De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Abstract

Actidnomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycie max IL.l Meff. cv. Harosoy 63) hypo-cotyls and in hypocotyls inoculated with the pthogeic fungus Phyto-phthra megasperma var. soine A. A. Hildb., but had lttle effect on protein synthesis witdn 6 hours. Blasticin S, conversely, inhibited protein synthesis in the hypocotyls without exIbing significant effects on messenger RNA synthesis. The normal culthvar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely ised by actino-mycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceoUin was concomitantly reduced. The effects of actnomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceolin both required denovo messenger RNA and protein synthesis early after infecton. Furthermore, actioomycin D and blasticidin S also were effective in suppressing resistance expression and glyceoUin production in soybean hypocotybs when inoculated with various Phyto-pbtorwa specles that were normally noapthogenic to the plants. This indicated that the mechanism of general resistance to these normaly nonpathogenic fungi also Involves de novo messenger RNA and protein synthesis and production of glyceofln. study (Tani et aL, personal communication) using an RNA synthesis inhibitor indicated that de novo RNA synthesis is also necessary for resistance expression in the oat crown rust disease. Hadwiger and co-workers (5, 7, 14) found that pisatin, a phyto-alexin produced in pea plants, could be induced by various chemicals which were presumed to affect the conformation of DNA; based on this and inhibitor experiments they (7, 14) suggested that pisatin production depended upon both de novo RNA and protein synthesis. To date, however, no systematic study that would link de novo RNA and protein synthesis, phytoalexin production, and disease resistance has been conducted. The expression of monogenic resistance in soybean (Glycine max [L.] Merr.) to a fungal pathogen Phytophthora megasperma var. sojae A. A. Hildb. has been attributed to the production of a phytoalexin (8-11, 23), glyceollin (13). Yoshikawa et aL (22) recently found that soybean hypocotyls resistant to P. megasperma var. sojae synthesized poly(A)-containing RNA about six times more rapidly than uninoculated plants early after infection and preceding the time of occurrence of resistant expression or phy-toalexin production. The poly(A)-containing RNA was later characterized to be in fact messenger RNA in the in vitro wheat germ translation system (Yoshikawa, unpublished data) and this provided the first evidence that the expression of disease resistance in plants is accompanied by activation of messenger RNA synthesis. The present experiments were designed to elucidate further whether de novo messenger RNA synthesis and protein synthesis are causally linked with the expression of resistance and the production of glyceollin by soybean hypocotyls. Disease resistance in several plant-fungal pathogen interactions has been suggested as being due to the inducible production of antibiotic molecules, phytoalexins (1, 12). The initial biochemical events which may occur soon after infection and lead to the subsequent expression of resistance or production of phytoalexins are not, however, currently well characterized. It has been proposed (2, 6) that the initiation of resistance responses is mediated by de novo gene activation resulting in the synthesis of new species of messenger RNAs and proteins possibly required for the production of phytoalexins. Quantitative and qualitative enhancements in protein metabolism appear to occur in some resistant-responding plants at early stages of infection (19, 21), but a more direct indication of the importance of de novo protein synthesis in disease resistance is the fact that certain protein synthesis inhibitors diminish plant resistance, thereby resulting in considerable pathogen growth (15, 17). Although possible alterations in messenger RNA metabolism in diseased plants have not been critically evaluated, a recent 'This research was supported in part by Grant 256040 from the Ministry of Education of Japan to M.Y. MATERIALS AND METHODS Chemicals. [G-3HJUridine and [U-'4Cllysine were obtained from The Radiochemical Centre, Amersham. Poly(U)-Sepharose (4B) was purchased from Pharmacia Fine Chemicals and actino-mycin D from Schwarz/Mann. Blasticidin S was donated by H. Sumi. Plant and Fungus. The soybean cultivar Harosoy 63 (resistant to race 1 of P. megasperma var. sojae) was grown as described previously (22) and used throughout the experiments unless otherwise specified. Race 1 of P. megasperma var. sojae was maintained on V-8 juice agar. For inoculum production, the fungus was grown for 3 to 5 days in a pea broth medium (10) and rinsed with deionized H20 prio. to inoculation. Seven-to 8-day-old plants were cut at the base of the hypocotyls and the cut ends placed in deionized H20 or aqueous solutions of either blasticidin S, a protein synthesis inhibitor (4), or actino-mycin D at various concentrations. A longitudinal slash wound (about I cm long) was made with a razor blade on each hypocotyl, approximately 1 cm below the cotyledonary node, and inoculated with a small piece of mycelium. Plants were maintained at a relative humidity of 100% after 314