A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity

Abstract

Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

Figure 1. The Fen kinase is responsible for the Rsb phenotype

a, Genome organization of the Pto family and Prf from Solanum pimpinellifolium. Rio Grande-PtoR (RG-PtoR) plants are wild type, RG-pto11 plants have a deleterious point mutation (yellow dot) in Pto, and RG-prf3 plants have a deletion in Prf. b, Growth of Pst strains delivering AvrPtoB (white) or AvrPtoB_1–509 (grey) in RG-PtoR, RG-pto11, RG-prf3 and RG-PtoR(hpPto) leaves. Error bars represent s.e.m. (n = 6). c.f.u., colony-forming units. c, Response after transient expression of Pto with AvrPto, AvrPtoB_1–387, mitogen-activated protein kinase kinase kinase (MAPKKK)α or Bax in N. benthamiana leaves silenced for genes in the Pto pathway (listed at the left) (see Supplementary Fig. 2). MEK, MAP kinase/ERK kinase; SIPK, salicylic-acid-induced protein kinase; WIPK, wound-induced protein kinase; dashes indicate no PCD. d, Pto family members or empty vector (ev) tested for interaction with forms of AvrPtoB in the yeast two-hybrid system. Blue patches show positive interactions. e, Response of RG-PtoS protoplasts after coexpression of AvrPtoB_1–387 with Pto (red columns), Fen (orange columns), PtoC (green columns) or empty vector (white columns). Data are presented as percentage cell death in experimental samples after subtraction of cell death percentages occurring with empty vector. Error bars represent s.e.m. (n = 3).

Figure 2. Fen is ubiquitinated in the presence of AvrPtoB

a, Pto family members or empty vector (ev) tested for interaction with AvrPtoB, or AvrPtoB proteins with compromised E3 ligase activity (Quad and E2BS) in the yeast two-hybrid system. Blue patches show positive interactions. b, Immunoblots of an in vitro ubiquitination assay with recombinant E1, E2, AvrPtoB, ubiquitin (Ub) and MBP fusions of the kinases shown. Polyubiquitination of Fen is indicated by (Fen–(Ub)_n). Polyubiquitinated forms of AvrPtoB (AvrPtoB–(Ub)_n) confirmed E3 ligase activity. An anti-Ub antibody detected the incorporation of ubiquitin in each assay.

Figure 3. In the presence of AvrPtoB, Fen is degraded in a proteasome-dependent manner

a, Immunoblot to detect accumulation of Fen, Pto or PtoC in RG-prf3 protoplasts in the presence of AvrPtoB or Quad. Visualization of ribulose 1,5-bisphosphate carboxylase–oxygenase (Rubisco) subunits by Coomassie blue confirmed equal loading. The Fen panel shows a roughly 20-s exposure, in contrast with about 10 s for the others; however, the images are from the same experiment. HA, haemagglutinin. b, As in a after treatment with MG132 (minus, dimethylsulphoxide (DMSO) only; plus, with 50 μM MG132). c, As in a with the addition of general plant protease inhibitor (PPI) cocktail (minus, DMSO only; plus, 0.2% PPI).

Figure 4. Rsb is present in many cultivated and wild species of tomato

a, Inoculation of cultivated tomato varieties MM, VFNT-C or AC with Pst strains. R and S indicate resistant or susceptible plants, respectively. b, Electrolyte leakage after coexpression of AC-fen, Fen or Pto with AvrPtoB_1–387 (orange), AvrPtoB_1–307 (green) or AvrPtoB (grey). The ratio of conductivity of samples coexpressing AvrPtoB_1–307, AvrPtoB_1–387 or AvrPtoB versus empty vector control is shown. Error bars represent s.e.m. (n = 3). c, Leaves from 3 of 21 wild tomato species shown to exhibit Rsb immunity in response to inoculation with Pst delivering AvrPtoB_1–387 (see Supplementary Table 1): left, disease; right, Rsb immunity.