The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes
1997; Springer Nature; Volume: 16; Issue: 11 Linguagem: Inglês
10.1093/emboj/16.11.3207
ISSN1460-2075
Autores Tópico(s)Phytoplasmas and Hemiptera pathogens
ResumoArticle1 June 1997free access The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes Jianmin Zhou Jianmin Zhou Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA Search for more papers by this author Xiaoyan Tang Xiaoyan Tang Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA Search for more papers by this author Gregory B. Martin Corresponding Author Gregory B. Martin Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA Search for more papers by this author Jianmin Zhou Jianmin Zhou Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA Search for more papers by this author Xiaoyan Tang Xiaoyan Tang Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA Search for more papers by this author Gregory B. Martin Corresponding Author Gregory B. Martin Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA Search for more papers by this author Author Information Jianmin Zhou1, Xiaoyan Tang1 and Gregory B. Martin 1 1Department of Agronomy, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, 47907-1150 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:3207-3218https://doi.org/10.1093/emboj/16.11.3207 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In tomato, the Pto kinase confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene, avrPto, in the pathogen Pseudomonas syringae pv. tomato. Using the yeast two-hybrid system, we have identified three genes, Pti4, Pti5 and Pti6, that encode proteins that physically interact with the Pto kinase. Pti4/5/6 each encode a protein with characteristics that are typical of transcription factors and are similar to the tobacco ethylene-responsive element-binding proteins (EREBPs). Using a gel mobility-shift assay, we demonstrate that, similarly to EREBPs, Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding 'pathogenesis-related' (PR) proteins. Expression of several PR genes and a tobacco EREBP gene is specifically enhanced upon Pto–avrPto recognition in tobacco. These observations establish a direct connection between a disease resistance gene and the specific activation of plant defense genes. Introduction It has long been recognized that transcriptional activation of a battery of plant defense-related genes is associated with pathogen invasion (reviewed by Lamb et al., 1989; Bowles, 1990; Dixon and Lamb, 1990). Well documented defense genes include those encoding pathogenesis-related proteins (PRs) (reviewed by Linthorst, 1991; Cutt and Klessig, 1992), hydroxyproline-rich glycoproteins, and enzymes for phytoalexin biosynthesis such as phenylalanine ammonia lyase (PAL) and chalcone synthase (Showalter et al., 1985; Bell et al., 1986). Although the role of these proteins in plant disease resistance has yet to be established, their enzymatic functions indicate that they are well suited for defense against pathogens. For example, two classes of PR proteins, β-1,3-glucanase and chitinase, are lytic enzymes capable of degrading polysaccharides found in the cell wall of many fungi and have antifungal activities when assayed in vitro (Mauch et al., 1988; Sela-Buurlage et al., 1993; Melchers et al., 1994; Niderman et al., 1995). Coordinated expression of nine classes of PR gene mRNAs is associated with the onset of systemic acquired resistance (SAR), a general resistance response induced by necrotizing pathogens (Ward et al., 1991). Overexpression of a bean chitinase in tobacco increased resistance to the soil-borne pathogen Rhizoctonia solani (Broglie et al., 1991), and overexpression of tobacco PR-1 in tobacco enhanced tolerance to Peronospora tabacina and Phytophthora parasitica (Alexander et al., 1993). Similarly, overexpression of a tobacco basic PR-5 in potato delayed the disease symptom produced by Phytophthora infestans (Liu et al., 1994). These results have spurred extensive investigations into the biological function of defense genes and the mechanisms by which they are activated. Much effort has been focused on the characterization