The Corresponding Pathogen Avirulence Gene

Published Date: 02 Nov 2017

HR involves the production of ROS, changes in ion fluxes, protein phosphorylation and dephosphorylation and gene activation. Studies with kinase inhibitors have indicated a role for protein kinases in HR (Zhang et al., 2000). Several R proteins and PAMP receptors are protein kinases and are thus potential targets of the kinase inhibitors (Gómez-Gómez & Boller, 2000). Moreover, inhibition of calcium-dependent protein kinases (CDPKs) and mitogen activated protein kinases (MAPKs) which are kinases functioning downstream of receptors in the signaling pathways could prevent the progression of HR cell death (Pedley & Martin, 2004). During HR, hydrogen peroxide is generated in a biphasic pattern. The first transient increase in hydrogen peroxide is triggered by PAMPs and some hours later the second, more persistent rise is induced by R-Avr interaction (Mur et al., 2008b; Mur et al. 2009). The first rise induces SA synthesis which then potentiates the second rise (Mur et al., 2009). In fact, in HR, SA functions mainly by augmenting ROS production (Lamb & Dixon, 1997). One mechanism by which SA can do so is the inhibition of mitochondrial ATP synthesis via an interruption of the electron flow through the respiratory transport chain (Xie & Chen, 2000).

Several cytological alterations have been noted during HR, including the shrinkage of plant protoplast (Heath, 2000). This can be the consequence of cytoskeletal rearrangements (Naton et al., 1996) and though the purpose of this during HR is not clear, it could promote the assembly of defense-related protein complexes and/or controlling the movement of cytoplasmic vesicles/organelles (Takemoto & Hardham, 2004). Moreover, cytoplasmic mobility rapidly stops (Naton et al., 1996) and this has been accounted to cytoskeleton depolymerization (Shimmen & Yokota, 2004). Plant protoplast shrinkage in HR is a feature reminiscent of cytoplasmic collapse in apoptosis (Heath, 2000). However, unlike apoptosis, apoptotic bodies are not formed at the end of HR and this has been explained by the presence of the plant cell wall. Just before HR-associated cellular collapse, there is an increased formation of large vesicles in the cytoplasm (Liu et al., 2005; Mur et al., 2008a). In one study, Mittler et al. (1997) found that these vesicles consisted of remains of the chloroplast with fragmented nuclear material left within the collapsing nucleus. The vesicles seemed to have mostly been formed from the vacuolar membrane but another source could be by plasma membrane invagination. In addition, early mitochondrial swelling is another cytological change which occurs during HR (Bestwick et al., 1995). This leads to a rapid loss of mitochondrial function. Studies have provided evidence of pore formation in mitochondria of cells undergoing HR (Yao et al., 2004) and there appears to be little production of ATP during the HR (Xie & Chen, 2000). Furthermore, several studies have reported cytochrome C release before cell death (Krause & Durner, 2004; Kiba et al., 2006) and this release could indicate the last phase of mitochondrial dysfunction. In fact, mitochondria could have a vital role in integrating the effects of multiple death signals (Jones et al., 2001; Amirsadeghi et al., 2006). Changes in the redox status of mitochondria may be important for cellular metabolic dysfunction which results in HR.

Inhibitor studies have demonstrated that proteases, especially the papain-class of cysteine proteases, are required for HR (Belenghi et al., 2003; Sanmartin et al., 2005). Papain proteases are not caspases. Two plant alternatives to caspases which show some homology to caspases are metacaspases and vacuolar processing enzymes (VPE) (Sanmartin et al., 2005). Though metacaspase has been shown to be induced by pathogens (Hoeberichts et al., 2003), no role in HR has been established. In contrast VPEs have been shown to be major effectors of HR (Hatsugai et al., 2004), suggesting that vacuolar processing and autophagy are key characteristics of HR. Indeed results have indicated that one role of autophagy is too highlight the sharp edge around the HR (Liu et al., 2005) and therefore autophagosomes could have as function to neutralize death-inducing signals emanating from the HR lesion. Apart from proteases, E3 enzymes also play a key role in HR. These enzymes have as function to add ubiquitin to specific proteins and target them for degradation by 26S proteasome (Viersta, 2003). It has been hypothesized that the targets of ubiquitinization are cell death suppressors which need to be destroyed to initiate HR.

Calcium influxes have been associated with cell death. Studies with Arabidopsis (Grant et al., 2000) and cowpea (Xu & Heath, 1998) have shown an increase in the cytosolic calcium concentration during HR. Moreover, the use of a calcium channel blocker (LaCl3) inhibited cell death in soybean cultures when inoculated with avirulent bacteria (Levine et al., 1996). Calcium ions were found to influence ROS generation through a reciprocal control mechanism (Grant et al., 2000). Oxidative burst has been correlated to an alkalinization-responsive peroxidase or amine oxidase (Bolwell et al., 2002). Studies have indicated that NADPH oxidases are major sources of ROS during HR. NADPH oxidases initiate ROS production in the apoplast indirectly via alkalinization-responsive peroxidases. One mechanism of action of ROS in HR is lipid peroxidation. For example, hydroxyl radicals remove a proton from phospholipids to start a lipid radical-lipid hydroperoxide (LOOH) chain reaction. LOOH is also formed by the action of lipoxygenase (LOX). LOX could serve to increase membrane damage (Croft et al., 1990) but it is also needed for production of defense products, some of which are cytotoxic (Alméras et al., 2003). The involvement of ROS-derived LOOH appears to be a more general characteristic of HR. However, increase in LOX expression has often been reported during HR (Porta & Rocha-Sosa, 2002) and this has been correlated with increased phospholipase A2 activity. This enzyme supplies polyunsaturated fatty acid to LOX from membrane phospholipids (Göbel et al., 2001). The production of ROS-derived LOOH and LOX-derived LOOH is under mutual regulation and the source of LOOH may not be essential to the HR (Göbel et al., 2003).

Another mechanism which leads to oxidative death is the alteration in levels or redox status of antioxidant pools. For instance levels of glutathione (GSH) have been reported to rise during HR (May et al., 1996; Vanacker et al., 1998) and they may exist mainly in the oxidized form. NO is known to play a crucial role in HR development (Zeier et al., 2004; Bocarra et al., 2005) though the mechanism of its production remains to be established. The effects of NO may overlap to a great extent with those of hydrogen peroxide (Zago et al., 2006). It also remains to be seen how far NO and ROS work independently or synergistically to bring about cell death. Some forms of HR and much ROS-dependent lipid peroxidation during HR may be light dependent (Montillet et al., 2005). The chloroplasts could contribute to ROS production by two mechanisms during HR. The first one includes photoproduction of ROS when photon intensity exceeds that required for carbon dioxide fixation (i.e. excess excitation energy) (Szabo et al., 2005). The second mechanism involves the ROS production associated with the release of porphyrin ring-containing chlorophyll products from the reaction centres as part of catabolic processes. Evidence has shown that excess excitation energy associated effects in HR may be pathosystem specific.