STRESS RESPONSES IN ALFALFA (MEDICAGO-SATIVA L) .7. INDUCTION OF DEFENSE RELATED MESSENGER-RNAS IN ELICITOR-TREATED CELL-SUSPENSION CULTURES

Polysomal mRNA was isolated from alfalfa (Medicago sativa L.) cell suspension cultures at various times after exposure to an elicitor preparation from the cell walls of the bean pathogen Colletotrichum lindemuthianum. Analysis of in vitro translated polypeptides by one- and two-dimensional gel electrophoresis revealed extensive changes in the pattern of mRNA translation products. These were classified as rapidly induced (with a lag of less than 1 h, class 1), inducibledelayed (lag of 2-3 h, class 2) or down-regulated by elicitor (class 3). Use of monospecific antisera to immunoprecipitate proteins synthesized in vitro revealed that translational activities of polysomal mRNAs encoding phenylalanine ammonia-lyase, 4-coumarate (4CL): CoA ligase and chalcone synthase (CHS) subunits were rapidly induced following exposure to elicitor. CHS subunits were the major newly synthesized polypeptides in class 1. A number of elicitor-induced isopolypeptide subunits of PAL and CHS were resolved by 2-D gel analysis of immunoprecipitates. A second major class 1 mRNA activity encoded a set of isopolypeptides of Mr 34 000. These polypeptides did not appear to be iso-forms of glucanase or chitinase. Northern blot analysis indicated that elicitor induced large increases in the steady state levels of PAL, 4CL and CHS transcripts. These observations provide the basis for cloning genes involved in the biosynthesis of the phytoalexin medicarpin, and perhaps other defence responses in alfalfa. © 1990.