An Atypical Kinase under Balancing Selection Confers Broad-Spectrum Disease Resistance in Arabidopsis

The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance in an agricultural context has led to the search for genes underlying quantitative resistance in plants. Such genes have been identified in only a few cases, all for fungal or nematode resistance, and encode diverse molecular functions. However, an understanding of the molecular mechanisms of quantitative resistance variation to other enemies and the associated evolutionary forces shaping this variation remain largely unknown. We report the identification, map-based cloning and functional validation of QRX3 (RKS1, Resistance related KinaSe 1), conferring broad-spectrum resistance to Xanthomonas campestris (Xc), a devastating worldwide bacterial vascular pathogen of crucifers. RKS1 encodes an atypical kinase that mediates a quantitative resistance mechanism in plants by restricting bacterial spread from the infection site. Nested Genome-Wide Association mapping revealed a major locus corresponding to an allelic series at RKS1 at the species level. An association between variation in resistance and RKS1 transcription was found using various transgenic lines as well as in natural accessions, suggesting that regulation of RKS1 expression is a major component of quantitative resistance to Xc. The co-existence of long lived RKS1 haplotypes in A. thaliana is shared with a variety of genes involved in pathogen recognition, suggesting common selective pressures. The identification of RKS1 constitutes a starting point for deciphering the mechanisms underlying broad spectrum quantitative disease resistance that is effective against a devastating and vascular crop pathogen. Because putative RKS1 orthologous have been found in other Brassica species, RKS1 provides an exciting opportunity for plant breeders to improve resistance to black rot in crops.</p

Localization and function analysis of Type III effector RipG7 of Ralstonia solanacearum

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An Atypical Kinase under Balancing Selection Confers Broad-Spectrum Disease Resistance in Arabidopsis

International audienceThe failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance in an agricultural context has led to the search for genes underlying quantitative resistance in plants. Such genes have been identified in only a few cases, all for fungal or nematode resistance, and encode diverse molecular functions. However, an understanding of the molecular mechanisms of quantitative resistance variation to other enemies and the associated evolutionary forces shaping this variation remain largely unknown. We report the identification, map-based cloning and functional validation of QRX3 (RKS1, Resistance related KinaSe 1), conferring broad-spectrum resistance to Xanthomonas campestris (Xc), a devastating worldwide bacterial vascular pathogen of crucifers. RKS1 encodes an atypical kinase that mediates a quantitative resistance mechanism in plants by restricting bacterial spread from the infection site. Nested Genome-Wide Association mapping revealed a major locus corresponding to an allelic series at RKS1 at the species level. An association between variation in resistance and RKS1 transcription was found using various transgenic lines as well as in natural accessions, suggesting that regulation of RKS1 expression is a major component of quantitative resistance to Xc. The co-existence of long lived RKS1 haplotypes in A. thaliana is shared with a variety of genes involved in pathogen recognition, suggesting common selective pressures. The identification of RKS1 constitutes a starting point for deciphering the mechanisms underlying broad spectrum quantitative disease resistance that is effective against a devastating and vascular crop pathogen. Because putative RKS1 orthologous have been found in other Brassica species, RKS1 provides an exciting opportunity for plant breeders to improve resistance to black rot in crops

Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum

The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts

<i>RKS1</i> allelic forms and expression in susceptible and resistant accessions of <i>Arabidopsis thaliana</i>.

<p>(A) Schematic representation of resistant (Col-0) and susceptible (Kas-1) <i>RKS1</i> allele polymorphisms. Sequence changes in both alleles are indicated. (B) Schematic representation of the most frequent 5′ and 3′ ends of <i>RKS1</i> transcripts found by 5′ and 3′ RACE experiments in resistant (Col-0, green) and susceptible (Kas-1, red) accessions. (C) <i>RKS1</i> gene expression evaluated by Q-RT-PCR in germinating seeds and in leaves, healthy or inoculated with <i>Xcc568</i>, from the resistant accession (Col-0, green) and the susceptible accession (Kas-1, red). Different primers are used to evaluate long (L) and long+short (L+S) transcripts (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766.s018" target="_blank">Table S2</a>) A.U.: arbitrary units. (D) Correlation between <i>RKS1</i> gene expression after infection with <i>Xcc568</i> and resistance phenotype. The dashed line indicates an exponentially decreasing function fitted on the median values of the 13 types of genetic line. Numbers in brackets indicate the number of representatives of each type of transgenic line.</p

<i>RKS1</i> confers resistance to multiple strains and races and pathovars of <i>Xcc</i>.

