Fig 1.
The xopAU gene of Xe strain 85–10 is conserved in multiple Xanthomonas species and encodes a protein kinase.
Phylogenetic tree of xopAU (A) and gyrB (B) homologs generated with Clustal X [68] using the standard neighbor joining phylogenetic tree definitions. NCBI xopAU and gyrB accession numbers and a sequence alignment of the xopAU genes included in the tree are reported in S1 Table and S1 Fig, respectively. Bootstrap values of 100 replications are shown on nodes. (C) In vitro kinase assay performed by incubating the indicated proteins in the presence of [γ-32P]ATP. Proteins were separated by SDS-PAGE and detected by autoradiography or Coomassie staining.
Fig 2.
Agrobacterium-mediated expression of XopAU induces plant immune responses.
(A-E) N. benthamiana leaves were infiltrated with Agrobacterium strains for the expression of His-XopAU or His-XopAUK240A driven by an estradiol-inducible system, or carrying an empty vector (EV), and treated with 17β-estradiol 24 h later. (A) Photographs of inoculated areas at 48 h after 17β-estradiol application. (B) Electrolyte leakage at 24 h with or without 17β-estradiol application. The box plot displays 25th, 50th (middle line) and 75th percentiles (n = 5). An asterisk indicates a significant difference (Mann-Whitney U test, p value <0.05) compared to EV. (C) Total protein was extracted from N. benthamiana leaves 8 h after 17β-estradiol application and immunoblotted with the indicated antibodies. Rbs, Rubisco loading control stained by Ponceau S. (D) Leaves of the pepper line ECW30R or the tomato line Hawaii 7981 were infiltrated with Agrobacterium strains as in (A) and photographed at 96 h after 17β-estradiol application. (E) Total protein was extracted from pepper leaves at 8 h after 17β-estradiol application and immunoblotted with the indicated antibodies. (F and G) N. benthamiana plants were infected with TRV, TRV:EDS1, TRV:NDR1 and TRV:RAR1. Leaves of silenced plants were inoculated with Agrobacterium (OD600 = 0.02) carrying an empty vector (EV), a vector for expression of His-XopAU from an estradiol-inducible system or Cf4/Avr4 driven by the CaMV 35S promoter. Inoculated leaves were treated with 17β-estradiol 24 h later. (F) Photographs of inoculated areas at 36 h after 17β-estradiol application. (G) Electrolyte leakage at 24 h after 17β-estradiol application. The box plot displays 25th, 50th (middle line) and 75th percentiles (n = 5). An asterisk indicates a significant difference (Mann-Whitney U test, p value <0.05) compared to EV. (A-G) Experiments were repeated at least three times with similar results.
Fig 3.
Phenotypic analysis of xopAU gene inactivation.
Leaves of the pepper line ECW30R were syringe-infiltrated with a 10 mM MgCl2 mock solution or with suspensions (1 x 107 CFU/ml) of the indicated strains. (A) Photograph of an inoculated leaf at 5 days post-inoculation (dpi). (B) Chlorophyll content relative to mock-inoculated areas at 5 dpi. The box plot displays 25th, 50th (middle line) and 75th percentiles (n = 4 or 5 biological repeats). An asterisk indicates a significant difference (Mann-Whitney U test, p value <0.05) relative to Xe avrBs2:KnR. (C) Total protein was extracted from the infected leaves at 3 days post -inoculation (dpi) and immunoblotted with the indicated antibodies. Rbs, Rubisco loading control stained by Ponceau S. (D) mRNA abundance of the PR-1 gene in the inoculated areas was measured by qRT-PCR at 72 h post-inoculation and calculated relative to areas inoculated with the Xe avrBs2:KnR strain. Values are means ± SD of three biological repeats. Asterisks indicate a significant difference (Student’s t test, p value < 0.05) relative to Xe avrBs2:KnR.
Fig 4.
Phenotypic analysis of Xe strains overexpressing XopAU.
Leaves of the pepper line ECW30R were syringe-infiltrated with a 10 mM MgCl2 mock solution or with suspensions (1 x 107 CFU/ml) of Xe strains carrying a vector either empty (EV) or for expression of XopAU-HA or XopAUK240A-HA. (A) Photograph of an inoculated leaf at 5 days post-inoculation (dpi). (B) Chlorophyll content relative to mock-inoculated areas at 3 and 5 dpi. (C) Electrolyte leakage in the inoculated areas at 3 and 5 dpi. In B and C, box plots display 25th, 50th (middle line) and 75th percentiles (n = 10). Asterisks indicate a significant difference (Mann-Whitney U test, p value <0.05) relative to Xe containing an empty vector. Experiments were repeated at least three times with similar results.
Fig 5.
Expression of defense and stress marker genes in leaves infected with Xe overexpressing XopAU.
Leaves of the pepper line ECW30R were syringe-infiltrated with a 10 mM MgCl2 mock solution (Mock) or with suspensions (1 x 107 CFU/ml) of Xe strains carrying a vector either empty (EV) or for expression of XopAU-HA or XopAUK240A-HA. (A) Total protein was extracted from the infected leaves at the indicated days post-inoculation (dpi) and immunoblotted with the indicated antibodies. Rbs, Rubisco loading control stained by Ponceau S. (B) mRNA abundance of the indicated genes in the inoculated areas was measured by qRT-PCR at 16 h post-inoculation and calculated relative to areas inoculated with the Xe strain carrying an empty vector. Values are means ± SE of three biological repeats. Asterisks indicate a significant difference (Student’s t test, p value < 0.05) relative to Xe containing an empty vector. (C) Leaves were infiltrated with bacterial suspensions (1 x 105 CFU/ml) of Xe strains carrying a vector either empty (EV) or for expression of XopAU-HA or XopAUK240A-HA, and bacterial growth was quantified at the indicated dpi. A box plot displays 25th, 50th (middle line) and 75th percentiles (n = 5). Asterisks indicate a significant difference (Mann-Whitney U test, p value <0.05) relative to Xe containing an empty vector. Experiments were repeated at least three times with similar results.
