Pto mutants differentially activate Prf-dependent, avrPto-independent resistance and gene-for-gene resistance

Abstract

Pto confers disease resistance to Pseudomonas syringae pv tomato carrying the cognate avrPto gene. Overexpression of Pto under the cauliflower mosaic virus 35S promoter activates spontaneous lesions and confers disease resistance in tomato (Lycopersicon esculentum) plants in the absence of avrPto. Here, we show that these AvrPto-independent defenses require a functional Prf gene. Several Pto-interacting (Pti) proteins are thought to play a role in Pto-mediated defense pathways. To test if interactions with Pti proteins are required for the AvrPto-independent defense responses by Pto overexpression, we isolated several Pto mutants that were unable to interact with one or more Pti proteins, but retained normal interaction with AvrPto. Overexpression of two mutants, Pto(G50S) and Pto(R150S), failed to activate AvrPto-independent defense responses or confer enhanced resistance to the virulent P. s. pv tomato. When introduced into plants carrying 35S::Pto, 35S::Pto(G50S) dominantly suppressed the AvrPto-independent resistance caused by former transgene. 35S::Pto(G50S) also blocked the induction of a number of defense genes by the wild-type 35S::Pto. However, 35S::Pto(G50S) and 35S::Pto(R150S) plants were completely resistant to P. s. pv tomato (avrPto), indicating a normal gene-for-gene resistance. Furthermore, 35S::Pto(G50S) plants exhibited normal induction of defense genes in recognition of avrPto. Thus, the AvrPto-independent defense activation and gene-for-gene resistance mediated by Pto are functionally separable.

Figure 1

A, Criteria used for reverse yeast two-hybrid screen. The number of pluses indicates relative β-galactosidase activity (see Table I). B, Equal expression of LexA fusion proteins in yeast. An anti-LexA polyclonal antibody was used to detect the presence of LexA-Pto fusion proteins in yeast strains carrying the wild-type Pto (WT) or different mutants. Approximately 50 μg of total soluble protein was loaded for western blot.

Figure 2

Spontaneous lesions and HR in transgenic plants overexpressing the wild-type and mutant Pto transgenes. Northern blot shows the accumulation of transgene transcripts in primary transgenic plants. Non-transgenic PtoS plants were used as a control. Lines 104, 160, 345, 93, 124, 135, 455, 456, 458, and 471 carrying respective transgenes were examined in the T₂ generation for the presence (+) or absence (−) of visible spontaneous lesions (SL) in leaves without infection. The plants were also tested for the presence (+) or absence (−) of HR in response to P. s. pv tomato (avrPto).

Figure 3

35S::Pto^G50S dominantly suppresses the 35S::Pto-dependent spontaneous lesions. A, Accumulation of autofluorescent materials in line 135 (35S::Pto/35S::Pto and 35S::Pto/_) and 104 (35S::Pto^G50S/35S::Pto^G50S and 35S::Pto^G50S/_), and line 104 × line 135 F₁ (35S::Pto/35S::Pto^G50S) was examined under a fluorescence microscope. The tested T₂ plants were self-pollinated and their homozygosity was determined in the T₃ generation. B, Total Pto transcripts (wild type and mutant) in homozygous 104 (35S::Pto^G50S), homozygous 135 (35S::Pto), and 35S::Pto/35S::Pto^G50S plants.

Figure 4

35S::Pto^G50S dominantly suppresses the 35S::Pto-mediated AvrPto-independent resistance, but confers gene-for-gene resistance. A, Accumulation of free SA. B, Bacterial growth assay of P. s. pv tomato in plants. C, Bacterial growth assay of P. s. pv tomato (avrPto) in plants. Each data point represents average of three replicates (three plants). Error bars indicate ses. Lines 104 and 135 were in the T₂ generation segregating for the transgenes. All plants used in the experiments had been confirmed for the presence of transgene by Southern analysis. Both homozygous and hemizygous plants of lines 104 and 135 were used. Different letters denote significant difference (P = 0.05) as determined by Student's t distribution.

Figure 5

Pto^G50S suppresses the 35S::Pto-induced defense gene expression, but mediates normal defense gene activation by AvrPto. A, Reverse northern analysis of gene expression in untreated non-transgenic PtoS and transgenic lines 104 (35S::Pto^G50S), 135 (35S::Pto), and line 104 × line 135 F₁ (35S::Pto/35S::Pto^G50S) plants. cDNA probes were hybridized to duplicated DNA blots containing PCR products of cDNA clones (Xiao et al., 2001). Lane 1 contains an actin cDNA as a constitutive control. Lanes 2 thorough 40 are 39 cDNA clones isolated previously (Xiao et al., 2001). The GenBank accession numbers of corresponding cDNA clones that showed a differential hybridization (+) between 35S::Pto and non-transgenic PtoS plants are: BG352044 (3), BG351997 (4), BG352022 (5), BG351998 (6), BG352013 (9), BG352014 (10), BG352025 (12), BG351999 (17), BG352007 (21), BG352005 (23), BG352012 (26), BG352008 (27), BG352006 (30), BG352009 (35), BG352020 (38), and BG352046 (40). B, Northern analysis of gene expression in PtoS (S), PtoR (R), and mutant 35S::Pto^G50S transgenic lines 104, 160, and 345 after inoculation with P. s. pv tomato (avrPto). Samples were collected at the indicated hours postinoculation, and RNA blots were hybridized with the indicated probes. Lines 104, 160, 345, and 135 were in the T₂ generation segregating for the transgenes. All plants used in the experiments had been confirmed for the presence of transgene by Southern analysis.

Figure 6

35S::Pto^R150S does not confer nonspecific resistance but confers normal gene-for-gene resistance to P. s. pv tomato (avrPto). Tomato lines PtoS, PtoR, wild-type 35S::Pto transgenic lines (93, 124, and 135), and 35S::Pto^R150S lines (455, 456, 458, and 471) were inoculated with the P. s. pv tomato (A) or P. s. pv tomato (avrPto) strain (B), and leaf bacterial populations were determined. Each data point is averaged from three replicates. Error bars indicate ses. Different letters denote significant difference (P = 0.05) as determined by Student's t distribution. All transgenic lines were in the T₂ generation segregating for the transgenes. All plants used in the experiments had been confirmed for the presence of transgene by Southern analysis.

Figure 7

prf-3 dominantly suppresses the 35S::Pto-dependent lesions and disease resistance. A, Accumulation of autofluorescent materials. B, Bacterial growth assay with P. s. pv tomato. C, Bacterial growth assay with P. s. pv tomato (avrPto). Plants used were PtoR × line 48 F₁ (35S::Pto/_ and Prf/Prf) and prf-3 × line 48 F₁ (35S::Pto/_ and Prf/prf). Each data point is averaged from three replicates. Different letters denote significant difference (P = 0.05) as determined by Student's t distribution.