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- American Society of Plant Biologists
Arbuscular mycorrhizas (AM) are widespread, ancient endosymbiotic associations that contribute significantly to soil nutrient uptake in plants. We have previously shown that initial fungal penetration of the host root is mediated via a specialized cytoplasmic assembly called the prepenetration apparatus (PPA), which directs AM hyphae through the epidermis (Genre et al., 2005). In vivo confocal microscopy studies performed on Medicago truncatula and Daucus carota, host plants with different patterns of AM colonization, now reveal that subsequent intracellular growth across the root outer cortex is also PPA dependent. On the other hand, inner root cortical colonization leading to arbuscule development involves more varied and complex PPA-related mechanisms. In particular, a striking alignment of polarized PPAs can be observed in adjacent inner cortical cells of D. carota, correlating with the intracellular root colonization strategy of this plant. Ultrastructural analysis of these PPA-containing cells reveals intense membrane trafficking coupled with nuclear enlargement and remodeling, typical features of arbusculated cells. Taken together, these findings imply that prepenetration responses are both conserved and modulated throughout the AM symbiosis as a function of the different stages of fungal accommodation and the host-specific pattern of root colonization. We propose a model for intracellular AM fungal accommodation integrating peri-arbuscular interface formation and the regulation of functional arbuscule development.
Arbuscular mycorrhizal (AM) fungi are the most widespread mutualistic symbionts of terrestrial plants and are considered to be essential for optimal plant growth, especially in nutrient-poor soils. AM fungi enhance the capacity of the root to scavenge water and mineral nutrients from the rhizosphere, while receiving in return photosynthates that are essential for the completion of their life cycle (Harrison, 2005). Nutrient exchange takes place within inner root tissues via intracellular branched fungal structures called arbuscules (Bonfante, 1984). The so-called accommodation of the microsymbiont involves invagination of the host plasma membrane around the fungus, thus creating an apoplastic space bordered by a specialized symbiotic interface (Bonfante, 2001; Parniske, 2004; Harrison, 2005). The intracellular accommodation of AM fungi represents an important paradigm for plant biotrophic interactions. Although the ultrastructural features of mature arbusculated cells have been relatively well documented, the cellular and molecular mechanisms responsible for the codifferentiation of the symbiotic partners within the root cortex are still poorly understood.
To reach the inner cortex, the AM fungus first needs to cross epidermal and outer cortical cell layers. Recent in vivo confocal imaging studies making use of fluorescent cellular markers have revealed that AM fungal penetration across epidermal cells of the model legume Medicago truncatula is immediately preceded by the transient formation of a novel intracellular assembly comprising cytoskeletal and endoplasmic reticulum (ER) components (Genre et al., 2005). This assembly, which we have named the prepenetration apparatus (PPA), includes a broad cytoplasmic bridge linking the site of fungal adhesion on the outer cell surface to the cell nucleus and defines the future intracellular path of hyphal penetration. This process also involves complex nuclear dynamics, including initial repositioning of the nucleus at the site of fungal adhesion and subsequent transcellular migration leading to the assembly of the PPA (Genre et al., 2005). We have hypothesized that the PPA is directly involved in the construction of the membrane/matrix interface surrounding the AM infection hypha. Together, these observations highlight the major role of the host plant during epidermal penetration by the fungal partner.
This discovery raises several important questions. First, is the PPA response observed in M. truncatula roots specific to legumes? This particular plant family possesses the unique capacity to form endosymbiotic relationships with both AM fungi and nitrogen-fixing rhizobia, and in both cases the microbe enters the root through an intracellular apoplastic compartment (Parniske, 2004). The characterization of mutants that block epidermal infection by both AM fungi and nitrogen-fixing rhizobia argues that, at least for this plant family, there is an active regulation of initial root entry for these symbiotic microbes (Parniske, 2004). Secondly, whereas AM hyphae simply traverse the epidermis to reach the root cortex, it is within inner cortical cells that the extensive fungal colonization of the root takes place, leading to the complex codifferentiation that generates the highly convoluted interface surrounding the ramified fungal branches of the functional arbuscule (Bonfante and Perotto, 1995; Gianinazzi-Pearson, 1996). It is therefore essential to determine whether prepenetration mechanisms similar to those described in epidermal cells also operate during the next stages of AM cortical colonization and peri-arbuscular interface formation (Smith et al., 2006).
Finally, it is now well established that cortical colonization patterns for AM fungi depend on the identity of the symbiotic partners, particularly the plant host. At one end of the so-called Arum-Paris continuum (Dickson, 2004), root cortical colonization occurs essentially via intercellular hyphal development, which is followed by intracellular infection and the formation of arbuscules within individual cortical cells. This type of colonization is typical of the M. truncatula/Gigaspora spp association. By contrast, Paris-type colonization involves the direct passage of intracellular hyphae from one inner cortical cell to the next, with the progressive formation of coils and intercalary arbuscules within adjacent cells. This pattern characterizes the interaction between Daucus carota and Gigaspora spp. Since the origin and biological significance of these differences in AM fungal colonization patterns are subjects of considerable debate, it is important to establish whether the underlying molecular and cellular mechanisms are related.
To address these questions, we have performed a detailed investigation of host cellular responses throughout AM fungal colonization for both M. truncatula and D. carota using an in vivo confocal microscopy approach similar to that described in Genre et al. (2005). This strategy has provided important information on the cellular dynamics that precede and accompany the two distinct modes of fungal colonization in both outer and inner root cortical tissues. In particular, we have been able to show that the formation of PPA-like intracellular assemblies not only precedes epidermal and outer cortical cell infection in both hosts, but that similar basic mechanisms also operate during arbuscule formation in the inner cortex. In addition, this in vivo cellular approach has allowed us to identify key stages during arbuscule development for both plant species and to reveal fundamental differences between colonization mechanisms in terms of cell-to-cell signaling within the inner cortex. Finally, advanced transmission electron microscopy (TEM) analysis of PPA-like structures formed during cortical colonization has provided strong evidence supporting the central role of this cytoplasmic assembly in symbiotic interface synthesis and has also revealed a striking remodeling of the pre-infection host cell nucleus. Based on these findings, we propose a model for intracellular fungal development and peri-arbuscular interface assembly that includes the regulation of functional arbuscule differentiation by the host cell.
Earlier studies of the host cellular dynamics associated with AM infection through the root epidermis used green fluorescent protein (GFP) cytoskeletal and ER cellular markers expressed in transformed roots of M. truncatula (Genre et al., 2005). To acquire confocal images deeper in the root and to follow subsequent cortical cell infection and colonization by the AM fungus in both M. truncatula and D. carota, we used the highly fluorescent ER marker GFP-HDEL (Haseloff et al., 1997). This marker labels the ER throughout the cytoplasm as well as at the nuclear periphery and hence allows precise nuclear localization within the cell. This is important since PPA formation is characterized by specific nuclear dynamics associated with strong ER labeling of the nuclear-linked cytoplasmic bridge that directs subsequent intracellular fungal infection. To further facilitate the identification of potential PPAs within the M. truncatula root, we used transgenic roots expressing GFP-HDEL driven by the Mt ENOD11 promoter (Genre et al., 2005). This promoter is rapidly and specifically induced during all stages of AM colonization (Chabaud et al., 2002), and its activation precedes PPA formation (Genre et al., 2005). Thus, PMtENOD11:GFP-HDEL serves as an excellent reporter for facilitating the identification of the earliest stages of cell activation preceding fungal infection within M. truncatula root tissues.
Colonization of the Root Outer Cortex Is PPA Dependent in M. truncatula
Different stages of outer cortical root infection for M. truncatula are illustrated in the in vivo confocal images presented in Figures 1A to 1F , where the GFP-labeled ER is visualized in green and the endogenous fluorescence of the Gigaspora hyphae in yellow/red. These images reveal that prepenetration responses in the outer cortical cells of M. truncatula closely resemble those previously described for epidermal cells (Genre et al., 2005). In Figures 1A and 1B, the adhesion of a fungal hyphopodium (also referred to as appressorium; Genre and Bonfante, 2007) on the root surface triggered the formation of a PPA, visualized as a broad ER bridge traversing the epidermal cell, and linking the hyphopodium contact point to the cell nucleus positioned at the opposite side of the cell. Note that the nucleus of the underlying outer cortical cell has migrated to a position opposite the epidermal cell nucleus and the terminal end of the PPA. Once the fungus entered and fully traversed the epidermal cell (Figures 1C and 1D), a second PPA was seen crossing the outer cortical cell and linking the hyphal tip to the nucleus that was then positioned at the opposite (inner) side of the outer cortical cell. Finally, as for epidermal infection, the outer cortical PPA disassembled once the fungus had traversed the cell, leaving a thin layer of residual ER around the intracellular hypha (Figures 1E and 1F). The image in Figure 1F also shows that hyphal ramification can occur in the outer cortex. Taken together, these results reveal that the cellular dynamics characteristic of epidermal infection involving nuclear migration and associated PPA formation and disassembly are conserved during subsequent fungal penetration of the outer cortical cell layer.
AM hyphae very occasionally penetrated the M. truncatula root by growing down through spaces between adjacent epidermal cells. In such cases, hyphae were observed in direct contact with the underlying outer cortical cells, subsequently leading to PPA-dependent intracellular infection (see Supplemental Figure 1A online). In no case did we observe intercellular infection through both the epidermis and outer cortex, suggesting that at least one step of intracellular infection is required for successful colonization of the inner root cortex.
