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Keywords:

  • plant immunity;
  • resistance gene;
  • PAMPs;
  • PAMP-triggered immunity

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants recognize the presence of attacking bacterial pathogens via the action of plasma membrane-localized pattern recognition receptors (PRRs) that detect specific microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) such as flagellin, lipopolysaccharide, cold shock protein, elongation factor TU (EF-Tu), and peptidoglycan (Abramovitch et al., 2006; Boller and Felix, 2009). These recognition events trigger an early response, termed PAMP-triggered immunity (PTI), which limits bacterial growth. The PTI response includes increased activity of mitogen-activated protein kinases (MAPKs), the generation of reactive oxygen species, a calcium burst, expression of transcription factors and PRR genes, and callose deposition at the cell wall (Chisholm et al., 2006; Boller and Felix, 2009). Bacterial pathogens overcome this first line of inducible defense by injecting into the plant cell, via the type III secretion system (T3SS), a repertoire of effector proteins that interfere with various steps of PTI (Chisholm et al., 2006; Boller and Felix, 2009). Plants, in turn, have evolved a second inducible defense system that relies on the ability of their resistance (R) proteins to recognize the presence of specific effectors and to thereby trigger a strong immune response typically associated with localized host programmed cell death, referred to as the hypersensitive response (HR). This effector-triggered immunity (ETI) presents a more robust layer of defense that restricts the proliferation of the pathogen. Recent studies show that certain pathogen effectors have successfully evolved to interdict ETI (Rosebrock et al., 2007). These complex multilayered interactions underlie the co-evolutionary ‘arms race’ between plants and pathogens.

The interaction between Pseudomonas syringae pv. tomato (Pst), the causative agent of bacterial speck disease, and its host plant tomato (Solanum lycopersicum) serves as a model system for elucidating the molecular mechanisms underlying bacterial pathogenesis and plant immunity. The sequenced genome of Pst strain DC3000 revealed approximately 30 effectors that are injected into the plant cell via the T3SS during infection (Cunnac et al., 2009). AvrPto, one of the best characterized of these effectors, is targeted to the plant plasma membrane via N-terminal myristoylation where it promotes bacterial virulence (Ronald et al., 1992; Chang et al., 2000; Shan et al., 2000b; Hauck et al., 2003; Wulf et al., 2004). Based on studies using Arabidopsis protoplasts it was hypothesized that AvrPto suppresses PTI by acting upstream of a MAPK cascade, possibly at the level of PAMP recognition (He et al., 2006). Two recent reports support this hypothesis. In one, AvrPto was found to bind the kinase domain of FLS2 and EFR, PRRs that binds flagellin and EF-Tu, respectively, thereby inhibiting their activity and disrupting their ability to activate PTI (Xiang et al., 2008). In the second, AvrPto was found to bind the kinase domain of BAK1 and to prevent the formation of the FLS2-BAK1 complex in vivo required for the activation of PTI (Shan et al., 2008). Another recent study shows that AvrPto suppresses the expression of certain miRNAs that are induced during PTI; this suppression may be a downstream effect of inhibition of PRR activities by AvrPto (Navarro et al., 2008).

AvrPto-like sequences are present in the genomes of many pathovars of P. syringae that infect a wide range of plant species (Ronald et al., 1992; Lin and Martin, 2007). In tomato, AvrPto is recognized by the product of the Pto gene, a member of a small gene family encoding cytoplasmic serine/threonine protein kinases (Martin et al., 1993). The Pto kinase physically interacts with AvrPto (and also another effector, AvrPtoB) in the plant cell, which triggers ETI (Scofield et al., 1996; Tang et al., 1996; Kim et al., 2002). This recognition event requires the presence of the nucleotide-binding leucine-rich repeat protein Prf (Salmeron et al., 1996; Mucyn et al., 2006). It has been postulated that the Pto kinase acts as a decoy of the true virulence targets of AvrPto and that the modification of Pto is detected by Prf to trigger ETI (Van Der Hoorn and Kamoun, 2008; Xiang et al., 2008; Cui et al., 2009).

