The hypersensitive response

Pathogens and herbivorous insects such as the RWA induce a broad spectrum of defence responses in the host plant. These defence responses range from transcriptional changes to macroscopic symptoms including alterations in growth, tissue death and chlorosis (Klingler et al., 2009). The HR was first defined by Stakman in 1915 as a mechanism for defence against infection by plant pathogens (Zidack, 1999). It is generally visible as necrotic lesions on the plant’s surface and is a widely occurring defence response in higher plants responding to all known groups of plant pathogens. This defence response is characterised by rapid and localised cell death at the point of pathogen attack (Keen, 1990). The main function of the HR is to limit the spread of a pathogen by breaking down the cell before it is colonised by

33 the pathogen (Pontier et al., 1998). The activation of the HR requires mechanisms that transmit signals via signal transduction pathways (Braun et al., 1997).

It is thought that two types of processes activate cell death. The first process is the change in cellular metabolism, which allows for compounds to accumulate that are toxic to both host plant cell and pathogen. The second process is the recognition of the pathogen which causes genetic reprogramming of the cell. This causes the activation of programmed cell death (Hammond-Kosack & Jones, 1996; Pontier et al., 1998).

The induction phase, the latent phase and the collapse phase collectively make up the HR. During the induction phase, pathogenic avr-gene expression is activated in the pathogen and the avr products are transported into the host cell. This phase involves a rapid reaction to close the wound which protects the plant from losing cellular components. It also prevents micro-organisms from entering the wound site into the plant tissue. Macroscopic symptoms occur during this phase as well as membrane damage associated with the HR. Photosynthetic protein synthesis is inhibited by arresting the translation of nuclear encoded photosynthetic genes.

During the final phase of the HR response, the host cell collapses (Jabs &

Slusarenko, 2000).

Cell death serves a number of purposes. In biotrophic pathogens, it may prevent the pathogen from accessing sufficient nutrients from the host plants. In necrotrophic and hemibiotrophic pathogens, the fact that the cells break up during the cell death process, may allow for the rapid accumulation of phytoalexins (which are antimicrobial peptides) to highly active concentrations (Hammond-Kosack & Jones, 1996; Osbourn,1996).

The HR, along with environmental factors such as UV stress, herbicide use and oxygen shortage, have been known to induce oxidative stress. Activation of the HR initiates an induced resistance response in the plant known as systemic acquired resistance (SAR) (Watanabe & Lam, 2006). The HR has been found to be the typical response of the plant to aphid infestation in antibiotic cultivars. Figure 2.6

34 summarises the HR and indicates how this response can be used to increase resistance to different pathogens or pests by genetically engineering a recognised avr-gene behind a pathogen inducible promoter.

Figure 2.6. Model of the hypersensitive response. The HR is triggered by the highly specific recognition of a pathogen-derived elicitor by a plant resistance gene product.

The powerful and concerted defence that constitutes the HR stops the pathogen (Adapted from Stuiver & Custers, 2001).

During the HR, other defence responses related to signalling, including ion fluxes, the generation of ROS and changes in protein phosphorylation also occur (Dixon et al., 1994).

35 2.3.6. Oxidative stress response

One of the earliest plant defence responses leading to the HR is the rapid production of ROS, known as the oxidative burst (Pontier et al., 1998). Both biotic and abiotic stresses cause the cell to undergo the HR. The oxidation of phenolic compounds that accumulate during this stress response seem to imply an increase in phenol oxidising enzymes. This causes the production of ROS, such as hydrogen peroxide (H2O2), the hydroxyl radical (OH^-) and the superoxide anion (O2

-), which results in a secondary oxidative stress in plants. Plants rapidly produce large amounts of O2

-(which is mostly dismutated via superoxide dismutase or SOD to form H2O2), this involves NAPDH oxidase. Both O2

and H2O2 are moderately active and are therefore converted to more active derivatives (Hammond-Kosack & Jones, 1996).

Hydroperoxyl radicals (HO2) are formed from the protonation of O2

-. These radicals can effectively cross biological membranes, resulting in the damaging of these membranes (Hammond-Kosack & Jones, 1996). H2O2 leads to an increase in benzoic acid hydroxylase activity which is necessary for the synthesis of salicylic acid, an important signalling hormone. In this way ROS plays an important role in the early phases of signalling during biotic and abiotic stresses (León et al., 1995).

Consecutively, H2O2 undergoes a reaction that yields the hydroxyl free radical (OH^-), which is extremely reactive and causes cellular damage to both the plant and the pathogen, ROS plays a critical role in the plant defence. It is a pH dependent process with an optimum at a neutral pH (Benhamou, 1996; Bolwell & Wojtaszek 1997; Lamb & Dixon, 1997).

Reactive oxygen enzymes may directly contribute to the defence response. Each of the reactive oxygen enzymes that are produced during the oxidative burst may both be toxic and aid in reactions that strengthen the cell wall through cell wall lignifications and cross-linking reactions. The influx of Ca²⁺ into the cell is controlled by the ROS H2O2. This has been shown to be an important factor in the development of reactive oxygen intermediate (ROI) mediated cell death (Levine et al., 1996).

36 H2O2 produced during the oxidative burst has been shown to function in the defence-signalling pathway. This leads to the expression of the HR. Oxidative deterioration is initiated by the degradation of a plant’s membrane lipids, resulting in free fatty acid. This process is initiated by providing a substrate for the enzyme lipoxygenase, which may contribute to the HR causing membrane peroxidation (Farmer, 1994; Goodman & Norvacky, 1994).

The ROS that are detected during the plant-pathogen interaction include O2

-, H2O2,

and OH^- (Wojtaszek, 1997). NADPH oxidase, oxalate oxidase, peroxidase and amine oxidase are enzymes that are generated during the elicited defence response (Benhamou, 1996; Bolwell & Wojtaszek 1997; Lamb & Dixon, 1997).