The edr2 mutants exhibit properties consistent with the assumption that EDR2 acts as a negative regulator of cell death ([16], this publication). The chlorosis and necrosis phenotypes do not develop spontaneously and do not develop in response to various abiotic stresses, such as wounding, heat stress, light stress, or drought stress. The chlorosis and necrosis were elicited only following inoculation with the pathogens G. cichoracearum, B. g. hordei or H. parasitica (Fig. 1D,1E, 2B; and [16]). These results confirm that EDR2 plays a role specifically in the cell death associated with plant-pathogen interactions and does not have a general role in cell death. These features distinguish the edr2 mutants from typical lesion mimic mutants such as the acd and lsd classes. In addition, the occurrence of chlorotic and necrotic tissue was restricted to inoculation sites and did not spread suggesting that EDR2 restricts the initiation of cell death rather than its spread (Fig. 1E).
Because both G. cichoracearum and H. parasitica are biotrophic pathogens, the chlorosis and necrosis that develop may be sufficient to account for the restricted growth of these pathogens in the edr2 mutants. However, the SA pathway, but not the ethylene/jasmonate pathway, appears to be somewhat deregulated in that PR1 transcript levels are elevated in edr2 mutants relative to wild type following elicitation by BTH or pathogen attack (Fig. 5; [16]). Furthermore, plants deficient in SA accumulation or signaling suppress both the development of chlorosis and necrosis as well as the disease resistance phenotypes of edr2 mutants (Fig. 1H,1I, 2E,2F; and [16]). Thus, it is also possible that SA-dependent defenses unrelated to cell death contribute to the disease resistance phenotype of edr2 mutants.
The SA signal transduction pathway is required for the HR elicited by incompatible plant-pathogen interactions. However, cell death is known to activate the SA signal transduction pathway in adjacent living tissue in a positive feedback loop that amplifies signal transduction via this defense pathway [12]. Thus, it is difficult to know whether EDR2 acts upstream of SA to limit SA activation of cell death or downstream of SA. PAD4 and EDS1 have homology to lipases and have been shown to be required for the accumulation of SA [20,21]. Given that EDR2 may bind lipid-like molecules via both its PH and START domains, EDR2 may have a direct or indirect inhibitory effect on PAD4 or EDS1 via a lipid-like intermediate. Candidates for this lipid-like intermediate could be a sphingolipid [10,11], phosphatidic acid [22-26] or oleic acid [27].
EDR2 encodes a novel protein with three predicted domains, a PH, a START and a DUF1336 domain. Three other predicted proteins with this domain structure occur in the Arabidopsis genome and three in the rice genome (XP_463792, NP_922009, ABB47745), but none have been assigned a function to date [28]. The DUF1336 domain appears to be plant-specific but 27 animal proteins contain PH and START domains including the human CERT (AAR26717), a splice variant of the Goodpasture antigen binding protein [28]. In the CERT protein, the PH domain binds PI-4-P as does the EDR2 PH domain [29]. In addition, the START domain of the CERT protein binds to ceramides. From these properties, Hanada et al. (2003) suggested that this protein acts to carry ceramides via a non-vesicle mediated transport mechanism from their site of synthesis in the endoplasmic reticulum to the Golgi where they are converted to syphingomyelin [29].
Plants also synthesize ceramide and more complex sphingolipids, some of which have been localized to detergent-resistant membrane domains [30-32]. Alterations in ceramides and/or sphingolipids or possibly the accumulation of their precursors stimulate cell death in plants and animals. The mechanism by which sphingolipids promote cell death is unknown and may be indirect via their impact on the functioning of cell death effectors found in lipid rafts such as ion channels [33]. It is tempting then to speculate that EDR2, like CERT, carries ceramides from the endoplasmic reticulum to another subcellular membrane, such as the plasma membrane or endosomes. Both membranes are labeled by EDR2:HA:GFP (Fig. 8). Presumably, the vesicle-mediated movement of ceramides among plant compartments is sufficient to support normal growth and development. If responses to pathogen attack demand additional ceramides or sphingolipids in a specific membrane, then non-vesicle-mediated transport via EDR2 may be required to supplement vesicle-mediated transport. This might explain why edr2 mutants do not constitutively exhibit lesions, as do acd5 and acd11. It is also possible that At5g45560, the closely related gene, is partially redundant to EDR2 but unable to meet extra demand in plants under pathogen attack.
Alternate models are possible. The function(s) of PH domains is not clear, but it is generally believed that they provide a way to selectively direct proteins to membranes [25]. However, the PH-domain can bind ligands other than phosphatidylinositols. For example, the PH-domains of the β-adrenergic receptor kinase and phospholipase C β bind to both a lipid and the Gβ,γ subunit of trimeric G-proteins [34,35]. Furthermore, it is conceivable that the PH-domain may interact co-operatively with the START-domain and that the concerted action of both domains influences the ligands bound to EDR2 and consequently EDR2 function, as has been observed with the insulin receptor substrate 1 [36]. It is equally possible that the DUF1336 domain plays a novel role in restricting cell death [16] and the PH and START domains serve to localize the EDR2 protein to the correct membrane following pathogen attack or during senescence. Determining the lipid or sterol molecule bound by the START domain would be an important step in unraveling the role of EDR2 in restricting cell death in plant-pathogen interactions.
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