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

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

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

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

Overexpression of <i>VAD1</i>, but not <i>VAD1</i> lacking the VASt domain, leads to enhanced susceptibility in <i>vad1-1</i> in response to the virulent strain <i>Pst</i> DC3000.

<p>(A) Four-week old wild type and 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 typical chlorosis 72h after inoculation with suspensions (5x10⁶ colony-forming units (CFU/mL) of <i>Pst</i> strain DC3000. <i>vad1-1</i> plants and transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct do not show (or very faint) symptoms. (B) Growth of <i>Pst</i> DC3000 in the different lines indicated. Leaves of four-week old plants were inoculated with a bacterial suspension (2x10⁵ CFU/mL) of <i>Pst</i> strain DC3000 and bacterial growth was measured 0 (grey bars) and 3 (black bars) days after inoculation. Data were collected from four independent experiments with five individual plants (four leaves/plant) per point. Statistical differences using Kruskal and Wallis one-way analysis of variance followed by the Conover's-test for multiple comparisons analysis of variance (P value < 0.05) are indicated by letters.</p

Structure-function analysis of <i>VAD1</i> effects on HR cell death and defense in response to bacterial avirulence effectors.

<p>(A) Schematic representation of VAD1 and constructs. GRAM (Glucosyltransferases, Rab-like GTPase Activators, Myotubularins) domain; VASt (VAD1 analog of START) domain; TM, transmembrane helix; CC: Coiled-coil. Residue number corresponds to amino acid position of VAD1 domains. (B) Observation of HR induced by AvrRpt2 after agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves alone or co-expressed with the constructs <i>35S</i>::<i>VAD1</i>, <i>35S</i>::<i>ΔGRAM</i>::<i>VAD1</i>, <i>35S</i>::<i>ΔVASt</i>::<i>VAD1</i> or <i>35S</i>::<i>SYMREM1</i>. Observations were made 24h post-infiltration. (C) Quantification of cell death by measuring electrolyte leakage 24h after agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves with the indicated strains (OD 0.3). Data are expressed relative to AvrRpt2 data at 1 h after sampling. Statistically significant differences were determined using Kruskal and Wallis one-way analysis of variance (letters indicate P < 0.05). (D and E) Expression analysis of the <i>PR1a</i> defense gene and <i>HSR203J</i> gene in <i>N</i>. <i>benthamiana</i> leaves after agroinfiltration with the indicated strains (OD 0.3) Data are presented as means ± SE (n ≥ 12), and error bars represent standard error. Letters indicate a significant difference between tested construct and control at P<0.05 by Kruskal and Wallis one-way analysis of variance test.</p

Robustness of plant quantitative disease resistance is provided by a decentralized immune network

International audienceQuantitative disease resistance (QDR) represents the predominant form of resistance in natural populations and crops. Surprisingly, very limited information exists on the biomolecular network of the signaling machineries underlying this form of plant immunity. This lack of information may result from its complex and quantitative nature. Here, we used an integrative approach including geno-mics, network reconstruction, and mutational analysis to identify and validate molecular networks that control QDR in Arabidopsis thaliana in response to the bacterial pathogen Xanthomonas cam-pestris. To tackle this challenge, we first performed a transcrip-tomic analysis focused on the early stages of infection and using transgenic lines deregulated for the expression of RKS1, a gene underlying a QTL conferring quantitative and broad-spectrum re-sistance to X. campestris. RKS1-dependent gene expression was shown to involve multiple cellular activities (signaling, transport, and metabolism processes), mainly distinct from effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses already characterized in A. thaliana. Protein-protein interaction network reconstitution then revealed a highly interconnected and distributed RKS1-dependent network, organized in five gene modules. Finally, knockout mutants for 41 genes belonging to the different functional modules of the network revealed that 76% of the genes and all gene modules par-ticipate partially in RKS1-mediated resistance. However, these func-tional modules exhibit differential robustness to genetic mutations, indicating that, within the decentralized structure of the QDR net-work, some modules are more resilient than others. In conclusion, our work sheds light on the complexity of QDR and provides com-prehensive understanding of a QDR immune network