G. Gaard

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.

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.

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.

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.

Distribution and appearance of alfalfa mosaic virus in infected plant cells

Abstract A virus isolated from Capsicum annuum L. was identified by host range, electron microscopy, and serology to be alfalfa mosaic virus (AMV). This isolate of AMV contained at least four components, Ta, Tb, M, and B with lengths 25, 40, 55, and 64 mμ, respectively. When subjected to high speed centrifugation, components Tb and M formed small crystalline arrays in the virus pellets. Similar crystalline AMV structures were found in some mesophyl cells of Capsicum , in sharply delimited epidermal areas of Vigna , but not in Nicotiana , where abundant uncrystallized virus was observed in the cytoplasm of infected cells. Measurements of AMV in the different hosts were compared with measurements of partially purified AMV. Components Tb and M were the only two components that could be recognized with certainty in the different hosts. We argue that the crystalline appearance of AMV in Capsicum and Vigna is a result of the loss of compartmentalization of certain basic proteins and some divalent cations that cause crystallization of AMV in vitro . The virus infection is responsible for a host reaction (necrosis) that results in loss of compartmentalization of these compounds. In Havana 38 tobacco, no necrosis occurred and no aggregation was observed. No change in component composition of the AMV population was detected after two passages through each of the three hosts.

Localization of some heme-containing enzymes in healthy and pea enation mosaic virus-infected plants

Abstract The localization of catalase and peroxidases (heme-proteins) by DAD (3,3′-diaminobenzidine) in pea enation mosaic virus-infected tissue is similar to that in healthy tissue. No cellular areas or organelles other than those known to contain these heme-proteins in healthy tissues seem to be responsible for the increased catalase and peroxidase activities in diseased tissue. Cell fractionation studies were carried out to confirm the results obtained by means of electron microscopy, but were inconclusive.