Diverse AvrPtoB homologs from several Pseudomonas syringae pathovars elicit Pto-dependent resistance and have similar virulence activities

Abstract

AvrPtoB is a type III effector protein from Pseudomonas syringae pv. tomato that physically interacts with the tomato Pto kinase and, depending on the host genotype, either elicits or suppresses programmed cell death associated with plant immunity. We reported previously that avrPtoB-related sequences are present in diverse gram-negative phytopathogenic bacteria. Here we describe characterization of avrPtoB homologs from P. syringae pv. tomato T1, PT23, and JL1065, P. syringae pv. syringae B728a, and P. syringae pv. maculicola ES4326. The avrPtoB homolog from P. syringae pv. maculicola, hopPmaL, was identified previously. The four new genes identified in this study are designated avrPtoB(T1), avrPtoB(PT23), avrPtoB(JL1065), and avrPtoB(B728a). The AvrPtoB homologs exhibit 52 to 66% amino acid identity with AvrPtoB. Transcripts of each of the avrPtoB homologs were detected in the Pseudomonas strains from which they were isolated. Proteins encoded by the homologs were detected in all strains except P. syringae pv. tomato T1, suggesting that T1 suppresses accumulation of AvrPtoB(T1). All of the homologs interacted with the Pto kinase in a yeast two-hybrid system and elicited a Pto-dependent defense response when they were delivered into leaf cells by DC3000DeltaavrPtoDeltaavrPtoB, a P. syringae pv. tomato strain with a deletion of both avrPto and avrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000DeltaavrPtoDeltaavrPtoB to form lesions on leaves of two susceptible tomato lines. With the exception of HopPmaL which lacks the C-terminal domain, all AvrPtoB homologs suppressed programmed cell death elicited by the AvrPto-Pto interaction in an Agrobacterium-mediated transient assay. Thus, despite their divergent sequences, AvrPtoB homologs from diverse P. syringae pathovars have conserved avirulence and virulence activities similar to AvrPtoB activity.

FIG. 1.

Sequence comparisons for AvrPtoB and AvrPtoB homologs. (A) Alignment of the amino acid sequences of AvrPtoB and five AvrPtoB homologs. Alignment was performed using ClustalW (40). Conserved residues are highlighted. The domain required for Pto interaction in a yeast two-hybrid system encompasses AvrPtoB residues 1 to 308. The domain required for the anti-PCD function consists of AvrPtoB residues 309 to 553 (1). The GenBank accession numbers are DQ133533 (avrPtoB_T1), DQ133534 (avrPtoB_PT23), and DQ133535 (avrPtoB_JL1065). (B) Neighbor-joining tree for AvrPtoB and eight other members of the AvrPtoB/VirPphA (HopAB) family, which was constructed using MEGA, version 2.1 (25). The horizontal branch lengths are proportional to the estimated numbers of amino acid substitutions, and bootstrap scores (expressed as percentages) are indicated at the nodes.

FIG. 2.

RT-PCR analysis of avrPtoB homolog transcripts. Total RNA was isolated from each Pseudomonas strain after growth without (lanes −) or with (lanes +) Hrp induction. RT-PCR was performed as described in Materials and Methods using gene-specific primers; primers for 16S rRNA were used as an internal control. The PCR products were separated on an agarose gel and stained with ethidium bromide. Transcripts for each of the AvrPtoB homologs could be detected in the strain from which the corresponding gene was isolated.

FIG. 3.

Analysis of protein accumulation and secretion of AvrPtoB and AvrPtoB homologs. (A) AvrPtoB and AvrPtoB homolog proteins were detected in cells of P. syringae strains by Western blotting using anti-AvrPtoB antibodies (see Materials and Methods). β-Lactamase was included as a loading control. Strains were grown without (lanes −) or with (lanes +) Hrp induction. AvrPtoB or AvrPtoB homolog proteins were detected in P. syringae pv. tomato DC3000, P. syringae pv. tomato PT23, P. syringae pv. tomato JL1065, and P. syringae pv. maculicola ES4326. (B) Secretion of AvrPtoB and AvrPtoB homologs into the culture medium in an Hrp-dependent manner was analyzed by Western blotting using anti-AvrPtoB antibodies. Secretion of AvrPtoB proteins was observed for all strains except T1 and B728a (see Results). Strains were grown in Hrp-inducing conditions. β-Lactamase antibodies were used as a control, and, as expected, no β-lactamase was detected in the culture medium. (C) Analysis of AvrPtoB-HA protein accumulation in strains DC3000ΔavrPtoΔavrPtoB and T1 by Western blotting using anti-HA antibodies. An AvrPtoB-HA-expressing plasmid was transformed into the two strains, and the strains were grown without (lanes −) or with (lanes +) Hrp induction. AvrPtoB was observed to accumulate in DC3000ΔavrPtoΔavrPtoB but not in T1. (D) Analysis of accumulation and secretion of AvrPto in strain T1. T1 and a derivative expressing AvrPto from a plasmid were grown without (lanes −) or with (lanes +) Hrp induction. Extracts were prepared from cells or from the culture medium (CM). Western blotting with anti-AvrPto antibodies detected AvrPto in both total cells and the culture medium, indicating that, as expected, this protein is expressed by and secreted from strain T1.