PPA-Related Dynamics Are Associated with Arbuscule Development in M. truncatula
Once hyphae reach the M. truncatula inner root cortex, there is a switch to intercellular hyphal growth through the apoplastic spaces of the cortical parenchyma before hyphae penetrate individual cells to initiate arbuscule development. Although confocal images are of lower resolution at this depth, the intensity of the GFP-HDEL ER marker proved sufficient for following intracellular dynamics throughout colonization and arbuscule differentiation (Figures 1G to 1J). Our results reveal a localized accumulation of ER within inner cortical cells contacted by intercellular hyphae (Figure 1G; see Supplemental Figures 2A and 2B online). This ER accumulation is indicative of cytoplasmic aggregation on the side of the cell contacted by the hypha and was always associated with nuclear repositioning as previously observed in epidermal cells following initial hyphopodium contact (Genre et al., 2005). However, in the case of the inner cortex, such repositioned nuclei were significantly enlarged in size compared with normal cortical cell nuclei (Figure 1G; see Supplemental Figure 2B online).
When intracellular infection occurred in the M. truncatula inner cortex, the enlarged nucleus had moved to a central position within the cell (Figure 1H) and there was a broad PPA-like cytoplasmic bridge enveloping the hypha and linking it to the nucleus. Initial hyphal growth into the inner cortical cell took place through this PPA-like bridge. However, in contrast with epidermal and outer cortical cell infection, once the initial phase of arbuscule development was completed with the formation of broad intracellular hyphae, localized aggregations of ER-rich cytoplasm organized at distinct sites along these so-called trunk hyphae (Figure 1I). Our observations suggest that these cytoplasmic aggregations predict the emergence of lateral hyphal branches. Finally, once arbuscule differentiation was complete, only a thin layer of ER-fluorescing cytoplasm remained around the initial hyphal trunk (Figure 1J). On the other hand, a dense three-dimensional meshwork of ER cisternae was present between the fine secondary branches of the arbuscule. Significantly, this pattern of ER distribution closely mirrors the organization of the peri-arbuscular actin cytoskeleton observed by Genre and Bonfante (1998) using immunofluorescence labeling.
In summary, these in vivo confocal studies have revealed four distinct stages of root inner cortical AM colonization for M. truncatula. Initially, intercellular hyphae trigger nuclear enlargement and repositioning, associated with cytoplasmic aggregation, in all contacted cells. Secondly, intracellular hyphal infection is preceded by nuclear movement from the fungal entry site toward the cell center and the assembly of a cytoplasmic bridge resembling the epidermal/outer cortical PPA. After hyphal infection along this PPA, the subsequent development of the arbuscular branches is preceded by the formation of prebranching cytoplasmic aggregations along the intracellular hypha. Finally, a layer of ER-rich cytoplasm surrounds the arbuscule branches. We therefore conclude that PPA-related intracellular accommodation mechanisms are associated with all the key stages of M. truncatula root colonization by AM fungi.
Long-Distance PPA Formation Prefigures Intracellular AM Colonization of D. carota
To investigate whether similar cellular mechanisms operate during AM infection of other host plants, we focused on the nonlegume D. carota. As stated earlier, the colonization pattern for carrot is radically different than that for M. truncatula. This is illustrated in Figure 2 , where the intercellular hyphal growth responsible for AM inner root colonization in M. truncatula is replaced by an exclusively intracellular cell-to-cell colonization pattern in carrot roots. Using the identical GFP-HDEL ER marker and a similar Agrobacterium rhizogenes–based root transformation approach, we initially studied Gigaspora infection across the outer cell layers of the D. carota root.
The image in Figure 3A shows that transient cytoplasmic assemblies resembling the M. truncatula root PPAs were also formed in carrot epidermal cells directly contacted by AM hyphopodia. As in M. truncatula, initial nuclear repositioning at the hyphal contact site is followed by nuclear migration across the cell and the progressive formation of the PPA cytoplasmic bridge that predicts the intracellular path subsequently taken by the penetration hypha. Similar pre-infection responses in the carrot outer cortex are shown in Figure 3B, where a transcellular PPA is positioned between the cell nucleus and the infecting hypha that had just penetrated across the overlying epidermal cell. Such observations lead us to conclude that pre-infection responses involving transient PPA formation in outer root tissues of D. carota are very similar to those observed for M. truncatula. Interestingly, AM hyphal penetration through spaces between root epidermal cells appeared to be more frequent for carrot compared with M. truncatula. However, as for M. truncatula, this always led to intracellular infection of the exposed outer cortical cell (see Supplemental Figure 1B online). Therefore, for both plant species, access of AM hyphae to inner root tissues always involves the intracellular crossing of at least one outer root cell layer.
Once infection hyphae reached the inner cortex, major differences in the cell prepenetration responses between M. truncatula and D. carota were revealed by these confocal microscopy studies. The images in Figures 3C and 3D illustrate the highly polarized intracellular organization observed within a linear file of inner cortical cells lying ahead of an advancing AM hypha. This polarization was characterized by an enlarged centrally located nucleus, flanked on one side by a broad, highly fluorescent cytoplasmic bridge directed toward the approaching fungal hypha. The nucleus was bordered on the other side by a narrow bridge linked to the opposite face of the cell and aligned with the cytoplasmic bridge in the adjacent cell (see Supplemental Figures 2C and 2D online). The cells furthest from the fungus had smaller aggregations, and the nucleus was often less enlarged and positioned close to the wall facing the fungus. This suggests that cell activation at a distance from the fungus is progressive and involves nuclear migration toward the center of the cell with the associated formation of broad and narrow cytoplasmic bridges. When the colonization site shown in Figures 3C and 3D was observed 16 h later, fungal infection had progressed along the entire row of adjacent activated cells, generating a series of hyphal coils (Figure 3E). The intracellular remodeling that predicted the fungal trajectory in these linear inner cortical cell files is again reminiscent of the PPA as defined by specific nuclear dynamics and the formation of a cytoplasmic bridge that determines the fungal intracellular pathway. However, PPA-dependent intracellular infection in the carrot inner cortex was not identical to that observed in M. truncatula due to the fact that in D. carota, both the PPA and the hypha fully traverse the cell.
Finally, as observed during arbuscule development in M. truncatula, dense ER aggregations also assembled at distinct sites along the intracellular hyphae within infected carrot inner cortical cells (Figure 3F; see Supplemental Figures 2E and 2F online), and these localized cytoplasmic aggregations were often associated with incipient lateral branches. In mature arbusculated coils, a network of ER surrounded both hyphal branches (Figure 3G) and the centrally located host cell nucleus (see Supplemental Figure 2F online). In conclusion, despite major differences in the strategies of inner root colonization, the basic host cellular mechanisms associated with AM infection appear to be conserved between M. truncatula and D. carota. For both colonization modes, PPA-related cellular responses precede and accompany inner cortical infection and arbuscule differentiation. However, whereas inner cortical PPA formation in M. truncatula depends on direct fungal contact, progressive intracellular colonization in D. carota is characterized by long-distance coordinated PPA development.
Ultrastructural Evidence for Intense Membrane Trafficking in PPA-Like Structures
For more detailed ultrastructural analysis, we focused on the PPAs formed during inner cortical infection of carrot roots because of the relative ease of initially identifying these linearly aligned cytoplasmic bridges using confocal microscopy. Thus, carrot root segments containing inner cortical PPAs were visualized using the fluorescent ER label and then prepared for TEM using a flat-embedding procedure. This approach is illustrated in Supplemental Figure 3 online, where aligned PPA-containing cells first observed in the confocal microscope (see Supplemental Figure 3A online) were then identified in embedded 0.5-μm-thick resin sections (see Supplemental Figure 3B online) prior to the cutting of 70-nm ultrathin sections for TEM observation (see Supplemental Figure 3C online).
TEM images revealed the cytoplasmic composition of the PPA (Figure 4A ; see Supplemental Figure 3C online). As expected from the strong GFP-HDEL fluorescent signal, large quantities of ER membranes were present within the cytoplasmic aggregation, particularly in the vicinity of the tonoplast and the nucleus (Figure 4B). In addition, Golgi stacks with electron-dense cisternae and either small (30 to 50 nm) dark-staining vesicles (Figures 4C and 4E) or larger (60 to 70 nm) light-staining vesicles (Figures 4D and 4F) were particularly abundant compared with control cortical cells (see Supplemental Figure 4 online). Furthermore, budding Golgi vesicles, often associated with microtubules (Figure 4C), fused into extensive tubular-vesicular trans-Golgi networks (TGNs; Figures 4E and 4F). The ubiquitous presence of such TGNs within the PPA argues that Golgi-derived membranes are one of the main components involved in perifungal membrane assembly. Multivesicular bodies (MVBs), most likely involved in membrane recycling (Tse et al., 2004; An et al., 2006), were also abundant, often in the vicinity of the tonoplast. These MVBs were either small with electron-dense vesicles (Figure 4G) or larger structures containing electron-transparent vesicles (Figure 4H). Similar MVBs were also present in colonized cells ahead of the developing hypha (Figure 4A). In conclusion, the simultaneous presence of all the main organelles associated with membrane trafficking in the PPA is a clear indicator of major endo- and exocytotic activities within inner cortical cells preparing for fungal penetration.
