G.A. De Zoeten

Interaction of bacteria and host cell walls: its relation to mechanisms of induced resistance☆

Abstract Cells of avirulent (B1) and incompatible (S210) strains of Pseudomonas solanacearum attach readily to the walls of tobacco mesophyll cells. By 4 h after infiltration of tobacco leaves with 10 8 bacterial cells/ml, fibrillar and granular material extruded from the host cell walls and bound by the outer wall layer envelop the attached bacteria. At the site of attachment, the host cell wall is frequently eroded, the plasmalemma separates from the cell wall and becomes convoluted and numerous membrane-bound vesicles accumulate in the space between the plasmalemma and the cell wall. With these bacteria, a hypersensitive reaction (HR) develops by 6 to 12 h after infiltration; as a result, the host cell collapses and organelles are deranged. In contrast, virulent strains of the pathogen (K60) are not attached and remain free to multiply in the intercellular fluid, causing no visible changes in organelle structure during the first 12 h after infiltration. Saprophytic bacteria ( Bacillus subtilis, Escherichia coli ) are attached and enveloped, but do not cause a visible HR. When heat-killed B1 cells are infiltrated into tobacco leaves, the dead bacteria attach in the same manner as avirulent, live cells and the initial host cell responses that envelop the bacteria are observed, but there is no cell collapse. After 24 h, challenge inoculation of the pretreated leaves with 10 8 live B1 cells/ml does not result in the HR and these bacteria do not attach to and are not enveloped by the host cell walls. Prevention of the HR appears to be related to this lack of attachment to the host cell walls.

Association of cowpea mosaic virus-induced double-stranded RNA with a cytopathological structure in infected cells.

Abstract A cytopathological structure in cowpea mosaic virus (CPMV) infected cowpea leaves 4 days after inoculation was characterized ultrastructurally. The fraction of a homogenate of infected leaves sedimenting at 1000 g was fractionated on a discontinuous sucrose gradient consisting of layers of 20, 45, and 60% sucrose. The chloroplast fraction, which was found at the interface of the 20% and 45% sucrose layers, known to contain 70–90% of all CPMV-RNA hybridizable material, was further fractionated on a discontinuous gradient consisting of layers of 37, 39, 41, 43, and 45% sucrose. The material at the interface of the 37% and 39% sucrose layers (Fraction I) and that at the interface of the 39% and 41% layers (fraction II) contained 90% of all structures resembling the cytopathological structures and less than 30% of all chloroplasts. Fractions I and II contained together approximately 90% of all CPMV-RNA hybridizable material. The fractions collected from the 43% and 45% sucrose layers and the pellet (fraction III) contained more than 70% of the chloroplasts, less than 10% of the cytopathological structures, and less than 10% of the CPMV-RNA hybridizable material. It was concluded that the CPMV-dsRNA is associated with the cytopathological structures in the cytoplasm rather than the chloroplasts. Autoradiography performed on sections of intact tissue and on sections of pellets of fractions I, II, and III, from tissue treated with 3 H-uridine, thymidine, and actinomycin D, presented strong evidence that CPMV-RNA replication is associated with the vesicular constituent of the cytopathological structures.

Nuclear vesiculation associated with pea enation mosaic virus-infected plant tissue.

Abstract Nuclei of pea enation mosaic virus-infected plant cells show, in the perinuclear space, vesicles that contain DNase-digestible fibrillar material. The vesicles in the perinuclear space are probably sloughed off from the nuclear envelope while they remain contained in a vesicular membrane which originated from the outer membrane of the nuclear envelope. The packaged vesicles can be visualized in the cytoplasm of all parenchymatic cell types and in the phloem elements of the above ground plant parts. A similar cytopathic effect could not be found in tissues of viruliferous pea aphids ( Acyrthosiphum pisi ). Virus was found only in the gut contents (lumen) of viruliferous aphids and could not be identified with certainty in any other part of the aphids.

Cultivated potato chloroplast DNA differs from the wild type by one deletion- Evidence and implications*

SummaryThe chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.

