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. 2012;8(6):e1002768.
doi: 10.1371/journal.ppat.1002768. Epub 2012 Jun 14.

Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence

Kyle W Taylor  1 , Jung-Gun Kim, Xue B Su, Chris D Aakre, Julie A Roden, Christopher M Adams, Mary Beth Mudgett
Affiliations

Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence

Kyle W Taylor et al. PLoS Pathog. 2012.

Abstract

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TFT1 is a PTI-induced gene in tomato.
(A) Susceptible VF36 tomato leaves were inoculated with 10 mM MgCl2 (white bars), 1×105 CFU/mL of Xcv (black bars), or Xcv ΔxopN (grey bars). (B) Susceptible VF36 tomato leaves were inoculated with 10 mM MgCl2 (white bars), 2×108 CFU/mL of Xcv (black bars), Xcv ΔhrpF (yellow bars), or Xcv ΔhrcV (green bars). Total RNA was isolated at 2, 4, 6, and 8 DPI (A) or 6 HPI (B). Q-PCR was performed to monitor TFT1 mRNA levels. Actin expression was used to normalize the expression value in each sample, and relative expression values were determined against the average value of the sample infiltrated with 10 mM MgCl2 at each time point. Error bars indicate SD for three (A) and four (B) plants. Asterisk indicates significant difference (t test, P<0.05) relative to the 10 mM MgCl2 control at 6 or 8 DPI (A) or 6 HPI (B).
Figure 2
Figure 2. Reduced TFT1 mRNA expression in VIGS tomato leaves promotes Xcv ΔhrpF and Xcv ΔxopN growth.
(A) Growth of Xcv, Xcv ΔhrpF, or Xcv ΔxopN in control (TRV2) and TFT1-silenced (TRV2-TFT1) susceptible VF36 tomato lines. Leaves were inoculated with 1×105 CFU/mL of pathogen. Bacterial growth was quantified at 0, 6, and 9 DPI. Data points represent mean CFU/cm2 ± SD of four plants. Asterisk indicates significant difference (t test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines. (B) Relative PR-1b1 mRNA levels in 4 control (TRV2) and 4 TFT1-silenced (TRV2-TFT1) tomato lines. Total RNA isolated from infected leaves in (A) on day 6 was used for Q-PCR. Actin mRNA expression was used to normalize the expression value in each sample. Error bars indicate SD for four plants. Asterisk indicates significant difference (t test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines.
Figure 3
Figure 3. Reduced TFT1 mRNA expression in VIGS tomato leaves correlates with reduced PTI marker mRNA abundance in response to Xcv infection.
Relative mRNA levels for four PTI marker genes (PTI5, GRAS4, WRKY28, and LRR22) in 4 control (TRV2) and 4 TFT1-silenced (TRV2-TFT1) tomato lines. Leaflets on the same branch were inoculated with 1×105 CFU/mL of Xcv or Xcv ΔhrpF. Total RNA isolated from inoculated leaves at 6 HPI was used for Q-PCR. Actin mRNA expression was used to normalize the expression value in each sample. Error bars indicate SD for four plants. Asterisk indicates significant difference (t test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines.
Figure 4
Figure 4. TFT1 associates with the C-terminal domain of XopN.
(A) Schematic of XopN protein and various deletion mutants. Wild type and mutant xopN were cloned into pXDGATcy86(GAL4-DNA binding domain) to create DBD-XopN fusion proteins: XopN, XopN 1–733; N, 1–349; C, 344–733; M4, 222–733; M5, 1–604; and M6, 1–514. Numbering refers to amino acid residues in wild type XopN (733 amino acids). (B) TFT1 interaction with XopN mutant proteins in yeast. Yeast strain AH109 carrying pXDGATcy86 containing vector, XopN, N, C, M3, M4, M5 and M6 were independently transformed with the following PREY constructs: pGADT7(GAL4 activation domain) alone (Vector) or pGADT7 containing TFT1. Strains were spotted on nonselective (SD-LT) and selective (SD-LTH) media ± 1 mM 3-amino-1,2,4-triazole and then incubated at 30°C for 3d. (C) Pull-down analysis of TFT1-HA and XopN-6His, XopN1–349-6His, or XopN345–733-6His in N. benthamiana extracts. N. benthamiana leaves were inoculated with a suspension of 