of cis-acting elements involved in elicitor- and pathogen-induced defense gene expression (bean chs, Choudhary et al., 1990; parsley PAL, Lois et al., 1989; tobacco PR-1a, Ohshima et al., 1990; van de Rhee and Bol, 1993; tobacco PR-2, Hennig et al., 1993; and Nicotiana plumbaginifolia gln1, Alonso et al., 1995). Using these DNA elements as probes, a few putative transcription factors involved in defense responses have been identified. For example, KAP-1 and KAP-2 bind to the H box of the bean chalcone synthase gene chs15 (Yu et al., 1993), parsley BPF1 binds to box P of the PAL gene (da Costa e Silva et al., 1993), parsley and Arabidopsis homeodomain proteins bind to the parsley pr2 gene promoter (Korfhage et al., 1994), and a tobacco 40 kDa DNA-binding protein is thought to be involved in salicylic acid-regulated PR gene expression (Goldsbrough et al., 1993). Many defense-related genes are induced in both compatible (susceptible) and incompatible (resistant) plant–pathogen interactions. However, the expression of many defense genes is more rapid and pronounced in a resistant plant challenged with an avirulent pathogen. In particular, incompatible interactions involving a plant resistance (R) gene and a corresponding pathogen avirulence (avr) gene (Flor, 1971) lead to accelerated plant defense gene expression. For example, inoculation of Arabidopsis carrying RPS2 with an avirulent Pseudomonas strain containing avrRpt2 rapidly induced PAL mRNA accumulation to a higher level compared with inoculation with a strain lacking the avr gene (Dong et al., 1991). Early induction of ELI3 (elicitor-activated gene) in Arabidopsis depends specifically on the expression of the RPM1 and avrB genes in the plant and pathogen, respectively (Kiedrowski et al., 1992). Similarly, AIG1 and AIG2 (avrRpt2-induced gene) mRNAs are induced in Arabidopsis carrying the RPS2 resistance gene 6 h after infection with Pseudomonas syringae pv. maculicola containing the corresponding avirulence gene avrRpt2 but are not induced until 12 h when either RPS2 or avrRpt2 is absent (Reuber and Ausubel, 1996). In addition, injection of an intercellular fluid containing the Avr9 and Avr2 peptides into tomato plants harboring Cf-9 or Cf-2 or of purified Avr9 peptide into Cf-9 plants induced the expression of two β-1,3-glucanase genes, but not in the plants lacking these Cf genes (Ashfield et al., 1994; Wubben et al., 1996). Many cloned R genes encode proteins that are likely to be involved either in the recognition of signals determined by avr genes or in the early steps of signal transduction (Martin et al., 1995). However, a direct link between any R gene and defense gene activation has yet to be established. The identification of signaling components leading to defense gene activation after R gene–avr gene recognition would be an important advance. Towards this goal, several Arabidopsis mutants have been identified that are either deficient in PR gene expression upon pathogen attack (npr1, Cao et al., 1994; nim1, Delaney et al., 1995), or constitutively express PR genes (cpr1, Bowling et al., 1994). Cloning of these genes and elucidation of the biochemical functions of these gene products will greatly advance our knowledge of plant defense. In tomato, resistance of plants carrying the Pto locus to Pseudomonas syringae pv. tomato strains expressing the avirulence gene avrPto (Ronald et al., 1992; Martin, 1995) is a model system to study signal transduction pathways mediated by a specific R gene. This system constitutes the only example of an R gene-mediated resistance pathway in which genes for multiple components have been cloned (Martin et al., 1993; Zhou et al., 1995; Salmeron et al., 1996). Currently, three components are known to be involved in the signaling pathway mediated by Pto: the serine/threonine protein kinase Pto, a second serine/threonine kinase Pti1 and the leucine-rich repeat type protein Prf. The Pto gene was discovered originally in Lycopersicon pimpinellifolium, a wild tomato species, and isolated by map-based cloning (Martin et al., 1993). Mutagenesis of a bacterial speck-resistant tomato line revealed a second