Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity - PubMed (original) (raw)

Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity

Robert B Abramovitch et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Microbial pathogens of both plants and animals employ virulence factors that suppress the host immune response. The tomato pathogen Pseudomonas syringae injects the AvrPtoB type III effector protein into the plant cell to suppress programmed cell death (PCD) associated with plant immunity. AvrPtoB also inhibits PCD in yeast, indicating that AvrPtoB manipulates a conserved component of eukaryotic PCD. To identify host targets of AvrPtoB, we performed a yeast two-hybrid screen and identified tomato ubiquitin (Ub) as a strong AvrPtoB interactor. AvrPtoB is ubiquitinated in vitro and exhibits E3 Ub ligase activity in the presence of recombinant E1 activating enzyme and specific E2 Ub-conjugating enzymes. The C terminus of AvrPtoB is sufficient for both anti-PCD and E3 Ub ligase activities, suggesting the two functions are associated. Indeed, mutation of AvrPtoB lysine residues in the C terminus, between K512 and K529, disrupts AvrPtoB-Ub interactions, decreases AvrPtoB-mediated anti-PCD activity, and abrogates P. syringae pathogenesis of susceptible tomato plants. Remarkably, quantitative decreases in AvrPtoB anti-PCD activity are correlated with decreases in AvrPtoB ubiquitination and E3 Ub ligase activity. Overall, these data reveal a unique bacterial pathogenesis strategy, where AvrPtoB manipulates the host Ub system and requires intrinsic E3 Ub ligase activity to suppress plant immunity.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

AvrPtoB interacts with ubiquitin. (A) AvrPtoB and the related protein VirPphA interact with Ub in a Y2H system. (B) AvrPtoB expressed in N. benthamiana is detected as multiple bands, separated by ≈8 kDa; this pattern is consistent with in vivo ubiquitination of AvrPtoB. AvrPtoB also causes an enhanced Ub smear. Numbers represent approximate size markers in kDa. ApB, AvrPtoB; Vec, empty-vector control; WB, Western blot.

Fig. 2.

AvrPtoB is ubiquitinated and acts as an E3 Ub ligase. (A) GST-AvrPtoB and AvrPtoB308–553 CTR are ubiquitinated in vitro when incubated with RRLs, Flag-Ub, and ATP. The asterisk highlights an upward shift of GST-AvrPtoB308–553 CTR. GST-AvrPtoB was separated on 8% SDS/PAGE. GST-NTR and GST-CTR were separated on 10% SDS/PAGE. (B) AvrPtoB without the GST tag is ubiquitinated in vitro. The asterisk highlights an upward shift of AvrPtoB. (C) In E3 Ub ligase assays, AvrPtoB autoubiquitinates specifically in the presence of the E2s UbcH5a, UbcH5c, or UbcH6. PD, pull down; WB, Western blot.

Fig. 3.

Specific AvrPtoB lysine residues between K512 and K529 are required for AvrPtoB–Ub interactions and AvrPtoB virulence function in tomato. (A) Y2H interactions of AvrPtoB K-to-R mutants with Ub and Pto. Interaction strength was quantified by using a liquid β-galactosidase assay. Error bars represent the SD of three replicates. Quad, K512R, K520R/K521R, K529R. (B) Agrobacterium_-mediated transient expression of AvrPtoB K-to-R mutants in RG–pto 11 tomato leaves. Three days postinoculation, leaves were cleared in ethanol, and PCD was observed to be staining in the infiltrated area. Samples were inoculated as indicated with the name of the substitutions or (i) K512R, K520R/K521R; (ii) K512R, K529R; (iii) K520R/K521R, K529R; (iv) quad, K512R, K520R/K521R, K529R, (v) AvrPtoB1–387 NTR. Loss of anti-PCD activity on RG–pto11 is highlighted by red circles surrounding the HR-based PCD. Browning present for AvrPtoB and the vector alone is the result of syringe damage and not indicative of HR-based PCD. (C) Growth 6 days postinoculation on tomato of DC3000::Δ_avrPtoB strains expressing the indicated K-to-R mutant genes from the native AvrPtoB promoter. Error bars represent SD of six samples. Growth measurements were repeated three times with similar results.

Fig. 4.

AvrPtoB anti-PCD activity depends on AvrPtoB ubiquitination and E3 Ub ligase activity. (A) AvrPtoB K-to-R mutants were expressed in N. benthamiana to determine whether proteins elicit or suppress PCD. For proteins expressed alone or coexpressed with AvrPto/Pto, cell death was assessed 7 or 5 days postinoculation, respectively. Photographs of these observations are supplied in Fig. 8. −, no cell death; +, weak, late onset cell death; ++, strong, early onset cell death. (B) Cell death in A was quantified by measuring conductivity associated with ion-leakage from leaves. Error bars represent the standard error about the mean of six samples. (C) Ubiquitination assays of AvrPtoB K-to-R mutants as described in the text. Proteins were incubated with RRL, Flag-Ub, and ATP. AvrPtoB was purified from the reaction and run on a 4–15% gradient SDS/PAGE. Schematic wedges below A and B demonstrate trends in the data of decreasing anti-PCD activity and ubiquitination, respectively. Quad, K512R, K520R/K521R, K529R mutant; PD, pull down; WB, Western blot.

Fig. 5.

AvrPtoB K-to-R mutants have decreased E3 Ub ligase activity. (A) To determine whether AvrPtoB K-to-R mutants have altered levels of E3 activity, the supernatants of E3 Ub ligase assays were examined for the presence of high-molecular-weight Ub conjugates. In this assay, the K-to-R mutants K512R, K529R, and the quad mutants have decreased E3 Ub ligase activity as compared with wild-type AvrPtoB. (B) Crystal structure of the AvrPtoB436–553 C-terminal domain reveals that the K512, K520, K521, and K529 are all spatially clustered near the conserved E2-binding-site residues F479, F525, and P533, suggesting that the K-to-R substitutions may be interfering with E3 activity by disrupting the E2-binding site.

References

1. Galan J. E., Collmer A. Science. 1999;284:1322–1328. - PubMed
1. Stebbins C. E., Galan J. E. Nature. 2001;412:701–705. - PubMed
1. Alfano J. R., Collmer A. Annu. Rev. Phytopathol. 2004;42:385–414. - PubMed
1. Abramovitch R. B., Martin G. B. Curr. Opin. Plant Biol. 2004;7:356–364. - PubMed
1. Pedley K. F., Martin G. B. Annu. Rev. Phytopathol. 2003;41:215–243. - PubMed

Type III effector AvrPtoB requires intrinsic E3 ubiquitin ligase activity to suppress plant cell death and immunity - PubMed (original) (raw)