Towards the elimination of ergot alkaloid biosynthesis genes in Neotyphodium coenophialum

• Published in: Phytopathology Vol. 98; no. 6; p. S54
• Main Authors: Florea, S, Machado, C, Panaccione, D, Schardl, C
• Format: Journal Article
• Language: English
• Published: 01.06.2008
• Subjects: Neotyphodium coenophialum
• ISSN: 0031-949X

Endophytes associated with cool season grasses elicit considerable ambivalence improving plant productivity and tolerance to biotic and abiotic stresses, but producing ergot alkaloids. Cattle and horses that graze on tall fescue pastures infested with N. coenophialum can suffer from fescue toxicosis, symptoms of which are similar to those caused by C. purpurea. N. coenophialum strain e19 from tall fescue cv. Kentucky 31 carries dmaW1 and dmaW2, two homologous genes that encode dimethylallyltryptophan synthase, the enzyme for the first step in ergot-alkaloid biosynthesis. The goal of this research is to have a modified strain of N. coenophialum that does not produce the toxic ergot alkaloids but retains all the beneficial characteristics which can be used in the field application. In our effort to disrupt both homologs and ultimately obtain marker-free mutants, we are using a marker-exchange strategy employing the Cre/loxP site-specific recombination system. Several transformation methods were used to replace dmaW2 with the mutant dmaW containing a hygromycin resistance gene (hph) flanked by loxP sites. Of 1522 transformants obtained and screened, three were likely dmaW2 disruptants because they gave no PCR product from the wild-type locus, but yielded the fragment from the disruption construct. The protoplasts of the dmaW2-knockouts have been transformed with pKAES175 plasmid containing Protub2cre and no resistance marker. The protoplasts were grown on unselective media and more than 500 putative transformants were screened for the excision of the hph gene located between the loxP sites. We identified 18 transformants that have the hph gene looped out. This result gives us the possibility to use the same marker for sequential knockout of dmaW1.