gene, Prf (Salmeron et al., 1994), that is required for both Pto-mediated resistance and fenthion sensitivity, a related phenotype mediated by the Fen gene (Martin et al., 1994). Recently, we and others have demonstrated that the AvrPto protein, presumably delivered into the plant cell by a type III secretion system of the bacterium, functions directly inside the plant cell (Scofield et al., 1996; Tang et al., 1996). The AvrPto and Pto proteins physically interact and this recognition event initiates the disease resistance response (Scofield et al., 1996; Tang et al., 1996). Using the yeast two-hybrid system with Pto as a bait, we had previously identified another protein kinase Pti1 that appears to act downstream of Pto and is involved in the hypersensitive response (HR; Zhou et al., 1995). Here we report the characterization of three additional Pto-interacting proteins, Pti4, Pti5 and Pti6, hereafter referred to as Pti4/5/6, that belong to a large family of plant transcription factors. These proteins bind to a cis-element that is widely conserved among PR genes and are implicated in the regulation of these genes during incompatible plant–pathogen interactions. Results Interaction of Pto with Pti4/5/6 Using the yeast two-hybrid system with Pto as a bait, we previously had screened a tomato cDNA library and isolated 149 clones encoding putative Pti proteins (Zhou et al., 1995). These cDNA clones belonged to 10 distinct classes, as indicated by cross-hybridization experiments. The cDNA clones Pti4, Pti5 and Pti6 represent three of the 10 distinct classes, and were characterized further. Independent clones varying in length that represent identical genes have been identified for each cDNA during the two-hybrid screening (12 for Pti4, nine for Pti5 and one for Pti6). Figure 1 shows the specific interaction of Pti4, Pti5 and Pti6 with Pto in yeast. Yeast strains carrying the Pto bait and a prey of Pti4, Pti5 or Pti6 grew in the absence of leucine, indicative of the LEU2 reporter gene activation (data not shown). When grown on X-Gal plates, these yeast cells were blue as a result of the lacZ reporter gene activation. As determined by the intensity of blue color, the strength of interaction of Pto with these three preys is in the order Pti6>Pti4>Pti5. In contrast, control yeast strains expressing the arbitrary bait Bicoid and any one of the three preys did not activate the LEU2 or the lacZ reporter genes. Previously, we have cloned the recessive pto allele from a tomato cultivar that is susceptible to P.s.tomato carrying avrPto (Jia et al., 1997). The amino acid sequence of pto is 87% identical to Pto. However, the pto bait did not interact with Pti4, Pti5 or Pti6 in yeast as shown by the lack of lacZ activation (Figure 1). The slight blue color of colonies expressing pto and Pti6 developed only after prolonged incubation and probably does not represent significant physical interaction of these proteins. In addition, other kinases such as Pti1 and Fen as baits did not interact with Pti4/5/6 (data not shown). These results indicate that the interactions of these Pti proteins with Pto were highly specific. Figure 1.Interaction of Pto with Pti4/5/6. EGY48 yeast cells containing a prey of Pti4, Pti5 or Pti6 (in pJG4-5), and a bait of Pto, pto or Bicoid (in pEG202) were grown on galactose Ura− His− Trp− X-Gal medium. The plates were incubated at 30°C for 3 days and photographed. Four independent, representative colonies are shown for each bait–prey combination. All bait proteins were expressed equally in yeast as determined by Western blots (Y.