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

PLOS ONE, Dec 2019

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 vad1-1 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 vad1-1. 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.

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

July An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens Mehdi Khafif☯ 0 1 Claudine Balague ☯ 0 1 Carine Huard-Chauveau 0 1 Dominique Roby 0 1 0 LIPM, Universit e de Toulouse, INRA, CNRS , Castanet-Tolosan , France 1 Editor: Hua Lu, University of Maryland Baltimore County , UNITED STATES 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 vad1-1 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 vad1-1. 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. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the French Laboratory of Excellence project "TULIP" (ANR-10LABX-41; ANR-11-IDEX-0002-02). Competing interests: The authors have declared that no competing interests exist. Introduction Programmed cell death (PCD) occurs in plants in a variety of cell types during development and to face environmental stresses such as pathogens or other stress signals [ 1, 2 ]. The Hypersensitive Response (HR) is one of the best characterized PCD, which is localized at the site of attempted pathogen invasion. In many plant-pathogen interactions, HR is triggered upon pathogen recognition. The plant immune system contains several layers [3]. The basal layer of defense, referred to as PAMP Triggered Immunity (PTI), is activated by the presence of pattern recognition receptors (PRRs) which recognize Microbe±or Pathogen-Associated Molecular Patterns (MAMPs or PAMPs). PRRs are plasma membrane receptors, that activate rapid and effective immune responses upon PAMP recognition [ 4 ]. However, pathogenic microorganisms can suppress PTI by producing effector proteins, thus leading to plant susceptibility to pathogen multiplication. However, a second and more robust layer of defense response, termed Effector Triggered Immunity (ETI) occurs upon recognition of pathogen effectors by a second type of plant immune receptors (R proteins). In many cases, ETI leads to the hypersensitive cell death, leading to the confinement of the pathogens at the attempted infection site. PCD is highly regulated at the genetic level as the control of this cell death programme is essential for the development of multicellular organisms. In plants, its fundamental role in response to pathogens has been revealed by the fact that many pathogens have evolved different strategies to suppress the HR. The molecular mechanisms leading to cell death after pathogen attack during ETI are not fully elucidated. However, some regulators or executioners of HR have been identified, such as BI-1 (BAX INHIBITOR 1, [ 5, 6 ], caspases and metacaspases [7] or MYB30 [ 8 ]. Characterization of mutants displaying spontaneous HR-like cell death on leaves, the so-called lesion mimic mutants (LMMs) has also been frequently reported, leading to the identification of genes encoding inhibitors of plant cell death, frequently associated with immunity [ 9, 10 ]. Demonstration of their implication in immunity control has been made in several cases, either by effector targeting [ 11, 12 ], by genetic interaction with immunity pathway components [ 13, 14, 15 ] or by suppressor identification [ 10 ]. In summary, numerous autoimmune or LMMs and their suppressors represent genes (potentially) involved in the control of the HR, and should be characterized more in depth [ 16 ]. We previously repor (...truncated)


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Mehdi Khafif, Claudine Balagué, Carine Huard-Chauveau, Dominique Roby. An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens, PLOS ONE, 2017, Volume 12, Issue 7, DOI: 10.1371/journal.pone.0179782