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. 2004 Oct;16(10):2809-21.
doi: 10.1105/tpc.104.024141. Epub 2004 Sep 14.

A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase

Ai-Jiuan Wu  1 , Vasilios M E Andriotis, Marcus C Durrant, John P Rathjen
Affiliations

A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase

Ai-Jiuan Wu et al. Plant Cell. 2004 Oct.

Abstract

Tomato (Lycopersicon esculentum) Pto kinase specifically recognizes the Pseudomonas effector proteins AvrPto and AvrPtoB, leading to induction of defense responses and hypersensitive cell death. Structural modeling of Pto combined with site-directed mutagenesis identified a patch of surface-exposed residues required for native regulation of signaling. Mutations in this area resulted in constitutive gain-of-function (CGF) forms of Pto that activated AvrPto-independent cell death via the cognate signaling pathway. The patch overlaps the peptide binding region of the kinase catalytic cleft and is part of a broader region required for interaction with bacterial effectors. We propose that the negative regulatory patch is normally occupied by a peptide that represses Pto signaling. Furthermore, we found that Pto kinase activity was required for Avr-dependent activation but dispensable for signaling by CGF forms of Pto. This suggests that Pto signals by a conformational change rather than phosphorylation of downstream substrates in the defense signaling pathway.

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Figures

Figure 1.
Figure 1.
Acidic Mutations throughout the Pto P+1 Loop Confer CGF Activity. (A) Expression of Pto P+1 loop mutants in N. benthamiana leaves. Leaves were infiltrated with A. tumefaciens containing each construct as indicated and the area of infiltration outlined with a pen. Leaves are shown 4 d after infiltration. (B) Accumulation of mutant proteins in planta. Total protein was extracted from leaves and blotted against polyclonal Pto antisera. Equivalent loadings were confirmed by Coomassie blue staining of the membrane (data not shown). Benth, noninfiltrated leaf tissue; EV, empty vector control. (C) Prf is required for cell death induced by P+1 loop mutants. Left, N. benthamiana leaves silenced for the Prf gene; right, leaves silenced with the empty VIGS vector. Top leaves were infiltrated with each Agrobacterium culture as indicated 4 weeks after inoculation with the tobacco rattle virus (TRV) silencing vector. Leaves were infiltrated identically on left and right.
Figure 2.
Figure 2.
Homology Model of Pto. Ribbon diagram of the Pto homology model in its active form, representing residues 20 to 321. The P+1 loop (amino acids 201 to 210) is shown in dark blue and the T-loop in cyan. The presumed catalytic residue Asp-164 is shown in yellow and bound ATP in red. Inset, the activation loop in its inactive conformation.
Figure 3.
Figure 3.
Kinase Activity Is Dispensable for Signaling by pto L205D. (A) In planta expression of Pto derivatives containing a substitution in the presumed catalytic residue (D164). Each mutant was expressed as indicated. (B) Pto mutant proteins capable of inducing an HR in vivo lack kinase activity. Proteins were expressed as MBP fusions in E. coli, purified and tested for the ability to autophosphorylate (top band) or transphosphorylate a GST-PtiI substrate (bottom band). The MBP-Pto fusions correspond to ∼77 K and GST-PtiK96N to ∼59 K. Bottom panel, Coomassie blue–stained gel showing equivalent loading of fusion proteins in the autoradiograph.
Figure 4.
Figure 4.
Surface-Exposed Residues Are Responsible for Native Regulation of Pto. (A) Expression of Ala substitution mutants in planta. Identity of each mutant is indicated. (B) Expression of Asp substitution mutants in planta. Identity of each mutant is indicated. (C) CGF mutants require Prf for signaling. Left, leaves silenced for Prf; right, leaves silenced for empty vector only. Pto mutants were expressed as indicated. (D) In planta recognition of AvrPto by non-CGF pto mutants. Mutants were expressed as indicated in N. benthamiana leaves carrying a dexamethasone-inducible avrPto transgene.
Figure 5.
Figure 5.
Mapping a Negative Regulatory Patch on the Surface of Pto. (A) A negative regulatory patch on Pto. Left, the P+1 loop residues 202 to 208 are dark red, catalytic residue D164 is yellow, and other CGF-conferring residues are red. Residues that retained the wild-type properties of Pto after mutation are shown in blue. Non-CGF mutants that did not respond to AvrPto in planta are colored gray. Right, a schematic of the same region defining the residues. Residues that are partially obscured in this projection are color coded as follows: 169, red; 206, orange; 210, yellow; 245, green; 253, blue. (B) Residues required for interaction of Pto with AvrPto and AvrPtoB in yeast. The view is comparable to (A). Residues required for interaction with AvrPto and AvrPtoB are shown in orange; dispensable residues are colored blue. K215 (magenta) is a unique binding determinant for AvrPtoB. Residues with inconclusive binding function are in gray. The catalytic residue D164 is yellow. (C) Overlap between regulatory and Avr-interaction patches. Overlay of (A) and (B). The negative regulatory patch directly overlaps the presumed Avr docking site (brown). Residues required solely for CGF activity (I214, red), Avr interaction (orange), or AvrPtoB binding (K215, magenta) are indicated.
Figure 6.
Figure 6.
Kinase Activity Is Dispensable for Signaling by Pto CGF Mutants. (A) Expression of Pto CGF mutants containing a second-site kinase knockout mutation in planta. Mutants were expressed as indicated. (B) Diminished kinase activity of most Pto CGF mutants. Mutant proteins were expressed as GST fusions and assayed for the ability to autophosphorylate or transphosphorylate in vitro. Radiolabeled species were detected by autoradiography after SDS-PAGE. The position of GST-Pto fusion proteins and cleaved Pt1K96N fusion proteins are indicated. Bottom panel, Coomassie stain of the autoradiograph gel showing equivalent loading.
Figure 7.
Figure 7.
Model of Pto Activation by AvrPto and AvrPtoB. Native Pto (blue) is regulated by an inhibitory peptide (red) that represses kinase activity. Upon secretion into the plant cell, AvrPto or AvrPtoB (green) interacts with Pto, displacing the regulatory peptide from the catalytic cleft and derepressing kinase activity. Activated Pto catalyzes a phosphorylation event(s) leading to a specific conformational change. An unknown effector protein recognizes the altered Pto conformation, leading to activation of the resistance pathway. Although the Pto-effector complex is shown dissociated from the Avr and inhibitor proteins, this remains speculative.
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References

    1. Axtell, M.J., and Staskawicz, B. (2003). Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112, 369–377. - PubMed
    1. Dangl, J.L., and Jones, J.D.G. (2001). Plant pathogens and integrated defence responses to infection. Nature 411, 826–833. - PubMed
    1. Ellingboe, A.H. (1981). Changing concepts in host-pathogen genetics. Annu. Rev. Phytopathol. 19, 125–143.
    1. Flor, H.H. (1971). Current status of the gene-for-gene concept. Annu. Rev. Phytopathol. 9, 275–298.
    1. Frederick, R.D., Thilmony, R.L., Sessa, G., and Martin, G.B. (1998). Recognition specificity for the bacterial avirulence protein AvrPto is determined by Thr-204 in the activation loop of the tomato Pto kinase. Mol. Cell 2, 241–245. - PubMed

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