Additional striking features of the inner cortical PPA included polymorphic mitochondria, often elongated and dividing (Figure 4I), as well as elongated and chromoplast-like plastids without starch granules and rich in electron-dense membranes and globules (Figures 4A and 4J). Such plastids, most likely involved in apocarotenoid synthesis (Schliemann et al., 2006), have been described in arbusculated cells (Scannerini and Bonfante, 1977; Fester et al., 2007). Finally, bundles of microtubules were clearly visible (Figure 4C), mostly oriented along the axis of the cytoplasmic bridge as previously observed for the M. truncatula epidermal PPA using the microtubule-specific fluorescent marker GFP:Map4-MBD (Genre et al., 2005). Together, these ultrastructural data on organelle remodeling highlight the dramatic change in metabolic activity of the host cell associated with the formation of the PPA and in particular the development of all the cellular machinery required for intense membrane trafficking.
Inner Cortical Cells Preparing for AM Infection Are Characterized by an Increase in Nuclear Size and Nuclear Remodeling
As indicated earlier in both M. truncatula and D. carota, there is a marked increase in nuclear dimensions associated with the inner cortical cells exhibiting prepenetration responses. We used confocal microscopy imaging to make a statistical study of the changes in nuclear size during AM root colonization of M. truncatula. The data presented in Figures 5A and 5B show that when the inner cortex is colonized only by intercellular hyphae (see Supplemental Figures 2A and 2B online) there is an additional population of large nuclei with an average diameter of 10 to 11 μm, compared with the 6- to 7-μm diameter typical of inner and outer cortical cell nuclei in uncolonized roots. This additional population of large nuclei corresponds to responding cells in direct contact with the intercellular hyphae (see Supplemental Figure 2B online).
In addition to a change in dimension, fluorescent propidium iodide staining of nuclei from sectioned root segments of both plants indicated that there is also a general remodeling of chromatin architecture. Figures 5C to 5F show that decondensed chromatin and large nucleoli were typical characteristics of the larger-size nuclei of PPA-containing inner cortical cells, in contrast with the denser nuclear content and small nucleoli in control inner cortical cells. Furthermore, TEM analysis showed that the dense heterochromatic regions present at the periphery of control nuclei (Figure 5G) disappear in the enlarged nuclei (Figure 5H; see Supplemental Figure 5A online) and that the nucleolus becomes highly organized with visible granular regions at the periphery (Figure 5H). A very similar nuclear morphology was also present in AM-infected inner cortical cells (see Supplemental Figure 5B online), in line with previous observations (Balestrini et al., 1992). In conclusion, the PPAs that developed in inner cortical cells of both M. truncatula and D. carota during AM colonization and prior to fungal penetration were associated with enlarged and remodeled nuclei, indicating intense transcriptional activity.
Comparative in vivo confocal microscopy studies performed on a legume (M. truncatula) and a nonlegume species (D. carota) displaying different patterns of AM root colonization (schematically outlined in Figure 6 ) have revealed that there is a common cellular mechanism underlying all phases of intracellular fungal accommodation. Prior to penetration, the host cell undergoes a major reorganization characterized by complex nuclear dynamics leading to the formation of a polarized cytoplasmic bridge termed the PPA, which defines the future hyphal path. Our results have shown that nuclear movement and PPA organization are modulated as a function of the root cell type and the nature of the subsequent intracellular fungal development. The initial stages of AM root colonization requires that the symbiotic fungus traverses both the epidermal and outer cortical tissues to reach the root inner cortex where the arbuscular symbiotic association is established during the subsequent growth and development of the fungus within and along the root (Figures 2 and 6). In this article, we have shown that prepenetration responses in the outer cortex of M. truncatula and D. carota are very similar to those described for the epidermis and are characterized by the formation of transcellular cytoplasmic bridges oriented toward the inner root tissues (Figures 1A, 1D, 3A, and 3B). Our studies further suggest that access to the inner root cortex requires at least one intracellular infection step, even in those cases where AM hyphae have bypassed the epidermal cell layer. Since intracellular accommodation via a PPA-dependent mechanism allows strict regulation by the host, it is therefore likely that the epidermal and outer cortical cell layers represent essential checkpoints for initial fungal entry (Novero et al., 2002; Demchenko et al., 2004).
When AM hyphae reach the root inner cortex, there is a fundamental change in the pattern of root colonization since in both plants the main direction of fungal growth has switched from radial to longitudinal. In the case of M. truncatula, inner cortical growth is essentially intercellular, whereas colonization of the carrot inner cortex remains intracellular (Figures 2 and 6). Nevertheless, despite these differences, intracellular infection leading to arbuscule development in both plant species was always preceded by cellular reorganization and the formation of cytoplasmic bridges that predict hyphal entry. The PPA assembled in the M. truncatula inner cortical cell results from direct contact with intercellular hyphae (Figures 1G and 1H; see Supplemental Figures 2A and 2B online), whereas prepenetration responses in the carrot inner cortex are characterized by aligned, polarized PPAs that anticipate the path of fungal growth along and within files of adjacent cells (Figures 3C and 3D; see Supplemental Figures 2C and 2D online). Thus, PPA formation and structure in the inner cortex clearly reflected the different AM colonization strategies adopted in the two species, which are examples of alternative colonization patterns from within the Arum-Paris continuum (Dickson, 2004).
The remarkable series of linear polarized PPAs formed in the D. carota inner cortex can extend over at least four to five adjacent cells ahead of the advancing fungus (Figure 3C). The alignment of cytoplasmic bridges at cell junctions (Figure 3D; see Supplemental Figure 2D online) argues in favor of sequential cell-to-cell signaling. Discovering the nature of this long-distance signaling mechanism that coordinates the cellular differentiation of PPAs and could involve both fungal and plant signals will be a major challenge for future studies.
Finally, it is very tempting to draw an analogy between these longitudinally aligned PPAs observed in the D. carota inner cortex and the radially aligned cytoplasmic bridges called pre-infection threads, which are formed across the width of the legume cortex prior to rhizobial infection of the root hair (van Brussel et al., 1992). Despite the different orientations within the root, in both cases the aligned bridges predict the intracellular apoplastic path taken by the respective symbiotic microorganisms. However, it remains to be determined whether this structural analogy is reflected by similar molecular and cellular mechanisms.
The Role of Nuclear Dynamics and Remodeling before Intracellular Accommodation of AM fungi
In vivo confocal studies have revealed that the host cell nucleus is a central player throughout AM root colonization, during both PPA formation and arbuscule differentiation. In the case of epidermal cell infection, we have previously shown that the earliest visible response to fungal adhesion is the repositioning of the nucleus directly opposite the contact site (Genre et al., 2005). Interestingly, the nuclear repositioning that precedes PPA formation in the M. truncatula outer cortex shown in Figure 1B clearly did not require direct physical contact with the AM fungus, since the hypha had not yet traversed the overlying epidermal cell. Furthermore, since the nucleus was positioned directly opposite the epidermal nucleus/PPA, it is likely that this early pre-infection response also resulted from direct cell-to-cell signaling between adjacent cells and suggests that the intracellular crossing of outer root tissues is a progressive cell-by-cell process. This also would be consistent with the proposed checkpoint role for the outer root tissues discussed earlier. Finally, it should be noted that nuclear repositioning is a well-known feature of several plant cell responses to both biotic and abiotic stimuli (Genre and Bonfante, 2007).
Our confocal studies have shown that when AM hyphae reach the M. truncatula inner cortex, intercellular hyphal progression along the root axis is systematically accompanied by nuclear repositioning opposite the site of fungal contact (Figure 1G; see Supplemental Figure 2A online). Furthermore, this migration of the nucleus toward the fungal contact site is accompanied by nuclear enlargement (see Supplemental Figure 2B online) and localized cytoplasmic aggregation. It should be noted that significant nuclear enlargement was not observed during PPA formation in the outer root tissues (cf. Figures 1B and 1F). On the other hand, an increase in nuclear dimensions is a well-documented feature of arbusculated cells (Balestrini et al., 1992); indeed, increases in nuclear ploidy have been reported in mycorrhizal roots, which could explain nuclear enlargement (Fusconi et al., 2005). Although we have no information on cell ploidy from our studies, our ultrastructural data show that the increase in nuclear size is associated with chromatin decondensation and the appearance of large, highly organized nucleoli, both suggesting intense transcriptional activity. Since only a proportion of the inner cortical cells contacted by the intercellular hypha will subsequently be infected and differentiate arbuscules, we consider that this particular cell activation status is an indicator of competence for fungal accommodation and arbuscule formation.
Enlarged nuclei with structural remodeling are also characteristic of PPA-containing inner cortical cells in carrot roots prior to fungal infection (Figures 4A and 5H; see Supplemental Figure 5A online), and these nuclei are similar to those of arbusculated cells (see Supplemental Figure 5B online). Thus, data from two host plants with different patterns of AM colonization clearly indicate that the preparation of inner cortical cells for arbuscule development involves major nuclear remodeling prior to fungal entry. This certainly is consistent with the intensive membrane and protein synthesis that necessarily accompanies PPA formation and subsequent arbuscule differentiation (see below).
A Cellular Model for the Coordinated Development of the Peri-Arbuscular Interface and Functional Arbuscule
