ELICITOR-INDUCED EXTENSIN INSOLUBILIZATION IN SUSPENSION-CULTURED TOMATO CELLS

Insolubilization of cell wall hydroxyproline-rich glycoproteins is a c ommon plant defence response when plants are challenged with either pa thogens or elicitors derived from pathogens.-h yeast elicitor that ins olubilized cell wall extensin of suspension-cultured tomato cells was purified by 80% ethanol precipitation of yeast extract followed by ani on-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 +/- 1 Da. Elicitor activit y was inactivated at 100 degrees for 10 min or after incubation with p ronase. No loss of elicitor activity was observed after periodate trea tment. Polyclonal antiserum raised against native tomato extensin was used to demonstrate extensin insolubilization in vivo. Salt-elutabilit y of extensin from tomato cells was dependent upon concentration and t ime of incubation of the cells with elicitor. SDS-PAGE/western blottin g of salt-eluted protein from elicited cells also demonstrated insolub ilization of HRGPs in the cell wall. The elicitor was found to stimula te cell wall peroxidase activity and extensin insolubilization in isol ated tomato cell walls and in intact cells. The elicitor also induced a transient oxidative burst which began after 5 min and was maximal af ter 20 min incubation with the cells. Other typical plant defence resp onses known to be triggered by elicitors, such as changes in H+, K+ or Ca2+ fluxes, enhanced activities of lipoxygenase, phenylalanine ammon ia lyase and superoxide dismutase, were not observed when tomato cells were challenged with this elicitor. The Ca2+ channel blocker verapami l 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 exte nsin insolubilization involve the cell wall without any apparent plasm a membrane participation. The above cell wall events maybe sufficient to form the early plant defence responses to microbial challenge. (C) 1997 Elsevier Science. All rights reserved.