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

Taylor KW, Kim JG, Su XB, Aakre CD, Roden JA, Adams CM, Mudgett MB - PLoS Pathog. (2012)

Bottom Line: XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato.Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence.This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, California, United States of America.

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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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.
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ppat-1002768-g006: 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.

Mentions: Examination of the C-terminal sequence of XopN revealed a putative Mode II recognition motif for 14-3-3 binding proteins [50] between amino acid residues 684–690, REHVSAP (Figure 6A). Mode II binding sites have the consensus sequence RXXXpS/TXP, where pS/pT represents phospho-serine or phospho-threonine and X can be any amino acids [50]. This suggested that serine residue 688 might be phosphorylated. To begin to address this, we first determined if XopN is phosphorylated in plant extracts using Phos-tag SDS-PAGE gels. His-tagged proteins were transiently expressed in N. benthamiana, purified using Ni+ affinity chromatography and then incubated with and without calf intestinal alkaline phosphatase (CIAP). Proteins were then separated in an 8% SDS-PAGE gel containing 50 µM Mn2+-Phos-tag and then analyzed by immunoblot analysis. XopN-6His treated with CIAP migrated faster in the Phos-tag gel compared to untreated XopN-6His indicating that XopN is phosphorylated in N. benthamiana extracts (Figure 7A).


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

Taylor KW, Kim JG, Su XB, Aakre CD, Roden JA, Adams CM, Mudgett MB - PLoS Pathog. (2012)

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.
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ppat-1002768-g006: 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.
Mentions: Examination of the C-terminal sequence of XopN revealed a putative Mode II recognition motif for 14-3-3 binding proteins [50] between amino acid residues 684–690, REHVSAP (Figure 6A). Mode II binding sites have the consensus sequence RXXXpS/TXP, where pS/pT represents phospho-serine or phospho-threonine and X can be any amino acids [50]. This suggested that serine residue 688 might be phosphorylated. To begin to address this, we first determined if XopN is phosphorylated in plant extracts using Phos-tag SDS-PAGE gels. His-tagged proteins were transiently expressed in N. benthamiana, purified using Ni+ affinity chromatography and then incubated with and without calf intestinal alkaline phosphatase (CIAP). Proteins were then separated in an 8% SDS-PAGE gel containing 50 µM Mn2+-Phos-tag and then analyzed by immunoblot analysis. XopN-6His treated with CIAP migrated faster in the Phos-tag gel compared to untreated XopN-6His indicating that XopN is phosphorylated in N. benthamiana extracts (Figure 7A).

Bottom Line: XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato.Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence.This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus