clerk Fiona Hamilton stated that if Gieruszak were to recuse himself from participating, there would not be a majority of council to vote and select a new councillor.
"That would not be sufficient for the appointment," she said. "Procedure dictates that any candidates with the fewest number of votes would be eliminated from voting and the next round of voting would begin."
She said that if Gieruszak were to recuse himself, a majority of council would not be able to properly appoint a new councillor until either of the candidates that Gieruszak and Peabody were in conflict of interest with were eliminated and one of those two members of council rejoin the proceedings.
Gieruszak, after hearing the information, explained that he would not recuse himself.
"The integrity commissioner has reviewed the situation at the airport and sent out an email yesterday (Jan. 20) indicating that there has been no transgression at the airport with respect to what (Englishman) thought there was there," he said.
The deputy mayor explained that there is a situation regarding access and trespass at the airport, which he sated is part of "regular business" and that no conflict of interest exists.
In response, Englishman thanked council for their decision to proceed and stated that his main reason for applying was to send a message to council.
"The main reason I applied for this position was to bring home the point to council, if I understand some of the issues, is that you decide to vote for your friends rather than for the issue. I won't go into particulars, but I hope if I get voted to council that I will vote for an issue, not (for what) the councillor next me wants if he is my friend," said Englishman.
He then stated that this has taken up more than his 10 minutes allotted to him and declined to continue speaking, stating that there were other candidates with more important things to say than he did.
Gieruszak stated that Englishman could have his entire 10 minutes and that the discussion and recess surrounding the conflict of issue would not be considered part of his time. Englishman, however, said he had taken the 10 minutes and that since council was under pressure to "get this done today, I'll just step back."
Following the exciting start to the meeting, the remaining candidates addressed council individually to explain why they are qualified to be the next member of council, as well as to answer any questions that council had of them.
After each candidate was finished, councillors Kym Hutcheon, James Lang, Steve Adams and Gieruszak completed the first round of voting.
The order of voting was determined by drawing a name from a hat, at which time the member of council marked their ballot and announced who they voted for.
In the first round, all but two candidates were eliminated from the ballot. Councillors Hutcheon, Lang and Adams voted for Elphick, and Gieruszak for Kuhnke.
With the elimination of five candidates (Bagnato, Englishman, McLean, Simpson and Fruin), Peabody was allowed to participate in the second round of voting.
In the second round, Hutcheon, Lang and Adams again voted for Elphick, with Peabody and Gieruszak voting for Kuhnke. With a 3-2 vote, Elphick was selected as the next councillor for Brockton.
Elphick, who works in professional labour relations and previously worked in the public service sector, stated in his address to council prior to voting that he has a strong passion for serving the public and is not afraid to stand up for his beliefs, citing his work in advocating for the Walkerton courthouse when the decision was made to move bail hearings to Owen Sound.
"(That was) a decision that would ultimately lead to another cut in the delivery of public services here in Brockton, impacting some of the most vulnerable citizens of our community; citizens whose voices often go unheard in an overbearing bureaucratic justice system," he said.
He added that he believes his youth and insight would be a welcome addition to council.
"I believe my youthful voice, insight and perspectives with having a young family will be a welcomed addition to our current council as we work to promote and retain families of all ages," said Elphick.
After being sworn in as the newest member of council, Elphick told the Herald-Times that he is looking forward to working for the citizens of Brockton, citing infrastructure and growth as priorities.
"Having a young family, (my priorities are) making sure that Brockton remains an attractive community of choice in the years ahead for both commerce and our residents, and ensuring that our infrastructure is appropriately managed, invested in, viable to continue us forward," he said.
Peabody thanked all of the candidates for applying, saying it was great to see such interest in the position.
"I would like to thank all of our candidates for applying. It's great to see so many people applying," he said. "It's certainly important to all of council that we did have interest and people who are willing serve the citizens of Brockton."
Elphick didn't get much time to celebrate following his appointment, as his first meeting as a member of council was on Tuesday night.


