The N-terminal region of Pseudomonas type III effector AvrPtoB elicits Pto-dependent immunity and has two distinct virulence determinants

Resistance to bacterial speck disease in tomato is activated by the physical interaction of the host Pto kinase with either of the sequence-dissimilar type III effector proteins AvrPto or AvrPtoB (HopAB2) from Pseudomonas syringae pv. tomato. Pto-mediated immunity requires Prf, a protein with a nucleotide-binding site and leucine-rich repeats. The N-terminal 307 amino acids of AvrPtoB were previously reported to interact with the Pto kinase, and we show here that this region (AvrPtoB(1-307)) is sufficient for eliciting Pto/Prf-dependent immunity against P. s. pv. tomato. AvrPtoB(1-307) was also found to be sufficient for a virulence activity that enhances ethylene production and increases growth of P. s. pv. tomato and severity of speck disease on susceptible tomato lines lacking either Pto or Prf. Moreover, we found that residues 308-387 of AvrPtoB are required for the previously reported ability of AvrPtoB to suppress pathogen-associated molecular patterns-induced basal defenses in Arabidopsis. Thus, the N-terminal region of AvrPtoB has two structurally distinct domains involved in different virulence-promoting mechanisms. Random and targeted mutagenesis identified five tightly clustered residues in AvrPtoB(1-307) that are required for interaction with Pto and for elicitation of immunity to P. s. pv. tomato. Mutation of one of the five clustered residues abolished the ethylene-associated virulence activity of AvrPtoB(1-307). However, individual mutations of the other four residues, despite abolishing interaction with Pto and avirulence activity, had no effect on AvrPtoB(1-307) virulence activity. None of these mutations affected the basal defense-suppressing activity of AvrPtoB(1-387). Based on sequence alignments, estimates of helical propensity, and the previously reported structure of AvrPto, we hypothesize that the Pto-interacting domains of AvrPto and AvrPtoB(1-307) have structural similarity. Together, these data support a model in which AvrPtoB(1-307) promotes ethylene-associated virulence by interaction not with Pto but with another unknown host protein.     

Pto- and Prf-mediated recognition of AvrPto and AvrPtoB restricts the ability of diverse pseudomonas syringae pathovars to infect tomato

The molecular basis underlying the ability of pathogens to infect certain plant species and not others is largely unknown. Pseudomonas syringae is a useful model species for investigating this phenomenon because it comprises more than 50 pathovars which have narrow host range specificities. Tomato (Solanum lycopersicum) is a host for P. syringae pv. tomato, the causative agent of bacterial speck disease, but is considered a nonhost for other P. syringae pathovars. Host resistance in tomato to bacterial speck disease is conferred by the Pto protein kinase which acts in concert with the Prf nucleotide-binding lucine-rich repeat protein to recognize P. syringae pv. tomato strains expressing the type III effectors AvrPto or AvrPtoB (HopAB2). The Pto and Prf genes were isolated from the wild tomato species S. pimpinellifolium and functional alleles of both of these genes now are known to exist in many species of tomato and in other Solanaceous species. Here, we extend earlier reports that avrPto and avrPtoB genes are widely distributed among pathovars of P. syringae which are considered nonhost pathogens of tomato. This observation prompted us to examine the possibility that recognition of these type III effectors by Pto or Prf might contribute to the inability of many P. syringae pathovars to infect tomato species. We show that 10 strains from presumed nonhost P. syringae pathovars are able to grow and cause pathovar-unique disease symptoms in tomato leaves lacking Pto or Prf, although they did not reach the population levels or cause symptoms as severe as a control P. syringae pv. tomato strain. Seven of these strains were found to express avrPto or avrPtoB. The AvrPto- and AvrPtoB-expressing strains elicited disease resistance on tomato leaves expressing Pto and Prf. Thus, a gene-for-gene recognition event may contribute to host range restriction of many P. syringae pathovars on tomato species. Furthermore, we conclude that the diverse disease symptoms caused by different Pseudomonas pathogens on their normal plant hosts are due largely to the array of virulence factors expressed by each pathovar and not to specific molecular or morphological attributes of the plant host.     

Plant Programmed Cell Death Caused by an Autoactive Form of Prf Is Suppressed by Co-Expression of the Prf LRR Domain

In tomato, the NBARC-LRR resistance (R) protein Prf acts in concert with the Pto or Fen kinase to determine immunity against Pseudomonas syringae pv. tomato (Pst). Prf-mediated defense signaling is initiated by the recognition of two sequence-unrelated Pst-secreted effector proteins, AvrPto and AvrPtoB, by tomato Pto or Fen. Prf detects these interactions and activates signaling leading to host defense responses including localized programmed cell death (PCD) that is associated with the arrest of Pst growth. We found that Prf variants with single amino acid substitutions at D1416 in the IHD motif (isoleucine-histidine-aspartic acid) in the NBARC domain cause effector-independent PCD when transiently expressed in leaves of Nicotiana benthamiana, suggesting D1416 plays an important role in activation of Prf. The N-terminal region of Prf (NPrf) and the LRR domain are required for this autoactive Prf cell death signaling but dispensable for accumulation of the Prf(D1416V) protein. Significantly, co-expression of the Prf LRR but not NPrf, with Prf(D1416V), AvrPto/Pto, AvrPtoB/Pto, an autoactive form of Pto (Pto(Y207D)), or Fen completely suppresses PCD. However, the Prf LRR does not interfere with PCD caused by Rpi-blb1(D475V), a distinct R protein-mediated PCD signaling event, or that caused by overexpression of MAPKKKα, a protein acting downstream of Prf. Furthermore, we found the Prf(D1416V) protein is unable to accumulate in plant cells when co-expressed with the Prf LRR domain, likely explaining the cell death suppression. The mechanism for the LRR-induced degradation of Prf(D1416V) is unknown but may involve interference in the intramolecular interactions of Prf or to binding of the unattached LRR to other host proteins that are needed for Prf stability.     

Structural analysis of Pseudomonas syringae AvrPtoB bound to host BAK1 reveals two similar kinase-interacting domains in a type III Effector

To infect plants, Pseudomonas syringae pv. tomato delivers ~30 type III effector proteins into host cells, many of which interfere with PAMP-triggered immunity (PTI). One effector, AvrPtoB, suppresses PTI using a central domain to bind host BAK1, a kinase that acts with several pattern recognition receptors to activate defense signaling. A second AvrPtoB domain binds and suppresses the PTI-associated kinase Bti9 but is conversely recognized by the protein kinase Pto to activate effector-triggered immunity. We report the crystal structure of the AvrPtoB-BAK1 complex, which revealed structural similarity between these two AvrPtoB domains, suggesting that they arose by intragenic duplication. The BAK1 kinase domain is structurally similar to Pto, and a conserved region within both BAK1 and Pto interacts with AvrPtoB. BAK1 kinase activity is inhibited by AvrPtoB, and mutations at the interaction interface disrupt AvrPtoB virulence activity. These results shed light on a structural mechanism underlying host-pathogen coevolution.     

An avrPto/avrPtoB mutant of Pseudomonas syringae pv. tomato DC3000 does not elicit Pto-mediated resistance and is less virulent on tomato

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 deltaavrPtodeltaavrPtoB 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 DC3000deltaavrPto and DC3000deltaavrPtoB each caused slightly less severe disease symptoms, although their growth rate was unaffected. However, DC3000deltaavr PtodeltaavrPtoB 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.     

Identification of MAPKs and their possible MAPK kinase activators involved in the Pto-mediated defense response of tomato

The Pto kinase mediates resistance to bacterial speck disease in tomato by activating host defenses upon recognition of Pseudomonas syringae pv. tomato strains expressing the AvrPto or AvrPtoB proteins. Previous gene-silencing experiments have indicated that mitogen activated protein kinase (MAPK) cascades play a key role downstream of the Pto kinase to activate host defense responses. Here we use biochemical methods to demonstrate that two tomato MAPKs, LeMPK2 and LeMPK3, are activated in leaves in a Pto-specific manner upon expression of AvrPto and AvrPtoB. We show that these same MAPKs are activated upon overexpression of LeMAPKKKalpha, a protein previously demonstrated to be involved in Pto-mediated immunity. We identified two phylogenetically unrelated MAPK kinases (LeMKK2 and LeMKK4) that when overexpressed in leaves elicit cell death and activate LeMPK2 and LeMPK3. In vitro analysis demonstrated that LeMKK2 and LeMKK4 each phosphorylate the same subset of three MAPKs. Together these data provide biochemical evidence for the involvement of MAPK cascades in Pto-mediated resistance.     