An acknowledged hallmark of AM accommodation in host inner cortical root cells is the de novo membrane synthesis related to the formation of the extensive perifungal interface. The structures of the various organelles that could be visualized in the TEM images of inner cortical PPAs are clear indicators of significant exo- and endocytotic activities (Figure 4), presumably related to the preparation for fungal accommodation. Interestingly, plasma membrane synthesis involving a vesicular-tubular stage is also associated with plant cell division and in particular the development of the cell plate during cytokinesis (Otegui et al., 2001; Bednarek and Falbel, 2002). It is therefore conceivable that perifungal interface development involves a similar mechanism, with Golgi vesicles and TGN tubules generating the membranous components that are later assembled through membrane fusion to create the apoplastic tunnel ahead of the advancing hyphal tip. If so, then the adaptation of essential cellular processes, such as cell division (Van Damme et al., 2007), could be responsible for the ancient evolutionary development of AM fungal accommodation dating back at least 400 million years. In addition to the major proliferation of the cell endomembranous systems, TEM analysis of PPAs in the carrot inner cortex also revealed extensive plastid modifications as well as mitochondrial enlargement and proliferation. The ability to use in vivo fluorescent markers, such as GFP-HDEL, to identify responding host cells in the root cortex will be invaluable for studying this intense metabolic activation in more detail using single-cell approaches such as laser microdissection (Balestrini et al., 2007).
Based on the results presented in this article, we propose a two-step model for the concerted development of the peri-arbuscular interface and the functional arbuscule. In this model, the PPA response that we have observed in inner cortical cells anticipates fungal entry and constructs the interface compartment around the broad trunk hyphae that initially develop in the colonized cell. Observations in both M. truncatula and D. carota roots revealed that the ER-rich cytoplasm surrounding these large hyphae was subsequently reassembled at localized sites in the form of small cytoplasmic aggregations (Figures 1I and 3F). Since these aggregations anticipated sites of lateral branch initiation, it is likely that they play a role in extending the periarbuscular interface, analogous to the functioning of the PPA. Interestingly, Javot et al. (2007) have shown that arbuscule development is prematurely aborted at the initial stage of trunk formation in AM-colonized M. truncatula plants defective in the host phosphate transporter gene Mt PT4. The authors deduce that functional arbuscule development, which involves additional hyphal branching, is dependent upon the capacity of the microsymbiont to supply phosphate to the host. Since our data suggest that the lateral branching of trunk hyphae depends upon the formation of the prebranching cytoplasmic aggregations, we propose that this critical step leading to major interface expansion could be the target for host regulation of functional arbuscule development. Furthermore, these prebranching aggregations might also be responsible for targeting host phosphate transporters to the perifungal membrane, since Mt PT4 is expressed only in arbusculated cells and localized at the peri-arbuscular interface. In this context, it is interesting to note that Drissner et al. (2007) have identified a potential membrane-associated lysophosphatidylcholine signal that can activate host phosphate transporter genes in potato roots.
The PPA therefore appears to be a highly versatile mechanism that can be modulated by the plant according to the specific needs of each cell. In epidermal and outer cortical cells, the transcellular radially oriented PPA is involved in the assembly of the interface compartment that surrounds the intracellular hypha. During longitudinal colonization of the inner cortex, a similar mechanism operates, but with the additional requirement of assembling the extensive periarbuscular interface around the complex three-dimensional branched structure of the arbuscule. Future research will establish whether this generalized host cellular mechanism that has evolved to accommodate AM fungi can be extended to other important plant–microbe biotrophic interactions.
Plant and Fungal Material
Experiments were performed with Agrobacterium rhizogenes–transformed root cultures derived from either Medicago truncatula Jemalong A17 or Daucus carota var Sativus. The AM fungal partner used in this study was Gigaspora gigantea. This particular AM fungus can be observed using confocal microscopy because of its yellowish cytoplasmic autofluorescence (Séjalon-Delmas et al., 1998). Spores of G. gigantea were produced using sand-grown clover that was surface-sterilized and stored at 4°C according to Bécard and Fortin (1988).
GFP-Tagged Cellular Markers Expressed in M. truncatula and D. carota Roots
The GFP tag used to monitor ER and nuclear cellular dynamics during AM fungal infection was GFP-HDEL, expressed using either the cauliflower mosaic virus 35S or Mt ENOD11 promoters as described by Genre et al. (2005). Only the 35S:GFP-HDEL construct was used in D. carota since the Mt ENOD11 promoter is not expressed during AM colonization in carrot roots. Vectors were transformed into the A. rhizogenes strain Arqua-1, which was then used to transform M. truncatula Jemalong A17 and D. carota using the protocols described by Boisson-Dernier et al. (2001) and Bécard and Fortin (1988), respectively. For both species, transformed roots with high levels of GFP fluorescence were excised, decontaminated, and then propagated in vitro on M medium (Boisson-Dernier et al., 2001). In each case, a single representative clone was chosen for further studies. Transgene expression in cloned roots was stable throughout in vitro root subculturing, and the presence of the transgenic constructs had no observable effects on root growth or on the interaction with the AM fungus.
In Vivo Microscopy Observation of AM Root Infection
The targeted AM inoculation technique for studying early stages of the symbiotic association between Gigaspora species and transformed root cultures, developed by Chabaud et al. (2002) and adapted for confocal observation by Genre et al. (2005), was applied to both plant species. Germinated spores of G. gigantea were transferred to vertically oriented Petri dishes containing a growing transgenic root explant. The root and fungus were covered with a thin (25 μm) gas-permeable plastic film (bioFOLIE 25; Sartorius) that allowed repeated and prolonged microscopic observation as well as minimizing potential contamination of the coculture.
Confocal Microscopy
Fungal colonization of the transformed root was monitored using a stereomicroscope. Potential infection sites were identified and followed in detail using a Leica TCS SP2 confocal microscope fitted with a long-distance ×40 water-immersion objective (HCX Apo 0.80). The argon laser band of 488 nm was used to excite both the GFP and the G. gigantea autofluorescence. The two signals were distinguished via specific emission windows: 500 to 525 nm for GFP and 590 to 630 nm for fungal autofluorescence. The latter channel was then false-colored in red to maximize the contrast in overlapping images.
Fungal cell wall staining with wheat germ agglutinin was performed on 100-μm-thick vibratome sections of colonized root segments. Sections were treated for 5 min in 1% NaClO, thoroughly washed in phosphate buffer, pH 6.8, incubated with tetramethylrhodamine-conjugated wheat germ agglutinin (Invitrogen) at a final concentration of 20 μg/mL (Vierheilig et al., 2005), and imaged with an excitation of 543 nm (argon laser) and a 590- to 630-nm emission window. Tetramethylrhodamine fluorescence was false-colored in green to maximize contrast with the weak plant cell wall autofluorescence, visualized using the same excitation wavelength and a 630- to 700-nm emission window. Vibratome sections were also used for staining plant nuclei. Sections were incubated with 50 ng/mL propidium iodide (Invitrogen) in phosphate buffer, pH 6.8, and imaged with an excitation of 488 nm and an emission window of 600 to 700 nm.
Nuclear diameters were estimated in living roots using the Leica LCS software to measure a transect across the nucleus based on the GFP-labeled nuclear periphery. The data in Figures 5A and 5B represent a total of 50 nuclei for each cell type (epidermis, outer cortex, and inner cortex) and for each condition (control and colonized roots).
Electron Microscopy
Carrot root segments with aligned PPAs in the inner cortex were initially identified under the confocal microscope. They were then excised under a stereomicroscope and rapidly fixed at room temperature for 2 h in 0.1 M phosphate-saline buffer, pH 6.8, containing 2% glutaraldehyde, 0.1 mM EGTA, 2 mg/mL tannic acid, and 0.1 mM MgCl2. Samples were then rinsed three times with phosphate buffer for 30 min and postfixed in 1% OsO4 in phosphate buffer for 2 h at 4°C. After several washes with phosphate buffer, followed by 1 h in distilled water, en-bloc staining with aqueous 0.5% uranyl acetate was performed for 2 h at room temperature. Samples were then washed in distilled water, dehydrated progressively in an ethanol series, incubated two times in absolute acetone, infiltrated in Epon-Araldite resin (Hoch, 1986), and flat-embedded in a thin resin layer between Teflon-coated glass slides (Howard and O'Donnell, 1987). The resin was polymerized for 24 h at 60°C.
Fungal colonization sites within flat-embedded samples were selected under an optical microscope, excised using a razor blade, and mounted on resin stubs prior to ultramicrotomy. Semithin sections of 0.5 μm were stained with 1% toluidine blue to check the sample contents using optical microscopy. Based on these observations, ultrathin (70 nm) sections were cut, counterstained with uranyl acetate and lead citrate (Reynolds, 1963), and then viewed with a Philips CM10 transmission electron microscope.
Accession Number
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AJ297721 (M. truncatula ENOD11 gene).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Intercellular Colonization of the Root Epidermis by G. gigantea.
Supplemental Figure 2. Prepenetration Responses in the Inner Cortex of M. truncatula and D. carota during AM Colonization.
Supplemental Figure 3. Ultrastructural Studies of D. carota Inner Cortical PPAs: From Confocal Microscopy to TEM.
Supplemental Figure 4. Ultrastructural Organization of a Control Cortical Cell from D. carota.
Supplemental Figure 5. Comparison of Nuclear Architecture in Inner Cortical Cells of D. carota
We thank Robert W. Roberson (Arizona State University, Tempe, AZ) for sharing his fixation protocol and for his advice concerning TEM sample preparation, Ton Timmers (Laboratory of Plant-Microbe Interactions) for advice and discussion about the use of GFP cellular markers, Alain Jauneau (Microscopy Platform, Institut Fédératif de Recherche 40) for his technical assistance concerning the confocal microscopy in Castanet Tolosan, and Joëlle Fournier (Laboratory of Plant-Microbe Interactions) for useful discussions. This research was supported by grants to P.B. from the Italian Ministry of Education (Progetti di Rilevanza Nazionale 2006), from the University of Torino (60%), Consiglio Nazionale delle Ricerche (Biodiversity Project), and the EU INTEGRAL Project. The San Paolo Company (Torino, Italy) is acknowledged for contributing to the acquisition of a Leica confocal microscope at the Laboratory of Advanced Microscopy in Torino.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Paola Bonfante (p.bonfante{at}ipp.cnr.it).
↵[W] Online version contains Web-only data.
- Received February 26, 2008.
- Revised April 15, 2008.
- Accepted May 7, 2008.
- Published May 30, 2008.


