Intracellular Localization and Expression of the Plasmid pAAT56 in the Japanese Pear Pathotype of Alternaria alternata.

Abstract

The circular DNA plasmid pAAT56 has been isolated from strain T88-56 of the Japanese pear pathotype of Alternaria alternata. The pAAT56 is 5354bp and possibly encodes two long open reading frames, designated ORF1 (1290bp) and ORF2 (1653bp). We have previously observed negative biological effects of pAAT56 on fungal phenotypes: reduction in pathogenicity and occasionally in vegetative growth. Here we analyzed the intracellular localization of pAAT56 and its expression at the RNA and protein levels. Mitochondria and nuclei were prepared from mycelia of strain T88-56 by subcellular fractionation. Total cellular DNA, mitochondrial DNA and nuclear DNA were subjected to gel blot analysis with pAAT56 probe. The analysis detected pAAT56 in total DNA and nuclear DNA, but not in mitochondrial DNA, indicating that the plasmid is not mitochondrial. RNA gel blot analysis with pAAT56 probe detected the presence of three RNAs of about 1.7, 2.7 and 5.4kb. Use of strand-specific RNA probes in RNA gel blot analysis demonstrated that all the transcripts are sense strands for ORF1 and ORF2. An ORF1-fusion protein was expressed in an Escherichia coli system and used for preparation of antiserum. Antiserum raised against the ORF1 product cross-reacted with six proteins (about 38, 49, 52, 62, 70 and 73kDa) in the pAAT56-carrying strain, but not in its pAAT56-cured derivatives. One of the proteins was identical in size to the predicted ORF1 product (49kDa). This result implies that the ORF1-related proteins are produced from pAAT56 in fungal cells, probably accompanied with posttranslational processing and/or interaction with fungal cellular protein (s).