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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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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.
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ppat-1002768-g007: 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.

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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375313&req=5

ppat-1002768-g007: 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.
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