A cluster of mutations disrupt the avirulence but not the virulence function of AvrPto

avrPto in Pseudomonas syringae pv. tomato encodes an avirulence protein that triggers race-specific resistance in tomato plants carrying Pto. The AvrPto protein is secreted from P. syringae pv. tomato to plant cells through the type III secretion pathway and activates race-specific resistance by a direct interaction with the Pto protein. Here we report that avrPto enhances the virulence of P. syringae pv. tomato in a strain-dependent manner in tomato plants lacking Pto. To determine whether the virulence function can be structurally separated from the avirulence function, we examined the virulence activity of a group of AvrPto mutants that carry single amino acid substitutions and lack the avirulence activity on tomato plants. Three mutants that were clustered in the center of AvrPto exhibited virulence activity in tomato plants with or without Pto. The rest of the mutations abolished the virulence. The identification of these mutants suggested that the avirulence function of AvrPto can be structurally separated from the virulence function.     

Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death

The AvrPtoB type III effector protein is conserved among diverse genera of plant pathogens suggesting it plays an important role in pathogenesis. Here we report that Pseudomonas AvrPtoB acts inside the plant cell to inhibit programmed cell death (PCD) initiated by the Pto and Cf9 disease resistance proteins and, remarkably, the pro-apoptotic mouse protein Bax. AvrPtoB also suppressed PCD in yeast, demonstrating that AvrPtoB functions as a cell death inhibitor across kingdoms. Using truncated AvrPtoB proteins, we identified distinct N- and C-terminal domains of AvrPtoB that are sufficient for host recognition and PCD inhibition, respectively. We also identified a novel resistance phenotype, Rsb, that is triggered by an AvrPtoB truncation disrupted in the anti-PCD domain. A Pseudomonas syringae pv. tomato DC3000 strain with a chromosomal mutation in the AvrPtoB C-terminus elicited Rsb-mediated immunity in previously susceptible tomato plants and disease was restored when full-length AvrPtoB was expressed in trans. Thus, our results indicate that a type III effector can induce plant susceptibility to bacterial infection by inhibiting host PCD.     

Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana

The Pto gene of tomato (Solanum lycopersicum) confers specific recognition of the unrelated bacterial effector proteins AvrPto and AvrPtoB. Pto resides in a constitutive molecular complex with the nucleotide binding site-leucine rich repeats protein Prf. Prf is absolutely required for specific recognition of both effectors. Here, using stable transgenic lines, we show that expression of Pto from its genomic promoter in susceptible tomatoes was sufficient to complement recognition of Pseudomonas syringae pv. tomato (Pst) bacteria expressing either avrPto or avrPtoB. Pto kinase activity was absolutely required for specific immunity. Expression of the Pto N-myristoylation mutant, pto(G2A), conferred recognition of Pst (avrPtoB), but not Pst (avrPto), although bacterial growth in these lines was intermediate between resistant and susceptible lines. Overexpression of pto(G2A) complemented recognition of avrPto. Transgenic tomato plants overexpressing wild-type Pto exhibited constitutive growth phenotypes, but these were absent in lines overexpressing pto(G2A). Therefore, Pto myristoylation is a quantitative factor for effector recognition in tomato, but is absolutely required for overexpression phenotypes. Native expression of Pto in the heterologous species Nicotiana benthamiana did not confer resistance to P. syringae pv. tabaci (Pta) expressing avrPto or avrPtoB, but recognition of both effectors was complemented by Prf co-expression. Thus, specific resistance conferred solely by Pto in N. benthamiana is an artefact of overexpression. Finally, pto(G2A) did not confer recognition of either avrPto or avrPtoB in N. benthamiana, regardless of the presence of Prf. Thus, co-expression of Prf in N. benthamiana complements many but not all aspects of normal Pto function.     

Host-mediated phosphorylation of type III effector AvrPto promotes Pseudomonas virulence and avirulence in tomato

The AvrPto protein from Pseudomonas syringae pv tomato is delivered into plant cells by the bacterial type III secretion system, where it either promotes host susceptibility or, in tomato plants expressing the Pto kinase, elicits disease resistance. Using two-dimensional gel electrophoresis, we obtained evidence that AvrPto is phosphorylated when expressed in plant leaves. In vitro phosphorylation of AvrPto by plant extracts occurs independently of Pto and is due to a kinase activity that is conserved in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three Ser residues clustered in the C-terminal 18 amino acids of AvrPto were identified in vitro as putative phosphorylation sites, and one site at S149 was directly confirmed as an in vivo phosphorylation site by mass spectrometry. Substitution of Ala for S149 significantly decreased the ability of AvrPto to enhance disease symptoms and promote growth of P. s. tomato in susceptible tomato leaves. In addition, S149A significantly decreased the avirulence activity of AvrPto in resistant tomato plants. Our observations support a model in which AvrPto has evolved to mimic a substrate of a highly conserved plant kinase to enhance its virulence activity. Furthermore, residues of AvrPto that promote virulence are also monitored by plant defenses.     