AvrPto is a small (18 kDa) hydrophilic protein (Ronald et al., 1992) that is the target of several host-mediated post-translational modifications including myristoylation and phosphorylation (Shan et al., 2000b; Anderson et al., 2006). Previous extensive mutagenesis studies have identified functional domains important for the virulence activity of AvrPto and for mediating its recognition by the Pto kinase (Shan et al., 2000b; Chang et al., 2001; Wulf et al., 2004; Xiang et al., 2008). The structure of the AvrPto core (amino acids 31–124) has been solved by NMR and x-ray crystallography and consists of four α-helices with a 19-residue omega (Ω) loop lying between helices C and D (Wulf et al., 2004; Xiang et al., 2008). Mutations in the ‘CD loop’ decrease the virulence activity of AvrPto by disrupting its ability to interact with components of PRR/BAK1 complexes (Shan et al., 2008; Xiang et al., 2008). The CD loop forms one of two AvrPto contact surfaces that are recognized and bound by the Pto kinase (Xing et al., 2007). Binding of AvrPto inhibits Pto kinase activity in vitro and leads to activation of Prf-mediated disease resistance (Mucyn et al., 2006; Xing et al., 2007). Substitutions altering the CD loop abolish the interaction with Pto, which, in turn, disrupts Pto/Prf-dependent ETI (Chang et al., 2001; Wulf et al., 2004; Xing et al., 2007).

Another important structural element of AvrPto, the C-terminal domain (CTD; amino acids 146–164), is phosphorylated in the plant cell by a host kinase activity (Anderson et al., 2006). This kinase activity is independent of either Pto or Prf and, in fact, is observed in diverse plant species including tomato, tobacco (Nicotiana tabacum), and Arabidopsis thaliana. Three serine residues in the CTD of AvrPto, S147, S149, and S153, were implicated as phosphorylation sites by in vitro experiments and S147 was confirmed in vivo (Anderson et al., 2006). Alanine substitutions at these phosphorylation sites reduce AvrPto virulence activity in tomato and weakly affect Pto-mediated recognition of the effector (Anderson et al., 2006). In a striking parallel with CD loop recognition by Pto, the CTD is also recognized by a putative R protein in cultivated tobacco (Thilmony et al., 1995; Shan et al., 2000b). It was speculated that structural features of the CTD rather than its phosphorylation status are recognized by the putative tobacco R protein (Anderson et al., 2006).

Here we have examined the mechanism by which CTD phosphorylation promotes virulence activity of AvrPto and have investigated the role of this post-translational modification in the recognition of AvrPto by tobacco. Our results shed light on a mechanism by which a pathogen effector undermines the host immune response and the way in which certain plant species have evolved to counteract this pathogen manipulation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The CTD contributes to AvrPto virulence activity to a similar degree as the CD loop

Both the CD loop and the CTD of AvrPto are known to contribute to the virulence activity of AvrPto (Shan et al., 2000a; Anderson et al., 2006). In order to understand the relationship between these two elements, we developed a series of AvrPto variants altered in either one or both of these domains. Because phosphorylation of S153 is known to play less of a role in virulence we focused on S147 and S149 for these experiments (Anderson et al., 2006). Each AvrPto variant was expressed in the Pst DC3000ΔavrPtoΔavrPtoB strain and their effects on disease severity and bacterial populations were assessed on susceptible tomato Rio Grande-prf3 plants (Figure 1a,b). When expressed under its native hrp promoter and delivered by the T3SS, AvrPto enhanced the growth of DC3000ΔavrPtoΔavrPtoB about tenfold more than a null mutant, AvrPto(G2A), that has a disrupted N-terminal myristoylation motif (Shan et al., 2000b). Both AvrPto(I96A), with an altered CD loop, and AvrPto(2xA; S147A/S149A), which disrupts CTD phosphorylation, had reduced virulence activity compared with AvrPto. When both the CD loop and the CTD were altered (AvrPto[I96A + 2xA]) the virulence activity of AvrPto was reduced to the level of AvrPto(G2A). Increased ethylene production was previously found to be associated with AvrPto virulence activity (Cohn and Martin, 2005) and so we also measured ethylene in susceptible plants infected with the same bacterial strains. Ethylene production was lower in plants inoculated with bacterial strains carrying AvrPto variants that affected either one of the domains and was reduced to the basal level with the strain carrying the CD loop/CTD-minus variant (Figure 1c). Protein expression and secretion of all AvrPto variants was similar to wild-type AvrPto (Figure 1d,e). Based on these results, we conclude that these two domains contribute additively to overall virulence activity of AvrPto.