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Worldwide phenomenon Frozen is part of the Huddersfield Festival of Light 2014 — and you've been invited to sing along to the Disney smash hit.
The Oscar-winning animated film will be screened in St George's Square, Huddersfield, at 2.45pm on Saturday November 29, and fans can also meet much-loved characters Anna, Elsa and Olaf.
To help the audience join in the fun, the film will play with lyrics on the screen — giving you no excuse not to sing along to the film's hit songs including Let It Go, Do You Want To Build A Snowman?, For The First Time In Forever and more.
The film, based on Hans Christian Andersen's The Snow Queen, has enchanted audiences around the world since its release last year, with memorabilia at the top of many youngsters' Christmas lists.
Many parents have no doubt been subjected to repeat viewings — but how well do you know the words to the Disney musical?
Get ready for the Festival of Light Frozen sing along and test your knowledge with our lyrics quiz.
Just complete the song lines!
Click below to play the quiz
How did you do? Are you an Elsa and Anna expert or do you need to watch Frozen again?
Don't forget to share your scores on Facebook and Twitter!
Looking for some festive film inspiration?
Check out our gallery of must-see Christmas classics

subset: commoncrawl


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Modern Design
Made with Horween Leather's legendary Shell Cordovan, the finish is hand-glazed, creating a rich and glossy look. In either the modern or traditional style, the leather looks and feels exceptional. It is soft, but has enough strength that it won't too easily get stretched or marred. Of course, those things will happen, but not as easily as one more overly soft.
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Each strap is fitted with the custom designed, metal injection molded (MIM) buckle and lugs. High tolerance fit and sharp details are created with this industry leading marine grade 316 stainless steel. Choose between silver or black hardware for your strap.


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Topeka Pizza is not just another
pizza place in Topeka, Kansas.
To be clear here, I have lived in Topeka, Kansas my entire life and I'm proud to be called a Topekan. Often I find myself watching the goes-on in our city from a distance. But when I see a good thing happening, I sometimes find myself speaking out.
Topeka, Kansas is a proud community. A community that cares deeply about the families that make up this great town we call home.
Whether you're a new resident, or a life-long Topekan, you come to realize the unique diversities of our community and how we so often rally together to help one another in time of need and for support of our local causes.
Which brings me to my reason for speaking out this time. For a thank you and a pat on the back.
In January of 2018, a new pizza restaurant was introduced to Topeka, Kansas. Topeka Pizza, located at 1630 SW Arvonia Place, opened their doors and quite literally, their hearts, to the Topeka community.
Owners, Justin and Brandon Kochan, also known as "The Buffalo Brothers", opened Topeka Pizza with the firm belief in becoming a true part of daily life here and of giving back to the community every chance possible.
Even before the restaurant opened, the brothers were busy with numerous charities, such as a Halloween Pumpkin Sale with proceeds going to Stormont-Vail Hospital and a Christmas Gift Drive for the Topeka Rescue Mission.
More recently, the Topeka Pizza Team has pledged to raise money for our Helping Hands Humane Society, a cause near and dear to Topekan's hearts, and to top it off TP is actually setting up at the HHHS to sell pizza and drinks, with proceeds going directly to the Topeka Helping Hands Humane Society that same day!
And to further their community efforts for the month, Topeka Pizza has also teamed up with Sole Reason to help our local Kids in need as well raising donations for children's sneakers.
As a Topeka Pizza customer and a life-long Topekan, I just want to take a minute to thank the TP Team for their community involvement and for the positive return Topeka has received since their opening in 2018.
It is truly heart warming to see a small locally owned business, who is just starting out, giving back to their community in the way Topeka Pizza has shown us.
Again, I'm proud to be a Topekan and I appreciate a local business who cares. I know where our pizza and buffalo wings are coming from next time we order up. Oh, and don't even get me started on the pizza rolls!
Keep up the great work Topeka Pizza! You are doing it,
The Buffalo Brothers Way!
Online Menu
Catering Services


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Chatterbox. 1897 [5 chromolithographed plates]
Boston: Estes & Lauriat, 1897. Hardcover. Wretched copy, wonderful breaker: 412p., humerous chromos staring boys mostly, two girls, and an elderly man, described below. Find profuse b&w wood-engraved plates, ads; clothbacked boards with pastedown illustration. A virtually unrestoreable exemplar; terrifically worn casing is detached from its terrifically shaken textblock; many..... More
Cat.No: 196625


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The Xbox 360 gets an update
As many users have noticed, an update rolled out for the Xbox 360 earlier this week. It is a fairly small update without many obvious changes since it takes care of "mostly back end stuff," according to a tweet from Major Nelson. The only noticeable change allows videos rented through the Xbox Video app on the Xbox 360 to be accessed and played on other devices such as a smartphone or laptop. Previously, movies rented through Xbox Video on the Xbox 360 could only be watched on the Xbox 360.
An update to the Xbox One should also release this month.
Source: TotalXbox.com


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Vancouver-based bakeries and pastry shops have embraced the spooky season by making a diverse selection of Halloween-inspired desserts. Indulge your sweet tooth and take this Halloween to the next level by devouring these unique treats.
Beaucoup BakeryPumpkin Lamington Cake from Beaucoup Bakery & Café
This October, Beaucoup Bakery's co-owner, Betty Hung, introduced a new cake that was inspired by the Australian desserts she had as a kid. Hung's Pumpkin Lamington Cake features a creamy cheese filling between layers of spiced pumpkin sponge, as well as a pumpkin white chocolate ganache coating with coconut, and crushed pumpkin seeds. The Lamington Cake's recipe is available online for those who are feeling confident in their baking abilities.
Where: 2150 Fir Street, Vancouver
Price: $3.25
BETA5 Chocolates Cream Puffs Trio from BETA5 Chocolates
Acclaimed Vancouver chocolate and pastry shop, BETA5, is celebrating Halloween in style with their new chocolate and decadent cream puffs. The cream puff trio is available from October 26 to 31 and includes a vanilla bean mummy, cocoa nib spider, and a Frankenstein head made from raspberry mousse. Now's the time to resolve your mummy issues.
Where: 413 Industrial Ave, Vancouver
Price: $5 per cream puff
PSL Petit GateauxPSL Petit Gateaux from Just Cakes Bakeshop
This Surrey-based bakeshop shop created an adorable creamy treat with their PSL Petit Gateaux. This fun-sized pastry consists of pumpkin spice, chocolate mousse and chocolate java. Now is your chance to eat a pumpkin while carving one.
Price: $7.50
Where: #5-7548 120 Street, Surrey
The Pie HoleHalloween Jack-O-Pumpkin or Skull Pumpkin Pie from The Pie Hole
The Pie Hole created a spooky good delight with their skull pumpkin pie. This nine-inch wonder was made from locally grown roasted pumpkins and the Pie Hole's signature double butter crust.
Where: 1864 West 4th Ave. Vancouver; 3497 Fraser St. Vancouver; 7832 6th Street, Burnaby
Price: $30.00 for a 9-inch
Temper Chocolate and Pastry XL pumpkin chocolate showpiece from Temper Chocolate and Pastry
Prepare to experience the largest sugar rush of your life with Temper's extra large chocolate showpiece. If you love chocolate, caramel and lollipops, this may be the perfect dessert for you.
Where: 2409 Marine Drive, West Vancouver
Price: $49


