Rapid generation of O 2 Ϫ and H 2 O 2 , which is reminiscent of the oxidative burst in neutrophils, is a central component of the resistance response of plants to pathogen challenge. Here, we report that the Arabidopsis rbohA ( for respiratory burst oxidase homolog A ) gene encodes a putative 108-kD protein, with a C-terminal region that shows pronounced similarity to the 69-kD apoprotein of the gp91 phox subunit of the neutrophil respiratory burst NADPH oxidase. The RbohA protein has a large hydrophilic N-terminal domain that is not present in gp91 phox . This domain contains two Ca 2 ϩ binding EF hand motifs and has extended similarity to the human RanGTPase-activating protein 1. rbohA , which is a member of a divergent gene family, generates transcripts of 3.6 and 4.0 kb that differ only in their polyadenylation sites. rbohA transcripts are most abundant in roots, with weaker expression in aerial organs and seedlings. Antibodies raised against a peptide near the RbohA C terminus detected a 105-kD protein that, unlike gp91 phox , does not appear to be highly glycosylated. Cell fractionation, two-phase partitioning, and detergent extraction indicate that RbohA is an intrinsic plasma membrane protein. We propose that plants have a plasma membrane enzyme similar to the neutrophil NADPH oxidase but with novel potential regulatory mechanisms for Ca 2 ϩ and G protein stimulation of O 2 Ϫ and H 2 O 2 production at the cell surface.
INTRODUCTIONRecognition of avirulent pathogens causes a burst of oxidative metabolism generating superoxide (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ) (Baker and Orlandi, 1995). Reactive oxygen intermediates are direct protective agents and also drive cross-linking of the cell wall before the activation of transcription-dependent defenses (Lamb and Dixon, 1997). Moreover, the oxidative burst can trigger the collapse of challenged host cells at the onset of the hypersensitive response and generate signals for defense gene induction (Levine et al., 1994; Jabs et al., 1997).The kinetics and defense functions of O 2 Ϫ and H 2 O 2 generation are reminiscent of the oxidative burst during neutrophil activation ( Tenhaken et al., 1995;Lamb and Dixon, 1997). The neutrophil oxidative burst involves the reaction O 2 ϩ NADPH → O 2 Ϫ ϩ NADP ϩ ϩ H ϩ catalyzed by a plasma membrane oxidase, followed by dismutation of O 2 Ϫ to H 2 O 2 (Taylor et al., 1993). The NADPH oxidase consists of two plasma membrane proteins, gp91 phox and p22 phox ( phox for phagocyte oxidase), which together form heterodimeric flavocytochrome b Ϫ 558 and three cytosolic regulatory proteins, p40 phox , p47 phox , and p67 phox , which translocate to the plasma membrane after stimulation to form the active complex (Bokoch, 1994). The small cytosolic GTPase Rac2 and possibly other G proteins also appear to be required for activation of the oxidase.A membrane-bound enzyme resembling the neutrophil NADPH oxidase may contribute to the pathogen-induced oxidative burst in plants. Thus, O 2 Ϫ generation can be observed in microsomal preparat...
