The tomato Pti4 gene encodes a transcription factor that was identified on the basis of its specific interaction with the product of the Pto disease resistance gene in a yeast two-hybrid system. We show here that the Pti4 protein specifically binds the GCC-box cis element, which is present in the promoter region of many pathogenesis-related ( PR ) genes. Expression of the Pti4 gene in tomato leaves was rapidly induced by ethylene and by infection with Pseudomonas syringae pv tomato, and this induction preceded expression of GCC-box-containing PR genes. Although salicylic acid also induced Pti4 gene expression, it did not induce GCC-box PR genes. Rather, salicylic acid antagonized ethylene-mediated expression of GCC-box PR genes. We demonstrate that the Pti4 protein is specifically phosphorylated by the Pto kinase and that this phosphorylation enhances binding of Pti4 to the GCC box. In addition, induced overexpression of Pto and Pti4 in tomato leaves resulted in a concomitant increase in GCC-box PR genes. Our results support a model in which phosphorylation of the Pti4 protein by the Pto kinase enhances the ability of Pti4 to activate expression of GCCbox PR genes in tomato. INTRODUCTIONA well-characterized plant defense response associated with pathogen attack is the expression of pathogenesis-related ( PR ) genes. PR genes are activated in both resistant and susceptible plants in response to pathogen attack. However, they often are expressed more rapidly and to a greater extent in incompatible interactions in which a resistant plant is challenged with an avirulent pathogen (Voisey and Slusarenko, 1989; van Kan et al., 1992;Jia and Martin, 1999). This implies that enhanced PR gene expression is mediated by a recognition event involving a disease resistance ( R ) gene and its cognate avirulence (avr) gene. The roles of PR genes in plant defense responses against pathogens have been widely investigated (Cutt and Klessig, 1992). The best-characterized plant defense genes include those that encode glucanases and chitinases. These enzymes appear to be antimicrobial, based on their ability to degrade cell wall components of pathogens (Mauch et al., 1988;Sela-Buurlage et al., 1993) and have been shown to enhance disease resistance when overexpressed in plants (Broglie et al., 1991; Zhu et al., 1994). Despite the established biological function in plant defense responses for some PR genes, the molecular mechanisms responsible for their expression in R-avr recognition remain largely unclear.The expression of many defense-related genes is regulated by signaling molecules such as salicylic acid (SA) and ethylene. Increases in SA and its conjugates have been associated with the activation of defense genes in many plant species (reviewed in Dempsey et al., 1999). Compelling evidence for a key role of SA in defense gene regulation comes from analysis of tobacco and Arabidopsis NahG transformants in which SA is converted to an inactive form. The NahG plants fail to induce several PR genes such as PR-1, PR-2, and PR-5 and sho...

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto , a resistance gene encoding a protein kinase. When introduced into P. s. tabaci , avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane. INTRODUCTIONAvirulence ( avr ) genes trigger gene-for-gene resistance in plants containing the corresponding disease resistance ( R ) genes . Numerous avr genes have been cloned from various bacterial pathogens. Recent studies suggest that bacterial Avr proteins are secreted into the host cells through the type III secretion pathway and subsequently are perceived by the corresponding R gene products (reviewed in Alfano and Collmer, 1997;Lindgren, 1997;He, 1998;Kjemtrup et al., 2000).The AvrBs3 protein represents one family of bacterial Avr proteins that share high sequence similarities. These proteins contain a variable number of nearly identical repeat domains in the internal portion of the protein (Leach and White, 1996). Deletion and mutagenesis analysis indicate that the internal repeat region determines the recognition specificity of the AvrBs3 protein (Herbers et al., 1992). AvrBs3 -like proteins contain a nuclear localization sequence and have been detected in plant nuclei (Yang and Gabriel, 1995;Van den Ackerveken et al., 1996;Zhu et al., 1998 Zhu et al., , 1999. The transcription activity of AvrXa10, a member of the AvrBs3 family, in eukaryotic cells is essential for its avirulence function (Zhu et al., 1999). The AvrBs3-like proteins may mediate the avirulence function by regulating plant gene expression.Other bacterial Avr proteins do not carry a characteristic sequence indicative of recognition specificity and in planta localization (Leach and White, 1996). The reciprocal interchanges between AvrB and AvrC, two Avr proteins that share considerable sequence similarity, have identified a large central region in AvrB and AvrC that determines the recognition specificity (Tamaki et al., 1991). The rest of the bacterial Avr proteins do not share much homology with each other or w...