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Filter By:
Beargrease is Salsa's carbon hotrod fat bike frame, designed to convert strength and determination into speed. The super light chassis paired with your choice of sure-footed 27.5 x 4 wheels and tires reinforce Beargrease's objective; to keep you in the express lane wherever you're headed. Point your front wheel towards the start line of a race on groomed snow or singletrack, challenge yourself on an overnight bikepacking trip, or just get your trail riding on.
Blackborow is Salsa's "dream big" cargo fat bike, best paired with an unlimited imagination. The extended wheelbase and rear stays offer huge carrying capacity via abundant frame and fork braze-ons, supporting room for four full-sized panniers on the beautifully engineered rear rack. Top-shelf, double and triple-butted, heat-treated 6066-T6 aluminum tubing is used throughout the frame. Additionally, the tubing is strategically shaped and formed for additional stiffness and strength optimization in key stress areas. The front end is compatible with all our rigid fat bike forks and also fat-specific suspension forks. When Blackborow is sensibly loaded, the bike still feels well balanced and is a pleasure to ride on winding trails compared to other long-tail cargo bikes. Featuring carbon construction with an aluminum steerer, the Kingpin Fork is lightweight and durable, and features an amazing array of accessory bosses to maximize its capabilities: dual sets of Three-Pack bosses on each leg accept bottle cages or our Anything Cage accessories, lowrider mounts work with our Down Under front rack for a traditional touring setup, and internal dynamo hub wire routing keeps the front end clean for those upgrading to dyno hubs. Blackborow is compatible with 26 x 3.8–4.33" tires on up to 100mm rim, 27.5 x 3.25–4.0", or 29 x 2.3–3.0". The ability to run multiple wheel and tire sizes allows you to customize the experience and fine-tune the ride height as needed. A removable direct-mount front derailleur bracket allows for a clean 1x build, but it's also compatible with 2x drivetrain setups. Plus, it's got internal routing for a dropper post. Blackborow is all about new possibilities, and owners will find themselves able to merge an array of other interests into their two-wheeled adventures. Salsa's Blackborow GX includes an array of high-quality components to support the remotest excursions. SRAM's GX Eagle drivetrain features strong and lightweight components as well as a wide-range 12-speed cassette for tackling quickly varying terrain. A set of SUN Ringle Mulefut rims have a plenty-wide 80mm footing, perfect for floating the 45NRTH Dillinger 27.5 x 3.8" tires. A suite of tried-and-true Salsa cockpit components and a WTB Volt saddle round out the build.
Fargo is Salsa's original drop bar mountain bike. A bit of a cult classic, it's designed for mixed surface riders and bikepackers who want the versatility to take any route and the capability to carry everything needed when they're out for days on end. The Fargo Steel's triple-butted Cobra Kai steel frame is as forgiving as it is reliable on weeks-long expeditions, while the carbon Firestarter fork pairs nicely with the frame and offers bosses and mount points for anything you'd want to attach to a bicycle fork. With an entire world of possible component setups, the Fargo Steel Frameset is a smooth-riding blank canvas you can build into the perfect tool for your two-wheeled needs.
The Fargo is Salsa's original drop bar mountain bike. A bit of a cult classic, it's designed for mixed surface riders and bikepackers that like variety in their routes, with the capability to carry everything needed when out for days on end. Carefully crafted in 3/2.5 seamless triple-butted Titanium tubing, paired with Salsa's new Firestarter Carbon boost fork, this frameset is ready for any dream-bike build. - Titanium tube set provides appropriate stiffness and compliance for fantastic ride quality - Alternator Dropouts v1.5; split-able version of the renowned Alternator Dropout for ultimate drivetrain versatility, including belt drive and singlespeed setups - Adjustable rear spacing can accommodate 27.5+ or 29-inch wheels, and on sizes Small to X-large, 29+ - Firestarter 110 Carbon Deluxe boost fork features high modulus carbon construction, tapered steer tube and four sets of Three-Pack mounts to offer incredible load-carrying possibilities. Mid-blade mounts for low rider racks. Internal routing for Boost spacing Dynamo hubs and fender mounts (compatible with 29 x 2.2-inch tire maximum clearance) - Fork now has a 51mm offset and is designed for 180mm maximum rotors, with tire clearance for 27.5 x 2.8-3.0-inch or 29 x 2.2 – 3.0-inch sizes - Frameset includes front and rear Salsa Deadbolt Ultralight thru-axles, Cane Creek 40 Headset and Salsa Lip-Lock seat collar
Beargrease does a lot of things well, but it does speed best. Lightweight, agile, and elegant, with all excesses removed, Beargrease will happily dispel the myth that fatbikes are slow. Fly through summer singletrack or blast out winter powder laps. Beargrease is your best bet for getting from point A to point B fast, all year, over whatever terrain stands between. With 4.0-inch wide 45NRTH Husker Du tires on 80mm Surly MOBD rims, and the burly Salsa Makwa aluminum fork, the Beargrease takes direction with undeviating response. A 20-speed SRAM X5 drivetrain lets you claw up the steepest pitches, and Hayes MX Comp mechanical disc brakes paired with the giant contact patch of fat tires creates surprising stopping power so you can maintain better control into turns and down techy descents. And, when a fatbike like this tips the scales at just over 31 pounds, the word "plow" is replaces with "float," and thoughts of blasting through fresh powder and monster-trucking through wild alpine singletrack will be running through your head.
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April 15, 2018 @ 12:00 am
50 Wacky Science with Thomas Westbrook
Thomas Westbrook is a YouTube creator and cohost of the Here and How Podcast. He answers the following questions, "who invented science?", "is the earth flat?", "how do you know that you are you?", and "where did life come from?".
Find Thomas and his friends at www.thehereandhow.com


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The tablet's lidar scanner works with an ultra-wide camera, motion sensors, studio-quality mics and apps to provide 'cutting-edge depth-sensing capabilities' that have 'never before possible on any mobile device,' Apple has said.
The custom-designed lidar scanner uses direct time-of-flight to measure reflected light from up to 5m away, both indoors and out.
New depth frameworks in Apple's iPadOS operating software combine depth points measured by the lidar and data from both cameras and motion sensors, which is then enhanced by computer vision algorithms on a A12Z Bionic chip for a more detailed understanding of a scene. The tight integration of these elements enables a 'whole new class of AR experiences' the company says.
Every existing ARKit app – Apple's AR developer app - will get instant AR placement to improve motion capture and people occlusion. Using the latest update to ARKit with a new Scene Geometry API, developers can use the new lidar scanner to 'unleash scenarios never before possible'.
One of the applications the lidar scanner targets is gaming – for example video game Hot Lava is launching a new AR mode later this year, which will transform living rooms into a lava-filled obstacle course. This technology allows the game to interact with the real world, blurring the lines between the physical and virtual world.


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Interface module for combined delivery power, data, and cooling at a network device
In one embodiment, an apparatus comprises a port module for coupling a cable providing mixed power, data, and cooling to…Read More
In one embodiment, an apparatus comprises a port module for coupling a cable providing mixed power, data, and cooling to…Read More
A cable, which can function as a mounting device for detectors, includes strands to be connected to the detectors. The…Read More
Halogen-free plastic composite materials, which are generally for use in sheathing and insulation software for wire and cable, are all…Read More
A cable cable containing a ground conductor and an insulated conductor wherein the insulated conductor includes a metallic conductor, an…Read More
A good example cable assembly includes: a plurality of conductors in a Litz cable arrangement; a layer of magnet wire…Read More
A system and process for enhanced magnet wire insulation is clarified. The technique comprises a sequence of operations to create…Read More
A conductive paste and method of fabricating thereof. The conductive paste comprises conductive particles dispersed in an organic medium, the…Read More
A running shear thinning gel makeup and ways of making such a composition are revealed. The running shear thinning gel…Read More
This patent record discloses fiber sensing techniques and apparatus for shape monitoring by utilizing single- and multi-core optical fiber implementations…Read More
The shape sensor system of the present invention contains a braided structure in which diameter bending members including two or…Read More


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What is a missional community?
Adapted from North American Mission Board
A missional community is a committed group of Jesus followers, the size of an extended family (12–25), empowered by the Spirit to participate in God's mission of redemption in a particular neighborhood and/or network.
There are seven key phrases in this definition that we want to describe further.
- Committed group. They are devoted to each other and to the mission of the community.
- Jesus followers. They are maturing disciples who are following Jesus' lead.
- Extended family. The group is small enough to care, yet large enough to dare.
- Empowered by the Spirit. They are formed and sent by the Spirit.
- Participate in God's mission. The missio Dei is the organizing principle of all they do.
- Of redemption. They will engage in both gospel proclamation and demonstration.
- Neighborhood or network. They are embedded in a neighborhood or network of relationships as an incarnational expression of the church.
How does Cedar City Church live out missional community?
CCC desires to live out the model of missional community both at set times throughout the week and every day in every place that we are. Currently, our missional community meets every Wednesday evening at 6:30pm either at the 12 New Street building or at someone's house. We post regularly about missional community gatherings on our social media accounts, so be sure to check our Facebook or Instagram for current info on where we are meeting.


