l h e inoculation of tobacco (Nicofiana fabacom L.) suspension cells with bacterial pathogens that elicit the hypersensitive response (HR) in leaves has been shown to elicit production of active oxygen. This response occurs in two phases, the second of which occurs 1 to 3 h after bacterial addition and is unique to HR-causing interactions. The relationship between the phase II active oxygen response and the HR was characterized using Pseudomonas syringae pv syringae and P. floorescens (pHIR1 l), which contains a cosmid clone of the brp/hrm region from P. syringae pv syringae. TnpboA mutations in complementation groups I1 through Xlll of the brp cluster blocked the phase II active oxygen response, whereas mutations in the group I brmA locus did not affect phase II. Despite the normal active oxygen response, bacteria with mutations in the brmA region did not cause the H R in intact tobacco leaves nor did they induce hypersensitive cell death in cell suspensions. The data indicate that the bacteria do not require the hrmA region to elicit active oxygen production, but a full and intact hrp/brm region is required to elicit hypersensitive cell death. Therefore, the phase II active oxygen response does not directly cause hypersensitive cell death nor is the response itself sufficient to trigger the HR.Our laboratory has been interested in plant recognition mechanisms involved in triggering plant defenses, specifically the HR. The first step in doing this was to identify the earliest possible plant responses unique to incompatible interactions that result in HR. Using cell suspensions of tobacco (Nicotiana tabaciim L.) and soybean, we have identified two plant responses that precede cell death by severa1 hours in incompatible interactions (Baker et al., , 1993aOrlandi et al., 1992). The XR involves a net uptake of Hf and efflux of Kf from the plant cell (Atkinson et al., 1985). The AO response involves two distinct phases of AO production: phase I is the immediate production of AO after the addition of compatible, incompatible, and saprophytic bacteria; phase I1 is elicited 1 to 3 h later only by , 1989; Baker et al., 1991,199313;Orlandi et al., 1992;Levine et al., 1994). The correlation among phase I1 AO production, the XR, and hypersensitive cell death has been consistent in severa1 plant/bacteria systems tested (for a review, see .The hrp/hrm genes of P.s.s. have been shown to govern the triggering of the HR and XR in tobacco. Transfer of the cosmid pHIR11, which contains the P.s.s. hrpbrm gene cluster, into P j . enabled the nonpathogen to elicit the HR and the XR (Huang et al., 1988). TnpkoA mutagenesis of pHIRll revealed 13 complementation groups (Huang et al., 1991). Mutations in 12 of the complementation groups resulted in the loss of the ability to elicit both the HR and the XR. It now appears that these 12 complementation groups compose the core krp region responsible for producing and exporting harpin,,,, which may be at least partially responsible for these responses (He et al., 1993).We report here that t...

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Stimulation of active oxygen metabolism occurs during the early stages of interactions involving bacteria and plant cell suspensions. Although many cellular processes are known to affect active oxygen metabolism in plants, it is not known which of these factors affect active oxygen levels during plant-bacteria interactions. Extracellular peroxidases have been shown to participate in both the production and utilization of active oxygen species such as H,O, and superoxide. Catalase and other scavenging mechanisms also affect the overall level of active oxygen. In this study the luminol-dependent chemiluminescent reaction previously used to measure H,O, levels in suspension cells was modified to allow the assay of both peroxidase and H,O,-scavenging activity. The early stages of the interactions between tobacco (Nicofiana fabacum) and Pseudomonas syringae pv syringae, as well as between soybean (Glycine max) and P. syringae pv glycinea, were investigated. This method of monitoring peroxidase and H,O,-scavenging activity proved to be rapid, sensitive, and nonintrusive, allowing the processing of multiple samples using intact cells or cell-free preparations. The results from the study demonstrate that the scavenging activities can be significant and must be considered when studying active oxygen production in biological interactions.As the study of plant-microbe interactions has become more molecular, there has been an increased use of specialized model systems to allow the study of specific phenomena. Studies of the bacteria-induced HR found that inoculation with incompatible species produces rapid plant cell death in cell suspensions similar to that found during the HR in whole plant tissues (Baker et al., 1993a;Baker and Mock, 1994). The use of suspension cells in addition to whole plants has allowed identification of early plant responses, which precede hypersensitive cell death (Atkinson et al., 1985b;Keppler et al., 1989;Baker et al., 1991Baker et al., , 1993a. One of these responses is the production of active oxygen. The response consists of a rapid, transient production a few minutes after addition of the incompatible bac-