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto , a resistance gene encoding a protein kinase. When introduced into P. s. tabaci , avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane. INTRODUCTIONAvirulence ( avr ) genes trigger gene-for-gene resistance in plants containing the corresponding disease resistance ( R ) genes . Numerous avr genes have been cloned from various bacterial pathogens. Recent studies suggest that bacterial Avr proteins are secreted into the host cells through the type III secretion pathway and subsequently are perceived by the corresponding R gene products (reviewed in Alfano and Collmer, 1997;Lindgren, 1997;He, 1998;Kjemtrup et al., 2000).The AvrBs3 protein represents one family of bacterial Avr proteins that share high sequence similarities. These proteins contain a variable number of nearly identical repeat domains in the internal portion of the protein (Leach and White, 1996). Deletion and mutagenesis analysis indicate that the internal repeat region determines the recognition specificity of the AvrBs3 protein (Herbers et al., 1992). AvrBs3 -like proteins contain a nuclear localization sequence and have been detected in plant nuclei (Yang and Gabriel, 1995;Van den Ackerveken et al., 1996;Zhu et al., 1998 Zhu et al., , 1999. The transcription activity of AvrXa10, a member of the AvrBs3 family, in eukaryotic cells is essential for its avirulence function (Zhu et al., 1999). The AvrBs3-like proteins may mediate the avirulence function by regulating plant gene expression.Other bacterial Avr proteins do not carry a characteristic sequence indicative of recognition specificity and in planta localization (Leach and White, 1996). The reciprocal interchanges between AvrB and AvrC, two Avr proteins that share considerable sequence similarity, have identified a large central region in AvrB and AvrC that determines the recognition specificity (Tamaki et al., 1991). The rest of the bacterial Avr proteins do not share much homology with each other or w...

The tomato Pti4 gene encodes a transcription factor that was identified on the basis of its specific interaction with the product of the Pto disease resistance gene in a yeast two-hybrid system. We show here that the Pti4 protein specifically binds the GCC-box cis element, which is present in the promoter region of many pathogenesis-related (PR) genes. Expression of the Pti4 gene in tomato leaves was rapidly induced by ethylene and by infection with Pseudomonas syringae pv tomato, and this induction preceded expression of GCC-box-containing PR genes. Although salicylic acid also induced Pti4 gene expression, it did not induce GCC-box PR genes. Rather, salicylic acid antagonized ethylene-mediated expression of GCC-box PR genes. We demonstrate that the Pti4 protein is specifically phosphorylated by the Pto kinase and that this phosphorylation enhances binding of Pti4 to the GCC box. In addition, induced overexpression of Pto and Pti4 in tomato leaves resulted in a concomitant increase in GCC-box PR genes. Our results support a model in which phosphorylation of the Pti4 protein by the Pto kinase enhances the ability of Pti4 to activate expression of GCC-box PR genes in tomato.

Summary The tomato genes Pti4 and Pti5 encode ethylene‐responsive element binding protein‐like transcription factors that bind to the GCC box, a conserved cis‐element in many defense‐related genes. The Pti proteins have previously been shown to interact with the tomato disease resistance protein Pto. Here we report that the expression of both Pti4 and Pti5 are induced by a virulent strain of Pseudomonas syringae pv tomato. The expression of Pti5 is further enhanced by the interaction of the Pto gene in tomato and the corresponding avrPto gene in the bacterium. The enhancement of Pti5 expression by Pto–avrPto interaction requires a functional Prf gene in the plant. Pti5 appears to be expressed specifically during biotic stresses, suggesting a specific role in plant defense. Pti4 and several EREBP‐like genes are induced by ethylene, salicylate and wounding. However, the Pseudomonas bacterium induced a wild‐type level of Pti4 and Pti5 transcripts in tomato plants carrying the nahG transgene, the Nr mutation, or the def1 mutation. In addition, the ethylene action inhibitor norbornadiene did not inhibit the induction of Pti4 and Pti5 either in the compatible or incompatible interactions. The results suggest that the Pseudomonas bacterium induces Pti4 and Pti5 expression through a pathway independent of salicylic acid, ethylene and jasmonic acid.