[show abstract][hide abstract] ABSTRACT: The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade ( and species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi ( species), a morning-glory symbiont (), and a bamboo pathogen (), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.
[show abstract][hide abstract] ABSTRACT: Interspecific hybridization is a common evolutionary process for the many epichloid fungi that consequently possess multiple gene copies accumulated from their parental strains. Serial gene manipulations in such strains are impeded by the limited availability of selectable resistance marker genes. Therefore, we developed a method for marker elimination suitable for a range of filamentous fungi that allows the reuse of the same marker for successive manipulations, and can also generate gene knockout mutants free of any foreign genes. For epichloae, the complete elimination of the marker gene from the genome would mitigate public concerns and regulatory hurdles to the use of such fungal strains in field experiments.
Methods in molecular biology (Clifton, N.J.) 01/2012; 824:371-9.
[show abstract][hide abstract] ABSTRACT: A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum,Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5–2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.