Development of a method for mRNA differential display in filamentous fungi: comparison of mRNA differnetial reverse transcription polymerase chain reaction and cDNA amplified fragment length polymorphism in Leptosphaeria maculans.

Kevin Gellatly, Gavin Ash, Janet Taylor

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

We modified a technique, cDNA-AFLP, for identifying differentially expressed genes in plants to work in the filamentous fungus Leptosphaeria maculans (Desmaz.) Ces. & De Not. The cDNA fragments generated by our method ranged in size from approximately 100 to 400 bps. On average, twice as many cDNA fragments were amplified per primer set with cDNA amplified fragment length polymorphism in comparison with mRNA differential display reverse transcription polymerase chain reaction. The DNA fragments of interest were excised from gels and analyzed by single-stranded conformation polymorphism to eliminate nondifferentially expressed cDNA contamination. The method was used to examine gene expression differences between cultures grown in the presence or absence of an analog of the Brassica phytoalexin brassinin. Eleven of the fourteen fragments examined were determined by reverse Northern blot to be differentially expressed. In examining gene expression differences between young cultures not producing sirodesmins and older cultures that were producing these phytotoxins, we found 17 of 25 fragments were differentially expressed. Northern blots with these fragments confirmed the results.
Original languageEnglish
Pages (from-to)955-960
Number of pages6
JournalCanadian Journal of Microbiology
Volume47
Issue number10
Publication statusPublished - 2001

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