The LRR domain is required to target XopAC to the plasma membrane of <i>N</i><i>. benthamiana</i> epidermal cells.

<p>YFPv-XopAC (A, E and F) and mutant variants (B, YFPv-XopAC-H469A; C, YFPv-XopAC∆fic and D, G, YFPv-XopAC∆LRR) were expressed using <i>Agrobacterium</i>-mediated transient transformation and imaged in epidermal cells by confocal laser microscopy 48 hours after inoculation. (A) The plasma membrane localized RLK-CFP fusion (At4g23740) and the nucleo-scytoplasmic marker MIEL1 (At5g18650) were co-expressed with YFPv-XopAC (E, F) or YFPv-XopAC∆LRR (G) and used as controls. The merged pictures are shown (E, F and G). Scale bars = 25 µm. White arrowheads indicate nuclei (N), cytosol (Cyto) and cytoplasmic strands (CS).</p

<i>xopAC</i> can confer avirulence to <i>Pst</i> strain DC3000 and <i>Rs</i> strain GMI100 on <i>Arabidopsis</i> ecotype Col-0.

<p>Four-week-old Col-0 plants were inoculated. (A, B) Wild-type and <i>hrcV</i> (<i>hrp</i>^-) mutant of <i>Rs</i> GMI1000 or strain derivatives carrying <i>xopAC</i> (+<i>xopAC</i>) or <i>xopAC-H469A</i> (+<i>H469A</i>) were inoculated by root dipping. (A) Pictures were taken at 11 days post-inoculation. (B) Bacterial populations in the aerial parts of the plants were determined at 5 dpi and expressed as log of colony-forming units per gram of fresh weight (cfu/gfw). For each strain, three samples of three plants each were analysed. Two independent experiments were performed. Statistical groups were determined using a Wilcoxon test (<i>P</i><0.003) and indicated by different letters. (C, D) Leaves were infiltrated with wild-type <i>Pst</i> DC3000 or derivatives carrying pEDV6-xopAC (+<i>xopAC</i>) or pEDV6-xopAC-H469A (+<i>H469A</i>). (C) Bacterial suspensions of <i>Pst</i> at 2x10⁷ cfu/ml or 5x10⁵ cfu/ml were used and pictures were taken 3 days post-inoculation. (D) Bacterial suspensions at 5x10⁵ cfu/ml were infiltrated in leaves. <i>In planta</i> bacterial populations in the inoculated areas were determined 0 and 3 days post-inoculation and expressed as log (cfu/cm²). Standard deviations were calculated on two independent experiments with three samples of two leaf discs from different plants for each strain. Statistical groups were determined using a Wilcoxon test (<i>P</i><0.012) and indicated by different letters. (E, F) Leaves were inoculated by hand infiltration with wild-type <i>Xcc</i> strain 8004 and 8004∆<i>xopAC</i>. (E) Bacterial suspensions of <i>Xcc</i> at 10⁸ cfu/ml or 10⁵ cfu/ml were used and pictures were taken 4 days post-inoculation. (F) <i>Xcc</i> strains were infiltrated at a bacterial density of 10⁵ cfu/ml. <i>In planta</i> bacterial populations in the inoculated areas were determined 0, 3 and 5 days post-inoculation and expressed as log (cfu/cm²). One representative experiment out of three is shown. Standard deviations were calculated on at least 4 biological samples. For each experiment, three samples of two leaf discs from different plants were collected for each strain. Statistical groups were determined using a Wilcoxon test (<i>P</i><0.021) and indicated by different letters.</p

The RLCK genes <i>RIPK-PIX8</i> and <i>PBL2</i> are required for <i>xopAC</i>-mediated avirulence of <i>Xcc</i> strain 8004.

<p>(A,B) Boxplot representation of pathogenicity of strain 8004 on Col-0 mutants and transgenics inoculated by piercing the central vein of the leaves is shown: middle bar = median; box limit = upper and lower quartile; extremes = Min and Max values. Kas was used as a susceptible control. Mutants in genes coding for the RIPK-RIN4/RPM1 complex (A) and various RLCK (B) were tested. Disease indices were scored 8 days post-inoculation: 0-1 no symptoms; 1-2 weak chlorosis, 2-3 strong chlorosis; 3-4 necrosis. N=3. Each time, at least 4 plants were inoculated on at least 3 leaves. Statistical groups were determined using a Tukey HSD test (<i>P</i><0.001) and are indicated by different letters. (C) A bacterial suspension (10⁵ cfu/ml) of <i>Xcc</i> strain 8004 was inoculated by piercing leaves of Col-0 mutants and transgenics. <i>In planta</i> bacterial populations in the inoculated areas were determined 0 and 4 days post-inoculation and expressed as log of colony-forming units per square cm (cfu/cm²). Standard deviations were calculated on two independent experiments. For each experiment, three samples of two leaf discs from different plants were collected for each strain. Statistical groups identified using a Wilcoxon test (<i>P</i><0.05) are indicated by different letters.</p

XopAC interacts with several members of the RLCK VIIa subfamily.

<p>(A) Neighbour-joining consensus tree of the 45 full-length <i>Arabidopsis</i> RLCK proteins aligned using Geneious alignment with default settings. AT1G24030 protein kinase was used to root the tree. Shaded areas define the two subfamilies VIIa and VIIb of RLK. PIX-RLCK proteins identified in the yeast two-hybrid screen with XopAC-H469A are indicated in red. Published protein names are indicated when available. (B, C, D) Yeast two-hybrid interaction tests between XopAC or its mutant allele H469A as bait and full-length PIX1, PIX7, PIX8-RIPK, PBL2 or BIK1 as prey. P53 was used as specificity control for the prey. Ten-fold serial dilutions of yeast transformants were spotted from left to right on minimal medium (-WL) and minimal medium without histidine (-WLH) or histidine and adenine (-WLHA) which were used to visualize prey/bait interaction. Pictures were taken 4 days after spotting.</p

The LRR and fic domains of XopAC are required for XopAC-triggered immunity in <i>Arabidopsis</i> ecotype Col-0.

<p>A boxplot representation of pathogenicity of wild-type <i>Xcc</i> strain 8004 and <i>xopAC</i> mutants (∆<i>xopAC</i>, ∆<i>LRR</i>, ∆<i>fic</i>, <i>xopAC-H469A</i>) complemented or not with pCZ917-xopAC_A (+<i>xopAC</i>) is shown: middle bar = median; box limit = upper and lower quartile; extremes = Min and Max values. Bacteria were inoculated by piercing the leaf central vein and infection symptoms were scored 7 days post-inoculation. Disease index indicates: 0-1 no symptoms; 1-2 weak chlorosis, 2-3 strong chlorosis; 3-4 necrosis. N=3. Each time, at least 4 plants were inoculated on at least 3 leaves. Statistical groups were determined using a Tukey HSD test (<i>P</i><0.001) and are indicated by a letter.</p