image

Figure 1.  The two functional domains of AvrPto contribute additively to its virulence activity. (a) Disease symptoms observed on susceptible tomato RG-prf3 (Pto/Pto prf/prf) plants 5 days after vacuum infiltration with DC3000ΔavrPtoΔavrPtoB delivering AvrPto or the indicated AvrPto variants [104 colony-forming units (c.f.u.) ml−1]. Red arrows point to enhanced disease symptoms compared with the G2A variant. (b) Bacterial populations in leaves of RG-prf3 at 0 and 3 days post-inoculation (dpi). Data are presented as c.f.u. cm−2 of leaf tissue. Letters above each bar represent groupings of statistical significance based on analysis of variance and comparisons for all pairs using Tukey–Kramer honestly significant difference (HSD;  0.05). Error bars indicate ± standard error (SE) (= 4). (c) Ethylene production in the same tomato plants as in (a) and (b) 3 days after bacterial inoculation. The amount of ethylene produced at earlier time points was close to zero (not shown). Letters above each bar represent groupings of statistical significance based on analysis of variance and comparisons for all pairs using Tukey–Kramer HSD ( 0.05). Error bars indicate ± SE (= 5). (d) Immunoblotting using an αAvrPto antibody was performed to detect expression of AvrPto proteins by Pseudomonas syringae pv. tomato (Pst) DC3000ΔavrPtoΔavrPtoB. (e) Immunoblotting using an αAvrPto antibody was performed to detect secretion of AvrPto from Pst DC3000ΔavrPtoΔavrPtoB.

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The AvrPto CTD promotes virulence in tomato using a mechanism other than the CD loop

In Arabidopsis the enhanced virulence attributable to the AvrPto CD loop is associated with suppression of PAMP-induced MAPK activation (He et al., 2006). To determine whether S147 and S149 play a role in this suppression in tomato, we assessed the effect of AvrPto variants on flg22-mediated MAPK activation using a protoplast system (Rosebrock et al., 2007). We observed that AvrPto suppresses flg22-induced activation of the tomato MAPK, SlMPK3. Consistent with the previous results from Arabidopsis (He et al., 2006), a substitution in the CD loop (I96A) reduced the ability of AvrPto to suppress this activation (Figure 2a). However, alanine substitutions at both S147 and S149 had no effect on the ability of AvrPto to suppress MAPK activity. We also tested the ability of AvrPto to suppress MAPK activation by another PAMP, chitin (Shan et al., 2008). Again, the CD loop and not the CTD was required to suppress MAPK activation resulting from host recognition of this PAMP (Figure 2b).

image

Figure 2.  The S147 and S149 residues of AvrPto are not required to suppress activation of tomato mitogen-activated protein kinase (SlMPK3) by flg22 or by chitin. (a) Hemagglutinin (HA)-tagged AvrPto proteins were expressed with HA-tagged SlMPK3 in tomato RG-PtoS (pto/pto Prf/Prf) protoplasts. Transfected protoplasts were incubated for 6 h and then treated with 100 nm flg22 for 10 min. EV indicates an empty vector control. (b) Tomato RG-prf3 protoplasts transfected with HA-tagged SlMPK3 and the HA-tagged AvrPto constructs were incubated and then treated with 50 μg ml−1 chitin for 10 min. Anti-HA antibodies were used for the immunoprecipitation of SlMPK3 and AvrPto. An in vitro assay was used to detect SlMPK3 phosphorylation of myelin basic protein (MBP; upper panel). The lower panel shows a protein blot verifying expression of SlMPK3-HA and AvrPto-HA using anti-HA antibodies.

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Phosphorylation of the CTD is required for its recognition by tobacco

Recognition of AvrPto by certain genotypes of either tomato or tobacco elicits resistance to P. syringae. In tomato, AvrPto is recognized via the direct interaction between the Pto kinase and the CD loop (Tang et al., 1996; Xing et al., 2007). In tobacco, the CD loop is not involved but instead the CTD is recognized by a putative R protein (Shan et al., 2000b). To further investigate the role of the CD loop and the CTD in allowing recognition of AvrPto we used Agrobacterium tumefaciens infiltration to transiently express a series of AvrPto variants in leaves of tomato and Nicotiana sylvestris (a parental species of amphidiploid tobacco) (Figure 3a). Wild-type AvrPto was recognized in both species, leading to visible cell death, and the G2A substitution in AvrPto abolished this response. The I96A substitution, which disrupts the interaction between AvrPto and Pto (Tang et al., 1996), abolished cell death in tomato RG-PtoR, as expected, but not in N. sylvestris.

