Small Molecules Involved In Signaling And Execution Of Hr Cell Death

One of the most rapid plant responses engaged following pathogen recognition is the oxidative burst, which constitutes the production of reactive oxygen intermediates (ROI), primarily superoxide (O2-) and hydrogen peroxide (H2O2), at the site of attempted invasion (Lamb and Dixon, 1997; Wojtaszek, 1997; Grant and Loake, 2000). It has been suggested that the oxidative burst and cognate redox signaling may play a central role in integration and coordination of the multitude of plant defense responses (Lamb and Dixon, 1997).

Superoxide anion generation in relation to HR was first reported for potato tuber slices inoculated with an avirulent race of Phytoph-thora infestans (Doke, 1983). Subsequently, the oxidative burst has been identified in numerous plant-pathogen interactions involving different kinds of pathogens. The origin of ROI generated during the oxidative burst is not unequivocally established, but candidate reactions are the action of a plasma membrane-located NADPH-depend-ent oxidase complex and cell wall peroxidases (Wojtaszek, 1997; Grant and Loake, 2000). The cytotoxicity and reactive nature of O2-requires its cellular concentration to be carefully controlled, which can be achieved by induction of antioxidant enzymes, such as glutathione S-transferase, glutathione peroxidase, or ascorbate peroxidase (Wojtaszek, 1997; Smirnoff, 2000).

ROI accumulation is a rapid event that precedes HR cell death in many plant-pathogen interactions showing ^-gene-triggered resistance (Kombrink and Somssich, 1995; Wojtaszek, 1997). Rapid and biphasic ROI accumulation has been observed in several cultured plant cell systems in response to bacterial or fungal elicitors, i.e., avr gene products (Levine et al., 1994; Jabs et al., 1997; Wojtaszek, 1997). While the first peak was considered nonspecific, the second sustained ROI burst was dependent on the pathogen race and only occurred with avirulent bacteria. Collectively, these data suggest a dual function for ROI in disease resistance: (1) direct participation in the development of host cell death during HR as well as direct inhibition of the pathogen, and (2) a role as a diffusable signal for induction of cellular protectants and defense responses in neighboring cells. Thus the strict spatial limitation of HR cell death may be the result of a dose-dependent action of ROI (Lamb and Dixon, 1997).

In animal cells, nitric oxide (NO) is known to act as second messenger in concert with ROI in processes such as the innate immune response, inflammation, and PCD. Recently it was shown that NO might also play an important role in the regulation of defense responses in plants. Infection of resistant, but not susceptible, tobacco plants with tobacco mosaic virus (TMV) resulted in enhanced NO synthase activity (Durner et al., 1998). Furthermore, external application of NO induced salicylic acid accumulation and PR gene expression, and NO inhibitors blocked both effects. This suggests that several critical players of animal NO signaling, such as cyclic GMP or cyclic AMP-ribose, are also operative in plants (Durner et al., 1998; Klessig et al., 2000). In cultured soybean (Glycine max L.) cells, it was demonstrated that the efficient induction of HR cell death required a balance between ROI and NO production, whereas unregulated NO production was not sufficient to induce HR cell death (Delledonne et al., 2001).

Other rapid changes observed following pathogen recognition are selective ion fluxes across the plasma membrane (Kombrink and Somssich, 1995; N├╝rnberger and Scheel, 2001). Although rapid responses have been extensively studied in appropriate model systems, such as cultured cells stimulated with defined elicitors, some debate concerns the precise temporal order in which they occur. In cultured parsley cells, it has been established that ion fluxes (H+, K+, Cl-,

Ca2+) precede the oxidative burst (Jabs et al., 1997), whereas in cultured soybean (Glycine max L.) cells, the oxidative burst apparently precedes and stimulates a rapid influx of Ca2+, which then leads to HR cell death (Levine et al., 1996; Morel and Dangl, 1997). The specific requirement of calcium signaling in HR cell death had been suggested from studies using ionophores and calcium-channel blockers (Jabs et al., 1997; N├╝rnberger and Scheel, 2001). Recent work suggests that this dependence on Ca2+ may involve specific isoforms of calmodulin, since HR-like cell death, PR protein gene expression, and broad-spectrum disease resistance were induced by transgenic expression of soybean calmodulin-encoding genes in tobacco (Heo et al., 1999). The molecular mechanisms of these calmodulin-induced responses are not yet known; however, it was recently demonstrated that calmodulin directly interacts with the barley Mlo protein and regulates defense against powdery mildew (Kim et al., 2002).

The function of salicylic acid as a crucial signal molecule that is involved in systemic acquired resistance and PR gene expression has been known for many years (Sticher et al., 1997). More recently, SA has also emerged as a positive feedback regulator of cell death during the HR (Feys and Parker, 2000). This was first suggested from studies with lesion mimic mutants of Arabidopsis (Weymann et al., 1995). The spontaneous cell death formation in the lsd6 and lsd7 mutants was suppressed by transgenic expression of the NahG gene, encoding a bacterial SA hydroxylase that degrades SA to catechol. Additional lesion-mimic mutants, ssil and acd6, that were recently isolated also show a SA-dependent HR phenotype (Rate et al., 1999; Shah et al., 1999). Furthermore, SA depletion in tobacco by NahG expression delayed HR cell death after infection with avirulent bacteria, and this delay was correlated with a reduced and delayed oxidative burst (Draper, 1997). Taken together, these data suggest that SA, in addition to SAR signaling, also has a role in early and local defense regulation by amplifying and sustaining the oxidative burst. In fact, SA might act in concert with ROI to define the threshold required for initiation of HR cell death.

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