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During a recent interview with Ring Rust Radio to promote the TNA Bound For Glory pay-per-view event, Matt and Jeff Hardy discussed the TNA sale rumors, the locker-room and more. You can read some highlights and listen to audio of the interview below:
Matt on what to expect at Bound for Glory: "At The Great War, you are going to see the culmination of this whole situation that has been building up and manifesting with the demonic Decay. On that evening, we will have an opportunity to win the tag team titles of the world and that's an opportunity and odyssey I sent Brother Nero out on. On October 2nd, this match will start in the Zone of Impact. It literally can go anywhere. It can go into the Universal Studies, it could go into Walt Disney, it could go into Tallahassee, it could go to Texas, it can go to Canada or it could go to South America. I might jump onto Skarsgard the Dilapidated Boat and sail all the way to Cuba. Who knows? This fight could go anywhere. When it's all said and done you're going to have a combination of a great wrestling match and an extreme wrestling match. Also something very Final Deletion, Delete or Decay way."
Matt on how the TNA's creative freedom has benefitted them: "That is a very interesting question. One thing that is very nice about Impact Wrestling is that whenever I come in and my broken universe follows me into the building along with Brother Nero, Rebecca, King Maxell, Senior Benjamin, the Scribe or Vanguard 1, I pretty much get to call my shots. That is something that is very unique to Impact Wrestling. I don't think that you would see the success that is genuinely created by letting someone who knows what they are and what they're doing do their own thing, and I think it worked out perfectly."
Matt on why TNA is a desirable destination for free-agent wrestlers: "I think something that has been very attractive for Impact Wrestling and a reason to come here is you are allowed much more creative freedom—as we spoke earlier in this conversation—then you are anywhere else. On top of that, you can work on Impact Wrestling television schedule and still have the freedom to do other things that you wish to do. Whether you're into acting, comedy, you just want to spend more time at home with the family, you want to go do autograph signings, conventions, wrestle for other promotions or whatever that may be, you can do that still. That is a very attractive feature. When you would work for the Vince Meekmahan show, obviously they own you lock, stock and barrel. Even in the honorable Ring, they are trying to do all they can to lock guys into exclusive contracts where you just work for them or New Japan Pro Wrestling or Pro Wrestling Guerrilla. The freedom allowed at Impact Wrestling is very special and very attractive to all these personalities. Right now, obviously, Impact is in the midst of a metamorphous and there is light at the end of the tunnel. Billy Corgan has a very positive vision for Impact Wrestling. I have faith in him and if we can get everything shifted to Billy Corgan, I think Impact Wrestling will be a force to be reckoned with going forward."
Matt on how sale rumors have affected the locker room: "I think to most of the wrestlers that maybe don't have the broken brilliance or the insight that I do currently, it may be troubling or unsettling to some. I think a lot of people have faith in Billy Corgan. If Billy Corgan would have the company, I think people would be very positive about it. I feel like Dixie Carter and the regime that came before is part of the reason there was a stain on Impact Wrestling. So I feel like moving past that and to a new ownership or someone that has a new majority or have more of a say so would be looked upon from all angles a positive thing in many, many ways. Obviously looking at his background and history as a rock star with the Smashing Pumpkins, Billy Corgan is someone who certainly has a vision. He is so consumed with wrestling and has such a love for wrestling at his core that his vision along with being a wrestling fan and along with listening to the right people he can make a huge Impact."
Jeff on the sale rumors: "I don't give a damn about selling TNA. We're talking about people like Bobby Lashley and nobody can mess with him. They couldn't stand a chance of being with him and the only one that would is the charismatic one Jeff Hardy. Bobby Lashley is going to be in trouble and you might have a new world champion on your hands. As far as that goes, I don't even think about that or waste one minute of my life worrying about who's going to sell or buy TNA. I don't care really. I just do what I do the best I can and that's it."
