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. 2009 Apr;21(4):1305-23.
doi: 10.1105/tpc.108.063123. Epub 2009 Apr 14.

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1

Jung-Gun Kim  1 , Xinyan Li, Julie Anne Roden, Kyle W Taylor, Chris D Aakre, Bessie Su, Sylvie Lalonde, Angela Kirik, Yanhui Chen, Gayathri Baranage, Heather McLane, Gregory B Martin, Mary Beth Mudgett
Affiliations

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1

Jung-Gun Kim et al. Plant Cell. 2009 Apr.

Abstract

XopN is a virulence factor from Xanthomonas campestris pathovar vesicatoria (Xcv) that is translocated into tomato (Solanum lycopersicum) leaf cells by the pathogen's type III secretion system. Xcv DeltaxopN mutants are impaired in growth and have reduced ability to elicit disease symptoms in susceptible tomato leaves. We show that XopN action in planta reduced pathogen-associated molecular pattern (PAMP)-induced gene expression and callose deposition in host tissue, indicating that XopN suppresses PAMP-triggered immune responses during Xcv infection. XopN is predicted to have irregular, alpha-helical repeats, suggesting multiple protein-protein interactions in planta. Consistent with this prediction, XopN interacted with the cytosolic domain of a Tomato Atypical Receptor-Like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) in yeast. XopN/TARK1 and XopN/TFT1 interactions were confirmed in planta by bimolecular fluorescence complementation and pull-down analysis. Xcv DeltaxopN virulence defects were partially suppressed in transgenic tomato leaves with reduced TARK1 mRNA levels, indicating that TARK1 plays an important role in the outcome of Xcv-tomato interactions. These data provide the basis for a model in which XopN binds to TARK1 to interfere with TARK1-dependent signaling events triggered in response to Xcv infection.

