Willem G. Langenberg

Immunohistochemical localization of barley stripe mosaic virions in infected wheat cells

Barley stripe mosaic virus particles were localized in ultrathin sections with colloidal gold-labeled specific IgG or antiserum followed by gold-labeled goat anti-rabbit IgG. On the average, 1.5 gold particles were attached per virus rod. A statistical analysis of counts of gold and virus particles showed that the staining procedure was highly reproducible from experiment to experiment and after several independently prepared colloidal gold solutions. The procedure should be useful for the intracellular localization of any protein to which an antibody can be prepared.

Chronology of appearance of barley stripe mosaic virus protein in infected wheat cells

By following sequential cell-to-cell infection from the meristematic point back toward older cells, the chronological appearance of viral protein in barley stripe mosaic virus (BSMV)-infected wheat root and shoot tips was examined by immunoelectronmicroscopy. In infected root tips, progression of infection was followed in a series of the first 20 infected cells and divided into four cytological stages. In stage I, peripheral vesicles in proplastids acted as infection initiation markers. No BSM virions were seen, but viral protein could be first detected on vesiculated proplastid membranes by gold—IgG complexes at late stage I. Sequentially, BSM virions appeared mostly at right angles to the proplastid membrane at stage II and to the endoplasmic reticulum at stage III. In stage IV, but not before or after, viral protein appeared in abundance in nuclei while few or no virions were present. Viral protein occurred only in the euchromatin region and within fibrillar centers in nucleoli, never in the heterochromatin region. By gold—nuclease staining, euchromatin was the main location of nuclear DNA. No excess viral protein could be detected in cell extracts of systemically infected tissue. The chronological appearance of viral protein in infected shoot tips followed four infection stages in root tips. The presence of peripheral vesicles in proplastids could be used as infection initiation markers in root tips but not in shoot tips of infected plants; vesiculation was a normal occurrence in young proplastids of healthy and infected shoot tissue, did not occur in mature chloroplasts of healthy shoots, and was rarely seen in fully developed chloroplasts in infected shoot tissue.

Chilling of tissue before glutaraldehyde fixation preserves fragile inclusions of several plant viruses

Glutaraldehyde—osmium fixation showed the presence of crystalline inclusions of brome mosaic virus (BMV), turnip yellow mosaic virus (TYMV) [and its protein], and potato virus X (PVX) in host tissues provided the tissue was vacuum-infiltrated with buffer and chilled a minimum of 2 hr before chilled glutaraldehyde was allowed to diffuse in. Tobacco mosaic virus (TMV) inclusions could only be preserved if osmium tetroxide postfixation was omitted. Cowpea mosaic virus (CPMV) crystalline inclusions were not observed with this method. Glutaraldehyde did not fix gelatin at concentrations below 2.5% unless the protein was first gelled at 5°C. Bovine serum albumin was not crosslinked by glutaraldehyde at concentrations of less than 3% at 5°C or of 4% at 25°C. Attempted fixation of low concentrations of gelatin and bovine serum albumin by glutaraldehyde at 5 or 25°C paralleled preservation or nonpreservation, respectively, of viral crystalline inclusions of TYMV, BMV, PVX, and TMV in expanded leaf tissue. The inability of glutaraldehyde to crosslink low concentrations of protein has consequences for the interpretation of the intracellular ultrastructural location of virus or virus-related products in expanded leaf tissues. It is proposed that a minimum protein concentration is required for glutaraldehyde to fix cells and that this minimum protein concentration decreases with decreases in temperature.

The ultrastructural appearance of cowpea mosaic virus in cowpea

Cowpea mosaic virions (CPMV) were present in large numbers in cowpea tissue, both in dispersed and crystalline aggregates. Virion aggregates were found after buffered glutaraldehyde or chromate fixation at pH 7.2. Five to 10 min in 5% glutaraldehyde was sufficient for good fixation and yielded sections of greater electron-density than prolonged glutaraldehyde fixation. Virions were seen in all types of cells of leaf tissue in the cytoplasm and in the central and cytoplasmic vacuoles, but not in cell organelles. Purified, fixed, and embedded virions and those in crystalline aggregates measured 19 nm. Negatively stained, purified virions were 28.5 nm and, after glutaraldehyde fixation, 26 nm. Viroplasms in CPMV-infected cells consisted of a dense, fine matrix in close contact with a membranous, endoplasmic-reticulum-derived body. The membranous system was also seen within the nucleus. In one instance, the fine dense matrix of the viroplasm had a regular gridlike appearance, with CPMV aligned along the grid pattern in a semicrystalline form.

Identification of barley yellow mosaic virus by immuno-electron microscopy in barley but not in Polymyxa graminis or Lagena radicicola

SamenvattingGerstegeelmozaïekvirus (BaYMV) werd in een perceel wintergerst (cv. Igri) gevonden by Wittem in Limburg, Nederland. Het virus werd met immuno-elektronen-microscopie geïdentificeerd in bladweefsel als BaYMV. In de wortels van geïnfecteerde planten werden alle vormen die van de schimmel-vector (Polymyxa graminis Led.) bekend zijn aangetroffen nl. cystosori, plasmodia en zoosporangia. Inwendig kon geen BaYMV worden aangetoond of waargenomen inPolymyxa ofLagena spp.

Disruptive influence of osmic acid and unbuffered chromic acid on inclusions of two plant viruses.

