AvrPto and AvrPtoB are type III effector proteins expressed by Pseudomonas syringae pv. tomato strain DC3000, a pathogen of both tomato and Arabidopsis spp. Each effector physically interacts with the tomato Pto kinase and elicits a hypersensitive response when expressed in tomato leaves containing Pto. An avrPto deletion mutant of DC3000 previously was shown to retain avirulence activity on Pto-expressing tomato plants. We developed an avrPtoB deletion mutant of DC3000 and found that it also retains Pto-specific avirulence on tomato. These observations suggested that avrPto and avrPtoB both contribute to avirulence. To test this hypothesis, we developed an ΔavrPtoΔavrPtoB double mutant in DC3000. This double mutant was able to cause disease on a Pto-expressing tomato line. Thus, avrPto and avrPtoB are the only avirulence genes in DC3000 that elicit Pto-mediated defense responses in tomato. When inoculated onto susceptible tomato leaves and compared with wild-type DC3000, the mutants DC3000ΔavrPto and DC3000ΔavrPtoB each caused slightly less severe disease symptoms, although their growth rate was unaffected. However, DC3000ΔavrPtoΔavrPtoB caused even less severe disease symptoms than the single mutants and grew more slowly than them on susceptible leaves. Our results indicate that AvrPto and AvrPtoB have phenotypically redundant avirulence activity on Pto-expressing tomato and additive virulence activities on susceptible tomato plants.
Summary The bacterial flagellin (FliC) epitopes flg22 and flg II ‐28 are microbe‐associated molecular patterns ( MAMP s). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS 2, neither the flg II ‐28 receptor nor the extent of flg II ‐28 recognition by different plant families is known. Here, we tested the significance of flg II ‐28 as a MAMP and the importance of allelic diversity in flg22 and flg II ‐28 in plant–pathogen interactions using purified peptides and a Pseudomonas syringae ∆ fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flg II ‐28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flg II ‐28 is restricted to a number of solanaceous species. Although the flg II ‐28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flg II ‐28 have FLS 2 ‐dependent effects on virulence. However, the expression of a tomato allele of FLS 2 does not confer to Nicotiana benthamiana the ability to detect flg II ‐28, and tomato plants silenced for FLS 2 are not altered in flg II ‐28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flg II ‐28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS 2 ‐dependent virulence effect in Arabidopsis.
In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein. In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule. Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathioneS-transferase fusion proteins, respectively. The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping. In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay. Moreover, an active glutathioneS-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto. Pti1 phosphorylation by Pto was also characterized and found to occur with aK m of 4.1 μm at sites similar to those autophosphorylated by Pti1. Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping. This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites. In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein. In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule. Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathioneS-transferase fusion proteins, respectively. The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping. In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay. Moreover, an active glutathioneS-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto. Pti1 phosphorylation by Pto was also characterized and found to occur with aK m of 4.1 μm at sites similar to those autophosphorylated by Pti1. Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping. This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites. Higher plants have evolved the ability to recognize and resist invading pathogens by the activation of defense mechanisms that inhibit pathogen growth and movement in the plant (1Hammond-Kosack K.E. Jones J.D.G. Plant Cell. 1996; 8: 1773-1791Crossref PubMed Scopus (1393) Google Scholar). In many plant-pathogen interactions, the rapid activation of the defense response is mediated by a specific recognition event involving the product of an avirulence (avr) 1The abbreviations used are: avr, avirulance gene; MBP, maltose-binding protein; GST, glutathioneS-transferase; R, resistance gene; PAGE, polyacrylamide gel electrophoresis; CDK, cyclin-dependent kinase. gene in the pathogen and the corresponding resistance (R) gene in the plant (2Flor H.H. Annu. Rev. Phytopathol. 