Quantitative disease resistance to the bacterial pathogen Xanthomonas campestris involves an Arabidopsis immune receptor pair and a gene of unknown function

Abstract

Although quantitative disease resistance (QDR) is a durable and broad-spectrum form of resistance in plants, the identification of the genes underlying QDR is still in its infancy. RKS1 (Resistance related KinaSe1) has been reported recently to confer QDR in Arabidopsis thaliana to most but not all races of the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc). We therefore explored the genetic bases of QDR in A. thaliana to diverse races of X. campestris (Xc). A nested genome-wide association mapping approach was used to finely map the genomic regions associated with QDR to Xcc12824 (race 2) and XccCFBP6943 (race 6). To identify the gene(s) implicated in QDR, insertional mutants (T-DNA) were selected for the candidate genes and phenotyped in response to Xc. We identified two major QTLs that confer resistance specifically to Xcc12824 and XccCFBP6943. Although QDR to Xcc12824 is conferred by At5g22540 encoding for a protein of unknown function, QDR to XccCFBP6943 involves the well-known immune receptor pair RRS1/RPS4. In addition to RKS1, this study reveals that three genes are involved in resistance to Xc with strikingly different ranges of specificity, suggesting that QDR to Xc involves a complex network integrating multiple response pathways triggered by distinct pathogen molecular determinants.

Keywords: Arabidopsis thaliana; GWA mapping; RRS1/RPS4; Xanthomonas campestris; mutant analysis; quantitative disease resistance.

Figure 1

The genetics of quantitative disease resistance to the strain X cc12824 identified by nested genome‐wide association (GWA) mapping. (a) Violin plots (i.e. box‐and‐whisker plot overlaid with a kernel density plot) of the phenotypic variation of our disease index. Whole‐genome scan of 214 051 single‐nucleotide polymorphisms (SNPs) for association with disease index at 10 days post‐inoculation (dpi) across (b) 380 accessions, (c) within the allelic group SNP‐5‐7481857‐C and (d) within the allelic group SNP‐5‐7481857‐A.

Figure 2

The genetics of quantitative disease resistance to the strain XccCFBP 6943 identified by nested genome‐wide association (GWA) mapping. (a) Violin plots (i.e. box‐and‐whisker plot overlaid with a kernel density plot) of the phenotypic variation of our disease index. Whole‐genome scan of 214 051 single‐nucleotide polymorphisms (SNPs) for association with disease index at 10 days post‐inoculation (dpi) across (b) 171 accessions, (c) within the allelic group SNP‐5‐18325565‐G and (d) within the allelic group SNP‐5‐18325565‐A.

Figure 3

The mutant mut540 (affected in the A t5g22540 gene) is susceptible to X cc12824. (a) Disease symptoms were observed at 7 and 10 days post‐inoculation (dpi) on leaves of wild‐type and mut540 mutant (SALK  113262C) plants inoculated with X cc12824 (race 2 defined by Fargier et al., 2011 and Vicente et al., 2001). (b) Time course evaluation of disease index was performed in the mut540 line (blue) relative to the susceptible accession Kas‐1 (red) and the resistant accession Col‐0 (green), after inoculation with X cc12824. Means and standard errors were calculated from 8–15 plants (three independent experiments). (c) Bacterial growth measurement [colony‐forming units (CFU)/cm² expressed on a log₁₀ scale] in leaves of the mut540 line relative to the wild‐type accession Col‐0. The susceptible accession Kas‐1 was included as a positive control. Bacterial growth was measured 0 dpi (grey bars) and 7 dpi (black bars) with X cc12824. Data were collected from two independent experiments; each time point corresponds to six independent measurements, each on three to five individual plants (four leaves/plant). *Statistically significant difference using Kruskal–Wallis test (P < 0.05).

Figure 4

The mutants rrs1‐1, rps4‐21, rps4‐21 rrs1‐1 and eds1‐1 are susceptible to XccCFBP 6943. (a) Disease symptoms were observed at 7 days post‐inoculation (dpi) on leaves of wild‐type plants (Ws‐0 and Col‐0) and mutants inoculated with the XccCFBP 6943 strain (race 6 defined by Fargier et al., 2011 and Vicente et al., 2001). (b) Time course evaluation of the disease index was performed in the mutants rrs1‐1 (blue cross), rps4‐21 (green circle), eds1‐1 (orange circle), rps4‐21 rrs1‐1 double mutant (rps4 rrs1, purple cross), the parental line Ws‐0 (blue square) and Col‐0 (green triangle) after inoculation with XccCFBP 6943. Means and standard errors were calculated from 10 plants (two independent experiments). (c) Bacterial growth measurement [colony‐forming units (CFU)/cm² expressed on a log₁₀ scale] in leaves of the mutants rrs1‐1, rsp4‐21, rps4‐21 rrs‐1 or eds1‐1 and the parental line Ws‐0. The susceptible accession Col‐0 was included as a positive control. Bacterial growth was measured 0 dpi (grey bars) and 7 dpi (black bars) with XccCFBP 6943. Data were collected from four independent experiments; each time point corresponds to six independent measurements, each on three to five individual plants (four leaves/plant). *Statistically significant difference using Kruskal–Wallis test (P < 0.05).

Figure 5

Quantitative resistance conferred by the genes RKS 1, A t5g22540 and RPS 4/ RRS 1 to the nine races of X anthomonas campestris pv. campestris (X cc) (as defined by Fargier et al., 2011 and Vicente et al., 2001), as well as to X anthomonas campestris pv. raphani (X cr). Top: ratio of disease index (DI) at 7 days post‐inoculation of mutant against disease index (DI) of corresponding wild‐type. Data correspond to one to three independent experiments, each on three to five individual plants (four leaves/plant). For each X . campestris strain, the ratios of rks1‐1/Col‐0, mut540/Col‐0 and rps4‐21 rrs1‐1/Ws‐0 are represented by red, blue and green circles, respectively. Filled circles indicate significant differences between a mutant line and its corresponding wild‐type after pairwise comparisons using a Tukey honestly significant difference (HSD) test (see Tables 1 and S3). The strains correspond to X cc1869 (race 1), X cc12824 (race 2), X cc568 (race 3), X cc147 (race 4), X cc1712 (race 5), X cc6943 (race 6), X cc4953 (race 7), X cc1124 (race 8), X cc8004 (race 9) and X cr756C (Xcr). Bottom: summary of broad‐spectrum resistance conferred by the genes RKS 1, A t5g22540 and RPS 4/ RRS 1. Filled squares indicate significant differences between a mutant line and its corresponding wild‐type after pairwise comparisons using a Tukey HSD test (see Tables 1 and S3).