Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci.

In planta RNAi against essential insect genes offers a promising route to control insect crop pests, but is constrained for many insect groups, notably phloem sap-feeding hemipterans, by poor RNAi efficacy. This study conducted on the phloem-feeding whitefly Bemisia tabaci reared on tomato plants investigated the causes of low RNAi efficacy and routes to ameliorate the problem. Experiments using tomato transgenic lines containing ds-GFP (green fluorescent protein) revealed that full-length dsRNA is phloem-mobile, ingested by the insects, and degraded in the insect. We identified B. tabaci homologs of nuclease genes (dsRNases) in other insects that degrade dsRNA, and demonstrated that degradation of ds-GFP in B. tabaci is suppressed by administration of dsRNA against these genes. dsRNA against the nuclease genes was co-administered with dsRNA against two insect genes, an aquaporin AQP1 and sucrase SUC1, that are predicted to protect B. tabaci against osmotic collapse. When dsRNA constructs for AQP1, SUC1, dsRNase1 and dsRNase2 were stacked, insect mortality was significantly elevated to 50% over 6 days on artificial diets. This effect was accompanied by significant reduction in gene expression of the target genes in surviving diet-fed insects. This study offers proof-of-principle that the efficacy of RNAi against insect pests can be enhanced by using dsRNA to suppress the activity of RNAi-suppressing nuclease genes, especially where multiple genes with related physiological function but different molecular function are targeted.

Phloem loading in the tulip tree. Mechanisms and evolutionary implications.

Minor vein ultrastructure and phloem loading were studied in leaves of the tulip tree (Liriodendron tulipifera; Magnoliaceae). Plasmodesmatal frequencies leading into minor vein companion cells are higher than in species known to load via the apoplast. However, these companion cells are not specialized as "intermediary cells" as they are in species in which the best evidence for symplastic phloem loading has been documented. Mesophyll cells plasmolyzed in 600 mM sorbitol, whereas sieve elements and companion cells did not plasmolyze even in 1.2 M sorbitol, indicating that solute accumulates in the phloem against a steep concentration gradient. Both [(14)C]sucrose and (14)C-labeled photo-assimilate accumulated in the minor vein network, as demonstrated by autoradiography. [(14)C]sucrose accumulation was prevented by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton cotransport from the apoplast. p-Chloromercuribenzenesulfonic acid largely, but not entirely, inhibited exudation of radiolabeled photoassimilate. The evidence is most consistent with the presence of an apoplastic component to phloem loading in this species, contrary to speculation that the more basal members of the angiosperms load by an entirely symplastic mechanism.

The evolution of minor vein phloem and phloem loading.

Phylogenetic analysis provides a rational basis for comparative studies of phloem structure and phloem loading. Although several types of minor vein companion cell have been identified, and progress has been made in correlating structural features of these cells with loading mechanisms, little is known about the phylogenetic relationships of the different types. To add to the available data on companion cells, we analyzed the ultrastructure of minor veins in Euonymus fortunei and Celastrus orbiculatis (Celastraceae) leaves and determined that in these species they are specialized as intermediary cells. This cell type has been implicated in symplastic phloem loading. The data were added to published data sets on minor vein phloem characteristics, which were then mapped to a well-supported molecular tree. The analysis indicates that extensive plasmodesmatal continuity between minor vein phloem and surrounding cells is ancestral in the angiosperms. Reduction in plasmodesmatal frequency at this interface is a general evolutionary trend, punctuated by instances of the reverse. This is especially true in the case of intermediary cells that have many plasmodesmata, but other distinguishing characteristics as well, and have arisen independently at least four, and probably six, times in derived lineages. The character of highly reduced plasmodesmatal frequency in minor vein phloem, common in crop plants, has several points of origin in the tree. Thus, caution should be exercised in generalizing results on apoplastic phloem loading obtained from model species. Transfer cells have many independent points of origin, not always from lineages with reduced plasmodesmatal frequency.

Minor vein structure and sugar transport in Arabidopsis thaliana.

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex.

Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter.

The definition of "minor" veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of the gusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.

Flow cytometric findings in the floral variant of follicular lymphoma.