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Our pot roast pheasant is a great recipe if you've never cooked pheasant before.
If you are looking for pheasant recipes look no further. You might be thinking roast pheasant sounds rather fancy, but this tasty bird is just as cheap as chicken. At only £1.58 per serving, this pot roast pheasant recipe is ideal if you're on a budget. And you might have thought that pheasant recipes may be difficult to cook and a bit too extravagant for a casual weekend, but this is one of the easiest pheasant recipes we've ever come across. In just two steps you can have this pot roast pheasant recipe on the table. It's a definite crowd-pleaser with the blend of delicious flavours – smoky bacon, fragrant garlic and rich Worcestershire sauce. Bacon lardons give this pheasant dish a smoky undertone which is delicious. And this is one of the healthier pheasant recipes we've come across as it won't break the diet! A portion contains only 290 calories and 7g of fat so it's a rather healthy family meal or dinner party treat. Brown the pheasant breasts before popping into a casserole dish. Let them cook the rest of the time in the broth for tender and flavoursome meat. Serve with new potatoes and softly fried leeks and enjoy. As pheasant recipes go, this is good one.
- 200g smoked bacon lardons
- 1tbsp oil
- A brace of oven-ready pheasant (a cock and a hen)
- 2 onions, peeled and cut into wedges
- 2 cloves garlic, peeled and chopped
- 4 celery sticks, chopped
- 400g can chopped tomatoes
- About 300ml hot chicken stock
- 2tbsp tomato purée or ketchup
- 2tsp Worcestershire sauce
- 2 tsp caster sugar
- Salt and freshly ground black pepper
- A few parsley leaves
- To serve:
- 400g small potatoes
- 2 leeks, trimmed and cut into chunks
Set the oven to 160°C/320°F/Gas Mark 3. Put the bacon and oil in a frying pan and cook over a med heat for a couple of minutes. Add the birds to the pan and cook them for a few mins on each side to brown the breasts. Put them in a casserole with the bacon.
Add the onion to the pan and cook for a couple of mins. Add the garlic and celery and fry for 2-3 mins. Pour in the tomatoes, stock, purée, Worcestershire sauce and sugar. Bring to the boil, season well and spoon into the casserole. Put the lid on and cook in the oven for 45 mins. Take the lid off, baste the pheasants and cook for another 15-25 mins, until the meat is tender. Leave for 10 mins before serving. Sprinkle with parsley leaves and serve with potatoes and leeks.
Top tip for making Pot roast pheasant
For ease of serving, take the birds out of the dish and when cool enough to handle, take the legs, breasts and all the meat off the bone. Put the dish on the hob, put the meat back and warm it through. If you don't want to use game, cook this recipe with a 1.5kg chicken, adding an extra 30-40 mins cooking time


text:
Galaxy S7 Oreo update issue - Home Button
I updated my Samsung Galaxy S7 to the At&t Oreo OS. Now my home button is not responding. Sometimes I have to press it like 20 times to get it to respond (It is not a hardware issue, because it sometimes responds on the first try). Any ways to fix it, or go back to old OS?
Accepted Solution
Official Solution
Hi there @maveledo,
Welcome to the Community Forums! Thank you for reaching out with your software concerns. I will be more than happy to point you in the right direction. When an update causes issues after being installed, there is a chance something went wrong with the install. I recommend performing a full factory data reset on the device and reinstall it. Here is an article for step – by – step instructions on how to complete this process.
I hope this helps. If you have additional questions, please feel free to reach back out to us. Take care and have a great day!
Denise, AT&T Community Specialist


text:
Pentax 560mm f5.6 HD DA ED AW Lens
The Pentax 560mm f5.6 HD DA ED AW Lens is a super-telephoto lens specially designed for use with the Pentax K-mount lenses and features a newly developed high-grade multi-layer lens coating to deliver clear, well-defined images of distant subjects. When mounted on a Pentax K-mount digital camera body, this super-telephoto lens offers an equivalent focal length of approximately 859mm in the 35mm ... More
Thinking of upgrading? Trade in today
1x Pentax 560mm f5.6 HD DA ED AW Lens Sorry, there are no more available.
Major features of the Pentax 560mm f5.6 HD DA ED AW Lens
1. New, high-grade HD Coating
This lens is treated with HD Coating*, a new high-grade multi-layer lens coating developed by PENTAX. Compared with conventional multi-layer coatings, it provides increased light transmittance with reduced reflection, to deliver sharp, clear images free from flare and ghost images, even under demanding lighting conditions.
2. Dustproof, weather-resistant construction
A new addition to the AW (All Weather) series, this lens features a dependable dustproof, weather-resistant construction with 29 special seals to effectively prevent the intrusion of dust and water into the lens interior. By coupling this lens with a dustproof, weather-resistant PENTAX interchangeable lens digital camera body such as the PENTAX K-5 or PENTAX K-30, the user is assured of a durable, reliable imaging system that performs superbly in demanding outdoor situations — even in rain or mist, or at locations prone to water splashes or spray.
3. New exterior design
The lens exterior has been painted an elegant white to minimize heat haze generated inside the lens barrel due to strong sun. Featured in the middle of the lens barrel is a red almite ring that clearly signifies the new HD PENTAX lens series. Strap lugs are made from aluminium to enhance its exclusive appearance.
4. Easy control of circular polarising filter
This lens comes with a built-in, removable filter holder in the barrel. By mounting the C-PL (circular polarising) filter (40.5mm in diameter; included as an exclusive accessory) to the holder, the user can easily obtain the desired polarising effect simply by rotating the filter with its control ring.
5. Other features
- Smooth autofocus operation using an AF driving motor (DC motor) installed in the lens barrel**
- Quick-Shift Focus system for instant switching to manual-focus operation after the subject is captured by the camera's AF system
- SP (Super Protect) coating applied to the front surface of the lens to effectively repel water and grease and to allow the user to easily remove dust and water stains
- Rounded diaphragm to produce a smooth, beautiful bokeh (out-of-focus rendition), while minimising the streaking effect of point light sources (coma)
* HD stands for High Definition, and signifies the lens's capacity to reproduce images that excel in transparency, definition, resolution and rendition.
** This lens is compatible with the K-5 II, K-5 II s, K-30, K-01, K-5, K-r, K-7, K-x, K-m, K20D, K200D, K100D Super and K10D camera bodies (with Ver. 1.30 or later firmware installed). When mounted on other camera bodies, the focus mode is automatically switched to manual.Back to top
Product Specification
|Aperture blades
|Lens mount
|Pentax K
|Camera Lens Type
|Telephoto Prime
|Filter thread (mm)
|Focal length (tele)
|Focal length (wide)
|Included Accessories
|C-PL filter, Mount cap, Lens case, Lens cap, Built-in lens hood
|Lens elements
|Lens groups
|Max aperture - tele
|Max aperture - wide
|Minimum aperture
|Minimum Focus Distance (m)
|130 x 521.7 mm
|Weight (g)
{{item.DisplayName}} for the Pentax 560mm f5.6 HD DA ED AW Lens
1x {{accessory.DisplayName}}
Spread the Cost
Spread the cost of this item over 12 months. With a 0% finance, no deposit and monthly repayments by direct debit this option is simple to manage. Please see the repayment example below to see how much purchasing this item on finance would cost.
|Finance Example
|Item Value (Cash Price)
|Agreement Duration
|12 Months
|Total Amount Credit
|Settlement Fee
|Settlement Payment
|Monthly Instalments
|11 x £374.92
|Final Instalments
|Total Amount Payable
|Annual Rate of Interest
|Representative APR
Are you eligible for finance?
Applicants must be:
- Aged 18 years or older from the application date
- In permanent UK residence for a minimum of three years
- Fulfil at least one of the following employment criteria:
- In regular, permanent employment.
- Retired from regular employment - With a minimum age of 50.
- Not working through disability.
- Self-employed.
Note: All applicants will be subject to a credit reference check by the lender.
Credit is subject to application and status. Warehouse Express Ltd trading as Wex Photo Video act as credit broker and not the lender. Barclays Partner Finance act as the lender.
How does it work?
Where a qualifying product is added to your basket, the full value of your basket, including any accessories purchases, will be eligible for interest free credit. This is subject to the value of the additional products not exceeding the value of the qualifying product. No deposit is required and payments will be collected by direct debit in monthly instalments, the first instalment being collected 30 days after shipment of the product.
What will I receive from the finance provider (lender)?
Barclays Partner Finance will send a loan agreement pack approximately four weeks after delivery of the goods, confirming that the account has been activated and, from this point, it is possible to make repayments against the loan.
For further information on how our finance product work, see our Finance help section.
To apply for finance, follow these three easy steps
† Once your application has been accepted, usually within minutes, we will immediately dispatch your equipment.