-T.Loh and G.B.Martin, unpublished results). Download figure Download PowerPoint Pti4/5/6 are members of a multigene family The longest Pti4/5/6 cDNAs isolated from the two-hybrid screen were 1.0, 0.8 and 0.8 kb, respectively. Each of these cDNAs was probed onto genomic blots containing DNA from tomato cultivar Rio Grande-PtoR (Figure 2A). Numerous fragments were detected by both Pti4 and Pti5 cDNA probes, while 2–3 fragments were detected by the Pti6 cDNA probe. The distinct restriction fragments detected by these probes indicated that the sequences of Pti4/5/6 are not identical. However, a cross-hybridization experiment indicated weak homology among the three classes of cDNAs (data not shown). Subsequent DNA sequence analysis confirmed this homology among Pti4/5/6 proteins (see below). Thus Pti4/5/6 appear to be members of a large gene family. Figure 2.Pti4/5/6 belong to a large gene family. (A) DNA gel blot analysis of tomato genomic DNA. Genomic DNA (5 μg/lane) from Rio Grande-PtoR plants was digested with the indicated restriction enzymes, and the DNA blot was hybridized to the Pti4/5/6 cDNA probes. (B) Nucleotide and deduced amino acid sequences of Pti4/5/6. Superscript letters denote the 5′ ends of cDNA clones recovered from the two-hybrid library screening. Amino acids with a single underline are acidic regions, whereas those with a double underline represent the conserved central basic region. Putative nuclear localization sequences are shown in bold. Nucleotide sequence data have been deposited in DDBJ/EMBL/GenBank (Pti4, accession No. U89255; Pti5, U89256; Pti6, U89257). Download figure Download PowerPoint We sequenced the inserts of Pti4/5/6 and found that each contained a single open reading frame that was fused in-frame to the activation domain of the prey plasmid (Figure 2B). To isolate full-length cDNA clones, we screened another tomato cDNA library with the Pti4/5/6 cDNA probes. This screen resulted in the isolation of longer cDNA clones for Pti6 but not for Pti4 and Pti5. The longest Pti6 cDNA clone is 1.4 kb and corresponds to the transcript size detected by a Northern blot, indicating that this Pti6 clone is probably full-length (data not shown). The original Pti4 and Pti5 clones are also likely to be full-length as they both contain a putative start codon at their 5′ ends (see below). The putative proteins encoded by Pti4/5/6 contain 234, 161 and 248 amino acid residues, respectively (Figure 2B). The Pti6 protein expressed from the original prey plasmid lacked the first 48 amino acid residues, indicating that these residues are not essential for interaction with Pto. Several features that are typical of transcription factors are present in the Pti4/5/6 proteins. Most strikingly, a central domain of 58 amino acids rich in basic residues is shared by all three proteins (62–81% identical), and is reminiscent of the DNA-binding domain of many transcription factors (Latchman, 1995). Pti4 and Pti6 contain short clusters of basic residues similar to known nuclear localization sequences (NLS; reviewed by Dingwall and Laskey, 1991). In addition, Pti4 and Pti5 each contain an acidic region N-terminal to the central region, and Pti6 contains acidic regions both N- and C-terminal to the central domain. Many transcription factors, such as the yeast GAL4 protein, the mammalian glucocorticoid receptor and the maize Viviporous-1 protein, contain acidic regions that function in transcriptional activation (McCarty et al., 1991; Latchman, 1995). When Pti5 and Pti6 were expressed independently as baits in the yeast strain EGY48, they showed strong autoactivation of the reporter genes in the absence of any prey (data not shown). This suggests that at least Pti5 and Pti6 contain functional transactivation domains. Pti4/5/6 bind the PR box that is conserved among many pathogenesis-related genes Using the BLAST program, we searched the DDBJ/EMBL/GenBank database (version 86.0) for proteins with similarity to Pti4/5/6. All protein sequences retrieved from the database shared significant homology with the basic, central domain of Pti4/5/6. These proteins include, in the order of similarity to Pti4/5/6, the tobacco ethylene-responsive element-binding proteins (EREBPs; Ohme-Takagi and Shinshi, 1995), and the Arabidopsis proteins TINY (Wilson et al., 1996), AP2 (Jofuku et al., 1994) and ANT (Elliott et al., 1996; Klucher et al., 1996). A number of expressed sequence tag (EST) sequences of unknown function from both Arabidopsis and rice also contain this central domain. EREBPs contain a single central domain that has been demonstrated recently to bind a cis-acting element conferring the ethylene responsiveness of the β-1,3-glucanase gene gln2 in tobacco (Ohme-Takagi and