The floral variant of follicular lymphoma (FL-F) is an unusual variant of follicular lymphoma. The malignant "follicles" have an unusual "floral" appearance due to the proliferation of surrounding and infiltrating small lymphocytes. This variant of malignant lymphoma has been supported by immunohistochemical immunophenotyping and gene-rearrangement studies; however, the flow cytometric findings have not previously been described. We report the flow cytometric findings in two cases of FL-F. The surrounding and infiltrating small lymphocytes represented mantle cells which, in one case, obscured monoclonality in the lymphocyte region. The monoclonal B-cells were CD10+, supporting a follicular center cell origin.

The absence of phloem loading in willow leaves.

Willow (Salix babylonica L.) is representative of a large group of plants that have extensive plasmodesmatal connections between minor vein phloem and adjoining cells. Because plasmodesmata provide a diffusion pathway for small molecules, it is unclear how sucrose could be loaded from the mesophyll into the phloem against a concentration gradient. In the studies reported here, the minor vein phloem of willow leaves plasmolyzed in approximately the same concentration of osmoticum as the mesophyll. Sucrose concentrations in mesophyll cells were greater than those reported in the literature for aphid stylet exudate from willow stems. Calculated turgor pressures in the mesophyll and minor vein phloem were greater than turgor reported in the literature for sieve elements in the stems of willow. Images of minor veins were not obtained in autoradiographs when attached leaves, or leaf pieces, were provided with 14CO2 or [14C]sucrose. Therefore, no evidence could be found for accumulation of sucrose against a concentration gradient in the minor vein phloem of willow. In these leaves, the mesophyll apparently acts as the "source" for long distance transport of sugar. The mechanism of translocation in willow, and the evolution of phloem loading, are discussed.

Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus.

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class III vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class III phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class III veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class III veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of viral entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.

The geminivirus BL1 movement protein is associated with endoplasmic reticulum-derived tubules in developing phloem cells.

Plant viruses encode movement proteins that are essential for systemic infection of their host but dispensable for replication and encapsidation. BL1, one of the two movement proteins encoded by the bipartite geminivirus squash leaf curl virus, was immunolocalized to unique approximately 40-nm tubules that extended up to and across the walls of procambial cells in systemically infected pumpkin leaves. These tubules were not found in procambial cells from pumpkin seedlings inoculated with BL1 mutants that are defective in movement. The tubules also specifically stained with antisera to binding protein (BiP), indicating that they were derived from the endoplasmic reticulum. Independent confirmation of this endoplasmic reticulum association was obtained by subcellular fractionation studies in which BL1 was localized to fractions that contained both endoplasmic reticulum membranes and BiP. Thus, squash leaf curl virus appears to recruit the endoplasmic reticulum as a conduit for cell-to-cell movement of the viral genome.

Laparoscopic cholecystectomy can disseminate in situ carcinoma of the gallbladder.

BACKGROUND: Early case reports suggest more frequent and rapid recurrences of carcinoma of the gallbladder after laparoscopic cholecystectomy (LC) than after open cholecystectomy. This cancer has a poor prognosis and occurs in 1 percent of patients who undergo cholecystectomies. STUDY DESIGN: A recent community hospital series of gallbladder carcinoma (GBC) was reviewed and the total reported experience of GBC after LC was compiled. Diagnostic findings were compared for patients with GBC and a consecutive series of 24 patients who had LC for benign disease. RESULTS: Nine patients with GBC were found among 928 patients who had undergone cholecystectomy (0.97 percent incidence). Compared to patients without GBC, patients with carcinoma were older, had thicker gallbladder walls, and had more abnormalities detected intraoperatively (all p < or = 0.05). Recurrence of GBC occurred more rapidly after LC, and in diffuse peritoneal and port sites when compared with recurrence patterns after open cholecystectomy. CONCLUSIONS: In patients with GBC, LC may be sufficient when the disease is confined to the gallbladder mucosa and the gallbladder is excised intact without bile spillage. However, patients whose gallbladders are torn during dissection or patients who have invasive tumors should undergo laparotomy and local reexcision. In situ GBC can be implanted if the organ is torn during dissection. When gallbladders with suspicious wall thickening or adhesions are noted at LC, especially in older patients, the procedure should be converted to open cholecystectomy.