Matt on if he's fulfilled working with TNA instead of WWE: "I am absolutely fulfilled working with Impact Wrestling. I am absolutely fulfilled if I never wrestle another day in my life. Right now, I'm in a position where I can give back and I can fight the Great War and not just literally, but figuratively. There are many things in the wrestling industry that needs to change and that is one of the things I am aiming to do day in and day out currently. The Great War doesn't just affect Impact Wrestling. It stretches to the Honorable Ring, the New Wrestling in Japan, the subterranean Lucha and the Vince Meekmahan show. We have to make sure this wonderful art form and wonderful magic known as pro wrestling continues to survive and thrive. It has to evolve in many, many ways because currently it isn't. Once my broken condition manifested, it has been a wonderful thing. Since the Final Deletion and Delete or Decay, every single week there's been more viewers coming back to Impact Wrestling and watching it for the first time and even the casual fans are tuning in. For the longest time, it was the diehard group of fans that would tune in each and every week. They were always there and usually negative in many ways as you guys know. You guys are on the Twitters and the Facebook and the social media platforms. Now the tide has turned some. That is something I'm looking to do, like incorporate things that can have the casual fans come back in and that is paramount for the business. We have to bring casual fans back to it. The casual fans have gone away ever since WCW was acquired by WWE, and that is something we must avoid at all costs. We won't have the awe and the magic of professional wrestling to continue to thrive."


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The result is a lot of stuff that appeals to the hunting instinct: Castile was already engaged in a race of exploration with Portugal to reach the Far East by sea when Columbus made his bold proposal to Isabella. The point is, perhaps regardless of whether this is easy to address or not, there may be a fundamental problem: One recent example worth highlighting here is Venezuela, where Hugo Chavez managed to reverse a coup against him.
Direct democracy may help prevent the perversion of democracy by those with power interests through the financing of parties and their various machines to garner votes. The managerial portion is surprisingly fun in its interlocking details. Although we are accustomed to hear about Muslim extremists pushing for relgious-based states in various Middle East countries, this example is one in a democracy where despite the principle of a separation of Church and State, Christian religious extremists push forward with their agenda, anyway.
Other natural resources include limited quantities of lignite, found in the southern part of the country, and phosphorite and gypsum, which are found throughout Moldova.
Democracies may create a more effective military It may seem ironic to many, considering that one principle underlying democracy is the desire for freedom, but democracies may create a more effective military.
Mammals include wild boar, wolves, badgers, wildcats, ermines, martins, and polecats. It had conquered Melilla inand further expansionism policy in North Africa was developed during the regency of Ferdinand the Catholic in Castile, stimulated by the Cardinal Cisneros.
You go up against the Hadean horde armed with your fists, six types of guns, and, our personal favorite, a chainsaw.
The Palestinian issue has been a symbol of the continuation of the Western monopoly on power Only a small percentage of Moldovan citizens identify themselves as Roma. Whether it is the mainstream media or the blogs, the analysis of the global security environment revolves around the mutual love-hate relationship between Western and Islamic countries.
Enemy Unknown UFO, also known as X-Com, is a turn-based, squad-level tactical combat game combined with a strategic management game. The main media outlet, RCTV, aggressively anti Chavez, was denied a renewal license innot because it was critical of Chavez policies, but because a pre-Chavez government law did not look too kindly on broadcasters encouraging coups after all, what government would.
The two became known as the Catholic Monarchswith their marriage a personal union that created a relationship between the Crown of Aragon and Castile, each with their own administrations, but ruled jointly by the two monarchs.
Perhaps… the greatest PC game ever made. There are fabulous views from the top, and once we have caught our breath the path continues gradually on steep tracks through forest, up to the top of the ridge.
Nicely rendered cut-scenes do a lot to further the plot, which is always nicely reflected by the gameplay. The Dniester uplands, their eastern slopes forming the high right bank of the Dniester River, border the central uplands on the east and northeast.
Managing your dungeon successfully takes great skill. Only a year later, Ferdinand became part of the Holy League against France, seeing a chance at taking both Milan — to which he held a dynastic claim — and Navarre. RCTV and their supporters tried to insist otherwise; that this was an issue of free speech.