Fig 6.
Cell death caused by delivery of XopAU in pepper cells by Xanthomonas campestris pv. campestris.
Leaves of the pepper line ECW30R were syringe-infiltrated with a 10 mM MgCl2 mock solution (Mock) or with suspensions (1 x 107 CFU/ml) of Xe or Xcc strains containing a vector for expression of XopAU-HA and XopAUK240A-HA, or an empty vector (EV). (A) Photograph of an inoculated leaf at 3 days post-inoculation (dpi). (B) Electrolyte leakage at 2 dpi. (C) Leaves were syringe-infiltrated with bacterial suspensions (1 x 105) of Xe and Xcc strains as in (A) and bacterial growth was quantified at the indicated dpi. In (B) and (C), box plots display 25th, 50th (middle line) and 75th percentiles (in A, n = 7 or 9; in C, n = 6). An asterisk indicates a significant difference (Mann-Whitney U test, p value <0.05) relative to Xe containing an empty vector. Experiments were repeated three times with similar results.
Fig 7.
Silencing of MEK2 in N. benthamiana reduces XopAU-induced cell death.
N. benthamiana plants were infected with TRV, TRV:MEK2, TRV:MAP3Kα and TRV:MAP3Kε. In (A), (B), and (C), leaves of silenced plants were inoculated with Agrobacterium (OD600 = 0.02) carrying a vector either empty (EV) or for expression of His-XopAU from an estradiol-inducible system, and treated with 17β-estradiol 24 h later. (A) Photographs of inoculated areas at 36 h after 17β-estradiol application. (B) Electrolyte leakage at 24 h after 17β-estradiol application. (C) Total proteins were extracted at 12 h after 17β-estradiol application and samples were immunoblotted with α:P-MAPK antibodies. Rbs, Rubisco loading control stained by Ponceau S. In (D) and (E), leaves of silenced plants were inoculated with mock or suspensions (5 x 107 CFU/ml) of Xcc carrying a vector either empty (EV) or for expression of XopAU-HA. (D) Photographs of inoculated areas at 48 h after Xcc inoculation. (E) Electrolyte leakage at 24 h after Xcc inoculation. In (B) and (E), box plots display 25th, 50th (middle line) and 75th percentiles. (in B, n = 10; in E, n = 5 or 7). Asterisks indicate a significant difference (Mann-Whitney U test, p value <0.05) relative to TRV empty control. Experiments were repeated three times with similar results.
Fig 8.
Expression of MKK2K99R suppresses cell death mediated by XopAU.
N. benthamiana leaves were inoculated with Agrobacterium to co-express His-XopAU or an empty vector (EV) with MKK2-HA, MKK2K99R-HA or GFP. Expression was driven by an estradiol-inducible system and 17β-estradiol was applied 24 h after agro-infiltration. (A) Photograph taken at 48 h after 17β-estradiol application. (B) Electrolyte leakage at 24 h and 48 h after 17β-estradiol application. A box plot displays 25th, 50th (middle line) and 75th percentiles. (n = 5). Asterisks indicate a significant difference (Mann-Whitney U test, p value <0.05) relative to co-expression of His-XopAU with a GFP control. The experiment was repeated three times with similar results.
Fig 9.
Co-expression of XopAU and MKK2 inhibits yeast growth.
Yeast cultures containing the pGML10 vector either empty (EV1) or for expression of XopAU and XopAUK240A, and the pGMU10 vector either empty (EV2) or for expression of MKK2 and MKK2K99R were normalized to OD600 = 0.1, and serial dilutions were spotted onto selective media containing 2% glucose or 2% galactose and 1% raffinose. Plates were incubated at 30°C for 72 h (2% glucose) or 96 h (2% galactose and 1% raffinose) and photographed. The experiment was repeated three times with similar results.
Fig 10.
Physical interaction of XopAU with components of plant MAPK cascades in yeast and in planta.
(A) Yeast expressing the indicated combinations of bait and prey were spotted on either selective medium (-HWUL) or non-selective medium (-HWU) with or without the addition of X-gal. (B) The indicated combinations of fusions proteins were co-expressed in epidermal cells of N. benthamiana leaves via Agrobacterium, and luciferase activity was quantified as relative luciferase units (RLU) at 48 h post-infiltration. C-LUC and N-LUC indicate the luciferase N-terminal (N-LUC) or C-terminal (C-LUC) region, respectively. A box plot displays 25th, 50th (middle line) and 75th percentiles. (n = at least 6). Asterisks indicate a significant difference (Mann-Whitney U test, p value <0.05) relative to C-LUC empty and C-LUC-BTI9. The experiment was repeated three times with similar results.
Fig 11.
In vitro kinase assays testing phosphorylation of XopAU-interacting proteins by XopAU (A), and MKK2K99R by XopAUK240A (B). The indicated proteins were incubated in a kinase assay in the presence of [γ-32P]ATP, fractionated by SDS-PAGE, and exposed to autoradiography (upper panel) or stained by Coomassie (lower panel). Asterisks in (A) mark bands corresponding to proteins that were tested as XopAU substrates. The experiments were repeated three times with similar results.
Table 1.
XopAU-mediated phosphorylation of MKK2 in planta.