Tomato Pto encodes a functional N-myristoylation motif that is required for signal transduction in Nicotiana benthamiana

Pto kinase of tomato (Lycopersicon esculentum) confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato expressing avrPto or avrPtoB. Pto interacts directly with these type-III secreted effectors, leading to induction of defence responses including the hypersensitive response (HR). Signalling by Pto requires the nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein Prf. Little is known of how Pto is controlled prior to or during stimulation, although kinase activity is required for Avr-dependent activation. Here we demonstrate a role for the N-terminus in signalling by Pto. N-terminal residues outside the kinase domain were required for induction of the HR in Nicotiana benthamiana. The N-terminus also contributed to both AvrPto-binding and phosphorylation abilities. Pto residues 1-10 comprise a consensus motif for covalent attachment of myristate, a hydrophobic 14-carbon saturated fatty acid, to the Gly-2 residue. Several lines of evidence indicate that this motif is important for Pto function. A heterologous N-myristoylation motif complemented N-terminal deletion mutants of Pto for Prf-dependent signalling. Signalling by wild-type and mutant forms of Pto was strictly dependent on the Gly-2 residue. The N-myristoylation motif of Pto complemented the cognate motif of AvrPto for avirulence function and membrane association. Furthermore, Pto was myristoylated in vivo dependent on the presence of Gly-2. The subcellular localization of Pto was independent of N-myristoylation, indicating that N-myristoylation is required for some function other than membrane affinity. Consistent with this idea, AvrPtoB was also found to be a soluble protein. The data indicate an important role(s) for the myristoylated N-terminus in Pto signalling.     

Prf immune complexes of tomato are oligomeric and contain multiple Pto‐like kinases that diversify effector recognition

Cytoplasmic recognition of pathogen virulence effectors by plant NB‐LRR proteins leads to strong induction of defence responses termed effector triggered immunity (ETI). In tomato, a protein complex containing the NB‐LRR protein Prf and the protein kinase Pto confers recognition of the Pseudomonas syringae effectors AvrPto and AvrPtoB. Although structurally unrelated, AvrPto and AvrPtoB interact with similar residues in the Pto catalytic cleft to activate ETI via an unknown mechanism. Here we show that the Prf complex is oligomeric, containing at least two molecules of Prf. Within the complex, Prf can associate with Pto or one of several Pto family members including Fen, Pth2, Pth3, or Pth5. The dimerization surface for Prf is the novel N‐terminal domain, which also coordinates an intramolecular interaction with the remainder of the molecule, and binds Pto kinase or a family member. Thus, association of two Prf N‐terminal domains brings the associated kinases into close promixity. Tomato lines containing Prf complexed with Pth proteins but not Pto possessed greater immunity against P. syringae than tomatoes lacking Prf. This demonstrates that incorporation of non‐Pto kinases into the Prf complex extends the number of effector proteins that can be recognized.     

Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity

The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto. Pto and AvrPto physically interact, and this interaction is required for activation of host resistance. We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens. AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses. AvrPtoB has little overall sequence similarity with AvrPto. However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto. Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.     

Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins

Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction.     

The HopPtoF locus of Pseudomonas syringae pv. tomato DC3000 encodes a type III chaperone and a cognate effector

Type III secretion systems are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcF(Pto) and HopF(Pto), that are preceded by a single hrp box promoter. We present evidence here to show that ShcF(Pto) and HopF(Pto) encode a type III chaperone and a cognate effector, respectively. ShcF(Pto) interacts with and stabilizes the HopF(Pto) protein in the bacterial cell. Translation of HopF(Pto) starts at a rare initiation codon ATA that limits the synthesis of the HopF(Pto) protein to a low level in bacterial cells.     

Effector-triggered immunity mediated by the Pto kinase

Pto was the first disease-resistance gene cloned from a plant that confers recognition of a specific pathogen. The intracellular protein kinase that it encodes activates an immune response in tomato (Solanum lycopersicum) to bacterial speck disease by interacting with either the AvrPto or AvrPtoB type III effector proteins that are delivered into the plant cell by Pseudomonas syringae pathovar tomato. This recognition event triggers signaling pathways leading to effector-triggered immunity (ETI), which inhibits pathogen growth. During the past 15 years, ~25 genes have been identified by loss-of-function studies to have a role in Pto-mediated ETI. Here, we review the experimental approaches that have been used in these studies, discuss the proteins that have been identified and characterized, and present a current model of Pto-mediated ETI.