The Spanish conquest of Oran was won with much bloodshed: Yet, unfortunately an Indian government report finds that its claims to religious integration and harmony are on far shakier grounds than previously believed.
The free press should act as a natural check against these issues in a functioning democracy, yet intertwined interests and agendas result in them often being mouthpieces of parties or just a press-release machine that unwittingly follows an agenda set by others resulting in limited analysis outside those boundaries.
Back at the village, we walk along the village road to our waiting transport. Instead, Darth Vader formally "knighted" Starkiller, officially recognizing him for the first time as his apprenticesomething that Starkiller always believed himself to be.
Levels are rather monotone and gloomy, and also rather cramped. Moldova receives highly variable amounts of precipitation—usually averaging about 20 inches mm annually, with totals a little lower in the south—but these figures conceal variations that may double the quantity in some years and result in prolonged dry spells in others.
In many parts of the country, especially in the rural areas, the necessities of life are procured by barter rather than by purchase. Huangyaguan is a small town situated in a beautiful valley in the heart of the Yanshan Mountains. After a fairly leisurely start (or driving from Beijing), we have our first experience walking on the Great Wall.
Tuesday evening, the U.S. Supreme Court granted a stay, halting implementation of the Environmental Protection Agency's Clean Power Plan pending the resolution of legal challenges to the program. Galen Marek (pronounced/ɡeɪlən ˈmæɹɛk/), codenamed Starkiller, was a Force-sensitive Human male who trained in the ways of the dark side of the Force as the secret apprentice of the Sith Lord Darth Vader.
The son of two Jedi Knights—Kento Marek and his wife, Mallie—Galen was born with an.
Imperial people share ideas, expertise and technology to find answers to the big scientific questions and tackle global challenges. The Japan scam: If you think it's hard getting to Japan, wait till you see how hard it is to leave.
The Great War was to temporarily increase British imperial control over Egypt. Almost immediately, the press was muzzled, nationalist meetings were prevented from occurring or broken up and the new Legislative Council was suspended.The challenges of imperial rule


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- Thread starter
- #1
I was wondering if anyone here has ever bred all of the breedable Pokemon as shiny pokemon without using an action reply device or game shark or any third party program and without RNG. As well as breeding them all with IV 6 perfects 31 stats. If not how close have you come if you have tried an under taking of this? Currently there are 665 non legendary breedable Pokemon, if you including Phione there are 666.


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NU Online News Service, March 4, 2003, 2:45 p.m. EDT – Aetna Inc., Hartford, has picked Connecture Inc., Atlanta, to provide automated sales and service support for small employer groups that buy Aetna health insurance.
Connecture and its systems will offer price quotes, generate proposals, underwrite prospective customers, enroll employees in plans and process renewals.
To handle the job, Connecture will have to integrate data from more than 20 different Aetna computer systems, Connecture says.
Connecture plans to start implementing the contract by using the Web to automate the renewal process for Aetna's small employer groups.


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French people are rude?
I want to visit France this summer, but I have heard of some friends that French people are considered to be very rude. Especially when you don't speak any French at all (like in my case). Should i be worried to travel in France now? cause this is the first time that i am travelling alone.... Anyone bad (or good) experiences with french people?
tnx,28 Mai 2007, 05:58 Nathalie
French People Suck
The French are incredibly arrogant, rude, deceitful and cheap. I had the misfortune of being involved with a French woman for a year, and she was the most difficult person I have ever had to deal with. She was mean and downright horrible. I have met her friends, and they were no different. They think they know everything and will argue their point to the death even if they have no clue what they are talking about. Also, they think all Americans are wasteful slobs.Americana 20 Apr 2009, 11:51 - Verstoß melden
A poor little frenchman lost in a big turmoil concerning his little homeland...
Hi everyone !
As a dirty foreigner from Paris, I make (obviously) the effort to be understood by the others. Sorry for the bad level of English.
