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, 12 (12), 2323-2338

The Pseudomonas AvrPto Protein Is Differentially Recognized by Tomato and Tobacco and Is Localized to the Plant Plasma Membrane

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The Pseudomonas AvrPto Protein Is Differentially Recognized by Tomato and Tobacco and Is Localized to the Plant Plasma Membrane

L Shan et al. Plant Cell.

Abstract

The avrPto gene of Pseudomonas syringae pv tomato triggers race-specific resistance in tomato plants carrying Pto, a resistance gene encoding a protein kinase. When introduced into P. s. tabaci, avrPto triggers resistance in tobacco W38 plants that carry the corresponding R gene. The AvrPto protein is believed to be secreted into host cells through the bacterial type III secretion pathway, where it activates disease resistance in tomato by interacting with Pto. We report here the identification of two distinct regions in AvrPto that determine the recognition specificity of this protein in tomato and tobacco. Point mutations in the central region disrupted the avirulence activity in tomato but not in tobacco. Conversely, point mutations in the C-terminal region abolished the avirulence in tobacco but not in tomato. We further report that AvrPto was localized to the plasma membrane of plant cells. Disrupting the membrane association by mutating a putative myristoylation motif of AvrPto abolished the avirulence activity in both tomato and tobacco. These findings demonstrate that AvrPto is recognized differently by the R genes in tomato and tobacco and that the recognition of AvrPto probably is associated with the plasma membrane.

