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Insolubilization of cell wall hydroxyproline-rich glycoproteins is a common plant defence response when plants are challenged with either pathogens or elicitors derived from pathogens. A yeast elicitor that insolubilized cell wall extensin of suspension-cultured tomato cells was purified by 80% ethanol precipitation of yeast extract followed by anion-exchange chromatography, gel-filtration chromatography and reverse phase HPLC. MALDI/TOF-mass spectrum analysis of the purified elicitor preparation generated one major peak at 410 ± Da. Elicitor activity was inactivated at 100° for 10 min or after incubation with pronase. No loss of elicitor activity was observed after periodate treatment. Polyclonal antiserum raised against native tomato extensin was used to demonstrate extensin insolubilization in vivo . Salt-elutability of extensin from tomato cells was dependent upon concentration and time of incubation of the cells with elicitor. SDS-PAGE/western blotting of salt-eluted protein from elicited cells also demonstrated insolubilization of HRGPs in the cell wall. The elicitor was found to stimulate cell wall peroxidase activity and extensin insolubilization in isolated tomato cell walls and in intact cells. The elicitor also induced a transient oxidative burst which began after 5 min and was maximal after 20 min incubation with the cells. Other typical plant defence responses known to be triggered by elicitors, such as changes in H + , K + or Ca 2+ fluxes, enhanced activities of lipoxygenase, phenylalanine ammonia lyase and superoxide dismutase, were not observed when tomato cells were challenged with this elicitor. The Ca 2+ channel blocker verapamil did not prevent elicitor-induced extensin insolubilization in whole cells. This study suggests that biochemical events, such as transient oxidative burst and enhanced peroxidase activity, which accompany extensin insolubilization involve the cell wall without any apparent plasma membrane participation. The above cell wall events may be sufficient to form the early plant defence responses to microbial challenge. © 1997 Elsevier Science Ltd. All rights reserved |
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