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Figure 3.  Tobacco recognizes the phosphorylated C-terminal domain of AvrPto. (a) AvrPto recognition assay in tomato (Solanum lycopersicum) and Nicotiana sylvestris using Agrobacterium tumefaciens-mediated transient expression. AvrPto, G2A, I96A, 2xA (S147A/S149A), 2xD (S147D/S149D), or ΔC30 were expressed in leaves of tomato RG-PtoR (Pto/Pto Prf/Prf) and N. sylvestris. EV indicates an empty vector control. Photographs were taken at 22 h (tomato) or 20 h (N. sylvestris) after inoculation. (b) Hypersensitive response (HR) assay in N. sylvestris‘TW136’, Nicotiana tabacum‘W38’, or Nicotiana tomentosa‘TW141’ using Pseudomonas syringae pv. tabaci delivering the AvrPto proteins. A high bacterial titer [4 × 107 colony-forming units (c.f.u.) ml−1] was used for inoculation. Images were taken at 20 h post-inoculation. (c) Immunoblotting using an αAvrPto antibody was performed to detect expression of AvrPto proteins by P. syringae pv. tabaci 11528R. (d) Immunoblotting using an αAvrPto antibody was performed to detect secretion of AvrPto from P. syringae pv. tabaci 11528R.

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AvrPto variants that either lack the CTD (Δ30) or have a CTD that is unable to be phosphorylated (S147A/S149A) did not elicit cell death in N. sylvestris (Figure 3a). Significantly, however, an AvrPto variant with aspartate substitutions at S147/S149, which mimic the negative charge of phosphorylated serine residues, does elicit cell death in N. sylvestris. AvrPto-mediated cell death in tomato RG-PtoR is unaffected in response to either the 2xA or ΔC30 alterations, indicating that the phosphorylation of the CTD does not markedly affect Pto-mediated recognition of AvrPto. Collectively, these results suggest that phosphorylation of S147 and S149 is required for recognition of AvrPto by N. sylvestris but not by tomato RG-PtoR. The same set of AvrPto variants were also tested in Nicotiana benthamiana and the results were similar to those from RG-PtoR tomato, suggesting that N. benthamiana possesses a Pto-like resistance specificity (Figure S1a in Supporting Information). Protein expression in plant cells was demonstrated for all the AvrPto variants using N. benthamiana leaves (Figure S1b).

Cultivated tobacco (N. tabacum) is thought to have arisen from an ancient hybridization event between N. sylvestris and Nicotiana tomentosa (Chase et al., 2003; Clarkson et al., 2004). To further characterize the recognition of AvrPto in these species we expressed the AvrPto variants in Pseudomonas syringae pv. tabaci and inoculated leaves of these two species and cultivated tobacco using a high bacterial titer for a HR assay [4 × 107 colony-forming units (c.f.u.) ml−1; Figure 3b). A HR was elicited in all three species by AvrPto and the phosphomimic AvrPto(S147D/S149D). In N. sylvestris and N. tabacum, the HR was abolished when AvrPto contained either the 2xA or ΔC18, whereas AvrPto with the I96A substitution still elicited a HR. In contrast, N. tomentosa did not recognize the CD loop variant but did respond to the AvrPto CTD-minus variants. All AvrPto variants were expressed and secreted similarly from P. syringae pv. tabaci (Figure 3c,d).

A disease assay using a lower titer (105 c.f.u. ml−1) of P. syringae pv. tabaci was performed in all three Nicotiana species used for the HR assay (Figure 4a). We only observed cell death associated with disease in interactions involving the AvrPto proteins that did not cause a HR. Consistent with the observations from the HR assay, AvrPto containing either 2xA or ΔC18 was not recognized by N. sylvestris or N. tabacum, leading to the development of disease symptoms, whereas the I96A substitution in AvrPto allowed disease formation in N. tomentosa.

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Figure 4.  The recognition of the phosphorylated-Ser at position 147 and 149 by Rpa shows quantitative effects and is independent from the CD loop structure. (a) Disease assays in Nicotiana sylvestris‘TW136’, Nicotiana tabacum‘W38’, or Nicotiana tomentosa‘TW141’ using Pseudomonas syringae pv. tabaci delivering the AvrPto proteins. Photographs of disease assays using a low bacterial inoculum [105 colony-forming units (c.f.u.) ml−1] were taken at 7 days post-inoculation (dpi). Disease-associated cell death indicates lack of recognition. (b) Bacterial population assays in N. sylvestris‘TW136’ at 2 dpi. Nicotiana sylvestris leaves were syringe-infiltrated with P. syringae pv. tabaci delivering the AvrPto variants at 105 c.f.u. ml−1. Data are presented as c.f.u. cm−2 of leaf tissue. Letters represent groupings of statistical significance based on analysis of variance and comparisons for all pairs using Tukey–Kramer honestly significant difference (HSD;  0.05). Error bars indicate ± SE (= 3).

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To further assess the effects of CTD phosphorylation on recognition by tobacco we measured bacterial populations in N. sylvestris leaves inoculated with the P. syringae pv. tabaci strains delivering AvrPto or the variants (Figure 4b). The phosphomimic protein, AvrPto(S147D/S149D), caused inhibition of bacterial growth to the same extent as wild-type AvrPto and AvrPto(I96A). Interestingly, there was a quantitative effect associated with individual substitutions at the S147 and S149 residues. Other alterations affecting the CTD abolished recognition by N. sylvestris. Together, these observations suggest that the phosphorylation status of the CTD, and not simply a structural feature (Anderson et al., 2006), is monitored by N. sylvestris and N. tabacum. We therefore now refer to the putative R protein in N. sylvestris and N. tabacum as Rpa (Resistance to phosphorylated AvrPto).

Rpa is conserved in several tobacco species but is not present in tomato species

We next investigated how widespread Rpa recognition specificity is among wild relatives of tobacco (Table 1). We found that Rpa is present in multiple accessions of both N. tabacum and N. sylvestris. Two tested accessions of the other presumed progenitor of tobacco, N. tomentosa, did not recognize the CTD but rather, like tomato, recognized the CD loop. Therefore, tobacco appears to have retained the Rpa specificity from N. sylvestris but not the Pto-like specificity of N. tomentosa. Two other diploid tobacco species, Nicotiana langsdorfii and Nicotiana rustica, also express an Rpa specificity, suggesting that this recognition mechanism arose before Nicotiana speciation. Diverse wild relatives of tomato were also tested, and none of them were found to recognize the CTD (Table S1).

Table 1.   Recognition of the C-terminal domain of AvrPto by wild species of tobacco using a disease assay. Leaves of each wild species were inoculated with Pseudomonas syringae pv. tabaci strains delivering empty vector, AvrPto, AvrPto(I96A), or AvrPto(2xA-S147A/S149A) (105 colony-forming units ml−1) and the host response was recorded 5 days after inoculation.
SpeciesAccessionsEmpty vectorAvrPtoAvrPto (I96A)AvrPto (2xA)
  1. R, no disease symptoms were observed; IS, an intermediate degree of disease was observed; S, extensive disease symptoms were observed.

  2. *Based on Agrobacterium tumefaciens-mediated cell death assay, N. benthamiana can recognize AvrPto and AvrPto (2xA), but not AvrPto (I96A).

Nicotiana tabacumBY2SRRS
Nicotiana tabacumKY14SRRS
Nicotiana tabacumPetite HavanaSRRS
Nicotiana tabacumSamsunSRRS
Nicotiana tabacumVirginia BrightSRRS
Nicotiana tabacumVirginia Gold LeafSRRS
Nicotiana tabacumW38SRRS
Nicotiana tabacumXanthiSRRS
Nicotiana sylvestrisTW136SRRS
Nicotiana sylvestrisTW137SRRS
Nicotiana sylvestrisTW138SRRS
Nicotiana tomentosaTW141SRSR
Nicotiana tomentosaTW140SISSIS
Nicotiana undulata SISISIS
Nicotiana rustica SISISS
Nicotiana benthamiana* SISISIS
Nicotiana langsdorfii SRRS

