Reduced <i>TFT1</i> mRNA expression in VIGS tomato leaves correlates with reduced PTI marker mRNA abundance in response to Xcv infection.

<p>Relative mRNA levels for four PTI marker genes (<i>PTI5</i>, <i>GRAS4</i>, <i>WRKY28</i>, and <i>LRR22</i>) in 4 control (TRV2) and 4 <i>TFT1</i>-silenced (TRV2-TFT1) tomato lines. Leaflets on the same branch were inoculated with 1×10⁵ CFU/mL of Xcv or Xcv <i>ΔhrpF</i>. Total RNA isolated from inoculated leaves at 6 HPI was used for Q-PCR. <i>Actin</i> mRNA expression was used to normalize the expression value in each sample. Error bars indicate SD for four plants. Asterisk indicates significant difference (<i>t</i> test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines.</p

Serine 688 in XopN is required for TFT1 binding in yeast but not <i>in planta</i>.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) XopN(S688A) and two phosphomimetic mutants, XopN(S688D) and XopN(S688E), interact with TFT1 in <i>N. benthamiana</i>. Leaves were hand-infiltrated with a suspension (8×10⁸ CFU/mL total) of two <i>A. tumefaciens</i> 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. (<b>D</b>) Growth of Xcv Δ<i>xopN</i> (vector), Xcv <i>ΔxopN</i> (XopN-HA), Xcv <i>ΔxopN</i> (XopN(S688A)-HA), Xcv <i>ΔxopN</i> (XopN(S688D)-HA, or Xcv <i>ΔxopN</i> (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. (<b>E</b>) Phenotype of tomato leaves inoculated with the strains described in (<b>D</b>). Leaves were photographed at 12 DPI. Analysis was repeated two times.</p

XopN is phosphorylated in plant extracts.

<p>(<b>A</b>) Phos-tag gel analysis of XopN-6His, XopN(L64A,L65A)-6His, XopN(S688A)-6His, or XopN(L64A,L65A,S688A)-6His purified from <i>N. benthamiana</i> 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>B</b>) MS analysis of a XopN phosphopeptide isolated from <i>N. benthamiana</i> 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.</p

XopN L64,L65 motif and S688 are required for TFT1 binding and XopN-dependent virulence.

<p>(<b>A</b>) Pull-down analysis of TFT1-HA and XopN(L64A,L65A,S688A)-6His, XopN(L64A,L65A)-6His, or XopN-6His in <i>N. benthamiana</i>. Leaves were infiltrated with a mixed suspension of <i>A. tumefaciens</i> expressing TFT1-HA (4×10⁸ CFU/mL) and <i>A. tumefaciens</i> 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>B</b>) BiFC analysis of XopN/TFT1 interactions in <i>N. benthamiana</i>. Leaves were hand-infiltrated with a suspension (8×10⁸ CFU/mL total) of two <i>A. tumefaciens</i> 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. (<b>C</b>) Growth of Xcv Δ<i>xopN</i> (vector), Xcv Δ<i>xopN</i> (L64A,L65A,S688A-HA), Xcv Δ<i>xopN</i> (L64A,L65A-HA), or Xcv Δ<i>xopN</i> (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. (<b>D</b>) Phenotype of tomato leaves inoculated with the strains described in (<b>C</b>). Leaves were photographed at 12 DPI. Similar phenotypes were observed in 3 independent experiments.</p

Reduced <i>TFT1</i> mRNA expression in VIGS tomato leaves promotes Xcv <i>ΔhrpF</i> and Xcv <i>ΔxopN</i> growth.

<p>(<b>A</b>) Growth of Xcv, Xcv <i>ΔhrpF</i>, or Xcv <i>ΔxopN</i> in control (TRV2) and <i>TFT1</i>-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 (<i>t</i> test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines. (<b>B</b>) Relative <i>PR-1b1</i> mRNA levels in 4 control (TRV2) and 4 <i>TFT1</i>-silenced (TRV2-TFT1) tomato lines. Total RNA isolated from infected leaves in (<b>A</b>) on day 6 was used for Q-PCR. <i>Actin</i> mRNA expression was used to normalize the expression value in each sample. Error bars indicate SD for four plants. Asterisk indicates significant difference (<i>t</i> test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines.</p

TFT1 associates with the C-terminal domain of XopN.

<p>(<b>A</b>) Schematic of XopN protein and various deletion mutants. Wild type and mutant <i>xopN</i> 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>B</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. (<b>C</b>) Pull-down analysis of TFT1-HA and XopN-6His, XopN_1–349-6His, or XopN_345–733-6His in <i>N. benthamiana</i> extracts. <i>N. benthamiana</i> leaves were inoculated with a suspension of 6×10⁸ CFU/mL of <i>A. tumefaciens</i> co-expressing TFT1-HA and XopN-6His, XopN_1–349-6His, or XopN_345–733-6His. 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; XopN_1–349-6His = 38.0 kDa; XopN_345–733-6His = 42.0 kDa; +, protein expressed; −, vector control. STD, molecular weight standard. (<b>D</b>) Pull-down analysis of TFT1-HA and XopN-6His or XopN_1–604-6His in <i>N. benthamiana</i> extracts. <i>N. benthamiana</i> leaves were hand-inoculated with a mixed suspension of 1×10⁸ CFU/mL of <i>A. tumefaciens</i> expressing TFT1-HA and 4×10⁸ CFU/mL of XopN-6His or XopN_1–604-6His. Samples were processed as described in (<b>C</b>). Expected protein MW: TFT1-HA = 29.3 kDa; XopN-6His = 78.7 kDa; XopN_1–604-6His = 64.9 kDa; +, protein expressed; −, vector control. STD, molecular weight standard.</p

XopN residues 605–733 are important for TFT1 binding and contribute to XopN-dependent virulence in tomato.

<p>(<b>A</b>) Growth of Xcv Δ<i>xopN</i> (vector) (grey bars), Xcv Δ<i>xopN</i> (XopN_1–604-HA) (blue bars), or Xcv Δ<i>xopN</i> (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>B</b>) Phenotype of tomato leaves inoculated with strains described in (<b>A</b>). Leaves were photographed at 12 DPI. Similar phenotypes were observed in 3 independent experiments.</p

<i>TFT1</i> is a PTI-induced gene in tomato.

<p>(<b>A</b>) Susceptible VF36 tomato leaves were inoculated with 10 mM MgCl₂ (white bars), 1×10⁵ CFU/mL of Xcv (black bars), or Xcv <i>ΔxopN</i> (grey bars). (<b>B</b>) Susceptible VF36 tomato leaves were inoculated with 10 mM MgCl₂ (white bars), 2×10⁸ CFU/mL of Xcv (black bars), Xcv <i>ΔhrpF</i> (yellow bars), or Xcv <i>ΔhrcV</i> (green bars). Total RNA was isolated at 2, 4, 6, and 8 DPI (<b>A</b>) or 6 HPI (<b>B</b>). Q-PCR was performed to monitor <i>TFT1</i> mRNA levels. <i>Actin</i> 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 (<b>A</b>) and four (<b>B</b>) plants. Asterisk indicates significant difference (<i>t</i> test, P<0.05) relative to the 10 mM MgCl₂ control at 6 or 8 DPI (<b>A</b>) or 6 HPI (<b>B</b>).</p