1971; 9: 275-296Crossref Google Scholar). Proteins encoded by R genes are postulated to function as receptors that bind the cognate avr gene product and activate defense mechanisms. Several R genes have been isolated to date, and structural characteristics of their encoded proteins support the proposed receptor function (3Baker B. Zambryski P. Staskawicz B. Dinesh-Kumar S.P. Science. 1997; 276: 726-733Crossref PubMed Scopus (801) Google Scholar). Most Rgenes encode cytoplasmic or transmembrane proteins containing a region of leucine-rich repeats of variable length and content, which might be involved in protein-protein interactions (4Jones D.A. Jones J.D.G. Adv. Bot. Res. Incorp. Adv. Plant Path. 1997; 24: 89-167Crossref Scopus (480) Google Scholar). Some leucine-rich repeat-containing R proteins, including those encoded by the tobaccoN, the flax L6, and the Arabidopsis RPP5 genes, also contain a region of homology with the cytoplasmic domains of the Drosophila Toll and the mammalian interleukin-1 receptor proteins (4Jones D.A. Jones J.D.G. Adv. Bot. Res. Incorp. Adv. Plant Path. 1997; 24: 89-167Crossref Scopus (480) Google Scholar). Another class of R genes is represented by Pto, which encodes a tomato serine/threonine protein kinase and confers resistance specifically to the bacterial pathogen Pseudomonas syringae pv. tomato expressing the avirulence geneavrPto (5Martin G.B. Brommonschenkel S. Chunwongse J. Frary A. Ganal M.W. Spivey R. Wu T. Earle E.D. Tanksley S.D. Science. 1993; 262: 1432-1436Crossref PubMed Scopus (1118) Google Scholar). Pto does not share motifs with other resistance genes, except for the kinase domain, which is also present in the product of the rice gene Xa21, conferring resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (6Song W.-Y. Wang G.-L. Chen L.-L. Kim H.-S. Pi L.-Y. Holsten T. Gardner J. Wang B. Zhai W.-X. Zhu L.-H. Fauquet C. Ronald P. Science. 1995; 270: 1804-1806Crossref PubMed Scopus (1772) Google Scholar). Recent studies have shown, using the yeast two-hybrid system, that a direct interaction occurs between the product of the tomatoPto resistance gene and the product of the P. syringae pv. tomato avrPto gene (7Tang X. Frederick R.D. Zhou J. Halterman D.A. Jia Y. Martin G.B. Science. 1996; 274: 2060-2063Crossref PubMed Scopus (513) Google Scholar, 8Scofield S.R. Tobias C.M. Rathjen J. Chang J.H. Lavelle D.T. Michelmore R.W. Staskawicz B.J. Science. 1996; 274: 2063-2065Crossref PubMed Scopus (435) Google Scholar). Mutations of Pto or AvrPto that interfere with this interaction in yeast also abolish disease resistance in plants. In addition, for resistance to take place, Pto and AvrPto must be present simultaneously inside the plant cell, strongly suggesting that the Pto-AvrPto recognition is an intracellular event. AvrPto and other bacterial avr gene products are thought to be delivered into the plant cell by a type III secretion system encoded by the bacterial pathogen (9Van den Ackerveken G. Bonas U. Trends Microbiol. 1997; 5: 394-398Abstract Full Text PDF PubMed Scopus (29) Google Scholar). In fact, a set of genes in P. syringae shows similarity to genes encoding components of the type III secretion system of mammalian pathogens, and is required both for resistance and pathogenicity (10Alfano J.R. Colmer A. Plant Cell. 1996; 8: 1683-1698Crossref PubMed Scopus (285) Google Scholar). The recognition event between the tomato Pto kinase and the bacterial AvrPto protein initiates a signal transduction pathway that involves downstream effectors and ultimately leads to disease resistance. ThePrf gene is required for Pto-mediated resistance and encodes a protein with a leucine zipper, a nucleotide binding site, and leucine-rich repeats, common motifs in other resistance gene products (11Salmeron J.M. Oldroyd G.E.D. Rommens C.M.T. Scofield S.R. Kim H.-S. Lavelle D.T. Dahlbeck D. Staskawicz B.J. Cell. 1996; 86: 123-133Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). However, the role of Prf in the Pto pathway remains unclear. Other putative effectors were identified by their specific interaction with the Pto kinase in the yeast two-hybrid system (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 13Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3207-3218Crossref PubMed Scopus (393) Google Scholar). Among them are Pti1, a serine/threonine protein kinase that is specifically phosphorylated in vitro by Pto and is involved in the hypersensitive response (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar), and Pti4, Pti5, and Pti6, putative transcription factors that are similar to the tobacco ethylene-responsive element-binding proteins (13Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3207-3218Crossref PubMed Scopus (393) Google Scholar, 14Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (943) Google Scholar). In mammals, autophosphorylation activity plays a central role in the regulation of receptor tyrosine kinases (15Heldin C.