Finding the missing pieces in the puzzle of plant disease resistance.

The molecular mechanisms involved in protecting plants from microbial infection have long remained a mystery. Intrigued by this fundamental biological question and the obvious benefits that the answers may have for improving agriculture and environmental protection, scientists have applied molecular, biochemical, and genetic approaches in studies of a number of plant systems to make significant progress in recent years in the field of plant pathology. The interaction between a potential phytopathogen and a plant can have different consequences. When a virulent pathogen infects a plant, it proliferates in planta, intracellularly or intercellularly, and generates disease symptoms. During the infection, plant genes designated as pathogenesis-related (PR) genes can be activated, and they may function to prevent the spread of the pathogen. However, when an avirulent pathogen infects a plant, it often triggers rapid death of cells and synthesis of antimicrobial compounds at the site of infection, which restricts the growth of the pathogen and renders it avirulent. The resulting visible necrosis is called a hypersensitive response (HR; refs. 1 and 2). The onset of an HR not only restricts the growth of the pathogen but also precedes (and may be the direct cause of) the activation of a signaling pathway that leads to systemic expression of a collection of PR genes and enhanced, lasting, and nonspecific resistance to a wide range of pathogens, known as systemic acquired resistance (SAR; refs. 3 and 4). Therefore, a plant exposed to an avirulent pathogen can be "immunized" against a variety of virulent pathogens. The signaling pathways that lead to the specific avirulent pathogen-induced HR and the onset of nonspecific systemic resistance are the focus of intense research (4-6). The specific interaction between an avirulent pathogen and its corresponding plant host that triggers an HR was described by Flor (7) with the gene-for-gene hypothesis. According to this hypothesis, an HR is induced when the product of an avirulence gene (avr gene) in the pathogen is recognized by a corresponding resistance gene (R gene) product in the plant host. The rapid HR prevents the growth of the pathogen and makes an otherwise virulent pathogen avirulent. Therefore, an HR is a dominant resistance response over a virulent infection. Several avirulence genes have been cloned from various plant pathogens; however, the biological functions of most of the gene products remain unknown (6). A recent breakthrough in the studies of resistance has been the cloning and analysis of several R genes that determine resistance in a number of plant systems to very different fungal, bacterial, and viral pathogens. Sequencing analysis shows that one R gene cloned from tomato (PTO) contains a serine/threonine protein kinase domain (8) and other R genes isolated from Arabidopsis, flax, tobacco, and tomato (RPS2, L6, N, and Cf9, respectively) share leucine-rich repeats (9-13), implicating these R gene products in signal transduction. It is intriguing that consensus regions are found in these presumed receptors that interact specifically with ligands produced by very different avirulence genes of very different pathogens; this suggests a possible common mechanism for the function of these receptors. The molecular events that occur after the specific recognition of a pathogenproduced ligand by the corresponding plant receptor appear to be nonspecific. The rapid cell death that follows pathogen recognition is a genetically determined event, since mutants have been isolated that spontaneously form HR-like necrotic lesions in the absence of pathogen infection (14, 15). The genes identified by these mutations (acd, for accelerated cell death, and lsd, for lesions simulating disease) probably function downstream of the R genes in triggering an HR. During an HR, a burst of reactive oxygen species, such as H202, is detected. They may function to directly inhibit microbial growth, to induce programmed plant cell death, to strengthen the walls of uninfected cells by crosslinking the wall structural proteins, and to induce the expression of a battery of PR genes. After an HR, there is also an increase in the level of salicylic acid (SA) at the site of HR lesions (10to 50-fold) and throughout the plant (2to 5-fold) (16, 17). Salicylic acid has been shown to be required for the induction of nonspecific resistance in the systemic tissue (SAR) and expression of those PR genes that are associated with SAR. A transgenic tobacco plant expressing a bacterial salicylate hydroxylase (that oxidizes SA to an inactive compound, catechol) fails to induce SAR after an avirulent pathogen infection and fails to express the PR genes (18). Further, exogenous application of SA can induce SAR and PR gene expression (19). The systemic signal that links the local HR with the activation of SAR has yet to be found. Grafting experiments between transgenic tobacco plants expressing salicylate hydroxylase and the wild-type plants showed that SA is probably not the systemic signal that moves from the site of the primary inoculation to the systemic tissues but that it is a local signal required for the induction of SAR (20). Also unknown is the mechanism(s) by which SA induces SAR and PR gene expression. Recent studies conducted in the laboratory of Daniel F. Klessig at Rutgers University have shed light on this question. By using 14C-labeled SA, Chen and Klessig (21) purified a soluble SA-binding protein (SABP) from tobacco that binds to SA with high affinity and specificity, suggesting that it is a receptor for SA (21). The protein has been identified as a catalase whose enzymatic activity, breaking down H202 to H20 and 02, is inhibited by SA (22). Chen et al. (22) have proposed that SA functions by increasing the intracellular level of reactive oxygen species, such as H202, and that reactive oxygen species may be involved in the activation of PR genes and in the induction of SAR. In this issue of the Proceedings, Conrath et al. (23) further strengthen this argument by showing that another chemical inducer of SAR, 2,6-dichloroisonicotinic acid (INA; ref. 24) also binds to catalase and inhibits its enzymatic activity with a dose-response curve similar to that of SA. Conrath et al. (23) have tested a number of INA analogs and SA derivatives and found correlation between the ability of these compounds to bind catalase and inhibit its activity with their ability to activate PR gene expression and to induce resistance. Furthermore, Conrath et al. (23) have shown that application of antioxidants suppresses INA-mediated PR gene expression. These data not only identify a cellular target for the action of INA but also strongly suggest