It's amazing to see how a little part of earth called France generate so much frenzy from american, British, Italian people and so much more... We French, are we so important that a lot of blogs or forum have to be made so that everyone can tell everyone what they think about 60 millions French inhabitants stretched between Spain, Italy, Switzerland, Germany, Luxemburg, Belgium, and Great-Britain ( Since 2003, "french bashing is a national sport, doesnt it)?.
Arrogant, rude... Always the same cliché ... You will have to give your signification in a sharpest way for these words because I think it doesn't have the same meaning on the two parts of the Atlantic Ocean or on the two parts of the British Channel.
Have you ever read the comments in a well read English tabloïds the week before France & England meet in a Rugby match (A kind of American Football with no protections where you have to score touch-downs, but you can't give the ball in front of you, to make it short )? It smells like they wanted the Hundred's Year War (1337-1453)to erupt again! They cry for hate against the F...in' froggies, You will never see the same in a French newspaper about English, American or anyone else because we will complain about this kind of uncivilized behaviour!
Does everyone love everyone in America ? If a foreigner have troubles with his rude american neighbour, would he be right to claim that all American people are rude ? It will be stupid of course!
Here in France, we make more and more efforts to accept that others are different. Yes we are not perfect, and we know it ! Yes, we know we have to accept that once powerful, we are not anymore the 1st or even 2nd greatest land in the world and we dont talk about the past "Grandeur de la France" with tears in the eyes ! Napoleon 1st is more a tyran than a heroe to us ! And we try to point out and contest some basic anti-americanism whenever it spreads through networks, because it's unbearable even in a land with freedom of speech.
And last but not least : ALL FRENCH KNOW (EVEN YOUNG GENERATION) WHAT THEY GET FROM THE SACRIFICE OF YOUNG AMERICAN SOLDIERS (WITH BRITISH,CANADIANS,POLISH AND EVEN FRENCH COMMANDOS)1944 JUNE THE 6TH ! COME IN NORMANDY AND IF YOUR FATHER, OR GRANDFATHER DIED ON OMAHA BEACH OR UTAH BEACH, BELIEVE ME, YOU WILL UNDERSTAND THAT IT STILL REALLY MEANS SOMETHING FOR US ...
At the opposit, no French will tell you about La Fayette and the "French help" to the Insurgents.
This succesful "help" doesn't give the French any right on Americans...Honestly, do you really believe that this helping hand from King Louis XVIII was for free in his mind ? Before helping the Insurgents, the most important was to break the growing power of the British and wipe the English out of its American colony. It's not as romantic as we would like it, but it's truth...
There are more reasons that could explain why relations are not easy between Americans & French.
It's like a couple : love & hate !
If you don't like French, I know there is nothing I can say that will change your mind...
Anyway : Long life to America.
RodolpheRodolphe 05 Mai 2009, 06:34 - Verstoß melden
Bye bye France
I got kick out. Now I got back to US but I lost evererthing changed. Peopele are angry and not friendly anymore.Ivan Abell aka big pain in the ass Cowboy 11 Mai 2009, 10:48 - Verstoß melden
Retards of Europe
Statistically speaking......the most retards are borne out of France. Maybe not the type you see in wheel chairs, not those kind of retards, but the ones who can't think logically. I don't need to understand the characteristics of the French, they over anaylze their food, and their laws, when in the in end, it's crap! Plain and simple. They struggle so hard to define themselves, when if something is so great, you simply can;t define it. BOUDIN!!!MODERN WORLD 23 Mai 2009, 03:02 - Verstoß melden
Another idiot....you are a wussy!AMERICAN 23 Mai 2009, 03:04 - Verstoß melden
lolhehe 23 Mai 2009, 04:41 - Verstoß melden
Some reasons
I'm a french guy from Bretagne, France.
I think that we haven't the same meaning of the rudeness. As sayed before, French people aren't ruder than any other people around the world. By the way, saying that french are rude is a stereotype as those we use for american (idiots, obeses) or italian (liers, thiefs).
Nevertheless, some facts which explain this stereotypes :
- French People, especially the olders, don't speak well english. They might don't speak with english speakers.