Shinshi, 1995). This ethylene-responsive element contains the core sequence GCCGCC. Since this sequence has been found in the promoters of many PR genes (see below), we refer to it as the PR box. TINY is an Arabidopsis gene encoding a protein with a single central domain and is involved in determining plant height, hypocotyl elongation and fertility (Wilson et al., 1996). AP2 and ANT are involved in flower development, and both contain two central domains that are also believed to bind DNA (Jofuku et al., 1994; Elliott et al., 1996; Klucher et al., 1996). Figure 3A shows an alignment of the amino acid sequences of Pti4/5/6 and the EREBPs. The homology among these sequences resides primarily in the DNA-binding domain (at least 67% identical). However, Pti4, EREBP-1 and EREBP-2 are highly similar (71–78% identity) to each other throughout their entire open reading frames. The N-terminus of Pti5 also shares significant homology with Pti4, EREBP-1 and EREBP-2, and the entire Pti5 protein is 43–48% identical to these three proteins. In contrast, Pti6, EREBP-3 and EREBP-4 are more distantly related, and their sequences outside the DNA-binding domain share little homology with any known proteins. Figure 3.Similarity of Pti4/5/6 to EREBP/AP2 transcription factors. (A) Alignment of Pti4/5/6 amino acid sequence with EREBPs from tobacco. The Pretty Box program (GCG package, version 7.0) was used to create the best alignment. Amino acids identical in at least four of the sequences are shaded in black and conservative substitutions are shaded in gray. (B) Phylogenetic tree of transcription factors in the EREBP/AP2 family. The putative DNA-binding region was used to create the best alignment with the Pileup program (GCG package, version 7.0), and the phylogram was created using the Neighbor-Joining method of the Growtree program (GCG package, version 7.0). The length of branches indicates the relative evolutionary distances. Sequences used in this analysis include the single DNA-binding region of Pti4/5/6, four tobacco EREBPs (Ohme-Takagi and Shinshi, 1995), two Arabidopsis EST clones (accession Nos R87001 and H37693), two rice EST clones (accession Nos D39914 and D47296), the two DNA-binding regions in Arabidopsis proteins AP2 (Jofuku et al., 1994) and ANT (Elliott et al., 1996; Klucher et al., 1996), the maize protein Zmmhcf1 (accesssion No. Z47554) and the second DNA-binding region of a rice EST clone (truncated at its first DNA-binding domain, accession No. D15799). Download figure Download PowerPoint To explore further the relationships of the proteins containing the EREBP DNA-binding domain, evolutionary distances among these sequences were calculated and a phylogenetic tree was constructed (Figure 3B). The sequences clearly divided into two major groups, with one consisting of proteins with a single DNA-binding domain, and the other of proteins with two DNA-binding domains. Pti4/5/6, EREBPs and several other EST sequences belong to the first group, whereas the proteins involved in flower development are in the second group. Thus, Pti4/5/6 are most closely related to EREBPs and are more distantly related to floral development regulators such as AP2 and ANT. It has been shown previously that the central, basic domain of the EREBPs binds the wild-type PR box of gln2 but not a mutated PR box harboring two G→T substitutions in the GCCGCC core sequence (Ohme-Takagi and Shinshi, 1995). The similarity of the Pti4/5/6 proteins and the EREBPs led us to hypothesize that Pti4/5/6 also bind to the PR box. To test this possibility, we expressed the full-length Pti6 and the C-terminal 141 amino acids of Pti5 as GST fusion proteins. For a positive control, we expressed the tobacco EREBP-2 protein as a GST fusion. The ability of GST–Pti5 and GST–Pti6 to bind the wild-type or the mutated gln2 PR boxes was tested using a mobility-shift gel assay. As shown in Figure 4, both GST–Pti5 and GST–Pti6 bound the wild-type PR box similarly to GST–EREBP-2. No binding was detected when the mutated PR box was used in the assay, indicating that binding of GST–Pti5 and GST–Pti6 to the PR box was highly specific. In contrast to GST–Pti5 and