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

Abstract

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.

Fig. 1. AvrPtoB-mediated inhibition of PCD in N.benthamiana leaves. (A) The proteins indicated were co-expressed in N.benthamiana using Agrobacterium-mediated transient expression. Leaves were agroinfiltrated within the marked circles and photos were taken 7 days after agroinfiltration. (B) AvrPtoB suppresses cell death initiated by AvrPto/Pto recognition in N.benthamiana. The N.benthamiana leaf was agroinfiltrated with avrPto and Pto and left to dry. On the left hand side, avrPtoB was then agroinfiltrated, and on the right hand side, an empty vector control was agroinfiltrated. After 7 days, an island of cell death suppressed tissue was observed in AvrPtoB-expressing cells. (C) Immunoblot analysis of AvrPto-HA, AvrPtoB-HA and Pto-HA co-expression in N.benthamiana. Lane 1: AvrPto, AvrPtoB, Pto; lane 2, AvrPtoB; lane 3, Pto; lane 4, AvrPto.

Fig. 2. AvrPtoB suppresses oxidative and heat stress-induced cell death in yeast. (A) AvrPtoB protects S.cerevisiae strain EGY48 from 3 mM H₂O₂-induced PCD. The agar plates show increased survival of yeast cells expressing AvrPtoB compared with the wild type after treatment with 3 mM H₂O₂. (B) AvrPtoB protects yeast from cell death triggered by: lane 1, 3 mM H₂O₂; lane 2, 5 mM H₂O₂; lane 3, 5 mM menadione; lane 4, 10 mM menadione; lane 5, heat shock at 50°C and lane 6, heat shock at 50°C with a 37°C pre-treatment. White bars represent wild-type yeast and black bars represent AvrPtoB-expressing yeast. Error bars show the standard deviation about the mean for three trials.

Fig. 3. Structural analysis of AvrPtoB recognition and anti-PCD activity. (A) A schematic representation of AvrPtoB truncations examined in this study and yeast two-hybrid analysis of physical interactions between AvrPtoB truncations and the Pto R protein. AvrPtoB truncations were cloned as bait fusions and tested against a Pto prey fusion. Constructs shaded black interacted strongly with Pto. (B) In planta transient expression of AvrPtoB truncations in tomato. RG-PtoR, RG-pto11 and RG-prf3 are isogenic tomato lines with the L.pimpenillifolium Pto haplotype and genotypes as indicated. RG-ptoS is a near-isogenic line with the L.esculentum Pto haplotype. *Note: a late-onset weak cell death phenotype was observed with Δ6 expression in RG-ptoS. +, cell death, –, no response.

Fig. 4. Recognition and anti-PCD activity of AvrPtoB truncations in N.benthamiana. (A) Full-length and truncated AvrPtoB constructs were transiently expressed: with AvrPto + Pto to test for anti-PCD activity (left); with Pto to test for Pto-mediated PCD (middle) and alone to test for Rsb-mediated PCD (right). Protein expression of each truncation is established by an observable phenotype. (B) Epistasis experiments examining the molecular basis of Δ6/Pto- and Δ7/Pto-initiated PCD and (C) Δ6-initiated PCD. Intact AvrPtoB suppressed PCD initiated by Δ6/Pto, Δ7/Pto and Δ6, suggesting an intermolecular mechanism of anti-PCD activity. Photos were taken 7 days after agroinfiltration.

Fig. 5. P.syringae pv. tomato DC3000 chromosomal mutants of avrPtoB and disease responses of inoculated tomato plants. (A) A schematic representation of avrPtoB chromosomal mutations in P.syringae pv. tomato DC3000, generated by insertion of the 6 kb pKnockout plasmid. Amino acid numbers correspond to the amino acid residue where the expressed mutant protein is interrupted by the insertion. (B) Disease responses of tomato plants inoculated with DC3000:mut mutants. Note that only DC3000::mut5 triggers immunity in RG-pto11 plants and this is the only mutant that expresses AvrPtoB with the Rsb triggering domain described in the text. The immunity observed in RG-PtoR plants is likely to be the result of AvrPto recognition. I, immunity; D, disease.

Fig. 6. AvrPtoB induces plant susceptibility to P.syringae pv. tomato DC3000 infection. (A) Disease symptoms or host immunity on tomato leaves 6 days after inoculation with indicated bacterial strains. Mutant DC3000::mut5 triggers immunity in RG-pto11 and expression of AvrPtoB in trans restores DC3000::mut5 pathogenicity. pDSK519 is a broad host range plasmid. I, immunity; D, disease. (B) Bacterial growth in leaves over a period of 6 days as measured by the number of c.f.u. per cm² of leaf tissue. Errors bars represent the standard deviation of bacterial counts.

Fig. 6. AvrPtoB induces plant susceptibility to P.syringae pv. tomato DC3000 infection. (A) Disease symptoms or host immunity on tomato leaves 6 days after inoculation with indicated bacterial strains. Mutant DC3000::mut5 triggers immunity in RG-pto11 and expression of AvrPtoB in trans restores DC3000::mut5 pathogenicity. pDSK519 is a broad host range plasmid. I, immunity; D, disease. (B) Bacterial growth in leaves over a period of 6 days as measured by the number of c.f.u. per cm² of leaf tissue. Errors bars represent the standard deviation of bacterial counts.

Fig. 7. A model for AvrPtoB recognition and PCD inhibition in tomato. The modular structure of AvrPtoB is depicted with the Pto-recognized N-terminal module shown as a brown circle, the anti-PCD C-terminal module shown as a red octagon, and the region recognized by Rsb shown as a blue connecting line. The black box represents an unknown factor predicted to act with Pto to suppress AvrPtoB anti-PCD function. Rsb-mediated cell death and immunity only occurs in the presence of the Δ6 truncation and the absence of Pto and intact AvrPtoB. Note: the gene(s) controlling the Rsb phenotype has not been identified; therefore, Rsb is presented in this model as a hypothetical R protein.