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Figures

Figure 1.
Figure 1.
Complementation of Xcv ΔxopN in Planta Growth Defect by Extrachromosomal Expression of XopN. (A) Growth of Xcv 85-10 (vector) (yellow bars), Xcv 85-10 ΔxopN (vector) (orange bars), and Xcv 85-10 ΔxopN (xopN-HA) (gray bars) strains in tomato VF36 leaves. Leaves were hand-infiltrated with a 105 cells/mL suspension of bacteria. The number of bacteria in each leaf was quantified at 0, 4, 6, 8, and 10 DAI. Data points represent mean log10 colony-forming units per cm2 ± sd of three independent experiments. Asterisks indicate statistically significant (t test, P < 0.05) differences between the bacterial numbers of Xcv 85-10 (vector) and Xcv 85-10 ΔxopN (vector). The difference between Xcv 85-10 (vector) and Xcv 85-10 ΔxopN (xopN-HA) was insignificant. Vector = pVSP61. (B) Phenotype of tomato leaves inoculated with the strains described in (A). Leaves were photographed at 12 DAI. Similar phenotypes were observed in at least three independent experiments.
Figure 2.
Figure 2.
XopN Suppresses mRNA Levels of PR Genes during Xcv Infection. Susceptible VF36 tomato leaves were hand-inoculated with 10 mM MgCl2 only (white bars) or with 105 cfu/mL of Xcv (black bars) or Xcv ΔxopN (gray bars). Total RNA was isolated at 4 and 6 DAI. Quantitative RT-PCR was performed for six different tomato PR genes as indicated on each panel. 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 4 DAI. Averages of two independent experiments are shown. Error bars indicate sd.
Figure 3.
Figure 3.
XopN Suppresses the mRNA Level of PTI Marker Genes in Xcv-Inoculated Tomato Leaves at an Early Time Point. Independent susceptible VF36 tomato leaves on the same branch were hand-inoculated with a 2 × 108 cfu/mL suspension of Xcv (wt, white bars), Xcv ΔxopN (light-gray bars), Xcv ΔxopN(xopN) (dark-gray bars), or Xcv ΔhrpF (black bars). Total RNA was isolated from individual leaves at 6 HAI. Quantitative RT-PCR was performed for four genes: PTI5, WRKY28, LRR22, and GRAS2. Actin expression was used to normalize the expression value in each sample. Relative expression levels were determined by comparing the mRNA abundance detected in each treatment [Xcv ΔxopN, Xcv ΔxopN(xopN), or Xcv ΔhrpF] to the respective levels detected in Xcv-inoculated leaves that were arbitrarily set to a value of 1. The average ± sd of four independent experiments is shown. Asterisk indicates significant difference (t test, P < 0.05) relative to wild-type Xcv-inoculated leaves. t test indicated that significance for the PTI5 marker was P < 0.06.
Figure 4.
Figure 4.
XopN Suppresses Callose Deposition in Leaves During Infection. (A) Callose deposition in susceptible VF36 tomato leaves. (B) Callose deposition in susceptible Arabidopsis Col-0 leaves. Leaves in (A) and (B) were hand-infiltrated with a 2 × 108 cfu/mL suspension of Pst DC3000 AvrRpt21-100, ΔCEL AvrRpt21-100, ΔCEL AvrRpt21-100-XopN, or ΔCEL AvrRpt21-100-XopN(LGAAA). Leaves were stained for callose after 12 HAI and then visualized by fluorescence microscopy. Average numbers of callose deposits ± sd per field of view (mm2) for six leaves from six plants are noted under each panel. Similar phenotypes were observed in at least three independent experiments. (C) Bacterial growth in susceptible Arabidopsis Col-0 leaves. Leaves were hand-infiltrated with a 105 cfu/mL suspension of bacteria described above. Number of bacteria in leaves was quantified at days 0 and 4. Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. Different letters at day 4 indicate statistically significant (Tukey's honestly significant difference (HSD) test, P < 0.05) differences between samples.
Figure 5.
Figure 5.
XopN Interacts with Tomato 14-3-3s and an Atypical Receptor-Like Kinase TARK1. (A) Schematic of XopN protein from Xcv. T3S signal resides between amino acids 1 and 179 (Roden et al., 2004). Putative α-helical repeats are shown in pink. (B) XopN-interacting proteins in yeast. Yeast strain EGY48, pSH18-34, pEG202-nls(xopN) (BAIT) was independently transformed with the following PREY constructs: pJG4-5 alone (vector) or pJG4-5 containing tomato TFT1, TFT3, TFT6, TARK1 amino acids 329 to 605 (TARK1329-605), or TARK1 cytosolic domain containing amino acids 269 to 605 (TARK1-CD). Strains were spotted on noninducing (GLC-UHT and GLC-UHTL) and inducing (GAL-UHT+X-Gal and GAL-UHTL) medium and then incubated at 30°C for 3 to 4 d. (C) XopN interaction with TARK1 cytosolic domain is specific. EGY48 pSH18-34, pEG202-nls(xopN) was transformed with pJG4-5 alone (vector) or pJG4-5 containing TARK1-CD, tomato BRI1 (Sl BRI1-KD), tomato FLS2 (Sl FLS2-CD), or tomato TARK1-like (TARK1-L-CD). Strains were plated on media as described in (B).
Figure 6.
Figure 6.
Microscopy Images Depicting XopN, TARK1, and TFT1 Subcellular Localization Patterns and Protein–Protein Interactions in N. benthamiana Leaves. (A) Agrobacterium-mediated transient expression of YFP, YFP-XopN, TARK1-GFP, and YFP-TFT1 in N. benthamiana leaves. Leaves were hand-infiltrated with a 6 × 108 cells/mL suspension of each A. tumefaciens strain. Leaf epidermal cells were imaged after 48 h by confocal microscopy at ×60. Bars = 50 μm. (B) BIFC analysis of XopN/TARK1 and XopN/TFT1 interactions in N. benthamiana leaves. Leaves were hand-infiltrated with a suspension (1.2 × 109 cells/mL total concentration) of two A. tumefaciens strains expressing two different fusion proteins (XopN-nYFP + TARK1-cCFP, XopN-nYFP + TFT1-cCFP, or XopN-nYFP + PLC2-cCFP) and then visualized as described in (A). PLC2 was used as a plasma membrane–associated protein control (see Supplemental Figure 3 online). A. tumefaciens–mediated transient expression of PLC2-YFP was performed as described in (A) and is shown in the right panel. Bars = 50 μm.