-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1445) Google Scholar). Hormones, or growth and differentiation factors, bind to receptors with tyrosine kinase activity and induce conformational alterations in the receptor extracellular domains causing oligomerization. Autophosphorylation by intermolecular phosphorylation in the receptor cytoplasmic domain then takes place and modulates the interaction between the activated receptor and cellular proteins (16Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). Induction of autophosphorylation activity by extracellular signals has also been observed in cytoplasmic protein kinases associated with receptors. For instance, autophosphorylation of the Janus kinases is activated by cytokines through cytokine receptors (17Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1145) Google Scholar), and components of the extracellular matrix determine the integrin-mediated activation of autophosphorylation of the focal adhesion kinase FAK (18Kornberg L. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar). Because the tomato Pto serine/threonine protein kinase is thought to function as an intracellular receptor or as part of a receptor complex for the AvrPto signal molecule, we were interested in determining the role of Pto autophosphorylation activity in the elicitation of the defense response by the Pto-AvrPto interaction. As a step toward testing the possibility that AvrPto mediates Pto dimerization and activation by intermolecular autophosphorylation, we characterized the biochemical properties of Pto autophosphorylation and phosphorylation of its putative effector Pti1. Pto and Pti1 were expressed inEscherichia coli as fusion proteins and assayed for their kinase activities. Pto and Pti1 autophosphorylated at multiple sites, and their autophosphorylation proceeded via an intramolecular mechanism. We also determined the kinetics of Pti1 phosphorylation by Pto and compared the tryptic digests of Pti1 phosphorylated by Pto and by the product of the recessive pto allele. For expression in bacteria, the Pto protein kinase and its mutagenized kinase-deficient form Pto(K69Q) were fused in frame to the C terminus of the maltose-binding protein (MBP), as described previously (19Loh Y.-T. Martin G.B. Plant Physiol. 1995; 108: 1735-1739Crossref PubMed Scopus (81) Google Scholar). The Pto protein was also expressed as a fusion to the C terminus of glutathioneS-transferase (GST). To develop the GST-Pto fusion, primers YTL8 (5′-ATGGGATCCAAGTATTCTAAGGCA-3′) and YTL5 (5′-CCCTGCAGTGAAAGAAGGATCCACAG-3′) were used to amplify a 1,057-base pair product encoding Pto from plasmid PTC3 (8Scofield S.R. Tobias C.M. Rathjen J. Chang J.H. Lavelle D.T. Michelmore R.W. Staskawicz B.J. Science. 1996; 274: 2063-2065Crossref PubMed Scopus (435) Google Scholar). Primer YTL8 introduced a BamHI restriction site at the N terminus of thePto gene, and primer YTL5 amplified a BamHI restriction site 70 base pairs after the TAA stop codon. The polymerase chain reaction product was digested by BamHI and inserted into vector pGEX-4T1 (Amersham Pharmacia Biotech) at the corresponding site. The constructs for expression of the kinase encoded by the recessive pto allele as a MBP fusion, and of the Pti1 protein kinase and its mutagenized form Pti1(K96N) as GST fusions, were prepared as described previously (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 20Jia Y. Loh Y.