RNA and protein synthesis during zoospore differentiation in synchronized cultures of Blastocladiella

Abstract Techniques are described for producing the highly synchronized differentiation of Blastocladiella zoospores in liquid culture. Under these conditions, the plants form discharge papillae at 17 1 2 hours, nuclear cap formation and zoospore cleavage occur just before 19 hours, and zoospore discharge is complete by 19 1 2 hours. The patterns of change in dry weight, RNA, DNA, and protein were examined before and after the induction of differentiation at the end of the exponential growth phase. After changing from the rich growth medium to a dilute salts medium, all synthetic rates decrease, and net increase in DNA, RNA, dry weight, and protein ceases at 16 1 2 , 16 1 2 , 17, and 17 1 2 hours, respectively. Significant RNA degradation occurs after 16 1 2 hours resulting in a final 35% loss in the total amount. Incorporation of externally added C14-uracil into RNA falls to a very low value by 17 hours, while C14-leucine incorporation into protein reaches a maximum between 16 1 2 and 17 1 2 hours, followed by a steady decline. Pulse labeling and density gradient analysis of the whole-cell RNA demonstrated that before 16 1 2 hours transfer-RNA and a “heavy” RNA fraction of high specific activity were rapidly labeled, followed by labeling in the ribosomal peaks. No uracil entered RNA during a 10-minute pulse of plants at, or after, 17 hours. The cessation of major RNA synthesis after 16 1 2 hours was accompanied by a significant decrease in apparent uracil pool size. In an experiment where the pools were heavily labeled before 16 hours, a small amount of uracil incorporation could be detected after 17 hours, but the extensive RNA degradation and failure of exogenous uracil to enter the pools prevented a reliable estimate of the actual quantity of new RNA produced. However, when the whole-cell RNA was randomly labeled during exponential growth and examined during differentiation, no evidence could be obtained for significant turnover of this RNA after 16 1 2 hours, despite a large loss in the total amount. These results provided direct evidence for the conservation of preexisting, exponential-phase ribosomes by aggregation to form the zoospore nuclear cap. Indirect evidence for the requirement of messenger-RNA synthesis during spore differentiation was obtained by the use of actinomycin D, puromycin, and p-fluorophenylalanine (PFP). Puromycin at 100 μg/ml was not effective in reducing leucine incorporation and did not inhibit papilla formation, but did prevent zoospore cleavage. The papillae formed in the presence of puromycin were, however, multiple and abnormal. Actinomycin D (25 μg/ml) and the amino acid analog PFP (0.005 M) were effective inhibitors for the incorporation of uracil and leucine, respectively. Both caused inhibition of papillae only if added to plants 30 minutes or more before these were formed. Both also prevented spore cleavage only when added to plants 1 hour before this event. From their identical patterns of inhibition, it was concluded that papilla formation and cleavage probably require the production of one or more short-lived messenger-RNA's 30 minutes and 1 hour, respectively, before they occur.