Figure 7.
Figure 7.
XopN Physically Associates with TARK1 and TFT1 in Plant Extracts. Pull-down analysis of XopN and TARK1 (A) and XopN and TFT1 (B). N. benthamiana leaves were hand-infiltrated with a suspension (6 × 108 cfu/mL) of A. tumefaciens coexpressing XopN-6xHis and TARK1-HA or XopN-6xHis and TFT1-HA. 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. This experiment was repeated at least three times. Expected protein molecular masses are as follows: XopN-6xHis = 78.7 kD; TARK1-HA = 67.9 kD; TFT1-HA = 29.3 kD. +, protein expressed; −, vector control.
Figure 8.
Figure 8.
XopN Binds to the LXXLL Domain in the Proximal Membrane Region of TARK1. (A) Schematic of TARK1 protein. SP, signal peptide; TMD, transmembrane domain; LEDLL, LXXLL motif in proximal membrane region. (B) Analysis of XopN interactions with select C-terminal domains of TARK1. EGY48 pSH18-34, pEG202-nls(lexA:xopN) was transformed with pJG4-5 alone (vector), pJG4-5 containing TARK1 cytosolic domain (TARK1-CD), the LXXLL motif + kinase domain (TARK1329-605), or the kinase domain (TARK1337-605). Strains were plated on inducing (GAL/-UHT/X-Gal or GAL/-UHTL) and noninducing (GLC/-UHT) medium and then incubated at 30°C for 3 to 4 d. (B) XopN interaction with TARK1 LXXLL site-directed mutants. The LEDLL motif was mutated by replacing the conserved Leu residues with Ala. EGY48 pSH18-34, pEG202-nls(xopN) was transformed with pJG4-5 containing no insert (vector), TARK1329-605 (LEDLL), or two Ala substitution mutants (LEDAA) and (AEDLL). Strains were analyzed as in (A).
Figure 9.
Figure 9.
An LXXLL Motif in the N terminus of XopN Is Required for TARK1 Binding and Contributes to XopN-Dependent Virulence in Tomato. (A) Schematic of XopN protein and various deletion mutants. XopN deletion mutants and an Ala substitution mutant were cloned into pEG202-nls to create truncated LexA-XopN fusion proteins: XopN, XopN 1 to 733; M5, 1 to 604; M6, 1 to 514; N = 1 to 349; M3, 178 to 733 and LGAAA, respectively. Numbering refers to the amino acid residue in the wild-type XopN protein (733 amino acids). LGALL is the LXXLL motif that resides between 61 and 65. (B) Analysis of XopN mutant proteins in yeast. Yeast strains EGY48, pSH18-34 carrying pJG4-5(TARK1-CD) were transformed with vector (pEG202-nls) or each XopN construct described in (A). Strains were plated on inducing (GAL/-UHT/X-Gal) medium and then incubated at 30°C for 3 to 4 days. This experiment was repeated at least three times. (C) XopN LXXLL motif is required for interaction with TARK1 in planta. Leaves were hand-infiltrated with a 6 × 108 cfu/mL suspension of A. tumefaciens coexpressing TARK1-HA and XopN-6xHis or TARK1-HA and LGAAA-6xHis. After 48 h, protein was extracted, His-tagged proteins were purified by Ni+ affinity chromatography, and then proteins were analyzed by protein gel blot analysis using anti-HA and anti-His sera. Expected protein molecular masses are as follows: XopN-6xHis wt and LGAAA-6xHis = 78.7 kD and TARK1-HA = 67.9 kD. The signal in the first lane of the input and pull-down protein blots probed with anti-His sera is nonspecific. (D) Growth of Xcv strains in VF36 tomato leaves. Leaves were hand-infiltrated with a 105 cells/mL suspension of Xcv ΔxopN expressing LGAAA-HA (green bars), XopN-HA (gray bars), or vector (orange bars). Bacterial growth was measured at 0, 4, 6, and 10 DAI. Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. Asterisk above the bars indicates statistically significant (t test, P < 0.05) differences between the bacterial numbers of Xcv ΔxopN expressing XopN and LGAAA-HA or vector.
Figure 10.
Figure 10.
Reduced TARK1 mRNA Expression in Transgenic Tomato Leaves Partially Suppresses the Xcv ΔxopN Growth Phenotype in Planta. (A) Relative TARK1 and TARK1-L mRNA levels in T2 VF36 tomato transgenic lines transformed with HP-TARK1. Total RNA isolated from tomato leaves was used in quantitative RT-PCR with primers specific for TARK1 and TARK1-L. Actin mRNA expression was used to normalize the expression value in each sample. C, control transgenic lines exhibiting TARK1 mRNA expression. Lines 1 and 2: two independent, silenced transgenic lines with reduced TARK1 mRNA expression. Error bars indicate sd representing technical error. (B) Growth curve analysis (left panels) and disease phenotypes (right panels) of the tomato lines described in (A) inoculated with Xcv (black bars) and Xcv ΔxopN (white bars). Leaves were hand-infiltrated with a 105 cells/mL suspension of Xcv and Xcv ΔxopN. Two leaves per plant were inoculated with each strain: Xcv-infected leaves are on the left and Xcv ΔxopN-infected leaves are on the right. Hole punches (shown in the photograph) were isolated and used to quantify bacterial growth at 0, 8, and 12 DAI. Data points represent mean log10 cfu per cm2 ± sd. Leaf symptoms were photographed at day 12. Similar results were observed in at least three independent experiments. (C) Statistical analysis of Xcv and Xcv ΔxopN growth in TARK1 silenced tomato plants from line 1. Three T2 individuals from line 1 were infected with Xcv and Xcv ΔxopN and bacterial growth was quantified as described in (B). Data points represent the mean log10 cfu/cm2 ± sd for three independent plants. Different letters at each time point indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P < 0.05) differences between the samples. (D) Relative TARK1 and TARK1-L mRNA levels in the tomato lines used in (C). mRNA levels were determined as described in (A). The average mRNA level in the three individual plants is shown. Error bars indicate sd.
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