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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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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.
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ppat-1002768-g003: 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.

Mentions: Reduced PR-1b1 mRNA levels in TFT1-silenced tomato leaves suggested that TFT1 may be required for other PTI marker gene expression in tomato. To examine this, we generated an independent set of control and TFT1-silenced tomato plants using VIGS. The plants were inoculated with a high titer (2×108 CFU/mL) of Xcv or Xcv ΔhrpF. Total RNA was isolated at 6 HPI and then the level of four mRNAs (i.e. PTI5, GRAS4, WRKY28, and LRR22) known to be associated with PTI in tomato [28], [49] was quantified by Q-PCR. TFT1 mRNA levels were significantly reduced (∼4-fold lower) in the TRV2-TFT1 leaves relative to the TRV2 control leaves (Figure S2). The mRNA levels for PTI5, GRAS4, WRKY28, and LRR22 were significantly higher in TRV2 control leaves inoculated with Xcv ΔhrpF compared to those inoculated with Xcv (Figure 3). This indicates that Xcv suppresses the accumulation of these PTI marker mRNAs in a type III-dependent manner. PTI5, GRAS4, WRKY28, and LRR22 mRNA levels in the TRV2-TFT1 leaves inoculated with Xcv ΔhrpF were significantly lower than those in the TRV2 leaves inoculated with Xcv ΔhrpF (Figure 3). PTI5, WRKY28, and LRR22 mRNA levels were also significantly reduced in the Xcv-infected TRV2-TFT1 leaves relative to the Xcv-infected control leaves. Taken together, these data show that TFT1 expression is required for the mRNA accumulation of these PTI marker genes in susceptible tomato leaves challenged with Xcv or Xcv ΔhrpF.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375313&req=5

ppat-1002768-g003: 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.
Mentions: Reduced PR-1b1 mRNA levels in TFT1-silenced tomato leaves suggested that TFT1 may be required for other PTI marker gene expression in tomato. To examine this, we generated an independent set of control and TFT1-silenced tomato plants using VIGS. The plants were inoculated with a high titer (2×108 CFU/mL) of Xcv or Xcv ΔhrpF. Total RNA was isolated at 6 HPI and then the level of four mRNAs (i.e. PTI5, GRAS4, WRKY28, and LRR22) known to be associated with PTI in tomato [28], [49] was quantified by Q-PCR. TFT1 mRNA levels were significantly reduced (∼4-fold lower) in the TRV2-TFT1 leaves relative to the TRV2 control leaves (Figure S2). The mRNA levels for PTI5, GRAS4, WRKY28, and LRR22 were significantly higher in TRV2 control leaves inoculated with Xcv ΔhrpF compared to those inoculated with Xcv (Figure 3). This indicates that Xcv suppresses the accumulation of these PTI marker mRNAs in a type III-dependent manner. PTI5, GRAS4, WRKY28, and LRR22 mRNA levels in the TRV2-TFT1 leaves inoculated with Xcv ΔhrpF were significantly lower than those in the TRV2 leaves inoculated with Xcv ΔhrpF (Figure 3). PTI5, WRKY28, and LRR22 mRNA levels were also significantly reduced in the Xcv-infected TRV2-TFT1 leaves relative to the Xcv-infected control leaves. Taken together, these data show that TFT1 expression is required for the mRNA accumulation of these PTI marker genes in susceptible tomato leaves challenged with Xcv or Xcv ΔhrpF.

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