Sexual asphyxiation: an unusual case involving four male adolescents.

A case of sexual asphyxiation is described involving children, adolescent males, homosexuality and two bizarre strangulations. Two adolescent males (ages 13 & 10) initially engaged in homosexual activity in conjunction with sexual asphyxiation. This unusual activity was by chance observed by one of the boy's younger brother and his friend. The younger children (aged 7 & 8) expressed a desire to join in the activity, which they did by letting ligatures be tied around their necks and engaging in anal intercourse to the point of fatal asphyxiation. The case is of interest in the reported way the younger boys discovered the practice (a sexually explicit magazine) and is unusual because of the young ages of the boys involved.

Phloem transport of antirrhinoside, an iridoid glycoside, inAsarina scandens (Scrophulariaceae).

Iridoid glycosides, terpene-derived compounds found in many plant families, protect the plant against generalist and nonadapted specialist insect herbivores, fungi, and bacteria. Antirrhinoside, a common iridoid glycoside in the tribe Antirrhineae (Scrophulariaceae), was rapidly labeled when mature leaves ofAsarina scandens were exposed to(14)CO2. Antirrhinoside was translocated in the phloem along with sucrose. Radiolabeled antirrhinoside appeared in the petiole of the labeled leaf within 20 min of the beginning of the labeling period. Antirrhinoside was also found in phloem sap obtained by the EDTA method.

The geminivirus BR1 movement protein binds single-stranded DNA and localizes to the cell nucleus.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.

Movement of virus and photoassimilate in the phloem: a comparative analysis.

Recent progress in the study of short-distance (cell-to-cell) movement of plant virus, facilitated by 'movement proteins', has led to a resurgence of interest in long-distance virus transport in the phloem. Relatively little is known about phloem-specific barriers to virus movement or about the form in which virus enters, travels within and exists this tissue. Progress in understanding virus and photoassimilate transport is limited by a paucity of information on the substructure and properties of plasmodesmata at specific interfaces. The direction of virus movement, once it has entered the phloem, can be understood by following photoassimilate translocation, a complex and dynamic process influenced by plant growth, development and vascular topology.

Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant.

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.

Effects of host plant development and genetic determinants on the long-distance movement of cauliflower mosaic virus in Arabidopsis.

During systemic infections, viruses move long distances through the plant vascular system. The long-distance movement of cauliflower mosaic virus (CaMV) in Arabidopsis has been examined using a whole plant in situ hybridization technique called plant skeleton hybridization. CaMV moves long distance through the phloem largely following the flow of photoassimilates from source to sink leaves. During the course of plant development, sink-source relationships change and the region of the plant that CaMV can invade is progressively reduced. In Arabidopsis, we have found that conditions that influence the rate of plant development dramatically impact the long-distance movement of CaMV, because under normal conditions the rate of plant development is closely matched to the kinetics of virus movement. Ecotypes and mutants of Arabidopsis that flower early show a form of resistance to systemic CaMV infection, which we call "developmental resistance." Developmental resistance results from the fact that the rosette leaves mature early in the life of an early flowering plant and become inaccessible to virus. On the other hand, if the development of early flowering plants is retarded by suboptimal growth conditions, inoculated plants appear more susceptible to the virus and systemic infections become more widespread. We have found that other Arabidopsis ecotypes, such as Enkheim-2 (En-2), show another form of resistance to virus movement that is not based on developmental or growth conditions. The virus resistance in ecotype En-2 is largely conditioned by a dominant trait at a single locus.

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : I. The Use of Steady-State Labeling.

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with (14)CO(2). The continuous assimilation of (14)CO(2) at constant specific activity produced stable tracer fluxes that facilitated straightforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of "empty" seed coats for perfusion.

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity.

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.

Sugar synthesis and phloem loading in Coleus blumei leaves.

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of (14)CO2 was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either (14)CO2 or [(14)C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [(14)C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to (14)CO2 for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of (14)CO2, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244-1249) indicate that the phloem loading pathway in Coleus is probably symplastic.