Susan B. Jones

Microscopy of the interaction of hrp mutants of Pseudomonas syringae pv. phaseolicola with a nonhost plant

Abstract Pseudomonas syringae pv. phaseolicola is a pathogen of green bean and induces localized, rapid plant cell death (the hypersensitive response or HR) when placed into leaves of the nonhost plant tobacco. The hrp genes control both pathogenicity and the ability to cause the HR. In this study we examined the interaction of the wild-type parent strain (NPS3121) and three independent, prototrophic hrp mutants (NPS4000, NPS4003, NPS4005) with tobacco by use of light and transmission electron microscopy. Even though the hrp mutants did not induce macroscopic symptoms on leaves by 48 h postinoculation, localized disruption of parenchyma cells did occur. The rapidity and severity of microscopic symptoms was related to the relative abilities of the mutants to cause macroscopic symptoms on green bean. Cells of the hrp mutants exhibited extensive surface blebbing. The increased surface blebbing of the mutants may reflect either outer membrane disorganization and/or a reduced ability to cope with an hypoosmotic apoplastic environment by production of periplasmic glucans. Unlike induction of a macroscopic HR, induction of more subtle responses by nonhost plant cells is not dependent on the presence of an intact hrp cluster.

Sedimentation field flow fractionation of mitochondrial and microsomal membranes from corn roots

Sedimentation field flow fractionation (sed-FFF) is shown to be a valuable procedure for analysis of a wide variety of subcellular particle preparations. The principles underlying this relatively new separation procedure are described. Separation is based on differences between particles in mass and/or density. As in chromatography, the procedure involves relating on-line or off-line measurements made on the effluent from the separation chamber to the elution (retention) time. In this work effluents were monitored for absorbance at 254, 280, and/or 320 nm; collected fractions were assayed for protein content, total ATPase activity, and/or marker enzyme activities and, when appropriate, were examined by electron microscopy. The ratio of the absorbances at 254 and 320 nm was found to provide a sensitive measure of partial resolution of subcellular particles. Preparations containing all of the subcellular particles of corn roots (exclusive of nuclei, cell walls, and ribosomes), and fractions thereof enriched in mitochondria, microsomes, Golgi membranes, or plasma membranes, were examined by sed-FFF. The subcellular particles appear to remain largely intact. All of the particles observed had a mass less than 2 X 10(11) g/mol. All of the preparations were grossly heterogeneous with respect to effective mass distribution. This is due in part to heterogeneity with respect to the organelle of origin. In microsome preparations, components of low, medium, and high density were present in the unretained peak; the retained region had comparatively more high density particles. Plasma membrane preparations had a very wide effective particle mass distribution. The observations suggest that, in addition to its utility for analytic purposes, sed-FFF is likely to prove useful for micro-preparative fractionation of some subcellular particle preparations. Sed-FFF and density gradient centrifugation can be utilized as complementary methods.

Stress metabolite accumulation, bacterial growth and bacterial immobilization during host and nonhost responses of soybean to bacteria

Abstract Hypersensitive response inducing (HR-inducing) strains of the heterologous (non-soybean) pathogens Pseudomonas syringae pv. phaseolicola and P. syringae pv. syringae attained populations in soybean leaves only three-fold to five-fold less than a compatible race of the soybean pathogen P. syringae pv. glycinea. Incompatible (HR-inducing) races of P. syringae pv. glycinea and a non-HR-inducing strain of the heterologous pathogen Corynebacterium flaccumfaciens pv. flaccumfaciens showed more restricted growth. A non-HR-inducing strain of the heterologous pathogen Erwinia carotovora subsp. atroseptica and a strain of the saprophyte Bacillus cereus did not grow in soybean leaves. Incompatible races of P. syringae pv. glycinea and the heterologous pseudomonads induced accumulation of isoflavonoids (daicizein, formononetin, genistein, glyceollin) and isoflavone glucosides (daidzin, genistin, ononin). Bacillus cereus induced accumulation of isoflavone glucosides alone, and C. flaccumfaciens pv. flaccumfaciens and E. carotovora subsp. astroseplica did not induce accumulation of either type of stress metabolite. Of the purified compounds tested, glyceollin alone had significant antibacterial activity, but only against Cram-positive bacteria. By use of in vitro bioassays, no evidence was obtained to indicate that induction of additional inhibitory compounds or inhibitory activity due to total isoflavonoid content of inoculated leaf tissue was responsible for resistance. Immobilization of bacteria by highly electron-dense material in intercellular spaces of soybean leaves occurred to the greatest extent with E. carotovora subsp. atroseptica and B. cereus, and to a lesser extent with C. flaccumfaciens pv. flaccumfaciens. No bacterial immobilization was evident at 48 h after inoculation with incompatible races of P. syringae pv. glycinea or heterologous pseudomonads.

Purification of cell wall fragments by sucrose gradient centrifugation

SummaryA procedure for purification of cell wall fragments was developed. The method utilizes sucrose density gradients to efficiently remove soluble enzyme and membrane contaminants from the cell wall. Purification at each stage was monitored biochemically by the removal of cytoplasmic associated markers and ultrastructurally by thorough electron microscopic examination of the isolated cell wall fractions. Cell walls purified by the procedure were compared to those purified by the more conventional multiple washing procedure.