-T. Zhou J. Martin G.B. Plant Cell. 1997; 9: 61-73PubMed Google Scholar). MBP and GST fusions were expressed in the E. coli strain PR745 (New England Biolabs) and affinity-purified by using amylose resin (New England Biolabs) and glutathione-agarose beads (Sigma), respectively. Autophosphorylation activity of MBP-Pto and GST-Pti1 fusion proteins was assayed at different protein concentrations in 25 μl of reaction buffer (50 mmTris-HCl, pH 7.0, 1 mm dithiothreitol, 10 mmMnCl2, 1 mg/ml bovine serum albumin, and 20 μm ATP), containing 4 μCi of [γ-32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech). Reactions were incubated for 15 min at room temperature and spotted on phosphocellulose P-81 filters (Whatman). The filters were extensively washed by three changes of 150 mm phosphoric acid, and by a final wash with ethanol. Phosphate incorporation was then measured using a scintillation counter (LS 6500, Beckman). The range of concentrations tested was from 0.72 to 17.9 μm and from 0.06 to 8.61 μm for MBP-Pto and GST-Pti1, respectively. Reactions containing the highest amount of enzyme were found to be linear over the entire reaction time (15 min). Assays to test Pto intermolecular autophosphorylation were performed with the GST-Pto fusion protein immobilized on glutathione-agarose beads and the other components dissolved in solution. GST-Pto fusion protein (2 μg) was mixed with MBP-Pto(K69Q) or GST-Pti1(K96N) fusion proteins (2 μg) in 30 μl of reaction buffer (described above), containing 3 μCi of [γ-32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech). Reactions were incubated for 10 min at room temperature and stopped by adding EDTA to a final concentration of 10 mm. Proteins were then fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and stained by Coomassie Brilliant Blue R250. The stained gel was dried and exposed to x-ray film. To determine V max and K mvalues of the MBP-Pto fusion for its substrate GST-Pti1(K96N) fusion protein, 0.5 μg of MBP-Pto was incubated at room temperature with different amounts of GST-Pti1(K96N) in 20 μl of kinase buffer containing 2 μCi of [γ-32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech). The range of GST-Pti1(K96N) concentrations tested was from 0.72 to 17.9 μm. Reactions were stopped after 10 min by adding EDTA to a final concentration of 10 mm. At this reaction time, phosphate incorporation was found to be linear for the highest substrate concentration used in the experiment. Proteins were then fractionated by SDS-PAGE, stained by Coomassie Brilliant Blue R250, and analyzed by Instant Imager (Packard Corp.). Values for the V max andK m parameters were estimated by nonlinear least squares fitting using SigmaPlot 4.0 for Windows software (SPSS Inc.). The procedure used for phosphopeptide analysis was essentially as described by Van der Geeret al. (21Van Der Geer P. Luo K. Sefton B.M. Hunter T. Hardie D.G. Protein Phosphorylation: A Practical Approach. IRL Press, New York1993: 31-59Google Scholar) with some modifications. The phosphorylated protein to be analyzed was fractionated on a SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane, and the corresponding piece of membrane was excised. After blocking with 100 mm acetic acid containing 0.5% polyvinyl pyrrolidone for 30 min, the membrane was extensively washed with water and twice with 50 mm ammonium bicarbonate, pH 8.2. The membrane-bound protein was then incubated in 200 μl of 50 mm ammonium bicarbonate, pH 8.2, with 10 μg ofN-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin for at least 4 h at 37 °C. The sample was lyophilized to dryness and oxidized by performic acid for 60 min at room temperature. After water dilution and evaporation of performic acid in a centrifugal vacuum concentrator, the sample was resuspended in electrophoresis buffer containing n-butanol, pyridine, acetic acid, and water at a ratio of 2:1:1:36, and separated for 40 min by thin layer electrophoresis at pH 4.7 and 1.0 kV on 20 × 20-cm cellulose TLC plates (EM Science). The first dimension fractionation was followed by ascending chromatography in phosphopeptide buffer containing n-butanol, pyridine, acetic acid, and water at a ratio of 15:10:3:12. After electrophoresis, the radioactive species were detected by autoradiography. Phosphopeptides were electrophoretically separated on alkaline 40% polyacrylamide gels as described by Dadd et al. (22Dadd C.A. Cook R.G. Allis C.D. BioTechniques. 1993; 14: 266-273PubMed Google Scholar). The Pto and Pti1 proteins are involved in resistance to bacterial speck disease in tomato plants and have been shown previously to be active serine/threonine kinases when tested in vitro(12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 19Loh Y.-T. Martin G.B. Plant Physiol. 