Luis Sequeira

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.

Increases in peroxidase activities are not directly involved in induced resistance in tobacco

Abstract Peroxidase activities and isozyme patterns were determined in tobacco leaves of the cultivar Bottom Special in which disease resistance had been induced by prior infiltration with heat-killed Pseudomonas solanacearum B1 cells. As compared with unprotected leaves, soluble peroxidase activity increased by 8 h after infiltration and an additional isozyme band (P1) appeared by 12 h. There were no changes in either ionically or covalently bound forms of peroxidase. Similar changes in peroxidase were observed in leaves that were infiltrated with killed bacteria and then shaded with aluminum foil, although protection was not obtained under these conditions. Similarly, in leaves infiltrated with heat-killed saprophytic bacteria (Escherichia coli B, Bacillus subtilis) which did not induce protection, peroxidase activity was increased and the P1 band appeared. Lipopolysaccharide from K60 cells of Pseudomonas solanacearum, which induced protection, also increased peroxidase activity and caused appearance of the P1 band. When leaf cells were wounded by injection with asbestos fibers, peroxidase activity increased but the P1 band was not visible. It was concluded that peroxidase increases are not directly involved in disease resistance, and probably result from injury caused by toxic compounds in the bacterial cell suspension.

Agglutination of avirulent strains of Pseudomonas sofanacearum by potato lectin

Fifty-five virulent isolates and 34 avirulent variants of Pseudomonas solanacearum from different geographic regions and representing all major races and biotypes were tested for their ability to bind to potato lectin. The lectin was extracted from “Katahdin” potato tubers and purified to homogeneity. All avirulent isolates agglutinated strongly with the lectin, whereas virulent isolates either failed to agglutinate or agglutinated only weakly and at much higher lectin concentrations. Failure of the virulent cells to bind to the lectin was correlated with the presence of extracellular polysaccharide (EPS) which is formed by virulent, but not avirulent, cells. Virulent cells were agglutinated strongly by potato lectin after most of the EPS was removed by repeated washing and centrifugation. Agglutination of avirulent cells or washed virulent cells could be totally prevented by the addition of EPS to the cells prior to addition of lectin. The weak agglutination exhibited by some unwashed virulent cells in suspension could also be prevented by addition of EPS. This indicates that the amount of EPS formed in culture and released in water suspensions determines the degree of agglutination obtained when lectin is added. Potato lectin, conjugated with fluorescein isothiocyanate, bound to rabbit erythrocytes and to avirulent, but not to virulent, cells of P. solanacearum . Moreover, binding of lectin to avirulent cells was hapten-specific and could be inhibited by chitin oligomers. Purified lipopolysaccharide (LPS) from P. solanacearum K60-B1 cells precipitated when mixed with purified potato lectin. Initial evidence indicates that binding sites for the lectin are present in the lipid A portion of the LPS.

induced resistance in tobacco leaves: The growth of Pseudomonas solanacearum in protected tissues?

Abstract Tobacco leaves develop systemic, nonspecific resistance to a wide variety of pathogens following infiltration with heat-killed cells of Pseudomonas solanacearum . The protective response was detected by challenge inoculations with a compatible strain of this bacterium at 10 9 cells/ml, carried out at0, 2, 4, 8 and 24 h after infiltration with heat-killed cells. When populations of bacteria in infiltrated tissues were determined, protection was detectable after 2 h as a lengthening of the lag phase, although growth rates at log phase remained basically unaltered. As the period between infiltration with heat-killed cells and challenge increased, the initial population introduced decreased rapidly and there was a concomitant reduction in the growth rate established after a protracted lag period. In fully protected leaves (24 h after infiltration) bacterial numbers decreased rapidly immediately following inoculation and populations leveled off at approximately 10 7 cells/ml, but tissues remained symptomless. Similar results were obtained at lower inoculum levels (10 6 cells/ml, bxcept that, after a sharp initial reduction in the number of cells and a protracted lag, populations increased and then stabilized at approximately 10 7 cells/ml. When an incompatible strain was used in the challenge inoculation, bacterial populations decreased more rapidly in the protected, symptomless tissues than in unprotected ones undergoing a hypersensitive reaction. These changes in bacterial populations indicate that protection may result from the release of inhibitory compounds into the intercellular spaces. In addition, cells of protected leaves appear to become more tolerant than normal cells to the presence of relatively high numbers of bacteria.

Comparison of soluble proteins associated with disease resistance induced by bacterial lipopolysaccharide and by viral necrosis

Abstract Infiltration of tobacco leaves with bacterial lipopolysaccharide (LPS) results in the acquisition of systemic resistance to subsequent bacterial infection. By means of gel electrophoresis, we have investigated the changes in soluble protein composition of Nicotiana tabacum cv. “Bottom Special” leaves after they were infiltrated with LPS from Pseudomonas solanacearum . A new protein band (approximate molecular weight 31800) was present in leaves infiltrated with LPS, but not in those infiltrated with phosphate buffer. In time course experiments, the accumulation of the new protein as well as the increase in relative content of at least two other proteins were correlated with the appearance of resistance to bacterial multiplication in tobacco tissues. The new protein band did not co-migrate with the “pathogenesis-related” (PR) proteins that accumulated in tobacco leaves infected with tobacco necrosis virus or tobacco mosaic virus (strain 1952-D). The PR proteins were present in tissues infiltrated with LPS as well as in extracts of tissues infiltrated with virulent and avirulent strains of P. solanaceamm , but their presence was not correlated with the acquisition of resistance to bacterial multiplication.

A hydroxyproline-rich bacterial agglutinin from potato: its localization by immunofluorescence

Abstract Potato tubers (cv. Katahdin) contain a hydroxyproline-rich glycoprotein (HPRG) that agglutinates certain avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum . This and similar agglutinins are thought to play an important role in the immobilization of incompatible bacteria in potato and tobacco tissues. The agglutinin from potato tubers was purified by ion exchange chromatography. Antisera to the intact or deglycosylated agglutinin were obtained from New Zealand white rabbits after multiple intradermal and intramuscular injections. Immunoglobulins were precipitated with (NH 4 ) 2 SO 4 and antibodies specific for the agglutinin were purified by affinity chromatography. Frozen sections of petiole or leaf tissue from tobacco and potato were treated firstly with sheep normal immunoglobulin and then with either anti-agglutinin antibodies or normal rabbit immunoglobulin for 20 min. The sections were rinsed and then treated with fluorescein isothiocyanate-conjugated sheep anti-rabbit immunoglobulin. When the sections were examined by fluorescence microscopy, it was determined that anti-agglutinin antibodies bound only to the cell walls, particularly those of parenchyma. Fluorescence was also evident on the cell walls of tobacco and potato xylem vessels, epidermis, and collenchyma. Control sections treated with normal rabbit immunoglobulin did not bind the labelled anti-rabbit immunoglobulin. Cell walls in tissue sections from non-solanaceous plants such as soybean, corn, or begonia, treated in the same manner, were also stained by the labelled antibodies. Antibodies to both intact and deglycosylated potato agglutinin bound to these plant cell walls, indicating that the receptors are proteins with antigenic determinants which are similar to those of proteins from potato or tobacco cell walls. Such proteins (HPRGs) are common components of plant cell walls and may play a role in immobilizing bacteria that gain access to the intercellular spaces.