FIG. 4.

AvrPtoB homologs interact with Pto and elicit Pto-dependent resistance. (A) Yeast two-hybrid interactions of Pto with AvrPto or the AvrPtoB homologs. Image 1, empty vector; image 2, AvrPtoB; image 3, AvrPtoB_T1; image 4, AvrPtoB_PT23; image 5, AvrPtoB_B728a; image 6, HopPmaL. (B) Elicitation of Pto-dependent resistance by infiltration of RG-PtoR leaves with 10⁴ CFU ml⁻¹ ΔavrPtoΔavrPtoB expressing the following genes: none (empty vector) (leaf 1), avrPtoB (leaf 2), avrPtoB_T1 (leaf 3), avrPtoB_PT23 (leaf 4), avrPtoB_B728a (leaf 5), and hopPmaL (leaf 6). Photographs were taken 6 days after inoculation. Disease developed 3 days after inoculation of RG-PtoR leaves with only ΔavrPtoΔavrPtoB(ev) (leaf 1). (C) Bacterial populations in RG-PtoR tomato leaves at 0, 2, and 4 days after inoculation with DC3000ΔavrPtoΔavrPtoB expressing the genes indicated. Leaves were vacuum infiltrated with 10⁴ CFU ml⁻¹ of bacteria. The error bars indicate the standard deviations for three replicates. Data analysis was performed using Duncan's multiple-range test. Means with the same letter above the bars are not different at a significance level of 5%. The experiments were performed twice, and similar results were obtained.

FIG. 5.

Lesion-forming ability on susceptible tomato leaves of DC3000ΔavrPtoΔavrPtoB strains expressing AvrPtoB or the AvrPtoB homologs. (A) Host defense response or disease symptoms in RG-PtoS, RG-pto11, and RG-prf3 plants 6 days after inoculation with DC3000ΔavrPtoΔavrPtoB strains expressing the following genes: none (empty vector) (leaf 1), avrPtoB (leaf 2), avrPtoB_T1 (leaf 3), avrPtoB_PT23 (leaf 4), avrPtoB_B728a (leaf 5), and hopPmaL (leaf 6). Photographs were taken under a dissecting microscope 6 days after inoculation, and each area shown is 1 cm². (B) Numbers of specks on tomato leaves inoculated with DC3000ΔavrPtoΔavrPtoB expressing the genes indicated. The numbers of specks per cm² were counted for nine independent areas of the leaflet, and the error bars indicate standard deviations. Leaflets at the same position of each plant were chosen for counting numbers of specks and photography. Means with the same letter above the bars are not different at a significance level of 5%. The experiments were performed twice, and similar results were obtained.

FIG. 6.

Growth of DC3000ΔavrPtoΔavrPtoB expressing AvrPtoB or the AvrPtoB homologs. (A) RG-PtoS. (B) RG-prf3. (C) RG-pto11. The bacterial strains were vacuum infiltrated into leaves of Rio-Grande tomato lines, and the bacterial populations were measured 0, 2, and 4 days after inoculation. The error bars indicate the standard deviations for three 1-cm-diameter leaf disks. Data analysis was performed using Duncan's multiple-range test. Means with the same letter above the bars are not different at a significance level of 5%. ev, empty vector pCPP45. The experiments were performed twice, and similar results were obtained.

FIG. 7.

Inhibition of programmed cell death by AvrPtoB in AvrPtoB homologs in N. benthamiana leaves. (A) The following proteins were coexpressed with AvrPto and Pto in mature leaves of N. benthamiana using Agrobacterium-mediated transient expression: none (empty vector) (leaf 1), AvrPtoB (leaf 2), AvrPtoB_T1 (leaf 3), AvrPtoB_PT23 (leaf 4), AvrPtoB_B728a (leaf 5), and HopPmaL (leaf 6). Photographs were taken 7 days after Agrobacterium infiltration. (B) Ion leakage in the area infiltrated with A. tumefaciens expressing AvrPto, Pto, AvrPtoB, or AvrPtoB homologs 1, 3, 5, and 7 days after infiltration. Conductivity is indicated on the y axis.