1995; 108: 1735-1739Crossref PubMed Scopus (81) Google Scholar). To further characterize their kinase activity, we tested whether Pto and Pti1 autophosphorylation mechanisms are intramolecular (first order with respect to enzyme concentration) or intermolecular (second order with respect to enzyme concentration). Pto and Pti1 were expressed in bacteria as MBP (MBP-Pto) and GST (GST-Pti1) fusion proteins, respectively, and the effect of various molar concentrations on the autophosphorylation reaction was studied. As shown in Figs. 1 A and 2 A, the rate of autophosphorylation was linear with respect to enzyme concentration for both MBP-Pto and GST-Pti1. In addition, the phosphate incorporation per molecule was constant when MBP-Pto concentration in the reaction varied by 60-fold (Fig. 1 B). Similarly, the phosphate incorporation per GST-Pti1 molecule varied by only 1.7 when the enzyme concentration in the reaction varied by 140-fold (Fig. 2 B). Finally, the van't Hoff plot of autophosphorylation (logarithm of phosphorylation rateversus logarithm of enzyme concentration), whose slope indicates the order of the reaction, had a slope of 1.11 ± 0.038 and 0.90 ± 0.010 for MBP-Pto and GST-Pti1, respectively (Figs. 1 C and 2 C). Taken together, these data indicate that both MBP-Pto and GST-Pti1 autophosphorylation occur predominantly via an intramolecular mechanism.Figure 2Effect of enzyme concentration on the autophosphorylation of GST-Pti1 fusion protein.Autophosphorylation activity of GST-Pti1 was tested at different enzyme concentrations in an in vitro kinase assay. The enzyme concentration varied from 0.06 to 8.61 μm. A, plot of phosphate incorporation rate versus GST-Pti1 concentration in the assay. B, specific activity of GST-Pti1 expressed as phosphate incorporation rate per picomole of GST-Pti1 present in the assay. C, van't Hoff plot of the logarithm of velocity versus the logarithm of GST-Pti1 concentration. Linear regression of the data in C estimated a slope of 0.90 ± 0.01 and a correlation coefficient of 0.99. InA–C, data are the mean ± S.E. (n = 4).View Large Image Figure ViewerDownload (PPT) To provide further evidence for intramolecular autophosphorylation by Pto, we tested if an active GST-Pto fusion protein can phosphorylate an inactive MBP-Pto molecule in which the invariant lysine residue in kinase subdomain II was substituted by a glutamine (19Loh Y.-T. Martin G.B. Plant Physiol. 1995; 108: 1735-1739Crossref PubMed Scopus (81) Google Scholar). As shown in Fig. 3, the GST-Pto fusion protein was able to autophosphorylate and to phosphorylate its substrate Pti1 as observed previously (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar). However, it failed to phosphorylate the inactive mutant protein MBP-Pto(K69Q), strongly supporting the notion that Pto autophosphorylation occurs through an intramolecular rather than intermolecular mechanism. The Pti1 protein kinase was shown previously to be specifically phosphorylatedin vitro by the Pto protein kinase (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar). To study the kinetics of this reaction, the initial velocity of the phosphorylation of a kinase-deficient mutant GST-Pti1(K96N) by Pto-MBP was analyzed at different substrate concentrations (Fig. 4). Nonlinear least squares fitting analysis of the data estimated K m andV max values for the GST-Pti1(K96N) substrate as 4.1 ± 0.6 μm and 0.55 ± 0.03 nmol/min/mg, respectively. To investigate Pto and Pti1 autophosphorylation sites in more detail, MBP-Pto and GST-Pti1 fusion proteins were autophosphorylated in vitroand digested with trypsin. The tryptic digests were resolved horizontally by thin layer electrophoresis at pH 4.7, and vertically by ascending chromatography. As shown in Fig. 5, both digestion of autophosphorylated MBP-Pto and GST-Pti1 generated one major and at least four minor phosphopeptides (Fig. 5, A and B). The ratio between the intensity of the major spot and minor spots was at least 5:1 for MBP-Pto digests, and 50:1 for GST-Pti1 digests. The variable intensity of the labeling might be a result of the degree of enzyme affinity for different sites and to the number of phosphorylation sites present in each peptide. Additional phosphopeptides were detectable after extensive exposures of the TLC plates to x-ray films and may indicate either the presence of sites in the proteins phosphorylated at a very low efficiency or, more likely, partial tryptic digestion products. It has been shown recently that Pti1 is a substrate not only for the Pto kinase, but also for the translation product of the recessive allele pto, which is a functional protein kinase but does not confer bacterial speck resistance to tomato plants (20Jia Y. Loh Y.