The Active Oxygen Response of Cell Suspensions to Incompatible Bacteria Is Not Sufficient to Cause Hypersensitive Cell Death

Abstract

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...

Measurement of Ca2+ Fluxes during Elicitation of the Oxidative Burst in Aequorin-transformed Tobacco Cells

“…a pathway commonly inaugurated by Ca 2ϩ channels (47). Finally, harpin, a heat stable protein from the bacteria E. amylovora was chosen, because it induces the oxidative burst (11,12) and may promote the hypersensitive response (42,48) in tobacco. Importantly, in addition to serving as potent inducers of the oxidative burst, all of the above elicitors are available in relatively pure form, confirming that any Ca 2ϩ transients observed will have been induced by the same component in the preparation that is responsible for stimulating the oxidative burst.…”

The hypersensitive response and the induction of cell death in plants

“…In contrast, using a different system (soybean cultured cells treated with H 2 O 2 ), Levine et al (1996) demonstrated that the oxidative burst precedes and stimulates a rapid influx of Ca 2+ , leading to cell death. Glazener et al (1996) showed that a mutant of Pseudomonas syringae pv syringae unable to trigger either an HR on tobacco leaves or cell death in cell suspensions was still able to elicit a normal XR and oxidative burst in cell suspensions. Thus the oxidative burst and the XR are probably necessary but may not be generally sufficient in each system to initiate the cell death process.…”

Death Don't Have No Mercy: Cell Death Programs in Plant-Microbe Interactions.

“…This response accompanies "incompatible interactions" and leads to disease resistance. As detailed below, the HR is programmed genetically in the plant and is a consequence of new host transcription and translation Godiard et al, 1994). The HR is a correlative feature of many but not all incompatible interactions controlled by classic disease resistance (R) genes (Dangl, 1995;Staskawicz et al, 1995; see also Bent, 1996, in this issue).…”