Characterisation of manganese superoxide dismutase from Phytophthora nicotianae

Abstract

Three polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium, zoospores and germinated cysts of Phytophthora nicotianae. Their relative molecular weights in non-denaturing gels were approximately 34.5, 36 and 50 kDa. No evidence for the presence of either iron or copper/zinc SODs was detected at any of the developmental stages examined. The level of activity of the MnSOD polypeptides was similar in mycelia and spores. Degenerate PCR was used to amplify partial genes of two different MnSODs, designated PnMnSODI and PnMnSOD2, from P. nicotianae. Southern blot analysis indicated that there are two PnMnSOD1 genes in the P. nicotianae genome. Full length sequence was obtained for one of these genes, PnMnSOD1a, from a P. nicotianae bacterial artificial chromosome (BAC) library. RNA blots probed with PnMnSOD1 showed similar levels of expression in vegetative and sporulating hyphae, lower levels in germinated cysts and no detectable expression in zoospores. PnMnSOD1a had 96%, 97 % and 99 % amino acid identity with homologous genes from P. ramorum, P. infestans and P. sojae, respectively. The second gene cloned from P. nicotianae, PnMnSOD2, had only 38 % amino acid identity with PnMnSOD1a and was homologous to MnSODs that possessed an N-terminal mitochondrial targeting sequence in Phytophthora species and other eukaryotes. Southern blots indicated that there is one copy of PnMnSOD2 in the P. nicotianae genome. PnMnSOD2 was expressed at similar levels in mycelia and germinated cysts but PnMnSOD2 transcripts were not detectable in zoospores.