Inhibition of the hypersensitive response in tobacco by pectate lyase digests of cell wall and of polygalacturonic acid

Abstract

A close association exists between induction of the bacterial hypersensitive response (HR) and stimulation of a prolonged net H + uptake/K + efflux in tobacco cells. The key role played by K + in cellular metabolism suggests that this specific loss of K + may play a critical role in HR. We previously reported that pectate lyase:(1) prevents the HR in tobacco leaves; and (2) stimulates a transient H + decrease (pH increase) and K + increase in supernatants of suspension cells. In addition we found that subsequent exposure of suspension cells to pectate lyase did not elicit additional transient K + H + responses. The latter result suggested that pectate lyase pretreatment may prevent HR by preventing cells from undergoing the prolonged K + H + response. The present study demonstrates that treatment of tobacco cell walls with this enzyme results in a heat stable digest which closely mimics the enzyme itself in ability to alter these plant responses. A similarly acting digest can be generated from polygalacturonic acid (PGA) by the same enzyme; digests with as little as 0·5 μg carbohydrate ml −1 will stimulate a transient pH response and 15 μg ml −1 will inhibit the HR. The magnitude of the transient pH increase is directly proportional to the concentration of digests of either walls or PGA; however, the increase in K + did not increase proportionally, suggesting that the mechanism of this response was different from the K + H + response associated with bacteria-induced HR. Once tobacco suspension culture cells have undergone a transient pH response, their response to further additions of the digest is greatly diminished. The effects of the PGA digest on the transient pH response of suspension cells and the HR of leaf tissue were compared to effects of heat-killed bacteria which classically have been demonstrated to inhibit the HR. Experiments with leaf discs pretreated with either PGA digest or heat-killed bacteria and subsequently challenged with an HR-causing bacteria, demonstrate that although HR is inhibited, the associated prolonged K + H + response is not. This suggests that the K + H + response in itself does not lead to cell death. Understanding the simplified model systems described here should provide new insight into the complex reactions involved in the hypersensitive response and plant/bacteria recognition.