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

AvrPto is a well-characterized effector, and yet it continues to reveal new information about how it manipulates the plant cell and, in turn, how the plant has evolved to counter these virulence activities (Navarro et al., 2008; Shan et al., 2008; Xiang et al., 2008). We have shown here that AvrPto has a modular structure and that its phosphorylation by a host kinase contributes in an additive fashion to its virulence activity and also to its recognition by a tobacco R protein. This modular structure consists first of an N-terminal region which is required for secretion of AvrPto into the plant cell and for its localization to the plant plasma membrane (Chang et al., 2000, 2001; Shan et al., 2000a,b). Beyond this, AvrPto contains two other structurally discrete domains, the CD loop and the CTD, each of which has a distinct virulence activity (Wulf et al., 2004; Xing et al., 2007). The CTD appears to exploit a host protein kinase in order to facilitate its virulence activity, but certain Nicotiana species have evolved to specifically detect this manipulation. Although the CTD enhances bacterial growth in tomato, to date no tomato species are known that are able to recognize this domain to activate immunity. The fact that certain Nicotiana species do recognize the phosphorylated CTD suggests this domain plays an important role in bacterial pathogenesis of Nicotiana.

Our initial observation that the CD loop and the CTD each contribute in an additive fashion to bacterial growth, disease symptoms, and ethylene production suggested that these two domains may act via different mechanisms. This possibility was tested by using an assay in which AvrPto suppresses flg22-mediated activation of a host MAPK. This suppression is known to involve the CD loop which is required for interaction with the kinase domains of both FLS2 and BAK1, thereby disrupting downstream signaling (Shan et al., 2008; Xiang et al., 2008). A previous report examined two alterations in the CTD (P146L and S147R) and found that they did not affect MAPK suppression activity (He et al., 2006) although neither of these alterations disrupted both S147 and S149. We found here that such a dual mutant (S147A/S149A) is able to fully suppress flg22-mediated MAPK activation and this now excludes a role for CTD phosphorylation in suppression of this PTI response. Furthermore, S147/S149 phosphorylation was not required for suppression of MAPK activation in response to another PAMP, chitin. These results indicate that the CTD promotes virulence in a manner distinct from the CD loop, which appears to interfere with multiple PTI pathways. The mechanism by which the CTD promotes bacterial virulence is unknown, and it remains an open question whether it also suppresses certain PTI responses or, alternatively, may act as a positive regulator to enhance a host process resulting in susceptibility.

Of the relatively few P. syringae type III effectors that have been studied in detail, three have now been shown to be phosphorylated by host kinases – AvrB, AvrPto, and AvrPtoB (Zipfel et al., 2004; Desveaux et al., 2007;Xiao et al., 2007a). Phosphorylation, along with myristoylation, therefore appears to be a common post-translational modification of effectors upon their delivery into the plant cell. Several possibilities exist for the molecular basis of CTD phosphorylation. For example, many host kinase genes are transcriptionally induced upon exposure of plant cells to bacterial PAMPs and AvrPto might take advantage of a PTI-induced kinase to phosphorylate its CTD (Navarro et al., 2004; Cohn and Martin, 2005). That kinase could, in turn, be a virulence target of AvrPto. It is also possible that the interaction between AvrPto and the CTD kinase leads to activation of downstream targets that promote AvrPto virulence. Ultimately, identification and characterization of the host kinase will be needed to shed light on the molecular basis of its interaction with AvrPto, its contribution to P. syringae virulence, and its normal role in plants when it is not phosphorylating AvrPto.

Plant recognition of a specific effector protein will obviously be more durable and effective if the host recognition mechanism targets the domain of the effector that is required for its virulence activity. There are now many cases where both the avirulence and virulence activities of an effector are coupled in this way; the contribution of the CD loop to both virulence and recognition by Pto exemplifies this relationship (Scofield et al., 1996; Tang et al., 1996; Kim et al., 2002; Xing et al., 2007). Remarkably, AvrPto now provides another example of this coupling in which the phosphorylated CTD plays both a role in virulence and is targeted by a recognition mechanism in tobacco. We are currently unable to test the virulence activity of the CTD in tobacco because we have not identified an accession that lacks Rpa-mediated resistance (and technical difficulties have prevented us from using N. tomentosa TW141 to address this point). However, based on the fact that the phosphorylated CTD promotes virulence in tomato we hypothesize that it will also do so in tobacco. Indeed, it is reasonable to assume that such virulence activity provided the selection pressure giving rise to Rpa in Nicotiana species.

