Fluorescein-conjugated Wheat Germ Agglutinin Research Articles

In situ observation of pinewood nematode in wood

In pine wilt disease, xylem dysfunction occurs in relation to nematode migration and proliferation in host tissue, but the detection accuracy of pinewood nematode (PWN), Bursaphelenchus xylophilus, in pine stem tissue remains unclear. This study describes the use of cryo-scanning electron microscopy (cryo-SEM) and fluorescein-conjugated wheat germ agglutinin (F-WGA) staining to detect PWN. After PWN invasion, the frequency of surface fluorescence in PWN increased in pine stems from the day of inoculation to 3 weeks after inoculation. However, the fluorescence frequency decreased significantly during the advanced disease stage after 5 weeks. Thus, detecting PWN based on fluorescent staining of the nematode body surface coat protein can be misleading when used to examine the correlation between the development of disease symptoms and the nematode population. In contrast, all cut body segments were fluorescent, and their fluorescent components were common in pine-stem cross sections, regardless of the timing after inoculation. In addition, PWN were observed under cryo-SEM only in empty resin canals and this distribution was confirmed by F-WGA staining of PWN cut in a cross section. Thus, PWN detection based on fluorescent staining of surface coat proteins by F-WGA was not reliable in longitudinal sections of pine stems because of changes in nematode stainability during disease progression. To detect PWN in infected plants, we concluded that a combination of both methods is most effective.

A Fluorescence Microscopic Study of the Infection Process of Discula theae-sinensis in Tea

The infection process of the anthracnose fungus Disculatheae-sinensis in tea was investigated in detail by fluorescence microscopy. Leaves of the susceptible tea variety ‘Yabukita’ spray inoculated with the conidial suspension were collected sequentially and examined after fluorescence staining with fluorescein-conjugated wheat germ agglutinin. Conidia adhering to the trichomes germinated and formed very short germ tubes. Penetration hyphae grew from the germ tube tips into the cell walls of the trichomes, and formed infection hyphae by 12 h after inoculation. Hyphae that had invaded the mesophyll were confined to the small round spots that formed surrounding the infected trichomes. However, hyphae that had reached the veins extended through the phloem, causing necrosis of the neighboring mesophylls and eventually the formation of large necrotic lesions.

Histological and anatomical responses in avocado, Persea americana, induced by the vascular wilt pathogen, Raffaelea lauricola

Raffaelea lauricola causes laurel wilt of avocado, Persea americana. Host × pathogen interactions were examined with light and scanning electron microscopy. The susceptible avocado cultivar ‘Simmonds’ was inoculated and examined 5 cm above the inoculation site 3, 7, 14, 21, and 42 days after inoculation (dai). No external symptoms were observed at 3 and 7 dai, and there were no anatomical differences when compared with the mock-inoculated plants. By 14 dai, external symptoms were present and dark discoloration had developed in sapwood. Tylose development increased significantly by 14 dai, and was positivity correlated with disease severity (P < 0.05). By 14 dai, gels formed in xylem vessels, fibers, and adjacent parenchyma cells; they were associated with xylem blockage and composed of phenols, pectin, and lipids, as suggested by, respectively, toluidine blue O, ruthenium red, and Sudan III stains. With a chitin-specific stain, fluorescein-conjugated wheat germ agglutinin, infrequent mycelia, and conidia of R. lauricola were visualized within xylem lumena and fibers, regardless of sample date. Understanding how avocado responds to the presence of this pathogen could assist the development of laurel wilt-resistant avocado genotypes and inform efforts to manage this disease with other measures.

Spatial Distribution of Raffaelea quercivora in Xylem of Naturally Infested and Inoculated Oak Trees

Mass mortality of Japanese oak caused by Raffaelea quercivora due to Japanese oak wilt (JOW) has been tremendous since the late 1980s in Japan. We investigated detailed distribution of R. quercivora hyphae in a naturally infected Quercus serrata tree using fluorescein-conjugated wheat germ agglutinin and also examined spatial relationships between hyphal distribution, water conduction loss, and host reactions in xylem in inoculated Q. crispula saplings. Hyphae of R. quercivora elongated longitudinally in vessels and transversely in ray tissues in both naturally infected and inoculated Quercus trees. Hyphae were confined within a relatively small area near the inoculation site. Vessel dysfunction was also confined and overlapped with hyphal distribution. The reaction zone consisting of two types of fluorescent substance was formed surrounding the outside of the hyphal area in xylem and was always found in non-water-conductive zone. These results suggest that wilting of Quercus trees caused by JOW may not be induced by dysfunction of a small number of vessels, but by that of many vessels, and it requires that R. quercivora hyphae spread from many galleries bored by beetles during mass attacks.

Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

Fluorescein-labeled wheat germ agglutinin stains the pine wood nematode, Bursaphelenchus xylophilus

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major threat to pine forests throughout East Asia. Nonetheless, its mechanism of invasion has not yet been described in detail. To better understand the pathology of this disease, it is important to examine the distribution of PWNs within pine tissue during the course of disease development. We attempted to stain nematodes with fluorescein-conjugated wheat germ agglutinin (F-WGA) as a means to locate and track the spread of PWNs. Although PWNs proliferated on Botrytis cinerea fungus were successfully stained only on their vulvas and spicule holes, PWNs extracted from inoculated Pinus thunbergii seedlings were stained on their surface. Stainability, or the percentage of PWNs stained with F-WGA over more than half of their surface, was about 20% by 1 day after inoculation, but increased to 80% at 10 days. The stainability of PWNs extracted from a 5-cm main stem segment that included the inoculation site was less than that of PWNs extracted from other parts of the main stem farther away (i.e., those that had dispersed). These results suggest that stainability is related to dispersal activity in time. Thus, to raise the stainability of PWNs at shorter timeframes after inoculation, PWNs with more than 80% stainability were re-inoculated into pine seedlings. This resulted in more than 70% stainability from 1 to 6 days after inoculation. In F-WGA stained thin paraffin sections of pine tissue of re-inoculated seedlings, PWNs brightly fluoresced under epifluorescence and were easily detected against the dark background of pine tissue. This staining technique with F-WGA is an excellent tool for detecting PWNs in pine tissue.

Pathophysiologic Implications of Reduced Podocyte Number in a Rat Model of Progressive Glomerular Injury

Changes in podocyte number or density have been suggested to play an important role in renal disease progression. Here, we investigated the temporal relationship between glomerular podocyte number and development of proteinuria and glomerulosclerosis in the male Munich Wistar Fromter (MWF) rat. We also assessed whether changes in podocyte number affect podocyte function and focused specifically on the slit diaphragm-associated protein nephrin. Age-matched Wistar rats were used as controls. Estimation of podocyte number per glomerulus was determined by digital morphometry of WT1-positive cells. MWF rats developed moderate hypertension, massive proteinuria, and glomerulosclerosis with age. Glomerular hypertrophy was already observed at 10 weeks of age and progressively increased thereafter. By contrast, mean podocyte number per glomerulus was lower than normal in young animals and further decreased with time. As a consequence, the capillary tuft volume per podocyte was more than threefold increased in older rats. Electron microscopy showed important changes in podocyte structure of MWF rats, with expansion of podocyte bodies surrounding glomerular filtration membrane. Glomerular nephrin expression was markedly altered in MWF rats and inversely correlated with both podocyte loss and proteinuria. Our findings suggest that reduction in podocyte number is an important determinant of podocyte dysfunction and progressive impairment of the glomerular permselectivity that lead to the development of massive proteinuria and ultimately to renal scarring.

Regulation of secondary cell wall development by cortical microtubules during tracheary element differentiation in Arabidopsis cell suspensions.

Cortical microtubules participate in the deposition of patterned secondary walls in tracheary element differentiation. In this study, we established a system to induce the differentiation of tracheary elements using a transgenic Arabidopsis (Arabidopsis thaliana) cell suspension stably expressing a green fluorescent protein-tubulin fusion protein. Approximately 30% of the cells differentiated into tracheary elements 96 h after culture in auxin-free media containing 1 mum brassinolide. With this differentiation system, we have been able to time-sequentially elucidate microtubule arrangement during secondary wall thickening. The development of secondary walls could be followed in living cells by staining with fluorescein-conjugated wheat germ agglutinin, and the three-dimensional structures of the secondary walls could be simultaneously analyzed. A single microtubule bundle first appeared beneath the narrow secondary wall and then developed into two separate bundles locating along both sides of the developing secondary wall. Microtubule inhibitors affected secondary wall thickening, suggesting that the pair of microtubule bundles adjacent to the secondary wall played a crucial role in the regulation of secondary wall development.