-T. Zhou J. Martin G.B. Plant Cell. 1997; 9: 61-73PubMed Google Scholar). To compare the Pti1 autophosphorylation sites to those phosphorylated by Pto or pto, the kinase-deficient mutant GST-Pti1(K96N) was phosphorylated by an MBP-Pto or MBP-pto fusion proteins and then digested by trypsin. Phosphopeptide maps of the Pti1 digestion products revealed the presence of one major phosphorylated peptide, similar to that observed in Pti1 autophosphorylation reactions (Fig. 5, B–D). To test whether this major spot was derived from the same peptide phosphorylated by Pti1 autophosphorylation and by Pto and recessive pto phosphorylation, tryptic digests from the three reactions were fractionated by alkaline electrophoresis. As shown in Fig. 6, the main phosphorylated peptide in all the reactions showed the same molecular weight and charge characteristics, suggesting that the same phosphorylation site(s) may be utilized by the three enzymes. Similar minor spots were observed in the phosphotryptic maps of autophosphorylated Pti1 and of Pti1 phosphorylated by Pto or recessive pto (Fig. 5,B–D). Their intensity was variable in different experiments, and analysis of their sequence will be required to determine whether they represent phosphorylation sites, degradation products, or partial digests of the main phosphorylated peptide. In this report, we characterized biochemical properties of two tomato serine/threonine protein kinases, Pto and Pti1, which are involved in the signaling pathway leading to resistance to the bacterial pathogen P. syringae pv. tomato expressing theavrPto gene. A specific recognition between the Pto kinase and the bacterial AvrPto protein occurs within the plant cell and triggers the activation of the pathway, defining the Pto kinase as an intracellular receptor or as part of a receptor complex (7Tang X. Frederick R.D. Zhou J. Halterman D.A. Jia Y. Martin G.B. Science. 1996; 274: 2060-2063Crossref PubMed Scopus (513) Google Scholar, 8Scofield S.R. Tobias C.M. Rathjen J. Chang J.H. Lavelle D.T. Michelmore R.W. Staskawicz B.J. Science. 1996; 274: 2063-2065Crossref PubMed Scopus (435) Google Scholar). In order to investigate the molecular mechanisms taking place in this interaction and the role of autophosphorylation in Pto kinase activation, we first examined the molecular characteristics of Pto autophosphorylation in vitro. We found that Pto autophosphorylates at several sites in the protein via an intramolecular process that is not affected by protein concentration. Induction of autophosphorylation activity by extracellular signals is well documented in mammals for receptors with tyrosine kinase activity (23Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (434) Google Scholar). Ligand binding to the extracellular domain of receptor tyrosine kinases induces receptor dimerization. Dimerization in turn activates autophosphorylation of the receptor catalytic domain, which is mediated by an intermolecular mechanism of phosphorylation. Dimerization and intermolecular autophosphorylation of a nuclear serine/threonine kinase from Arabidopsis thaliana have been shown recently (24Roe J.L. Durfee T. Zupan J.R. Repetti P.P. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). However, the mechanism of activation for this protein kinase, essential for leaf and flower morphogenesis, is still unknown. In order to determine whether Pto dimerization and activation is plausible in the interaction between the signal molecule AvrPto and the Pto kinase, we tested in vitro the mechanism of Pto autophosphorylation. We found that Pto autophosphorylation occurs via an intramolecular reaction, making it unlikely that oligomerization is required for Pto activation. Alternative modes of activation remain to be tested to elucidate the molecular mechanisms taking place during the Pto-AvrPto interaction. It is possible that AvrPto might activate Pto by causing conformational changes that possibly expose certain domains, which were not available previously, to autophosphorylation or to phosphorylation by an additional protein kinase. Such a mechanism occurs during activation of cyclin-dependent kinases (CDKs) by cyclins (25Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). The activity of CDK2, for example, which is involved in regulation of events in the eukaryotic cell cycle, is stimulated by a two-step mechanism of activation. First, the regulatory subunit cyclin A associates with CDK2, causing conformational changes in the kinase catalytic sites that make Thr-160 more accessible for phosphorylation by the CDK-activating kinase. Second, CDK-activating kinase phosphorylation of Thr-160 determines full activation of CDK2 (26Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar). Even in such a scenario, autophosphorylation activity of Pto still may represent a prerequisite for the interaction with AvrPto. In fact, it has been observed that forms of Pto mutated in residues essential for kinase activity do not interact with AvrPto in the two-hybrid system (7Tang X. Frederick R.D. Zhou J. Halterman D.A. Jia Y. Martin G.B. Science. 