Project Number: WVA00161
CRIS Number: 0189386
Toxin biosynthesis genes in ergopeptine-producing fungi
Investigators: Panaccione, D. G., Schardl, C. L.
Performing Department: Plant & Soil Sciences -- 1825
Start Date: 09/01/2001
Termination Date: 08/31/2004
Reporting period: 09/01/2001 to 12/31/2001
Progress Report:
During these first few months of the project, research focused on the biochemical consequences of knocking out lysergyl peptide synthetase to produce an ergovaline-deficient grass endophyte. The concentrations of the alkaloids ergine and ergonovine (the two most likely sinks for lysergic acid in a lysergyl peptide synthetase mutant) were measured in relevant grass-endophyte symbiota. The symbiota assayed included perennial ryegrass (Lolium perenne) infected with: wild-type, ergovaline-producing Neotyphodium sp. Lp1; an ergovaline-deficient, lysergyl peptide synthetase knockout mutant derived from Lp1; and, an ergovaline-producing strain of Lp1 carrying an ectopic integration of the gene knockout construct. In HPLC analyses, we were able to detect ergine and ergonovine standards. However, we were unable to resolve these two polar lysergyl compounds with our isocratic HPLC system and, thus, they eluted as a single peak. Analyses of the ergovaline-deficient gene knockout-containing symbiota and the ergovaline-producing control symbiota resulted in peaks of similar magnitude in the ergine/ergonovine area of the chromatograms. Thus, although we cannot currently distinguish between ergine, ergonovine, or a combination of the two compounds, there was no significant increase in the accumulation of these polar lysergyl derivatives, collectively, in ergovaline-deficient, knockout-containing symbiota. More detailed instrumental analyses are planned to confirm and extend these findings. Since the results indicate that, in the lysergyl peptide synthetase knockout strains, lysergic acid is not being shunted into ergine and ergonovine, it is likely that there is some type of feedback inhibition mechanism operating in the pathway. We analyzed one potential mechanism of feedback inhibition -- reduction in accumulation of mRNA from dmaW, the gene that encodes the first committed step in the ergot alkaloid pathway and which has been shown to be transcriptionally regulated by growth conditions. The accumulation of dmaW transcripts in the endophytic fungus growing within its perennial ryegrass host was assessed by competitive PCR. The amount of dmaW transcript was quantified by comparison to an internal 'mimic' DNA molecule that was present in increasing concentrations in each of several reactions. The dmaW values were then normalized to values obtained from a control gene (beta-tubulin) that were obtained in a similar series of reactions. The comparison to beta-tubulin was necessary to account for potential differences in total fungal mRNA quantities isolated from different symbiota. Our results showed that there were approximately equal levels of dmaW transcript in all endophytes examined. These results indicate that the feedback inhibition inferred from the HPLC analyses is acting at some level other than transcript accumulation of dmaW.
Publications: (No publications.)
Impact:
Ergovaline is in an important mycotoxin that accumulates in grasses infected with certain plant-mutualistic species of Neotyphodium. Ergovaline may be eliminated from plant-fungus symbiota by knocking out the fungal lysergyl peptide synthetase gene. Before considering practical applications of eliminating ergovaline by this strategy, it is important to understand the effects that such a knockout mutation have on the accumulation of related alkaloids that are synthesized from common precursors.