Analysis of ENOD40 expression in alb1, a symbiotic mutant of Lotus japonicus that forms empty nodules with incompletely developed nodule vascular bundles.

The alb1 mutant of Lotus japonicus (Ljsym74) forms empty nodules in which most of the bacteria remain in abnormally enlarged infection threads and fail to enter the host plant cells. The alb1 mutant was also found to be defective in differentiation of ramified nodule vascular bundles; only a single vascular bundle differentiates at the proximal end of the alb1 nodules and it fails to differentiate further. Histochemical analysis using fluorescein-conjugated wheat-germ agglutinin (F-WGA) indicated that the mutation in the ALB1 gene specifically affects the differentiation of vascular bundles in nodules. Analysis of nodulin gene expression revealed that the expression of an early nodulin gene, ENOD40, was very low in alb1 nodules. At early developmental stages of alb1 nodules, the pattern of ENOD40 transcription was essentially the same as that in wild-type nodules; transcripts were localized in dividing cortical cells and in the pericycle of the root stele opposite nodule primordia, as in wild-type nodules. However, mature alb1 nodules exhibited very weak or no expression of ENOD40 in the peripheral cells of the undeveloped nodule vascular bundle. The ENOD40 expression pattern in alb1 nodules is distinct from that in another ineffective mutant, fen1 (Ljsym76), in which ENOD40 expression persists prior to premature senescence. These findings lead us to speculate that ENOD40 may play a role in the differentiation of nodule vascular bundles.

Increased number of cardiomyocytes in cross-sections from tachycardia-induced cardiomyopathic hearts.

Based on positive identification of DNA replication and mitotic division in cardiomyocytes isolated from failing hearts, it has been proposed that adult ventricular cardiomyocytes can gain the capacity to proliferate with progression of heart failure. However, due to the lack of a reliable method to distinctly image individual cardiac cells within the myocardial syntitium, such a concept still remains largely controversial. In the present study, we used laser confocal microscopy, to image cross-sections of intact myocardium stained with fluorescein-conjugated wheat germ agglutinin and propidium iodide. This approach allowed to clearly separate the profile of individual myocytes within cardiac tissue sections. We found that in the left ventricles of dogs, subjected to tachycardia-induced cardiomyopathy, the number of cells was significantly increased in both longitudinal and transversal sections. Treatment with the angiotensin-converting enzyme inhibitor, enalapril, reversed these changes to values similar to those found in controls. Therefore, this study provides evidence, at the in situ level, for cellular hyperplasia in heart failure. This supports the more general notion that adult cardiomyocytes may not be terminally differentiated, and that an increase in cell number could contribute to the increase in left ventricular mass observed with progression of disease.

Accumulation of WGA Receptors in the Cleavage Furrow during Cytokinesis of Sea Urchin Eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.

The interaction of avian spermatozoa with the perivitelline layer in vitro and in vivo.

The differences in the hydrolytic activity of chicken spermatozoa towards the inner perivitelline layer in situ and when isolated from the ovum were investigated. Points of hydrolysis produced by chicken spermatozoa on the inner perivitelline layer of laid and freshly ovulated eggs were viewed as dark holes after staining with fluorescein-conjugated wheat-germ agglutinin. In laid eggs that had been fertilized in vivo, these holes appeared more frequently on the perivitelline layer overlying the blastodisc at the animal pole than on that overlying the vegetal pole. However, in an assay in which fragments of the inner perivitelline layer from freshly ovulated eggs were incubated with spermatozoa in vitro, points of hydrolysis appeared with similar frequency on the perivitelline layer from over the germinal disc at the animal pole or from that over the vegetal pole. No difference could be demonstrated in the electrophoretic profile of peptides extracted from the inner perivitelline layer at the animal or vegetal pole. The factor(s) responsible for either increased attraction of spermatozoa to, or for increased activity at, the perivitelline layer of the animal pole does not appear to be associated with the perivitelline layer itself.

Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins

Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.

The effect of oral contraceptives on rat platelet membrane glycoproteins

Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of 125I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets.

The purification and immunocytochemical localization of the major iodinatable cell surface glycoproteins of Chinese hamster ovary cells.

ABSTRACT A group of high molecular weight, acidic glycoproteins (HMWAG) are enriched in a plasma membrane subfraction of low density that can be isolated from Chinese hamster ovary (CHO) cells. The HMWAG represent the major components labelled by either lactoperoxidase-catalysed iodination of whole cells or by 125I-labelled wheat germ agglutinin (WGA) overlays of the membrane fraction after it had been analysed by 2-dimensional electrophoresis. The HMWAG were solubilized from membrane in Triton X100/borate buffer and purified by affinity chromatography on WGA-Sepharose. Antisera prepared against these glycoproteins in mice bound to several CHO cell lines including the WGA-resistant line Pro-5 WGAB1. Indirect immunofluorescence experiments performed at 4 °C showed that the glycoproteins had a fairly uniform but punctate distribution over the cell edge and dorsal surface of substratum-attached cells. Filopodia projecting along the substratum were also intensely labelled. After permeabilizing the cells the HMWAG were also shown to be located in cytoplasmic vesicles and in a perinuclear zone, which is assumed to be Golgi-associated. Electron microscopic observations using indirect immunoferritin procedures confirmed that the cell surface HMWAG were heavily concentrated on microvilli. The distribution of label seen with fluorescein-conjugated WGA was similar to that noted with fluorescent antibody, except that WGA was more uniformly distributed on the cell surface and also bound to the nuclear envelope in permeabilized cells. However, if cells labelled at 4 °C were warmed to 37 °C for 30 min the patterns of internalization of WGA and antibody were distinct. With the lectin, label rapidly became concentrated in the perinuclear zone, whereas with antibody the microvilli became denuded of label, which was then internalized into cytoplasmic vacuoles just beneath the plasma membrane. These results confirm that the HMWAG are concentrated on regions of the cell surface that are likely to produce vesicles readily upon cell breakage. They also suggest that pattern of internalization of a cell surface glycoprotein may be influenced by the nature of the ligand that binds to it.

Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D. Hyphal tips and septae were strongly labelled by short pulses of [3H] N-acetyl-D-glucosamine ([3H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA). The dynamics of wall formation was followed by ‘pulse and chase’ as well as by ‘pulse and wash’ treatments in which the colony was shortly exposed to [3H]GlcNAc and then freed from the radioactive chitin precursor. Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis. FITC-WGA binding, [3H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.

Hyphal walls of isolated lichen fungi: autoradiographic localization of precursor incorporation and binding of fluorescein-conjugated lectins.

The hyphal walls of three mycobionts, isolated from the lichens Xanthoria parietina, Tornabenia intricata and Sarcogyne sp. were investigated by two techniques: microautoradiography of fungal colonies exposed to radioactive carbohydrate precursors: and binding, in vivo, of fluorescein conjugated lectins to hyphal walls of such colonies. N-[3H] acetylglucosamine was readily incorporated into tips, young hyphal walls and septa of the three mycobionts and the free-living fungus Trichoderma viride, but not into Phytophthora citrophthora, indicating that chitin is a major component of the mycobionts' hyphal walls. All three mycobionts, but neither of the free-living fungi, incorporated [3H] mannose and [3H] mannitol into their hyphal walls. Fluorescein-conjugated wheat germ agglutinin was bound to the hyphal walls of the three mycobionts and T. viride, but not to the walls of P. citrophthora; the binding pattern was similar to the grain pattern obtained in autoradiographs after short N-[3H]acetylglucosamine labelling. As wheat germ agglutinin binds specifically to chitin oligomers, the lectin binding tests further confirmed that chitin is a mycobiont hyphal wall component. Binding characteristics of several fluorescein-conjugated lectins to the three mycobionts indicated that this technique can yield useful information concerning the chemical composition of hyphal wall surfaces.