1996; 274: 2060-2063Crossref PubMed Scopus (513) Google Scholar, 8Scofield S.R. Tobias C.M. Rathjen J. Chang J.H. Lavelle D.T. Michelmore R.W. Staskawicz B.J. Science. 1996; 274: 2063-2065Crossref PubMed Scopus (435) Google Scholar). The requirement of a phosphorylated residue for the interaction between a protein kinase and a regulatory subunit has been observed for the cyclic AMP-dependent protein kinase (27Levin L.R. Zoller M.J. Mol. Cell. Biol. 1990; 10: 1066-1075Crossref PubMed Scopus (50) Google Scholar). Cyclic AMP-dependent protein kinase requires phosphorylation of a conserved threonine residue for the interaction with an associated regulatory subunit which represses its activity. Similarly, theArabidopsis serine/threonine receptor kinase RLK5 interactsin vitro with the KAPP type 2C protein phosphatase only in its autophosphorylated form (28Stone J.M. Collinge M.A. Smith R.D. Horn M.A. Walker J.C. Science. 1994; 266: 793-795Crossref PubMed Scopus (234) Google Scholar). In order to define the role of Pto autophosphorylation in vivo, it will be necessary to identify the residues that are phosphorylated and examine the importance of these sites on the ability of Pto to confer disease resistance. In several instances, it has been shown that sites phosphorylated in vitro by autophosphorylation or cross-phosphorylation mechanisms are also phosphorylated in vivo (29Cheng M. Zhen E. Robinson M.J. Ebert D. Goldsmith E. Cobb M.H. J. Biol. Chem. 1996; 271: 12057-12062Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30Colwill K. Pawson T. Andrews B. Prasad J. Manley J.L. Bell J.C. Duncan P.I. EMBO J. 1996; 15: 265-275Crossref PubMed Scopus (475) Google Scholar). Finally, examination of the quantitative and qualitative effect of AvrPto on Pto autophosphorylation may shed light on the mechanism of Pto activation. The recognition event between Pto and AvrPto is postulated to result in activation of Pto effectors including the serine/threonine kinase Pti1 (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar) and putative transcription factors (13Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3207-3218Crossref PubMed Scopus (393) Google Scholar). Here, we have shown that Pti1, similar to Pto, autophosphorylates intramolecularly at one major and a few minor phosphorylation sites. The functional relevance of this mechanism of autophosphorylation in the Pto-signaling pathway has yet to be determined. Autophosphorylation of serine/threonine protein kinases in plants has been shown to occur intermolecularly or intramolecularly, but neither of the two mechanisms has been related to a specific form of regulation of activity (24Roe J.L. Durfee T. Zupan J.R. Repetti P.P. McLean B.G. Zambryski P.C. J. Biol. Chem. 1997; 272: 5838-5845Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 31Horn M.A. Walker J.C. Biochim. Biophys. Acta. 1994; 1208: 65-74Crossref PubMed Scopus (81) Google Scholar, 32Schulze-Muth P. Irmler S. Schröder G. Schröder J. J. Biol. Chem. 1996; 271: 26684-26689Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Pti1 has been shown previously to be phosphorylated in vitroby Pto and proposed as an in vivo substrate for Pto (12Zhou J. Loh Y.-T. Bressan R.A. Martin G.B. Cell. 1995; 83: 925-935Abstract Full Text PDF PubMed Scopus (322) Google Scholar). Here, we further characterized this in vitro interaction and estimated the K m of Pto for Pti1 to be 4.1 μm. This K m is in the range of values observed in the interaction between kinases from different signaling pathways and their physiological or synthetic substrates. For example, the K m value of Raf-1 for MEK is 0.8 μm (33Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar), cAMP-dependent protein kinase fromDictyostelium discoideum phosphorylates a synthetic heptapeptide with a K m value of 12 μm, and the ERK-2 mitogen-activated protein kinase shows aK m value of 0.12 μm for Raf-1 (34Lee R., M. Cobb M.H. Blackshear P.J. J. Biol. Chem. 1992; 267: 1088-1092Abstract Full Text PDF PubMed Google Scholar). A recessive pto allele that does not confer speck disease resistance was isolated recently and shown to encode a functional protein kinase (20Jia Y. Loh Y.-T. Zhou J. Martin G.B. Plant Cell. 1997; 9: 61-73PubMed Google Scholar). Although the pto kinase is able to use Pti1 as a substrate for phosphorylation, it shows very poor physical interaction with Pti1 and three additional Pto-interacting proteins in the yeast two-hybrid system (20Jia Y. Loh Y.