GST–Pti6, neither GST–Pti1 nor GST itself bound to the PR box. These results further confirmed the specificity of binding of Pti5 and Pti6 to the gln2 PR box. Thus the DNA-binding domain of both Pti5 and Pti6 is functionally homologous to that of EREBPs. We have been unable to express Pti4 in Escherichia coli and were therefore unable to test its activity in this assay. However, given the high degree of homology between Pti4 and EREBP-2, Pti4 is also likely to bind to the PR box. Figure 4.Pti5 and Pti6 bind the PR box. Fifty ng of purified GST fusion proteins were mixed with the wild-type PR box probe (GCC) or the mutated PR box probe (mGCC), and the gel mobility-shift assay was performed as described in Materials and methods. Download figure Download PowerPoint Interaction of Pto with tobacco EREBP-2 The sequence similarity and PR box-binding properties of Pti/4/5/6 and the tobacco EREBPs strongly suggest that these proteins are functionally homologous. Introduction of the tomato Pto gene into tobacco is known to enhance defense responses specifically upon inoculation with P.s.tabaci expressing avrPto (Thilmony et al., 1995). In addition, tobacco cultivar W-38 contains a functional, endogenous Pto gene that also specifically recognizes the expression of avrPto in P.s.tabaci (Thilmony et al., 1995). Together these observations imply that the Pto-mediated signaling pathway is conserved in both tomato and tobacco and that the tomato Pto protein is compatible with other signaling components from tobacco. We thus predicted that at least some of the tobacco EREBPs would interact with the tomato Pto protein. The high degree of homology shared between EREBP-2 and Pti4 suggests that EREBP-2 is a functional homolog of Pti4. We therefore tested the ability of EREBP-2 to interact with Pto. Expression of EREBP-2 as a prey in yeast strain EGY48 carrying the tomato Pto bait strongly activated both the lacZ and the LEU2 reporter genes, indicating interaction of Pto with EREBP-2 (Figure 5; J.Zhou and G.B.Martin, unpublished results). This interaction is highly specific, as the pto bait and the Bicoid bait failed to interact with EREBP-2. This experiment thus supports our hypothesis that the tobacco EREBP-2 protein is a functional homolog of Pti4/5/6. Figure 5.Interaction of EREBP-2 with Pto. EGY48 yeast cells containing the EREBP-2 prey and a bait of either Pto, pto or Bicoid (in pEG202) were grown on galactose Ura− His− Trp− X-Gal medium. The plates were incubated at 30°C for 3 days and photographed. Eight independent, representative colonies are shown for each bait–prey combination. Download figure Download PowerPoint Activation of PR gene expression upon Pto–avrPto recognition Our results suggest that Pti4/5/6 and EREBPs are transcription factors involved in the regulation of gene expression by binding to the PR box. A search for plant promoter sequences containing the PR box core sequence (GCCGCC) uncovered a number of predominantly basic PR genes from bean, tobacco, potato, Arabidopsis and tomato (Table I). It is thus plausible that Pto regulates PR gene expression in tomato via interaction with PR box-binding proteins such as Pti4/5/6 and EREBPs. Table 1. Occurrence of PR-box in plant defense genes Plant Gene name Protein encoded Reference N.plumbaginifolia gn1 β-1, 3-glucanase Castresana et al. (1990) N.plumbaginifolia gn2 basic β-1,3-glucanase Gheysen et al. (1990) N.tabacum CHN50 basic chitinase Fukuda et al. (1991) N.tabacum CHN17 basic chitinase Shinshi et al. (1990) N.tabacum CHN14 basic chitinase van Buuren et al. (1992) N.tabacum GLA (gln2) basic β-1,3-glucanase Sperisen et al. (1991) and Ohme-Takagi(1990) and Shinshi (1990) N.tabacum GLB basic β-1,3-glucanase Sperisen et al. (1991) N.tabacum PRP1 basic PR-1 Payne et al. (1989) N.tabacum prb-1b basic PR-1 Meller et al. (1993) N.tabacum Osmotin basic PR-5 Liu et al. (1995) N.tabacum OPL neutral PR-5 Sato et al. (1996) N.tabacum chi-v class V chitinase Melchers et al. (1994) S.tuberosum WIN2 wound inducible (PR-4-like) Stanford et al. (1988) S.tuberosum STPRINPSG protease inhibitor Y.Choi et al. (DDBJ/EMBL/GenBank Z12824) S.commersonii pOSML13 basic PR-5 