text:
Taken in a coffee bean plantation in 2009. Santa Ana, El Salvador. (5 years and 1048 days ago)
IMO, this just doesn't have the quality you'd expect from NG
Not by far...
I couldn´t agree more with both of you, but in my defense, this was taken with a disposable camera, plus, more then the quality of the picture is the statement.
Howdie stranger!
If you want to rate this picture or participate in this contest, just:
LOGIN HERE or REGISTER FOR FREE


text:
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text:
Former Jharkhand chief minister and JVM-P leader Babulal Marandi has said holding elections in the state is the only alternative.
"Only elections are the alternative in Jharkhand as no party or group has come to stake claim for the formation of a government," the founder and national president of Jharkhand Vikas Morcha (Prajatantrik) said at a function.
Jharkhand came under President's rule for the second time on June 1 after all the parties had refused to form government.
Marandi blamed the Congress for the state administration's decision to withdraw certain facilities to former Jharkhand chief ministers.
"The decision has unmasked the Congress' anti-tribal face. The decision to withdraw facilities was taken as all the four ex-CMs are tribals," claimed Marandi, whose party has a pre-poll tie-up with the Congress. Several other states have the provision of providing facilities to ex-CMs, he added.
Last week, the Governor's advisory council had taken a decision to redraw facilities enjoyed by the ex-CMs.
Besides Marandi, JMM president Shibu Soren, BJP's general secretary Arjun Munda and Madhu Koda have been at the helm of affairs at different times and enjoy sprawling accommodation and various other benefits.


text:
Disparo, Wounded Pig And Graves Announce Three New Zealand Shows
Australian fastcore outfit Disparo are heading back to New Zealand for three shows in July and are bringing with them compatriot grindcore group Wounded Pig. The two trans-Tasman acts will also be joined by Auckland hardcore band Graves for the tour, which will also see the three acts joined by a raft of fantastic local support while they make their way around the North Island. Here are the details...
Disparo, Wounded Pig And Graves
Thursday 16th July, Lucha Lounge, Auckland w/ Parents and Markdown
Friday 17th July 17th, Nivara Lounge, Hamilton w/ Wizz Kids, Shitripper and Spiteful Urinator
Saturday 18th July, Valhalla, Wellington w/ Cephalopod and One Outs
Here's a video from Disparo for 'DIY Bother', stitched together with footage of their tour through USA, UK and all over Europe....
And here is a video of Wounded Pig playing live in Australia late last year...
Support UTR
You can show your support to keep UnderTheRadar running by making a contribution. Any small amount can make a huge difference and keep us bringing you the best, comprehensive local content. Support UTR


text:
Turbo Studio (formerly known as Spoon Studio) – with this app you can combine files needed to make an app run into a package that is ready for use without deployment. The application allows you to create a virtual container and run programs in a virtual environment without the installation and make changes in registry and system.
Features: – Click Here
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Dear friends you have to successfully install this software on your computer or laptop. If you have to any problem regarding to installation process on your computer or laptop, then please you can share your problem and difficulty with me in the comment box. So I have to solve your problem regarding installation this software.
1. This Program Provided For Only Education Purposes.
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3. If You Like This Program, Support Developers, Buy It! They Deserved It
4. I am not responsibility if any problem regarding to install this program on your computer.
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text: After Khomeini: Iran under His Successors By Saïd Amir Arjomand No cover image After Khomeini: Iran under His Successors By Saïd Amir Arjomand Read preview Synopsis For many Americans, Iran is our most dangerous enemy--part of George W. Bush's "axis of evil" even before the appearance of Ahmadinejad. But what is the reality? How did Ahmadinejad rise to power, and how much power does he really have? What are the chances of normalizing relations with Iran? In After Khomeini, Saïd Amir Arjomand paints a subtle and perceptive portrait of contemporary Iran. This work, a sequel to Arjomand's acclaimed The Turban for the Crown, examines Iran under the successors of Ayatollah Khomeini up to the present day. He begins, as the Islamic Republic did, with Khomeini, offering a brilliant capsule biography of the man who masterminded the revolution that overthrew the Shah. Arjomand draws clear distinctions between the moderates of the initial phrase of the revolution, radicals, pragmatists, and hardliners, the latter best exemplified by Mahmud Ahmadinejad. Taking a chronological and thematic approach, he traces the emergence and consolidation of the present system of collective rule by clerical councils and the peaceful transition to dual leadership by the ayatollah as the supreme guide and the subordinate president of the Islamic Republic of Iran. He explains the internal political quarrels among Khomeini's heirs as a struggle over his revolutionary legacy. And he outlines how the ruling clerical elite and the nation's security forces are interdependent politically and economically, speculating on the potential future role of the Revolutionary Guards. Bringing the work up to current political events, Arjomand analyzes Iran's foreign policy as well, including the impact of the fall of Communism on Iran and Ahmadinejad's nuclear policy. Few countries loom larger in American foreign relations than Iran. In this rich and insightful account, an expert on Iranian society and politics untangles the complexities of a nation still riding the turbulent wake of one of history's great revolutions. Excerpt Iran has not ceased to surprise the world since the American ambassador's famous "thinking the unthinkable" 1978 cable about the imminent fall of the Shah and the coming of the Islamic revolution. The apparent sequence of moderate government under President Akbar Hashemi-Rafsanjani (1989–1997) and democratic reform under President Mohammad Khatami (1997–2005) was followed by a spectacular reversal no one foresaw. The hardliners returned to capture the Majles (Iranian parliament) in the national elections of 2004, and one of their leaders, Mahmoud Ahmadinejad, beat a former President (Hashemi-Rafsanjani) and two much better known reformist candidates in the presidential elections of 2005. President Ahmadinejad (2005–2009) has revived the revolutionary populism of old, now coupled with an aggressive foreign policy, including a nuclear program. Iran's political regime has proved remarkably resilient through all these changes, despite the disaffection of the younger half of the population. And the regime has become all the more robust and defiant internationally, partly as a result of the Bush administration's ill-advised bluff about regime change from 2002 onward. The greatest misunderstanding concerning Iran after the revolution stems from the assumption that the revolution was over, either with the victory of pragmatism and Hashemi-Rafsanjani's program of economic reconstruction in 1989, following the Iran–Iraq war, or with the rise of the reform movement under Khatami in 1997. The truth is that the death of Ayatollah Ruhollah . . . Related books and articles Books Journals Magazines Newspapers Encyclopedia Iran since the Revolution By Sepehr Zabih Routledge, 2011 Read preview Overview Ghosts of Revolution: Rekindled Memories of Imprisonment in Iran By Shahla Talebi Stanford University Press, 2011 PSPRIMARY SOURCE A primary source is a work that is being studied, or that provides first-hand or direct evidence on a topic. Common types of primary sources include works of literature, historical documents, original philosophical writings, and religious texts. Read preview Overview Immortal: A Military History of Iran and Its Armed Forces By Steven R. Ward Georgetown University Press, 2009 Read preview Overview MODERN HISTORY AND POLITICS-The Oil Kings: How the U.S., Iran and Saudi Arabia Changed the Balance of Power in the Middle East By Long, David E. The Middle East Journal, Vol. 66, No. 1, Winter 2012 PRPEER-REVIEWED PERIODICAL Peer-reviewed publications on Questia are publications containing articles which were subject to evaluation for accuracy and substance by professional peers of the article's author(s). Read preview Overview KURDS: The Kurds in Iran: The Past, Present and Future By Gunter, Michael M. The Middle East Journal, Vol. 61, No. 3, Summer 2007 PRPEER-REVIEWED PERIODICAL Peer-reviewed publications on Questia are publications containing articles which were subject to evaluation for accuracy and substance by professional peers of the article's author(s). Read preview Overview IRAN: Eternal Iran: Continuity and Chaos By Sadri, Houman A. The Middle East Journal, Vol. 60, No. 3, Summer 2006 PRPEER-REVIEWED PERIODICAL Peer-reviewed publications on Questia are publications containing articles which were subject to evaluation for accuracy and substance by professional peers of the article's author(s). Read preview Overview Persian Pilgrimages: Journeys across Iran By Galford, Hugh S. Washington Report on Middle East Affairs, Vol. 22, No. 1, January/February 2003 Read preview Overview Experts Discuss Iran's Strategic Concerns and U.S. Interests By Hamedani, Nina Washington Report on Middle East Affairs, Vol. 27, No. 3, April 2008 Read preview Overview Letter from Iran By Molavi, Afshin The Nation, Vol. 269, No. 3, July 27, 1999 Read preview Overview The Royal Burgh; EXCLUSIVE Lady in Red Singer Chris Has Another Claim to Fame - He's Related to Robert the Bruce. Now He's Set to Make History of His Own by Being the First Western Pop Star to Play in Iran in 30 Years By Dingwall, John Daily Record (Glasgow, Scotland), March 22, 2008 Read preview Overview Iran's Election Results Seen Too Difficult to Call By Sands, David R. The Washington Times (Washington, DC), February 16, 2000 Read preview Overview U.S. Relations with Iran Haunted by History By Lee, Matthew Telegraph - Herald (Dubuque), November 20, 2014 Read preview Overview VIEW ALL RELATED BOOKS AND ARTICLES