Pseudomonas syringae type III effector AvrPtoB is phosphorylated in plant cells on serine 258, promoting its virulence activity

The Pseudomonas syringae pv. tomato protein AvrPtoB is translocated into plant cells via the bacterial type III secretion system. In resistant tomato leaves, AvrPtoB acts as an avirulence protein by interacting with the host Pto kinase and eliciting the host immune response. Pto-mediated immunity requires Prf, a Pto-interacting protein with a putative nucleotide-binding site and a region of leucine-rich repeats. In susceptible tomato plants, which lack either Pto or Prf, AvrPtoB acts as a virulence protein by promoting P. syringae pv. tomato growth and enhancing symptoms associated with bacterial speck disease. The N-terminal 307 amino acids of AvrPtoB (designated AvrPtoB(1-307)) are sufficient for these virulence activities and for Pto-mediated avirulence. We report that AvrPtoB is phosphorylated by a Pto- and Prf-independent kinase activity that is conserved in several plant species, including tomato (Solanum lycopersicum), Nicotiana benthamiana, and Arabidopsis thaliana. AvrPtoB(1-307) was phosphorylated in tomato protoplasts, and mass spectrometry identified serine 258 as the major in vivo phosphorylation site of this protein. An alanine substitution of Ser(258) resulted in the loss of virulence and the diminution of avirulence activity of AvrPtoB(1-307), whereas a phosphomimetic S258D mutant had activities similar to wild type AvrPtoB(1-307). These observations suggest that AvrPtoB has evolved to mimic a substrate of a conserved plant kinase, leading to enhancement of its virulence and avirulence activities in the host cell.     

Crystal Structure of the Complex between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9Å and of the AvrPtoB_121-205–Pto complex at a resolution of 3.3 Å. AvrPtoB_121-205 exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.     

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.     

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.     

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.     

Phosphorylation of the Pseudomonas syringae effector AvrPto is required for FLS2/BAK1-independent virulence activity and recognition by tobacco

The type III effector protein AvrPto from Pseudomonas syringae pv. tomato is secreted into plant cells where it promotes bacterial growth and enhances symptoms of speck disease on susceptible tomato plants. The virulence activity of AvrPto is due, in part, to its interaction with components of host pattern recognition receptor complexes, which disrupts pathogen-associated molecular pattern-triggered immunity. This disruption mechanism requires a structural element of the AvrPto protein, the CD loop, which is also required for triggering Pto/Prf-mediated resistance in tomato. We have shown previously that the carboxyl-terminal domain (CTD) of AvrPto is phosphorylated and also contributes to bacterial virulence. Here we report that phosphorylation of the CTD on S147 and S149 promotes bacterial virulence in an FLS2/BAK1-independent manner, which is mechanistically distinct from the CD loop. In a striking corollary with Pto recognition of the CD loop in tomato, the tobacco species Nicotiana sylvestris and Nicotiana tabacum have a recognition mechanism that specifically detects the phosphorylation status of the CTD. Thus different species in the Solanaceae family have evolved distinct recognition mechanisms to monitor the same type III effector.     

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.     

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.     

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.     

The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.     

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.     

The solution structure of type III effector protein AvrPto reveals conformational and dynamic features important for plant pathogenesis

Pseudomonas syringae pv. tomato, the causative agent of bacterial speck disease of tomato, uses a type III secretion system (TTSS) to deliver effector proteins into the host cell. In resistant plants, the bacterial effector protein AvrPto physically interacts with the host Pto kinase and elicits antibacterial defense responses. In susceptible plants, which lack the Pto kinase, AvrPto acts as a virulence factor to promote bacterial growth. The solution structure of AvrPto reveals a functional core consisting of a three-helix bundle motif flanked by disordered N- and C-terminal tails. Residues required for Pto binding lie in a 19 residue Omega loop. Modeling suggests a hydrophobic patch involving the activation loop of Pto forms a contact surface with the AvrPto Omega loop and that helix packing mediates interactions between AvrPto and putative virulence targets Api2 and Api3. The AvrPto structure has a low stability that may facilitate chaperone-independent secretion by the TTSS.     

Functional analysis of the plant disease resistance gene Pto using DNA shuffling

Pto is a serine/threonine kinase that mediates resistance in tomato to strains of Pseudomonas syringae pv. tomato expressing the (a)virulence proteins AvrPto or AvrPtoB. DNA shuffling was used as a combinatorial in vitro genetic approach to dissect the functional regions of Pto. The Pto gene was shuffled with four of its paralogs from a resistant haplotype to create a library of recombinant products that was screened for interaction with AvrPto in yeast. All interacting clones and a representative sample of noninteracting clones were sequenced, and their ability to signal downstream was tested by the elicitation of a hypersensitive response in an AvrPto-dependent or -independent manner in planta. Eight candidate regions important for binding to AvrPto or for downstream signaling were identified by statistical correlations between individual amino acid positions and phenotype. A subset of the regions had previously been identified as important for recognition, confirming the validity of the shuffling approach. Three novel regions important for Pto function were validated by site-directed mutagenesis. Several chimeras and point mutants exhibited a differential interaction with (a)virulence proteins in the AvrPto and VirPphA family, demonstrating distinct binding requirements for different ligands. Additionally, the identification of chimeras that are both constitutively active as well as capable of binding AvrPto indicates that elicitation of downstream signaling does not involve a conformational change that precludes binding of AvrPto, as previously hypothesized. The correlations between phenotypes and variation generated by DNA shuffling paralleled natural variation observed between orthologs of Pto from Lycopersicon spp.     

Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto.

Resistance in tomato to Pseudomonas syringae pv tomato (avrPto) is conferred by the gene Pto in a gene-for-gene relationship. A hypersensitive disease resistance response (HR) is elicited when Pto and avrPto are expressed experimentally within the same plant cell. The kinase capability of Pto was required for AvrPto-dependent HR induction. Systematic mutagenesis of the activation segment of Pto kinase confirmed the homologous P+1 loop as an AvrPto-binding determinant. Specific amino acid substitutions in this region led to constitutive induction of HR upon expression in the plant cell in the absence of AvrPto. Constitutively active Pto mutants required kinase capability for activity, and were unable to interact with proteins previously shown to bind to wild-type Pto. The constitutive gain-of-function phenotype was dependent on a functional Prf gene, demonstrating activation of the cognate disease resistance pathway and precluding a role for Prf upstream of Pto.     

Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.