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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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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).
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ppat-1002768-g001: 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).

Mentions: To determine if Xcv infection alters TFT1 mRNA abundance, we monitored TFT1 mRNA levels in 4-week old VF36 tomato leaflets inoculated with 10 mM MgCl2 or a 10 mM MgCl2 suspension containing a low titer (1×105 colony forming units (CFU)/mL) of Xcv or Xcv ΔxopN. These conditions mirror those used in bacterial growth curves to monitor changes in pathogen multiplication and disease symptom development over a two-week period. Quantitative real-time RT-PCR (Q-PCR) revealed that the relative level of TFT1 mRNA was similar in all leaf samples at 2 and 4 days post-inoculation (DPI). At 6 and 8 DPI, TFT1 mRNA levels significantly increased in both the Xcv- and Xcv ΔxopN-infected leaf tissues (Figure 1A). This is the time period when Xcv and Xcv ΔxopN titers begin to differ significantly within the leaf tissue due to the impact of PTI in tomato [28]. It is also the time point when XopN-dependent suppression of PTI marker genes occurs [28]. These data indicate that Xcv infection induces TFT1 mRNA levels tomato leaves in a XopN-independent manner.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002768-g001: 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).
Mentions: To determine if Xcv infection alters TFT1 mRNA abundance, we monitored TFT1 mRNA levels in 4-week old VF36 tomato leaflets inoculated with 10 mM MgCl2 or a 10 mM MgCl2 suspension containing a low titer (1×105 colony forming units (CFU)/mL) of Xcv or Xcv ΔxopN. These conditions mirror those used in bacterial growth curves to monitor changes in pathogen multiplication and disease symptom development over a two-week period. Quantitative real-time RT-PCR (Q-PCR) revealed that the relative level of TFT1 mRNA was similar in all leaf samples at 2 and 4 days post-inoculation (DPI). At 6 and 8 DPI, TFT1 mRNA levels significantly increased in both the Xcv- and Xcv ΔxopN-infected leaf tissues (Figure 1A). This is the time period when Xcv and Xcv ΔxopN titers begin to differ significantly within the leaf tissue due to the impact of PTI in tomato [28]. It is also the time point when XopN-dependent suppression of PTI marker genes occurs [28]. These data indicate that Xcv infection induces TFT1 mRNA levels tomato leaves in a XopN-independent manner.

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