Nicotiana tabacum is a complex amphidiploid that is thought to have originated from a natural hybridization event between the two diploid species, N. sylvestris and N. tomentosa (Chase et al., 2003). Since N. sylvestris expresses a specificity for the CTD (Rpa) and N. tomentosa recognizes the CD loop (i.e. it has a Pto-like activity), N. tabacum might be expected to have both Rpa and Pto-like specificities. However, we observed only Rpa specificity in the eight N. tabacum accessions we examined. It is possible that the specific N. tomentosa plant involved in the original hybridization event leading to tobacco did not have Pto. Alternatively, it is possible that Pto was lost from N. tabacum, either randomly or because Rpa alone was sufficient to provide durable resistance against bacterial pathogens delivering AvrPto (note that only one of the specificities, Pto, is observed among the accessions of wild tomato species). In the future, identification of the Rpa gene from N. sylvestris and the putative Pto gene from N. tomentosa should allow a deeper understanding of the evolutionary history of and mechanistic differences between these two recognition specificities.

AvrPto has both remarkable similarities and striking differences with AvrPtoB, the other type III effector recognized by the tomato Pto kinase. Both effectors make physical contact with the Pto P loop although they each have another unique contact surface involved in binding to Pto (Xing et al., 2007; Dong et al., 2009). AvrPto is only 18 kDa while AvrPtoB is 60 kDa, and their structures are markedly different (Kim et al., 2002; Wulf et al., 2004; Janjusevic et al., 2006; Xing et al., 2007; Dong et al., 2009). However, the virulence activity of both effectors is enhanced by a host-mediated phosphorylation event, although it is not known whether this modification is unnecessary for AvrPtoB disruption of the FLS2/BAK1 complex as it is for AvrPto (Anderson et al., 2006; Xiao et al., 2007a). Finally, each effector is known to be recognized by two R proteins – Pto and Fen in the case of AvrPtoB (Kim et al., 2002; Rosebrock et al., 2007) and Pto and Rpa in the case of AvrPto (Martin et al., 1993; Shan et al., 2000b).

It is striking that AvrPto, like AvrPtoB, has a modular structure with discrete domains having distinct virulence activities, which together are required for its full virulence activity. Another well-studied example of an effector with multiple domains each contributing distinct virulence function is the Salmonella effector SptP that consists of both a tyrosine phosphatase and a GTPase-activating protein (Kaniga et al., 1996; Stebbins and Galan, 2000). In addition, the HopX effector expressed by P. syringae pv. phaseolicola is a modular type III effector protein that has two domains involved in its avirulence activity and possibly contributing to its virulence (Nimchuk et al., 2007). The extent to which type III effectors as a whole have multiple domains contributing to their virulence (and possibly host recognition) is currently unknown. However, the fact that even a small type III effector protein such as AvrPto is modular and has diverse activities might indicate that modularity is a common feature of effectors that reflects multiple selective pressures on their corresponding genes during the course of host-microbe evolution.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Protoplast assays for MAP kinase suppression

Three- to four-week-old tomato Rio Grande-prf3 (Pto/Pto prf/prf) or Rio Grande-PtoS (pto/pto Prf/Prf) leaves were used for protoplast isolation. Protoplasts were transformed using a polyethylene glycol protocol described previously ((Rosebrock et al., 2007; Xiao et al., 2007b); http://genetics.mgh.harvard.edu/sheenweb/). The hemagglutinin (HA)-tagged SlMPK3 gene (Holley et al., 2003) was expressed in protoplasts by using the pTEX CaMV 35S promoter expression cassette (Xiao et al., 2007b). AvrPto variants were expressed with a CaMV 35S promoter in vector pJD301 (Anderson et al., 2006). The avrPto gene from Pst strain JL1065 was used as the template for all experiments in this paper. Ten micrograms of pTEX::SlMPK3 and 7 μg of pJD301::avrPto plasmids were used in each transformation. After 6 h of incubation, PAMP-treatments were as follows: 100 nm flg22 (GenScrip) or 50 μg ml−1 chitin (Sigma-Aldrich, http://www.sigmaaldrich.com/). Ten minutes after PAMP treatment, the protoplasts were collected by centrifugation at 100 g for 2 min. Detection of the AvrPto-mediated suppression of PAMP-induced MAPK activity was performed as described previously (He et al., 2006). Data shown are representative of a minimum of three independent experiments.

