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. 2013 Aug 9;8(8):e73469.
doi: 10.1371/journal.pone.0073469. eCollection 2013.

xopAC-triggered immunity against Xanthomonas depends on Arabidopsis receptor-like cytoplasmic kinase genes PBL2 and RIPK

Endrick Guy  1 , Martine Lautier, Matthieu Chabannes, Brice Roux, Emmanuelle Lauber, Matthieu Arlat, Laurent D Noël
Affiliations

xopAC-triggered immunity against Xanthomonas depends on Arabidopsis receptor-like cytoplasmic kinase genes PBL2 and RIPK

Endrick Guy et al. PLoS One. .

Abstract

Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The LRR and fic domains of XopAC are required for XopAC-triggered immunity in Arabidopsis ecotype Col-0.
A boxplot representation of pathogenicity of wild-type Xcc strain 8004 and xopAC mutants (∆xopAC, ∆LRR, ∆fic, xopAC-H469A) complemented or not with pCZ917-xopACA (+xopAC) 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 (P<0.001) and are indicated by a letter.
Figure 2
Figure 2. xopAC can confer avirulence to Pst strain DC3000 and Rs strain GMI100 on Arabidopsis ecotype Col-0.
Four-week-old Col-0 plants were inoculated. (A, B) Wild-type and hrcV (hrp -) mutant of Rs GMI1000 or strain derivatives carrying xopAC (+xopAC) or xopAC-H469A (+H469A) 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 (P<0.003) and indicated by different letters. (C, D) Leaves were infiltrated with wild-type Pst DC3000 or derivatives carrying pEDV6-xopAC (+xopAC) or pEDV6-xopAC-H469A (+H469A). (C) Bacterial suspensions of Pst 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. In planta 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 (P<0.012) and indicated by different letters. (E, F) Leaves were inoculated by hand infiltration with wild-type Xcc strain 8004 and 8004∆xopAC. (E) Bacterial suspensions of Xcc at 108 cfu/ml or 105 cfu/ml were used and pictures were taken 4 days post-inoculation. (F) Xcc strains were infiltrated at a bacterial density of 105 cfu/ml. In planta 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 (P<0.021) and indicated by different letters.
Figure 3
Figure 3. The LRR domain is required to target XopAC to the plasma membrane of N . benthamiana epidermal cells.
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 Agrobacterium -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).
Figure 4
Figure 4. XopAC interacts with several members of the RLCK VIIa subfamily.
(A) Neighbour-joining consensus tree of the 45 full-length Arabidopsis 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.
Figure 5
Figure 5. The RLCK genes RIPK-PIX8 and PBL2 are required for xopAC-mediated avirulence of Xcc strain 8004.
(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 (P<0.001) and are indicated by different letters. (C) A bacterial suspension (105 cfu/ml) of Xcc strain 8004 was inoculated by piercing leaves of Col-0 mutants and transgenics. In planta 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 (P<0.05) are indicated by different letters.
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