The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. tnifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 ,ug per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by N03 or NH4', and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-0-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (0-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.Establishment of an effective Rhizobium-legume symbiosis can be viewed as a process of cellular recognition and compatibility between bacterial and plant cells. The infection process involves bacterial attachment, root hair deformation, bacterial penetration of the root hair wall, formation and sustained development of the infection thread, bacterial release from infection threads within emerging root nodule cells, and bacterial differentiation into nitrogen-fixing bacteroids.The lipopolysaccharides (LPS) of rhizobia are likely to be involved in the infection process. They are major glycoconjugates on the surface of Rhizobium leguminosarum biovars * Corresponding author. t Present address: Laboratoire des Relationes Plantes-Microorganismes,

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC ؊ mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC ؊ mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.

Suspension cell cultures of Nicotiana tabacum L. inoculated with the incompatible pathogen Pseudomonas syringae pv pisi undergo a hypersensitive reaction. Addition of the singlet oxygen quencher bixin to cell suspensions had no effect on hypersensitive cell death. Addition of the singlet oxygen quencher 1,4-diazabicyclo octane (DABCO) increased the medium pH and delayed the onset of cell death. This delay was eliminated when cell suspensions were buffered, and could also be induced by increasing medium pH with KOH. Bixin and DABCO also did not suppress the hypersensitive reaction in tobacco leaves. These data do not support a role for singlet oxygen in the hypersensitive reaction. Medium pH, however, appears to be a critical factor in cell suspension cultures.Several reports have suggested that active oxygen-induced lipid peroxidation may play a role in the HR,3 a defense reaction commonly induced by plant pathogens following inoculation of incompatible hosts. Keppler and Novacky (10) reported increases in lipid peroxidation during the HR in inoculated cucumber cotyledons and postulated that this increased lipid peroxidation leads to the typical loss of electrolytes during the HR. They further demonstrated that the HRinduced lipid peroxidation could be inhibited by the addition of SOD (1 1 ('02) is an excited state of molecular oxygen which can be generated in a number of ways including the spontaneous dismutation of two 2-radicals, and by the interaction of 02 with the triplet state of photosensitizing compounds such as Chl (7, 13). '02 is a very potent inducer of lipid peroxidation and membrane damage (13). There has been one brief report that 3-carotene (a singlet oxygen quencher) increased bacterial populations in an incompatible host when coinjected into leaves, suggesting an involvement of '02 in the HR (16). The purpose ofthis study was to further investigate a possible role of '02 in the HR.In this study we investigated the effects oftwo singlet oxygen quenchers, DABCO and the carotenoid carboxylic acid bixin, on the HR induced in leaves and in cell suspension cultures of tobacco following inoculation with the incompatible pathogen Pseudomonas syringae pv pisi. Both ofthese compounds are potent 102 quenchers and have been shown to protect tobacco cell suspension cultures against photosensitizer-generated 102 (3). Suspension cultured cells have been used in previous studies of bacterially induced HR (1,2). In this study, changes occurring in cells undergoing the HR were followed by using the fluorescent vital stain FDA (15, 19). Decreased staining of cells by FDA has been correlated with progress of the HR in cell suspension cultures (9). MATERIALS AND METHODS BacteriaCultures ofPseudomonas syringae pv pisi Young, Dye, and Wilkie were originally obtained from R. N. Goodman, University of Missouri. Cultures were grown for 24 h at 28°C on nutrient agar (Difco) with 0.5% yeast extract and 1.0% dextrose (NYDA) at pH 7.0. Bacteria were suspended in sterile water or 50 mM Mes buffer (pH 6.0) and the c...

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