No 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.

Calcium acts as a second messenger in many higher plant cellular activities including cell elongation, cell division, protoplasmic streaming, and enzyme secretion (Marmé, 1988). Many of the effects of Ca2+ are mediated by CaBPs that undergo conformational activation upon association with one to four Ca2+ ions. Structural analyses of parvalbumin and other CaBPs (Kretsinger and Nockolds, 1973; Babu et al., 1985; Herzberg and James, 1985) defined a Ca2+-binding "EF-hand" domain consisting of a loop of 12 residues flanked on each side by a 12-residue a-helix. CaBPs are known to possess one or more copies of this conserved EF-hand domain (Kressinger, 1976). Calmodulin is the most ubiquitous and highly conserved CaBP (Anderson et al., 1980; Watterson et al., 1980), whereas others, including troponin C (muscle), calcineurin (brain), and parvalbumin (muscle), transduce Ca2+ signals in a tissueand species-specific manner. Sequence homology between them is largely restricted to the calcium-binding domains.

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.