-T. Zhou J. Martin G.B. Plant Cell. 1997; 9: 61-73PubMed Google Scholar). Here, we found by phosphopeptide analysis that Pti1 phosphorylation by Pto or pto and Pti autophosphorylation occur at similar sites. This observation raises questions about how the activities of different components in the speck disease resistance signaling pathway are regulated. These results also suggest that the inability of the pto kinase to mediate a resistance response toP. syringae pv. tomato expressing the avrPto gene is not related to an altered recognition specificity for Pti1 phosphorylation sites. We thank Drs. C. L. Ashendel, Y.-Q. Gu, and A. T. S. Taylor for helpful comments on the manuscript, and Dr. W. E. Nyquist for assistance with the statistical analysis of the data.
The tomato Pto kinase confers resistance to bacterial speck disease caused by strains of Pseudomonas syringae pv. tomato that express the avirulence gene avrPto. Pto contains a putative myristylation site at its amino terminus that was hypothesized to play a role in localizing Pto in the plant cell. Site-directed mutagenesis was used to change the invariant glycine residue in the myristylation motif to an alanine. Transgenes encoding the mutant Pto(G2A) and wild-type Pto were placed behind the cauliflower mosaic virus 35S promoter and transformed into tomato plants that are susceptible to bacterial speck disease. Both the mutant and wild-type forms of Pto conferred resistance to a strain of P. syringae pv. tomato expressing avrPto. These results indicate that the myristylation motif of Pto is not required for bacterial speck disease resistance.
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.
Tomato (Solanum lycopersicum), along with many other economically valuable species, belongs to the Solanaceae family. Understanding how plants in this family defend themselves against pathogens offers the opportunity of improving yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques that allow determination of changes in transcript abundance during pathogen attack. Such changes can implicate the affected gene as participating in plant defense. Testing the involvement of these candidate genes in defense has relied largely on posttranscriptional gene silencing, particularly virus-induced gene silencing. We discuss how functional genomics has played a key role in our current understanding of the defense response in tomato and related species and what are the challenges and opportunities for the future.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMicrotiter plate binding assay for cholinergic compounds utilizing the nicotinic acetylcholine receptorLu. Chen, Glenn B. Martin, and Garry A. RechnitzCite this: Anal. Chem. 1992, 64, 23, 3018–3023Publication Date (Print):December 1, 1992Publication History Published online1 May 2002Published inissue 1 December 1992https://pubs.acs.org/doi/10.1021/ac00047a025https://doi.org/10.1021/ac00047a025research-articleACS PublicationsRequest reuse permissionsArticle Views118Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Inducible NO synthase (iNOS) activity is induced upon pathogen inoculation in resistant, but not susceptible, tobacco and Arabidopsis plants. It was shown recently that a variant form of the Arabidopsis P protein (AtvarP) has iNOS activity. P protein is part of the glycine decarboxylase complex (GDC). It is unclear whether P protein also has iNOS activity and, if so, whether AtvarP, P, or both, play a role in plant defense. Here, we show that iNOS activity is induced in both resistant and susceptible tomato leaves upon inoculation with the Pseudomonas syringae pv. tomato strain DC3000. Virus-induced gene-silencing targeting LevarP , a putative tomato ortholog of AtvarP , led to complete suppression of DC3000-induced iNOS activation and an ≈80% reduction in GDC activity; it also increased disease-symptom severity and DC3000 growth in both resistant and susceptible tomato. To determine whether enhanced susceptibility exhibited by LevarP -silenced, susceptible tomato was due to loss of ( i ) iNOS activity, ( ii ) GDC activity, or ( iii ) both, GDC activity was inhibited with or without concurrent suppression of iNOS. Treatment with methotrexate inhibited both iNOS and GDC activities and resulted in increased susceptibility, comparable with that observed in LevarP -silenced plants. When normal iNOS activity was maintained in the presence of methotrexate by the addition of tetrahydrobiopterin, there was no change in susceptibility, despite a dramatic reduction in GDC activity. Together, these results indicate that iNOS contributes to host defense response against DC3000.
