A method for making directed changes to the Fusarium graminearum genome without leaving markers or other extraneous...

A method is described which allows exact targeted changes to the Fusarium graminearum genome, including changes of as little as one particular base pair to gene-size insertions, replacements or modifications. The technique leaves no other DNA in the genome, such as marker genes, and can be used serially to effect multiple complex changes in any desired chromosomal locations. The method is based on our previous finding that after transformation, DNA with homology to F. graminearum DNA recombines itself into the genome in a predictable manner involving multiple tandem copies. We designed a cloning vehicle with a built-in hygromycin-resistance marker (hygB) which can be used to transform the fungus, and with cloning sites to carry DNA with any desired genomic modifications. To effect a desired genomic change the sequence changes of interest are incorporated between two adjacent borders homologous to F. graminearum DNA which will target them to the desired location. This modified DNA is attached within the cloning sites within the vehicle. Transformants are readily obtained in which tandem copies of the vehicle plus insert are inserted between the two genomic border sequence homologues. Progeny of a transformant are subsequently screened for those with a decreased resistance to the antibiotic, and then for those which have completely lost the marker and the entire vehicle, leaving only the desired sequence modifications between the two genomic border sequences which were targeted. This method is demonstrated by exactly replacing the trichodiene synthase (tri5) gene coding sequence (CDS) with that of a green fluorescence protein (gfp) gene with no other genomic changes. This derivative was then re-engineered to replace the gfp CDS with that of the wild type, exactly regenerating the original F. graminearum genome.