Zhu et al. (1995) S.commersonii pOSML81 basic PR-5 Zhu et al. (1995) L.esculentum CHN basic chitinase cited by Hart et al. (1993) P.vulgaris CH5B basic chitinase Broglie et al. (1989) A.thaliana CHA2 basic chitinase Samac et al. (1990) A.thaliana PAL3 phenylalanine ammonia-lyase Wanner et al. (1995) B.napus Bp10 ascorbate oxidase Albani et al. (1992) Next we examined whether there was a correlation between avrPto–Pto recognition and induction of PR genes containing the PR box. We chose tobacco to address this question because PR gene expression is easily monitored in this species, and a number of PR gene promoters have been analyzed in detail in tobacco. In addition, the Pto-mediated signal transduction pathway is conserved between tomato and tobacco (Rommens et al., 1995; Thilmony et al., 1995). Tobacco leaves (W-38) either with or without the tomato Pto transgene were injected with P.s.tabaci strains with or without avrPto (at a level of 106 c.f.u./ml). We examined the mRNA levels of three PR genes that contain PR boxes in their promoter: the PRP1 gene encoding a basic PR-1 protein (Payne et al., 1989), the basic chitinase gene CHN50 (Fukuda et al., 1991) and the Osmotin gene encoding a basic PR-5 protein (Liu et al., 1995). Figure 6 shows that transcripts of the three genes accumulated after inoculation with Pseudomonas bacteria. In the non-transgenic plants, inoculation of P.s.tabaci containing avrPto induced the three transcripts at 18 h. In contrast, injection of P.s.tabaci without the avrPto gene in the same plants delayed transcript accumulation. The early induction of PR genes by avirulent bacteria in the non-transgenic plants is likely to be a result of the recognition of avrPto by the endogenous Pto homolog in this particular tobacco cultivar (Thilmony et al., 1995). In the Pto transgenic plants, this early induction by P.s.tabaci carrying avrPto was accelerated further, with the earliest expression observed at 9 h after inoculation. Surprisingly, the induction of the PR genes in the Pto transgenic plants by virulent bacteria (P.s.tabaci without avrPto) was also accelerated and occurred at 18 h after infection. One possible explanation for this induction is that an unknown avr gene is present in the P.s.tabaci strain and is recognized by the tomato Pto gene. Alternatively, overexpression of the tomato Pto gene in tobacco may potentiate PR gene induction by Pseudomonas bacteria. Together, these results support a role for Pto in rapid expression of PR genes containing the PR box. Figure 6.Induction of tobacco PR gene expression upon Pto–avrPto recognition. Tobacco leaves, either non-transgenic (W-38) or with the Pto transgene (35S::Pto), were injected with P.s.tabaci at 106 c.f.u./ml. Leaf tissue was harvested at the indicated times post-inoculation, and RNA was extracted. Duplicates of RNA gel blots were hybridized to tobacco PRP1, CHN50, Osmotin or to rDNA probes. The experiment was repeated three times with the same results. Download figure Download PowerPoint To test further the functional relevance of EREBPs in resistance responses, we examined mRNA accumulation of EREBP-1 and EREBP-2, which have the highest homology to Pti4 and Pti5. We were unable to detect reliably the expression of EREBP-2 mRNA. However, we found that EREBP-1 mRNA was induced in the Pto transgenic tobacco plants at a high level 9 h after infection with P.s.tabaci expressing avrPto (at an inoculum level of 106 c.f.u./ml; Figure 7A). In contrast, EREBP-1 mRNA did not accumulate until 18 h after inoculation with P.s.tabaci lacking avrPto. Similar results were observed with non-transgenic tobacco plants, suggesting that the endogenous Pto homolog is sufficient for the EREBP-1 activation. These experiments indicated that EREBP-1 mRNA accumulated earlier than that of the PR mRNAs during incompatible interactions. To investigate this observation further, we inoculated the Pto transgenic tobacco leaves with high inocula (108 c.f.u./ml) of P.s.tabaci with or without avrPto and examined the expression of CHN50, Osmotin and the EREBP-1 mRNAs. Figure 7B shows that the pre
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