The hypersensitive necrosis response (HR) elicited by incompatible plant-pathogen interactions is thought to be a form of programmed cell death. Several of the features diagnostic for programmed cell death, such as nuclear condensation, DNA fragmentation and cytoplast shrinkage have been observed in plants cells undergoing HR [1].
Searches of sequenced plant genomes for plant orthologs of animal programmed cell death genes have identified only one gene that resembles its animal counterpart, the BAX INHIBITOR 1 gene, suggesting that components of the regulation and execution of programmed cell death differ substantially between animals and plants [2]. In spite of this conclusion, several observations suggest that plant and animal programmed cell death processes share some properties. Expression of the BAX pro-apoptotic factor in plants causes cell death and the plant BAX INHIBITOR 1 suppresses this cell death [3]. Inhibitors known to block the action of caspases in animals are effective at limiting HR in plants [4]. Recently, vacuolar processing enzyme gamma was identified as the functional equivalent of animal caspases [5,6]. In addition, BECLIN1, an ortholog of the yeast and animal autophagy genes ATG6/BECLIN1, was identified by the run-away cell death observed in beclin1-deficient mutants following pathogen attack. The ability of plant BECLIN1 to restrict cell death was dependent on several other autophagy-related genes providing another point of similarity between plant and animal programmed cell death [7]. Finally, sphingolipids have been implicated in cell death in both plants and animals. The fungal toxin fumonisin B1, which blocks ceramide biosynthesis in animals and elicits programmed cell death response, exerts a similar effect on plants [8]. Similarly, AAL toxin, a host-selective toxin produced by Alternaria alternata f. sp. lycopersici (a pathogen of tomato) causes cell death in both plants and animals and appears to target the same step in ceramide biosynthesis as fumonisin B1 [8,9]. In addition, the acd5 and acd11 mutants, which exhibit constitutive cell death, carry mutations in genes encoding a ceramide kinase and a sphingosine transfer protein, respectively [10,11].
These similarities are not sufficient to provide a complete understanding of programmed cell death or the HR in plants. Lesion mimic mutants, displaying spontaneous lesions, have been recovered in screens for mutants with deregulated cell death and have arisen in screens for mutants with altered disease resistance properties [1,12]. Among the cloned genes are those that resemble resistance genes (SSI1, SSI4) that appear to be constitutively activated. COP (copine, a Ca+2 binding and phospholipid binding protein), LSD1 (Zn-finger domain, putative transcription factor), and barley MLO (a negative regulator of defenses against powdery mildews) may be involved in the signaling of cell death. Also, mutations in several metabolic genes (DND1 [cyclic nucleotide gated channel 2], HLM1 [cyclic nucleotide gated channel 4], SSI2 (=FAB2) [stearoyl-ACP desaturase], LIN2 [coproporphyrinogen III oxidase], ACD2 [red chlorophyll catabolite reductase]) exhibit spontaneous lesions. Notable among these metabolic genes are the sphingolipid metabolism genes ACD5 and ACD11 mentioned above. In addition, mutations of genes encoding a number of novel proteins (ACD6 [ankyrin-repeat containing protein], SVN1 [GRAM domain containing membrane protein], and CPR5 [transmembrane protein]) lead to spontaneous lesioning phenotypes.
In addition to the lesion mimic mutants, a few mutants have been described that do not develop spontaneous lesions but rather display HR-like lesions only in response to a stimulus such as pathogen attack. enhanced disease resistant 1 (edr1)-edr3 are examples of such mutants [13-16]. edr1 and edr2, but not edr3, also show elevated defense responses (e.g., PR1 expression) following powdery mildew attack. These phenotypes were suppressed in mutants with defects in the salicylic acid (SA) signal transduction pathway (e.g., pad4, npr1, eds1) but not by those with defects in the ethylene/jasmonate pathway (i.e., ein2), suggesting that these mutants are hypersensitive to or have a lower threshold for responding to stress and activating the SA pathway. EDR1 encodes a CTR-like kinase, EDR2 a novel protein, and EDR3 a dynamin-like protein (DRP1E) [14,15,17]. The edr1 and edr2 mutants have a second phenotype that is SA-independent; they are hyper-sensitive to ethylene-induced senescence, implicating these two genes in the regulation of senescence as well as defense signaling [14,17]. The diverse nature of processes interrupted in these mutants suggests that much remains to be uncovered about the mechanisms controlling cell death in plants.
We initiated a screen for mutants that developed an exaggerated cell death response following inoculation with the powdery mildew fungus, Golovinomyces cichoracearum (=Erysiphe cichoracearum) as a means of identifying components of the HR programmed cell death. Lesion mimic mutants with spontaneous lesions were discarded from this screen to minimize the likelihood of recovering mutants with a metabolic dysfunction or that were compromised in the mechanisms protecting plants from the oxidative stress that arises during photosynthesis. These mutants were named mildew-induced lesions (mil) mutants and below we describe the characterization of the mil1 mutant and cloning of MIL1 gene. During the course of this work, EDR2 was cloned and as described below shown to be the gene compromised in the mil1 mutant [16]. For this reason, we have renamed mil1 edr2-6.
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