<p>Time course evaluation of disease index in lines differing only by the presence of <i>RKS1</i> gene (<i>rks1-1</i> mutant (blue), complemented <i>rks1-1</i> mutant (purple) and the parental lines Col-0 (green) and Kas-1 (red) after inoculation with different strains of (A) <i>Xcc</i> belonging to races as defined by Vicente <i>et al.. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766-Vicente2" target="_blank">[49]</a> and Fargier <i>et al.. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003766#pgen.1003766-Fargier2" target="_blank">[50]</a>, and of (B) <i>Xc</i> pathovars <i>raphani</i> (<i>Xcr</i>), <i>armoriaceae</i> (<i>Xca</i>), <i>vesicatoria</i> (<i>Xcv</i>) and <i>incanae</i> (<i>Xci</i>).</p

The genetics of <i>Xcc568</i> quantitative resistance at the species level identified by nested GWA mapping.

<p>(A) Violin plots (i.e. box-and-whisker plot overlaid with a kernel density plot) of phenotypic variation of our disease index. Whole-genome scan of 214,051 SNPs for association with disease index at 10 dpi across (B) 381 accessions, (C) within the allelic group SNP-3-21386192-T and (D) within the allelic group SNP-3-21386192-C.</p

Genetic evidence that <i>RKS1</i> is causal for <i>QRX3</i> QTL.

<p>Disease symptoms were observed on leaves of wild-type plants, mutants, HIF lines or lines complemented with the <i>RKS1</i> gene, at 7 (C) or 10 (A and B) days post-inoculation. Time course evaluation of our disease index was performed after inoculation with <i>Xcc568</i> under the same conditions. (A) Mutant complementation (lines #9, #E9, #F9). (B) amiRNA silencing (lines #23 and #24). (C) HIF line complementation (lines #105 and #106 for the susceptible HIF (HIF685), lines #107 and #110 for the resistant HIF (HIF1011)). Means and standard errors were calculated for 16–60 plants (4–9 independent experiments). (D) Bacterial growth measurement (colony forming unit (CFU)/cm² expressed in a log10 scale) in leaves of lines differing only by the presence of <i>RKS1</i> gene (wild type (Col-0), <i>rks1-1</i> mutant, and the complemented mutant line (#9)). The susceptible accession Kas-1 has been included as a positive control. Bacterial growth has been measured 0 (grey bars) and 7 (black bars) days after inoculation with <i>Xcc</i> strain 568. Data were collected from two independent experiments, each timepoint corresponds to 6 independent measurements, each on 3–5 individual plants (four leaves/plant).</p

Identification and mapping of the major QTL, <i>QRX3</i>, for resistance to <i>Xcc</i>.

<p>(A and B) Phenotype of susceptible (Kas-1) and resistant (Col-0) accessions (Col-5 and Col-0 (used here) show similar phenotypes) : (A) symptoms 7 days post-inoculation (dpi) and (B) bacterial invasion of leaf tissue using an <i>Xcc568</i> reporter strain that carries the <i>Photorhabdus luminescens</i> lux operon. (C) QTL maps of resistance to <i>Xcc</i> in the Col-5 x Kas-1 recombinant inbred line population at four inoculation times: yellow, 3 dpi; orange, 5 dpi; red, 7 dpi and purple, 10 dpi. The horizontal dotted line represents the significance threshold for the LOD score (average = 2.50). (D to F) Map-based isolation of the <i>QRX3</i> locus. (D) Genetic map of chromosome III is shown between markers <i>T04109</i> and <i>MS005</i> with the defined target interval for <i>QRX3</i> (in red). (E) A number of additional markers and recombinant lines were used to reduce the <i>QRX3</i> locus to a 44.9 kb region between the markers <i>3-57670</i> and <i>3-57810</i>. (F) The corresponding physical interval contains 17 open reading frames (ORFs). Genes are represented by arrows. The black arrows correspond to a cluster of putative kinase genes, the red arrow corresponds to <i>RKS1</i>.</p

Phenotypic analysis of insertional mutants corresponding to genes of the <i>QRX3</i> locus.

<p>(A) Structure of the kinase cluster contained within the <i>QRX3</i> locus and positions of the insertional mutations are indicated with vertical lines. (B) Disease symptoms were observed on leaves of mutant and wild-type plants, 10 days post-inoculation with a bacterial suspension adjusted to 2×10⁸ cfu/mL. (C) Time course evaluation of disease index after inoculation with <i>Xcc568</i> under the same conditions. Means and standard errors were calculated from 3–8 plants.</p