The Pto gene encodes a serine/threonine kinase that confers resistance to bacterial speck disease in tomato. Using the yeast two-hybrid system, we identified a second serine/threonine kinase, Pto-interacting 1 (Pti1), that physically interacts with Pto. Cross-phosphorylation assays revealed that Pto specifically phosphorylates Pti1 and that Pti1 does not phosphorylate Pto. Fen, another serine/threonine kinase from tomato that is closely related to Pto, was unable to phosphorylate Pti1 and was not phosphorylated by Pti1. Expression of a Pti1 transgene in tobacco plants enhanced the hypersensitive response to a P. syringae pv. tabaci strain carrying the avirulence gene avrPto. These findings indicate that Pti1 is involved in a Pto-mediated signaling pathway, probably by acting as a component downstream of Pto in a phosphorylation cascade.

The Pti4, Pti5, and Pti6 proteins from tomato were identified based on their interaction with the product of the Pto disease resistance gene, a Ser-Thr protein kinase. They belong to the ethylene-response factor (ERF) family of plantunique transcription factors and bind specifically to the GCC-box cis element present in the promoters of many pathogenesis-related ( PR ) genes. Here, we show that these tomato ERFs are localized to the nucleus and function in vivo as transcription activators that regulate the expression of GCC box-containing PR genes. Expression of Pti4 , Pti5 , or Pti 6 in Arabidopsis activated the expression of the salicylic acid-regulated genes PR1 and PR2 . Expression of jasmonic acid-and ethylene-regulated genes, such as PR3 , PR4 , PDF1.2 , and Thi2.1 , was affected differently by each of the three tomato ERFs, with Arabidopsis -Pti4 plants having very high levels of PDF1.2 transcripts. Exogenous application of salicylic acid to Arabidopsis-Pti4 plants suppressed the increased expression of PDF1.2 but further stimulated PR1 expression. Arabidopsis plants expressing Pti4 displayed increased resistance to the fungal pathogen Erysiphe orontii and increased tolerance to the bacterial pathogen Pseudomonas syringae pv tomato . These results indicate that Pti4, Pti5, and Pti6 activate the expression of a wide array of PR genes and play important and distinct roles in plant defense. INTRODUCTIONPlants respond to pathogen attack by activating multiple defense mechanisms to protect themselves from infection. These rapid cellular responses often are triggered by the recognition of specific pathogens and the activation of highly regulated signal transduction pathways. A major target of these pathways is the cell nucleus, where signals lead to the transcriptional activation of a large array of defense genes (Maleck et al., 2000;Schenk et al., 2000). The products of these genes include pathogenesis-related (PR) proteins as well as enzymes involved in the biosynthesis of protective secondary metabolites. Although the functions of many PR proteins remain unknown, some PR proteins, such as ␤ -1,3-glucanase (PR2) and chitinase (PR3), are hydrolytic enzymes that have been shown to degrade fungal cell walls and to inhibit fungal growth both in vivo and in vitro (Broglie et al., 1991;Sela-Buurlage et al., 1993; Zhu et al., 1994). It was shown recently that osmotin (PR5) induces apoptosis in yeast, and it may act similarly toward plant fungal pathogens (Narasimhan et al., 2001).Several signaling molecules, such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), have been shown to be important components of defense response pathways (Dong, 1998;Reymond and Farmer, 1998; Dempsey et al., 1999;Pieterse and van Loon, 1999). Infection by microbial pathogens results in an increase in the levels of these molecules in plants, and many PR genes that are induced upon pathogen infection also are upregulated by one or more of these signaling molecules (Malamy et al., 1990; Dempsey et al., 1999). The SA-dependent...

The octadecanoid signaling pathway has been shown to play an important role in plant defense against various chewing insects and some pathogenic fungi. Here, we examined the interaction of a cell-content feeding arachnid herbivore, the two-spotted spider mite (Tetranychus urticae Koch), with cultivated tomato (Lycopersicon esculentum) and an isogenic mutant line (defenseless-1 [def-1]) that is deficient in the biosynthesis of the octadecanoid pathway-derived signal, jasmonic acid (JA). Spider mite feeding and fecundity on def-1 plants was significantly greater than on wild-type plants. Decreased resistance of def-1 plants was correlated with reduced JA accumulation and expression of defensive proteinase inhibitor (PI) genes, which were induced in mite-damaged wild-type leaves. Treatment of def-1 plants with methyl-JA restored resistance to spider mite feeding and reduced the fecundity of female mites. Plants expressing a 35S::prosystemin transgene that constitutively activates the octadecanoid pathway in a Def-1-dependent manner were highly resistant to attack by spider mites and western flower thrips (Frankliniella occidentalis), another cell-content feeder of economic importance. These findings indicate that activation of the octadecanoid signaling pathway promotes resistance of tomato to a broad spectrum of herbivores. The techniques of amplified fragment length polymorphism (AFLP) and bulk segregant analysis were used to map the Def-1 gene to a region on the long arm of chromosome 3 that is genetically separable from the map position of known JA biosynthetic genes. Tight linkage of Def-1 to a T-DNA insertion harboring the maize (Zea mays) Dissociation transposable element suggests a strategy for directed transposon tagging of the gene.Plant resistance to arthropod herbivores is often mediated by phytochemicals that negatively affect the feeding, growth, or reproduction of the attacking pest (Karban and Baldwin, 1997; Walling, 2000). Although many defensive compounds have been identified from diverse plant species, relatively little is known about the underlying genetic mechanisms that control their biosynthesis in response to developmental and environmental cues. Lycopersicon spp. provide an attractive model system to address this question. Cultivated tomato (Lycopersicon esculentum) is a natural host to over 100 arthropod herbivores that feed on roots, leaves, or fruit (Lange and Bronson, 1981). Included among the major pests of tomato are adult and larval stages of Coleoptera (beetles), Lepidoptera (moths), Diptera (flies), Thysanoptera (thrips), Heteroptera (true bugs), Homoptera (aphids and whiteflies), and Acari (spider mites).Natural resistance of tomato to many herbivores is attributed to both constitutive and inducible defensive phytochemicals (Farrar and Kennedy, 1992). Among the most thoroughly studied inducible defenses in tomato are proteinase inhibitor (PI) proteins that inhibit digestive enzymes in the gut of some insect herbivores (Green and Ryan, 1972; Broadway and Duffey, 1986). Wound-induc...

The catalytic activity and amino acid specificity of the tomato Pto and Fen kinases were investigated. The Pto and Fen genes were fused to the carboxyl terminus of the maltose-binding protein and expressed in Escbericbia coli. lncubation of the purified fusion proteins with [y-3ZP]ATP in an in vitro assay showed that both proteins were capable of autophosphorylation. Mutant fusion proteins in which the conserved lysine residue of subdomain li was changed to a glutamine were unable to autophosphorylate. Phosphoamino analysis of the active fusion proteins indicated that both kinases phosphorylate serine and threonine residues but not tyrosine.

The Pto gene was derived originally from the wíld tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. The Fen gene is also derived from L. pimpinellifolium and confers sensitivity to the insecticide fenthion. We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L. esculentum, and designated them pfo and fen. High conservation of genome organization between the two tomato species allowed us to ídentífy the pto and fen alleles from among the cluster of closely related Pfo gene family members. The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively. In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases. In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Ptil serine/ threonine kinase. However, the pto kinase shows impaired interaction with Ptil and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system. The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto-and fen-mediated signal transduction pathways.