Figures

Figure 1.
Figure 1.
Mutations of AvrPto That Disrupt the Pto Interaction Abolish the Avirulence Activity in Tomato Plants. AvrPto mutants that abolished the Pto interaction (Table 1) were introduced into P. s. tomato strain T1, and the resulting strains (104 colony-forming units [cfu]/mL) were inoculated individually into 6-week-old tomato PtoR plants by vacuum infiltration. P. s. tomato strains T1(−avrPto) and T1(avrPto) were inoculated as controls. The bacterial numbers were measured at 0 and 4 days after inoculation. Six leaf discs (1 cm2) from three inoculated plants were divided into three tubes and ground separately. Bacteria were diluted and plated on King's B (KB) medium containing appropriate antibiotics, and cfu were counted. The number for each time point represents the average of three measurements. Error bars indicate se.
Figure 2.
Figure 2.
Some AvrPto Mutants Disrupt Avirulence in Tomato but Not in Tobacco. The AvrPto mutants described in Table 1 were introduced into P. s. tabaci strain 11528R, and the resulting strains were injected separately into tobacco W38 leaves. Inoculum of 108 cfu/mL was used for the HR assay. Inoculum of 105 cfu/mL was used to assay for disease symptoms and to measure bacterial growth in tobacco. (A) Disease symptoms in W38 plants caused by strain 11528R carrying avrPto mutants. Inoculated leaves were photographed 4 days after inoculation. (B) The HR in W38 plant caused by strain 11528R carrying avrPto mutants. Inoculated leaves were photographed 18 hr after inoculation. (C) Growth of strain 11528R carrying avrPto mutants in W38 plants. Error bars indicate se.
Figure 3.
Figure 3.
AvrPto Mutants Exhibit Different Protein Stability in P. s. tomato Strain T1. P. s. tomato strain T1 carrying no avrPto (−avrPto), the wild-type avrPto (avrPto), or avrPto mutants was grown overnight in KB medium with appropriate antibiotics. The bacterial cells were washed with minimal medium (Huynh et al., 1989) and grown in minimal medium overnight at room temperature. Bacteria were separated from the liquid medium by centrifugation. Bacterial cells (B) and supernatant (S) were examined for the presence of the AvrPto protein by means of protein gel blot analysis. (A) Protein gel blot analysis with anti-AvrPto antibodies. (B) Protein gel blot analysis with antibodies for the kanamycin-resistance protein. (The −avrPto T1 strain did not contain the kanamycin-resistance gene.)
Figure 4.
Figure 4.
Some AvrPto Mutants Recognize Pto but Not the Tobacco R Gene. (A) HR induction on W38 and W38(35S::Pto) plants by P. s. tabaci strain 11528R carrying avrPto mutants. (B) Disease symptoms in W38 and W38(35S::Pto) plants caused by P. s. tabaci strain 11528R carrying avrPto mutants. (C) Growth of P. s. tabaci strain 11528R carrying avrPto mutants in tobacco W38 plants. (D) Growth of P. s. tabaci strain T1 carrying avrPto mutants in tomato PtoR plants. Plant inoculation and bacterial measurement were as described in Figures 1 and 2. Error bars indicate se.
Figure 5.
Figure 5.
The C-Terminal Deletion in AvrPto Specifically Abolishes Recognition by the Tobacco R Gene. (A) Growth of P. s. tabaci strain 11528R carrying avrPto deletion mutants in tobacco W38 plants. (B) Growth of P. s. tomato strain T1 carrying avrPto deletion mutants in tomato PtoR plants. Inoculation and bacterial measurement were as described in Figure 4. Error bars indicate se.
Figure 6.
Figure 6.
AvrPto Protein Is Localized in the Plasma Membrane. (A) Tetracycline-induced HR. Transgenic tobacco plants (W38) carrying the tetracycline repressor (TetR) transgene plus an empty vector (vector) or the avrPto transgene were injected with either 10 mM MgCl2 (Buffer) or 1 mg/L tetracycline (Tc). Leaves were photographed 17 hr after tetracycline treatment. (B) Membrane localization of AvrPto. (Left) Tobacco transgenic plants containing empty vector (vec) or the avrPto transgene (avr) were injected with 10 mM MgCl2 (Tc, −) or 1 mg/L tetracycline (Tc, +). Leaf tissues were collected 10 hr after injection. Total protein was extracted as described in Methods, separated by SDS-PAGE, and analyzed by protein gel blotting with anti-AvrPto antibodies. (Right) Total protein was extracted from leaves containing the plain vector or the wild-type avrPto gene and separated by centrifugation into soluble (S) and membrane (M) fractions. Protein in the membrane fraction was resuspended in an equal volume of protein extraction buffer containing 1% Triton X-100, separated by SDS-PAGE, and analyzed by protein gel blotting with anti-AvrPto antibodies. Arrowheads indicate AvrPto. (C) Localization of AvrPto to the plasma membrane. Plasma membrane was separated from intracellular membrane by the two-phase partitioning system. (Top) Localization of the AvrPto protein. Lanes 1 through 4: soluble fraction, plasma membrane, intracellular membrane, and total membrane, respectively. Lanes 1 and 4 were loaded with 20 μg of protein; lanes 2 and 3 received 10 μg of protein. (Bottom) Marker enzyme activities in plasma and intracellular membranes. PM and INTRA denote plasma and intracellular membranes, respectively. The activity of vanadate-sensitive ATPase is expressed as nmol phosphate·min−1·mg−1 protein. The activity of cytochrome (Cyt) c oxidase and NADH-dependent cytochrome c reductase is expressed as μmol cytochrome c·min−1·mg−1 protein.
Figure 7.
Figure 7.
The G2A Mutation Disrupts Membrane Localization and the Avirulence Function of AvrPto. (A) The G2A mutation disrupts the membrane association of AvrPto. Tobacco transgenic plants containing the avrPto gene or the G2A mutant were inoculated with 1 mg/L tetracycline. Leaf tissues were collected 10 hr after injection. Membrane protein was separated from soluble protein by centrifugation and examined by using anti-AvrPto antibodies. S, soluble protein; M, membrane protein. (B) The myristoylation mutation disrupts the avirulence activity in tomato. T1 strains carrying no avrPto (−avrPto), the wild-type avrPto (avrPto), or the mutant avrPto (G2A) were vacuum-infiltrated into tomato PtoR plants at 104 cfu/mL. Disease symptoms were documented 4 days after inoculation. (C) The G2A mutation disrupts the avirulence activity in tobacco. P. s. tabaci 11528R strains carrying no avrPto (−avrPto), the wild-type avrPto (avrPto), or the mutant avrPto (G2A) were inoculated into W38 plants at a concentration of 108 cfu/mL. The HR was photographed 18 hr after inoculation. (D) The G2A mutation does not affect the secretion of AvrPto. P. s. tomato T1 strains carrying no avrPto (−avrPto), the wild-type avrPto (avrPto), or the mutant avrPto (G2A) were grown in minimal medium. Bacterial cells (B) and supernatant (S) were examined for the presence of the AvrPto protein by using protein gel blot analysis. (E) The secretion of the G2A protein is Hrp-dependent. ΔavrPto/G2A designates the DC3000ΔavrPto strain containing the G2A mutant; hrcC/G2A designates the hrcC mutant strain of DC3000 containing the G2A mutant; hrcC designates the hrcC mutant strain of DC3000. Secretion of AvrPto and the G2A protein were measured as described in (D). (F) Direct expression of the G2A mutant protein in plant cells does not cause the HR. The wild-type avrPto and mutants G2A and 1603 (Table 2) were cloned into a plant expression vector and introduced into the Agrobacterium EHA105 strain. The resulting strains were injected into W38 plants. The HR was documented 3 days after injection. (G) The G2A mutation does not affect the Pto interaction. The G2A mutant was cloned into the prey vector pJG4-5, and the interaction with Pto was assayed with the yeast two-hybrid system. A noninteracting member of the Pto family (pto; Jia et al., 1997) was included as a control.

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