Regulation of poly(A) polymerase activity and poly(A) + RNA in germinated wheat embryos under conditions of pathogenesis

Abstract

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A) + RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [ 3H]AMP from [ 3H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [ 3H]ATP by other labelled nucleoside triphosphates, such as [ 3H]GTP, [ 3H]UTP or [α- 32P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The 3H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T 1 treatment. In addition, the nearest-neighbour frequency analysis of the 32P-labelled reaction product predominantly yielded [ 32P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [ 3H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen ( Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A) + RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A) + RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.