6×108 CFU/mL of A. tumefaciens co-expressing TFT1-HA and XopN-6His, XopN1–349-6His, or XopN345–733-6His. After 48 h, protein was extracted, purified by Ni+ affinity chromatography, and then analyzed by protein gel blot analysis using anti-His and anti-HA sera. Expected protein MW: TFT1-HA = 29.3 kDa; XopN-6His = 78.7 kDa; XopN1–349-6His = 38.0 kDa; XopN345–733-6His = 42.0 kDa; +, protein expressed; −, vector control. STD, molecular weight standard. (D) Pull-down analysis of TFT1-HA and XopN-6His or XopN1–604-6His in N. benthamiana extracts. N. benthamiana leaves were hand-inoculated with a mixed suspension of 1×108 CFU/mL of A. tumefaciens expressing TFT1-HA and 4×108 CFU/mL of XopN-6His or XopN1–604-6His. Samples were processed as described in (C). Expected protein MW: TFT1-HA = 29.3 kDa; XopN-6His = 78.7 kDa; XopN1–604-6His = 64.9 kDa; +, protein expressed; −, vector control. STD, molecular weight standard.
Figure 5
Figure 5. XopN residues 605–733 are important for TFT1 binding and contribute to XopN-dependent virulence in tomato.
(A) Growth of Xcv ΔxopN (vector) (grey bars), Xcv ΔxopN (XopN1–604-HA) (blue bars), or Xcv ΔxopN (XopN-HA) (black bars) in susceptible tomato VF36 leaves. Leaves were inoculated with a 1×105 CFU/mL suspension of bacteria. Number of bacteria in each leaf was quantified at 0, 6 and 8 DPI. Data points represent mean CFU/cm2 ± SD of three plants. Analysis was repeated at least three times. Vector = pVSP61. Different letters at day 8 indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P<0.05) differences between the samples. (B) Phenotype of tomato leaves inoculated with strains described in (A). Leaves were photographed at 12 DPI. Similar phenotypes were observed in 3 independent experiments.
Figure 6
Figure 6. Serine 688 in XopN is required for TFT1 binding in yeast but not in planta.
(A) Schematic of putative 14-3-3 motifs in XopN protein. Black boxes represent regions for putative Mode I and II 14-3-3 binding motifs. Mode II site contains S688. PEST domain is underlined. N-terminal leucines (L64, L65) required for TARK1-binding are labeled. (B) XopN(S688A) mutant does not interact with TFT1 in yeast. Serine 688 in XopN was mutated to alanine. Yeast strain AH109 carrying pXDGATcy86(GAL4-DNA binding domain) containing XopN, and XopN(S688A) was transformed with the following PREY constructs: pGADT7(GAL4 activation domain) alone (Vector) or pGADT7 containing TFT1. Strains were spotted on nonselective (SD-LT) and selective (SD-LTH) medium and then incubated at 30°C for 3d. (C) XopN(S688A) and two phosphomimetic mutants, XopN(S688D) and XopN(S688E), interact with TFT1 in N. benthamiana. Leaves were hand-infiltrated with a suspension (8×108 CFU/mL total) of two A. tumefaciens strains expressing TFT1-HA and XopN-6His or XopN(S688A)-6His or XopN(688D)-6His or XopN(688E)-6His. After 48 h, protein was extracted, purified by Ni+ affinity chromatography, and analyzed by protein gel blot analysis using anti-His and anti-HA sera. Expected protein MW: XopN-6His, S688A-6xHis, S688D-6His, and S688E-6His = 78.7 kDa; TFT1-HA = 29.3 kDa. +, protein expressed; −, vector control. (D) Growth of Xcv ΔxopN (vector), Xcv ΔxopN (XopN-HA), Xcv ΔxopN (XopN(S688A)-HA), Xcv ΔxopN (XopN(S688D)-HA, or Xcv ΔxopN (XopN(S688E)-HA in susceptible tomato VF36 leaves. Leaves were inoculated with a 1×105 CFU/mL suspension of bacteria. Number of bacteria in each leaf was quantified at 0 and 10 DPI. Data points represent mean CFU/cm2 ± SD of four plants. Different letters at day 10 indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P<0.05) differences between the samples. Vector = pVSP61. (E) Phenotype of tomato leaves inoculated with the strains described in (D). Leaves were photographed at 12 DPI. Analysis was repeated two times.
Figure 7
Figure 7. XopN is phosphorylated in plant extracts.
