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

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

<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

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

Putative pathogenicity elements identified in the genome of <i>Xam</i> CIO151.

1<p>Number indicates CDS identified by key word searches in iANT.</p

Phylogeny of conserved effectors in the genus <i>Xanthomonas</i>.

<p>Phylogenetic tree of concatenated conserved effector protein sequences of AvrBs2, XopK, XopL, XopN, XopQ XopR families and the Hpa1 protein, obtained with a Bayesian approach. Numbers on branches indicate Bayesian support values. Length of branches indicates the number of amino acid substitutions per site.</p

Circular representation of the genome sequence of <i>Xam</i> CIO151.

<p>From outside to inside: first circle in blue indicates CDS predicted in the positive strands for the scaffolds classified as probable chromosomal regions. Second circle in red indicates the CDS predicted in the negative strand. Red spots in the black third circle indicate the region identified with atypical nucleotide composition. The fourth circle indicates the deviation pattern from the average G+C content. Inner circle shows GC skew values, positive values are shown in purple and negative values are shown in orange. Numbers correspond to scaffold IDs.</p

Comparison of the genomic structure of <i>Xam</i> CIO151 with that of closely related members from the genus <i>Xanthomonas</i>.

<p>Scaffolds of <i>Xam</i> CIO151 were ordered based on the alignment with the complete genome sequence of <i>X. euvesicatoria</i>, <b><i>Xeu</i></b>, and then genome comparisons were performed using MUMmer (<b>A</b>). Alignment of ordered scaffolds of <i>Xam</i> CIO151 with the complete genome sequences of <i>X. axonopodis</i> pv. citri str. 306, <b><i>Xac</i></b> (<b>B</b>); <i>X. campestris</i> pv. campestris str. 8004, <b><i>Xcc</i></b> (<b>C</b>); <i>X. albilineans</i>, <b><i>Xal</i></b> (<b>D</b>); and <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> PXO99^A, <b><i>Xoo</i></b> (<b>E</b>) chromosomes. Scaffolds classified as parts of the chromosome of <i>Xam</i> CIO151 are shown in the y-axis. Red dots represent conserved segments while blue dots represent inverted regions.</p

Characteristics of VNTR loci for 65 <i>Xam</i> draft genome sequences.

1<p>: Repeat unit sizes are given in bp.</p>2 and 3<p>: Minimal and maximal numbers of repeats (only those in integer numbers) are given.</p>4<p>Number of samples with a complete VNTR locus in the draft genome sequence is given.</p>5<p>Number of different VNTR patterns (haplotypes) is given.</p>6<p>Hunter-Gaston discriminatory index (HGDI) scores are given.</p