Plant virus uncoating as a result of virus-cell wall interactions

The fate of tobacco rattle virus (TRV) particles was studied after leaf panel infiltration. It was found that end-on virus attachment to cell walls of hosts (Nicotiana tobacum L. var. Xanthi-nc) and nonhosts (Zea mays L.) occurred, and that a virus degradative phase commenced immediately after attachment. Length of TRV particles changed drastically following infiltration. The normal length of the particles became smaller than that of the particle carrying the coat protein gene (108 nm). Five days after infiltration, no particles could be detected on the walls of cells bordering intercellular spaces. Virus attachment and degradation was thus shown to be nonspecific. Autoradiographic studies showed that iodinated viral coat protein or a breakdown product of this protein ((125I-label in coat protein of complete virus) and not virus particles are transported to the vascular bundle after infiltration. Tobacco mosaic virus (TMV) and TRV were infiltrated into their respective local lesion hosts. There was an initial time interval early after the infiltration during which wounding (pin pricking) the infiltrated leaf panels resulted in local lesion formation. The results are discussed with regard to the phenomenon of viral genome release in the plant virus infection process.

In situ localization of pea enation mosaic virus double-stranded ribonucleic acid.

Abstract Precursor ([ 3 H]uridine) incorporation studies in the presence or absence of actinomycin D (AMD) showed that AMD-insensitive nucleic acid synthesis occurred only in the nuclei in pea enation mosaic virus (PEMV)-infected pea plant tissues. Ferritin-labeled antibody studies showed that ds-RNA was present only in nuclei. In vitro hybridization of the nucleic acid from various infected or healthy cell fractions with [ 125 I]PEMV-ss-RNA indicated that PEMV-ds-RNA was primarily associated with nuclei-enriched fractions from infected cells. An in situ hybridization technique, which utilized autoradiography to detect the subcellular location of material which hybridized with [ 125 I]PEMV-ss-RNA, was developed. This method confirmed that PEMV-ds-RNA was localized in the nuclei of PEMV-infected tissues.

The localization of pea enation mosaic virus-induced RNA-dependent RNA polymerase in infected peas

Abstract A procedure was developed for preparing cell fractions rich in chloroplasts, nuclei, and pea enation mosaic virus (PEMV)-induced cytopathological structures (vesicles). Those fractions from infected pea plants which contained nuclei or vesicles also contained actinomycin D-insensitive RNA polymerase activity and PEMV-specific hybridizable RNA. The fraction from infected plants containing predominantly chloroplasts had little of this polymerase activity or RNA, as was the case with all fractions from healthy plants. The significance of the polymerase activity in the nuclei and vesicles is discussed, as well as the potential role of the vesicles in the virus infection cycle.

The presence of viral antigen in the apoplast of systemically virus-infected plants

Abstract In healthy and tobacco mosaic virus (TMV)-infected tissues infiltrated with TMV, there are quantitative and qualitative differences in virus cell wall attachment, and in the retention of virus or its coat protein. An 125 I-labeled antibody technique was combined with cell wall digestion to determine antigen content of Nicotiana silvestris cell walls from light green (LG) and dark green (DG) areas of infected leaf tissues. The values (expressed in μg of bound 125 I-labeled TMV-U 1 -IgG/mg cell wall) ranged between 1 and 1.5 for light green (LG) and from 0.2 to 0.5 for dark green (DG) tissues of systemically infected leaves. Autoradiography with 125 I-labeled anti-TMV-U 1 -Fab fragments of IgG showed that Fab fragments can penetrate cell walls of tobacco leaf tissue, since viral antigen was shown to be present in cell walls of cells sustaining virus multiplication by this method.

Purification and properties of the replicative forms and replicative intermediates of pea enation mosaic virus

Abstract Pea enation mosiac virus consists of two nucleoprotein components with sedimentation coefficients of 95 and 115 S. Three single-stranded RNAs were obtained from purified preparations of virus. Replicative forms and replicative intermediates have been isolated from diseased tissue. These molecules were characterized with respect to buoyant density, melting properties, approximate molecular weight and length distribution. Ribonucleic acid isolated from purified virus was labeled in vitro with 125 I and the product was used in molecular hybridization experiments. It was shown that replicative form RNA hybridizes to virus RNA with greater specificity than to controls.

Observations on ectodesmata and the virus infection process

Ectodesmata were visualized in outer epidermal cell walls of Nicotiana tabacum (L.) var. T.I. 787 by means of a silver nitrate stain and electron microscopy. After inoculation of potato virus X (PVX), neither whole virus nor viral RNA could be observed in ectodesmata by means of direct observations in the electron microscope. Fully assembled virus particles were seen attached to, but not penetrating, the cuticle of inoculated tobacco leaves.