Molecular Components for Cell Death Control during Plant Pathogen Interactions

In animal apoptosis, a central switch involving three classes of proteins is now accepted as the key regulator of cell death in diverse species (Raff, 1998). The enzymatic component of this tripartite switch is a specialized class of cysteine proteases called caspases, the name of which derives from their specificity of cleaving after an asparatic acid at the P1 position of their substrate recognition sites. When activated, these proteases begin a proteolytic cascade that irreversibly commits the cell to undergo cell death. The activity of caspases is under stringent control by paired pro-apoptotic and pro-survival signals through the positive regulator called CED4/APAF-1 and the negative regulator BCL2 and its related proteins (BLPs). CED4/APAF-1 proteins apparently activate caspases through proteinprotein interactions while BLPs can regulate caspases through multiple mechanisms (Raff, 1998). In mammals, a large family of BLPs has been shown to control PCD and is comprised of genes showing pro-apoptotic (i.e. Bax, Bak and Bid) as well as pro-survival (i.e. Bcl2 and Bcl-xL) activities. No BLPhomologue has been detected in plants yet. However, two recent studies involving the ectopic expression of BLPs in plants suggest the existence of similar regulatory mechanisms in the two kingdoms. In transgenic tobacco plants expressing Bcl-xL or Ced-9, HR cell death as well as cell death induced by paraquat treatment or UV-B irradiation is delayed (Mitsuhara et al., 1999). Lacomme and Santa-Cruz (1999) found that Bax expression activates cell death in plants and that its localization in mitochondria is required. A similar result has been observed also in yeast in which no BLP has been found in the sequenced genome. In animals, BLPs are known to activate or repress the leakage of cytochrome c from the mitochondria, a crucial step in the cell death signaling cascade, but BLPs can also regulate caspase activity directly. The analogy between the phenotypes induced by Bax expression in plants and in yeasts, and the required localization of Bax in the mitochondria in both of these cases are in favor of a crucial role for the mitochondria in Bax-induced cell death (Lam et al., 1999). Besides the BLPs, caspases have been shown to play a crucial role in the final cascade leading to cell execution in animals (Raff, 1998). In plants, specific peptide inhibitors of caspases have been shown to abolish HR cell death during the incompatible interaction between tobacco and Pseudomonas syringae pv. phaseolicola (del Pozo and Lam, 1998). Furthermore, caspase activity has been detected during the early stages of TMV-induced HR cell death. These data raised for the first time the possibility that caspase-like proteins may play a regulatory role during cell death in plants. Other proteases may also be involved in HR cell death regulation as well. Cell death induced by a xylanase from Trichoderma viride in tobacco cells can be abolished by inhibitors of serine proteases (Yano et al., 1999). In soybean cells, cysteine protease activity is activated during H2O2-induced cell death and the expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibits both protease activity and cell death induced by ROS or by avirulent bacterial pathogens (Solomon et al., 1999). Proteases with different substrate specificity may thus play an important role in cell death control during plant-pathogen interactions.

It is well established that HR is an active process, but the intrinsic program activated during plant-pathogen interactions remains largely unknown. It was thought that a genetic approach should open up new insights into components of the regulatory cascade since alterations in this genetic program would lead to aberrant activation of cell death. Indeed, many mutants showing spontaneous cell death that resembles HR or disease symptoms, the so-called lesion mimic mutants, have been isolated in several species (Dangl et al., 1996; Buschges et al., 1997; Buckner et al., 1998). Although several of these genes have been cloned, no clear conservation of gene structure has emerged, unlike what has been found for the disease resistance genes where conserved motifs have been revealed. For example, the Arabidopsis lsd1 gene whose mutation leads to a propagation lesion mimic phenotype encodes a novel zinc-finger protein that could act as a transcription factor, whereas the maize lls1 gene defining a similar mutant phenotype encodes a probable dioxygenase whose substrate remains unknown (Dietrich et al., 1997; Gray et al., 1997). Other examples are the mlo gene from barley corresponding to a novel transmembrane protein and Les22 from maize that encodes uroporphyrinogen decarboxylase, a key enzyme in heme biosynthesis (Buschges et al., 1997; Hu et al., 1998). This diversity suggests that perturbations in very different metabolic pathways can lead to spontaneous cell death. In order to ascertain whether similar pathways of cell death may be activated by these mutations, extragenic suppressors that identify critical loci downstream from these lesion mimic genes will be important tools (Jabs et al., 1996).

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