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

An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens

Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid -binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant vadl-1 has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response -like lesions along the vascular system, associated with defense gene expression and increased resistance to Pseudomonas strains. To test the potential of ectopic expression of VAD1 to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure -function analysis of VAD1 by transient over -expression in Nicotiana benthamiana and by complementation of the mutant vadl-1. We found that (i) overexpression of VAD1 controls negatively the HR cell death and defense expression either transiently in Nicotiana benthamania or in Arabidopsis plants in response to avirulent strains of Pseudomonas syringae, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD

Ethylene is one of the key elements for cell death and defense response control in the Arabidopsis lesion mimic mutant vad1

Pas de clé UTAlthough ethylene is involved in the complex cross talk of signaling pathways regulating plant defense responses to microbial attack, its functions remain to be elucidated. The lesion mimic mutant vad1-1 (for vascular associated death), which exhibits the light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, is a good model for studying the role of ethylene in programmed cell death and defense. Here, we demonstrate that expression of genes associated with ethylene synthesis and signaling is enhanced in vad1-1 under lesion-promoting conditions and after plant-pathogen interaction. Analyses of the progeny from crosses between vad1-1 plants and either 35SERF1 transgenic plants or ein2-1, ein3-1, ein4-1, ctr1-1, or eto2-1 mutants revealed that the vad1-1 cell death and defense phenotypes are dependent on ethylene biosynthesis and signaling. In contrast, whereas vad1-1-dependent increased resistance was abolished by ein2, ein3, and ein4 mutations, positive regulation of ethylene biosynthesis (eto2-1) or ethylene responses (35SERF1) did not exacerbate this phenotype. In addition, VAD1 expression in response to a hypersensitive response-inducing bacterial pathogen is dependent on ethylene perception and signaling. These results, together with previous data, suggest that VAD1 could act as an integrative node in hormonal signaling, with ethylene acting in concert with salicylic acid as a positive regulator of cell death propagation

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

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 relatedKinaSe1) 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

The genetic architecture of the adaptive potential of Arabidopsis thaliana in response to Pseudomonas syringae strains isolated from south-west France

International audiencePhytopathogens are a threat for global food production and security. Emergence or re-emergence of plant pathogens is highly dependent on the environmental conditions affecting pathogen spread and survival. Under climate change, a geographic expansion of pathogen distribution poleward has been observed, potentially resulting in disease outbreaks on crops and wild plants. Therefore, estimating the adaptive potential of plants to novel epidemics and describing the underlying genetic architecture is a primary need to propose agricultural management strategies reducing pathogen outbreaks and to breed novel plant cultivars adapted to pathogens that might spread under climate change. To address this challenge, we inoculated Pseudomonas syringae strains isolated from Arabidopsis thaliana populations from south-west of France on the highly genetically polymorphic TOU-A A. thaliana population from north-east France. While no adaptive potential was identified in response to most P. syringae strains, the TOU-A population displayed a variable disease response to the JACO-CL strain belonging to the P. syringae phylogroup 7 (PG7). This strain carried a reduced type III secretion system (T3SS) characteristic of the PG7 as well as flexible genomic traits and potential novel effectors. Genome-wide association mapping on 192 TOU-A accessions revealed a polygenic architecture of disease response to JACO-CL. The main quantitative trait locus (QTL) region encompasses two R genes and the AT5G18310 gene encoding ubiquitin hydrolase, a target of the AvrRpt2 P. syringae effector. Altogether, our results pave the way for a better understanding of the genetic and molecular basis of the adaptive potential in an ecologically relevant A. thaliana-P. syringae pathosystem

An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens

<div><p>Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid-binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant <i>vad1-1</i> has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, associated with defense gene expression and increased resistance to <i>Pseudomonas</i> strains. To test the potential of ectopic expression of <i>VAD1</i> to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure-function analysis of VAD1 by transient over-expression in <i>Nicotiana benthamiana</i> and by complementation of the mutant <i>vad1-1</i>. We found that (i) overexpression of <i>VAD1</i> controls negatively the HR cell death and defense expression either transiently in <i>Nicotiana benthamania</i> or in Arabidopsis plants in response to avirulent strains of <i>Pseudomonas syringae</i>, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.</p></div

Overexpression of <i>VAD1</i>, but not <i>VAD1</i> lacking the VASt domain, leads to suppression of cell death and defense phenotypes in <i>vad1-1</i> during plant development and in response to bacterial avirulent pathogens.

<p>(A) Four-week old <i>vad1-1</i> plants and transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct show typical cell death lesions (marked by white arrows), while transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>VAD1</i> construct or the <i>35S</i>::<i>RFP</i>::<i>ΔGRAM</i> construct show a wild type phenotype. (B-C) Five-week old plants have been inoculated with suspensions (2.10⁶ colony-forming units (CFU/mL) of <i>Pst</i> strain DC3000 expressing AvrRpm1. Wild type and transgenic <i>35S</i>::<i>RFP</i>::<i>VAD1</i> and <i>35S</i>::<i>RFP</i>::<i>ΔGRAM</i> plants showed typical HR lesions, while <i>vad1-1</i> and transgenic plants containing the construct <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct showed run away cell death. These phenotypes were observed 3 days post-inoculation (B) or 6 days post-inoculation (C). (D) Quantification of cell death by measuring electrolyte leakage 24 (grey bars) and 48h (black bars) after infiltration of leaves with <i>Pst</i> strain DC3000 AvrRpm1 (2x10⁷ colony-forming units (CFU/mL). Data are expressed relative to WS-4 1h after sampling. One transgenic line is shown per construct, out of 2–3 analyzed with similar results. Statistically significant differences were determined using Kruskal and Wallis one-way analysis of variance followed by nonparametric multiple comparison (* indicates P < 0.05).</p

VAD1 localizes in multiple subcellular compartments and a VASt domain deletion excludes VAD1 from the nucleus.

<p>(A) Transient co-expression by agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves of <i>35S</i>::<i>RFP</i>::<i>VAD1</i> construct (left panels), with subcellular markers (central panels, ERD2–GFP for the Golgi, γ-TIP–GFP for the tonoplast, GFP for the cytoplasm, MYB30-GFP for the nucleus, PMA4–GFP for the plasma membrane). Co-localization of VAD1 with the different subcellular markers is shown in the merge panel (right). Confocal images were observed two days after agroinfiltration. Scale bars = 20μM. (B) Transient co-expression by agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves of <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct (left panels), with subcellular markers (central panels, ERD2–GFP labels the Golgi, γ-TIP–GFP labels the tonoplast, GFP labels the cytoplasm, MYB30-GFP labels the nucleus, PMA4–GFP labels the plasma membrane (Plasma m.). Co-localization of VAD1 with the different subcellular markers is shown in the merge panel (right). Localization of <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> is not observed in the nucleus. Confocal images were observed two days after agroinfiltration. Bars = 20 μM.</p

Percentage of leaves with lesions in the different plant lines, at several times (17, 21, 24 and 28 days post-transplanting) of plant development, under normal growth conditions.

<p>Percentage of leaves with lesions in the different plant lines, at several times (17, 21, 24 and 28 days post-transplanting) of plant development, under normal growth conditions.</p