The various components of Daltons chrome-osmium were used separately and in sequence. Results were compared with chrome-formalin-osmium, formaldehyde-osmium, and glutaraldehyde-osmium fixed tissues. Wheat streak mosaic virus (WSMW) and tobacco mosaic virus (TMV) inclusions were stable if tissues were fixed with 0.008 M chromate, buffered with phosphate at pH 7.2, but not with unbuffered chromate. Osmic acid post-fixation did not change WSMV inclusions but dissolved TMV inclusions. Osmic acid prefixation destroyed viral inclusion integrity for both viruses. It is shown that when plant tissues are fixed with Daltons chrome-osmium, osmium prefixation and dichromate postfixation occur. This fixative initially contains a mixture of osmic acid and chromate. With our chrome-osmium fixative, chromate prefixation, and osmic acid postfixation take place. The sequence of fixation of the tissue and pH control, were all important in the maintenance of the viral crystal structure of WSMV and TMV. Glutaraldehyde-osmium fixed tissues appeared leached compared with the chrome—osmium and formaldehyde—osmium fixed tissue and viral inclusions were not preserved.

Soil-borne wheat mosaic virus protein interactions with wheat streak mosaic virus cylindrical inclusions☆

Abstract Cylindrical inclusions (CI) of wheat streak mosaic virus (WSMV) became labeled with antibody to soil-borne wheat mosaic virus (SBWMV) in thin sections of wheat leaf cells infected with both viruses. Either the capsid protein or one of the other two proteins coded by RNA II of SBWMV must bind to the WSMV inclusion body. However, the WSMV inclusions did not become labeled with antibody to SBWMV in leaf cells with a mixed infection of WSMV and an RNA II deletion mutant of SBWMV. The deleted RNA II codes for capsid (19.7K) and 28K proteins, but not for the 90K protein found with wild-type SBWMV infections. All three proteins react with antibodies to virions. It is tentatively concluded that the 90K protein of SBWMV binds to WSMV inclusion bodies in cells infected with both viruses. Fewer WSMV cylindrical inclusions were aligned penpendicular to cell walls in cells with mixed infections and in which the inclusions were labeled with SBWMV antibody than in either singly or doubly infected cells where the inclusions did not become so labeled.

Transgenic tobacco plants expressing the bacterial mc gene resist virus infection

A bacterial rnc gene coding for a double-stranded RNA-dependent RNase III endoribonuclease and a mutant, rnc70, were expressed in tobacco plants. The RNase III protein produced in the transgenic plants was the same size as the bacterial protein. Expression of the wild-type gene could cause stunting in some plant lines, but not in others. Expression of the mutant protein did not affect normal growth and development of the transgenic plants. Transgenic plants of the R1 and R2 generations, expressing the wild type, as well as a mutant protein, were resistant to infection by three disparate RNA plant viruses with a divided genome but not against two viruses with a single-stranded RNA genome. Introduction of the rnc gene in crop plants may provide resistance to economically important virus diseases.

THE NEAR‐SPINELESS TRACHELOMONAS GRANDIS (EUGLENOPHYCEAE) SUPERFICIALLY APPEARS SPINY BY ATTRACTING BACTERIA TO ITS SURFACE1,2

Trachelomonas grandis Singh has a mucilaginous, highly porous mineralized lorica (envelope) generally without ornamentation except occasionally for a few short, tapered, anterior or posterior spines. However, as our first cultures of this species aged, rod‐shaped structures appeared on the loricas. That these surface projections were bacteria was determined by scanning and transmission electron microscopy. The bacteria, 2‐6 μm long with rounded apices, were oriented perpendicular to the exterior lorica surface and were attached on one end by apically produced tie‐down fibers. The bacteria also secreted fibers over their entire surface, forming a network between them that collapsed during specimen preparation for scanning electron microscopy. The density of the surface bacteria increased with time until the lorica took on a “spiny” appearance superficially similar to lorica extensions of algal origin. In mature algal specimens, an estimated 1200‐1800 bacteria per lorica occurred as a monolayer, the maximum number related to the surface area of the lorica available for bacterial colonization. The bacteria, always motionless while attached, showed putative evidence of budding. Fission formed short chains of up to three cells on the lorica. Our cultures maintained this association for 8 years (1972‐1979). However, cultures ordered for further study in the past year have failed to develop loricas with more than just a few bacterial cells, and most have none.

Sorghum chlorotic spot virus binds to potyvirus cylindrical inclusions in tobacco leaf cells

Abstract Nicotiana benthamiana can be doubly infected with either potato virus Y or tobacco etch virus and sorghum chlorotic spot virus (SCSV). Immunogold labeling showed that cylindrical inclusions of either potyvirus bind virions of the unrelated rod-shaped furovirus SCSV. Not all cells in doubly infected N. benthamiana plants contained both viruses. In cells infected by the potyviruses but not by SCSV, cylindrical inclusions did not label with the antiserum to SCSV. Numbers of cells infected with SCSV did not increase in doubly infected plants compared to those in plants infected with SCSV alone. Systemic infection of N. benthamiana by either potyvirus was not prevented by SCSV infections. This provides further evidence that unrelated rod-shaped viruses can bind to potyvirus cylindrical inclusion bodies, and that this phenomenon is not limited to graminaceous hosts.