Pathogenesis assays in tomato and tobacco

For the pathogenesis assays in tomato, the avrPto variants were cloned into the broad-host-range vector pCPP45 (Lin and Martin, 2005) and transformed into Pst DC3000ΔavrPtoΔavrPtoB by electroporation. For the pathogenesis assays in tobacco, the avrPto variants were cloned into the broad-host-range vector pDSK519 (Anderson et al., 2006), and transformed into P. syringae pv. tabaci 11528R. Expression and secretion assays of the AvrPto variants for both Pst and P. syringae pv. tabaci were performed using published protocols (Shan et al., 2000b). Site-directed mutagenesis of avrPto was performed using the Quickchange protocol and Pfu Turbo DNA polymerase (Stratagene, http://www.stratagene.com/). Primers are listed in Table S2. The antibodies used for immunoblotting were anti-AvrPto (Shan et al., 2000a) and anti-NptII (US Biological Corp., http://usbio.net/).

Five- to six-week-old plants of tomato Rio Grande-prf3 (Pto/Pto, prf/prf) were vacuum-infiltrated with different Pst DC3000 strains at an inoculum of 104 c.f.u. ml−1 and maintained in a climate-controlled growth chamber as described previously (Anderson et al., 2006). Bacterial populations in tomato leaves were measured at 2 or 3 days after infiltration. Analysis of variance (anova) and comparisons for all pairs using Tukey–Kramer honestly significant difference (HSD) were performed using JMP7 (SAS Institute Inc., http://www.sas.com/). The least significance difference at a 0.05 probability level was used to test the differences between means. Error bars indicate standard error (= 4). Disease symptoms were photographed 5 days after inoculation.

Four- to five-week-old tobacco plants (N. sylvestris, N. tomentosa, or N. tabacum) were used for inoculating different P. syringae pv. tabaci 11528R strains by syringe infiltration. For disease assays, an inoculum of 105 c.f.u. ml−1 was used, and for HR assays an inoculum of 4 × 107 c.f.u. ml−1 was used. Bacterial populations were measured in the disease assay 2 or 3 days after inoculation. JMP7 was used for statistical analysis with the least significance difference at a 0.05 probability level. Error bars indicate standard error (= 3). Disease symptoms were photographed 5 days after inoculation. Plant responses were photographed at 7 days after inoculation for the disease assays and at 20 h after inoculation for the HR. Data shown represent a minimum of three independent experiments.

Agrobacterium-mediated transient expression

Agrobacterium tumefaciens strain GV2260 was used to deliver the pCAMBIA2300 with a CaMV 35S promoter expression cassette for transient gene expression. All AvrPto variants contained a C-terminal HA epitope tag. Presence or absence of cell death caused by overexpressing AvrPto protein was determined at 20 h post-inoculation (hpi) for N. sylvestris and N. tabacum, 22 hpi for S. lycopersicum, and 30 hpi for N. benthamiana. Data shown represent a minimum of three independent experiments. Expression of AvrPto proteins was confirmed in N. benthamiana 24 hpi, prior to the appearance of visible cell death. The antibodies used for immunoblotting were anti-AvrPto (Shan et al., 2000a) or anti-HA (Roche Applied Science, http://www.roche-applied-science.com/).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Kathy Munkvold and Chang-Sik Oh for critical reading of the manuscript, and Andre Velasquez for statistical analysis. This work was supported, in part, by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant 2005-35301-15675 and National Science Foundation grant DBI-0605059 (GBM).

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1.Agrobacterium-mediated transient expression assay in leaf of N.  benthamiana. (a) AvrPto, AvrPto(G2A), AvrPto(I96A), AvrPto(2xA; S147A/S149A), AvrPto (2xD; S147D/S149D) or AvrPto(ΔC30) were expressed in N.  benthamiana leaf and a photograph was taken at 30 h later. (b) Expression of the AvrPto proteins was examined at 24 h after infiltration by using an immunoblot assay with an αAvrPto antibody.

Table S1. Recognition of AvrPto by wild species of tomato using a disease assay. Leaves of each species were inoculated with P. s. pv. tomato T1 strains delivering empty vector, AvrPto, AvrPto(I96A), or AvrPto(&Dgr;C18) (5 × 104 c.f.u. ml−1). The host response to each strain was recorded 7–10 days after inoculation as either resistance (R) or susceptibility (S).

Table S2. Primers used in this study.

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