(A) Phos-tag gel analysis of XopN-6His, XopN(L64A,L65A)-6His, XopN(S688A)-6His, or XopN(L64A,L65A,S688A)-6His purified from N. benthamiana leaves at 48 HPI by Ni+ affinity chromatography. Protein was treated without or with CIAP for 60 min and then separated on 8% SDS-PAGE gels containing 50 µM Mn2+-Phos-tag. Gels were analyzed by immunoblot analysis using anti-His sera. (B) MS analysis of a XopN phosphopeptide isolated from N. benthamiana leaf extracts. The graph shows the fragmentation spectrum of the phosphopeptide EHVSAPpSSPNR. Serine 688 is phosphorylated. Major identified b- and y- ions are labeled. The m/z value for each b- and y- ion is shown.
Figure 8
Figure 8. XopN L64,L65 motif and S688 are required for TFT1 binding and XopN-dependent virulence.
(A) Pull-down analysis of TFT1-HA and XopN(L64A,L65A,S688A)-6His, XopN(L64A,L65A)-6His, or XopN-6His in N. benthamiana. Leaves were infiltrated with a mixed suspension of A. tumefaciens expressing TFT1-HA (4×108 CFU/mL) and A. tumefaciens expressing XopN(L64A,L65A)-6His, XopN(L64A,L65A,S688A)-6His or XopN-6His (4×108 CFU/mL). After 48 h, protein was extracted, purified by Ni+ affinity chromatography, and then analyzed by protein gel blot analysis using anti-His and anti-HA sera. Expected protein MW: TFT1-HA = 29.3 kDa; XopN(L64A,L65A)-6His, XopN(L64A,L65A,S688A)-6His and XopN-6His = 78.7 kDa. +, protein expressed; −, vector control. STD, molecular weight standard. (B) BiFC analysis of XopN/TFT1 interactions in N. benthamiana. Leaves were hand-infiltrated with a suspension (8×108 CFU/mL total) of two A. tumefaciens strains expressing different fusion proteins (XopN-nYFP+TFT1-cCFP; L64A,L65A-nYFP+TFT1-cCFP; S688A-nYFP+TFT1-cCFP; L64A,L65A,S688A-nYFP+TFT1-cCFP; or GUS-nYFP+TFT1-cCFP) and then visualized by confocal microscopy at 48 HPI at 63X. White bar = 25 µm. (C) Growth of Xcv ΔxopN (vector), Xcv ΔxopN (L64A,L65A,S688A-HA), Xcv ΔxopN (L64A,L65A-HA), or Xcv ΔxopN (XopN-HA) strains in susceptible VF36 tomato leaves. Leaves were hand-infiltrated with a 1×105 CFU/mL suspension of bacteria. Number of bacteria in each leaf was quantified at 0 and 8 DPI. Data points represent mean CFU/cm2 ± SD of three plants. Different letters at day 8 indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P<0.05) differences between the samples. Vector = pVSP61. Analysis was repeated at least three times. (D) Phenotype of tomato leaves inoculated with the strains described in (C). Leaves were photographed at 12 DPI. Similar phenotypes were observed in 3 independent experiments.
Figure 9
Figure 9. XopN promotes TARK1 and TFT1 binding in N. benthamiana.
(A-E) BiFC analysis of TARK1/TFT1 interactions in N. benthamiana leaves in the presence of wild type and mutant XopN protein. Leaves were hand-infiltrated with a suspension (6×108 CFU/mL total) of three A. tumefaciens strains expressing different fusion proteins: (A) GUS-6His+TARK1-SCFP3Ac+TFT1-VenusN; (B) XopN-6His+TARK1-SCFP3Ac+TFT1-VenusN; (C) XopN(L64A,L65A)-6His+TARK1-SCFP3Ac+TFT1-VenusN; (D) XopN(S688A)-6His+TARK1-SCFP3Ac+TFT1-VenusN; (E) XopN-(L64A,L65A,S688A)-6His+TARK1-SCFP3Ac+TFT1-VenusN. Cells were visualized by confocal microscopy at 48 HPI at 63X. White bar = 40 µm. (F) GST-TFT1 affinity purification of TARK1 and XopN in vitro. GST or GST-TFT1 was incubated with N. benthamiana leaf extracts containing TARK1-HA ± vector, XopN-6His or mutant XopN-6His (i.e. XopN(L64A,L65A)-6His, XopN(S688A)-6His, or XopN(L64A,L65A,S688A)-6His). Proteins were purified using glutathione sepharose and analyzed by immunoblot analysis using anti-His, anti-HA, and anti-GST sera. Protein input levels are shown on the left. GST and GST-TFT1 pull-downs are shown on right. Expected protein MW: GST = 28 kDa; GST-TFT1 = 56.5 kDa; TARK1-HA = 67.9 KDa; XopN(L64A,L65A)-6His, XopN(L64A,L65A,S688A)-6His and XopN-6His = 78.7 kDa. +, protein expressed; −, vector control. STD, molecular weight standard.
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