- A number of workers, such as bus drivers, waiters, policemen, are quite rudes, especially in big city (Paris), even with french.
- It's true that we give importance to such words : Bonjour (Hello), S'il vous plait (Please), Merci (Thank), Au revoir(Good Bye).
_ Lot's of French people are racists. It's very sad. Very Shameful. But it's true.
Hope that'll help.gwen 26 Mai 2009, 02:54 - Verstoß melden
The pathetically rude French
My heart goes out to you people because yes, its true, the French tend to be an exquisitely rude and aggressive people.I am a student living in France for the year and am looking foward with avid anticipation to leaving it.I have visited many other countries including America and have never encountered any problems like I have in France. Their level of rudeness and arrogance is shocking. I am a polite person and am constantly overwhelmed by the level of rudeness and general ill- will on the street, in shops, in everyday life. Also, the level of unwanted attention from men is absolutely repulsive. I dress in a classy way pearls etc. and just because I am seen as attractive I get men shouting comments out of car windows in a very aggressive manner. I have never experienced this in my life prior to coming to France. By the way, I never way skirts or dresses and I dress in a very conservative style. I am saddened because I had thought France was all about culture and elegance and I loved studying the French language. Now, I question my career with the French language as I cannot bear to associate myself with a language belonging to such an arrogant, aggressive people. It does'nt take Einstein to get a glimpse into the French mentality: Look at Nicolas Sarkozy who has illustrated himself as an aggressive, imfamously tempermental statesman across world media.Maire 26 Mai 2009, 07:53 - Verstoß melden
this strang county makes me sad!
I am not European but as a university scholar lived in England (3 years) and Germany (4 years) and now I am living in Paris, for last year. I can talk Farsi, Urdu, Arabic, English, German all flunet and I know French for everyday life.
France is rather strange country and I think the average french knows extermly little about even Europ! Average french person cannot talk any other languages and they tent to correct your french without considering they put pressure on non-french native speaker! One might notice a huge ignorance of people in the main cities and great deal of conservative way of communicating with other people.
Average French might seems rude as they don't show any degree of tolerance to other people. One might wildly observe dual rules for french and non-french although it is against the law. It is hard for forginer to get solve the legal problems and you would feel very huge discrimination.
In not big cities people are more down to the earth and try to understand you a lot better!
In general, I certainly dislike to live in France and looking forward to go away from it.farzad 27 Mai 2009, 03:35 - Verstoß melden
Not sure, but there must be a reason as to why so many people think of us as rude etc. I have had many opportunities to meet french people outside France, and can add a few more adjectives to the above. Cheap is one of them, they just don't like to part with their money and think services are just owed to them no matter what. They do not mix with the locals even after many years in a foreign country. Must be coming from the colonies mentality. As far as pompous intelectuals, make no mistake they are still there and very active. Unfortunately.artus 11 Jun 2009, 11:53 - Verstoß melden
and the oscar goes to...
Ivan. Seriously, what an imagination. I got lost a couple of time in the plot, the several origins of his father, the royal blood, the police, the mayor, etc. Did the movie come out yet?artus 11 Jun 2009, 12:22 - Verstoß melden
Hola isabel
Yo tambien soy de Venezuela, si vienes solo por una temporada chevere, sino, preparate psicologicamente porque son super complicados etc ...
puedes escribirme al correo
[email removed] y hablamos. Suerte!anonimo 20 Jun 2009, 02:24 - Verstoß melden
Why THE French ?
There are 65 millions of French, why should be the same and then all be rude ? Some are rude, especially in Paris (I belong to them)! People of Paris have the same kind of life than in any other 10 millions inhabitant cities in the world, like in NYC or in London, they must sometimes be rude.
PS : Anyway, people who say or thing "THE" French must be considered as idiots and treated rudely !
PS2 : Charlotte shows herself an example of French idiot, please be rude with her !Nicolas from Paris 22 Jul 2009, 02:37 - Verstoß melden
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