RNAi in the perennial ryegrass fungal endophyte Neotyphodium lolii

TIAN, P.; FELITTI, S.A.; RABINOVICH, M.; KAUR, J.; GUTHRIDGE, K.M.; SAWBRIDGE, T.; SPANGENBERG, G.C.

Buenos Aires

Simposio; The 6th International Symposium on the Molecular Breeding of Forage and Turf -MBFT 2010; 2010

The association between the grass endophyte Neotyphodium lolii and its host perennial ryegrass (Lolium perenne L.) is of agronomic importance for temperate pastoral agriculture. To further understand the association, tools for reverse genetics in the fungal endophyte have been developed. Vectors for RNAi mediated gene silencing in grass endophytes were generated based on the Multisite GatewayTM recombination system. Intron hairpin RNA (ihRNA) vectors to gfp were introduced into green fluorescent protein (GFP) expressing transgenic reporter endophyte and gfp silencing was demonstrated. The establishment of an Agrobacterium-mediated transformation system for Neotyphodium lolii brings the ability to undertake high throughput gene knockouts using RNAi based system. The genome sequence of Neotyphodium lolii not only provides a resource of genes to study, but also offers the opportunity to study the RNAi gene complement in an asexual endophytic fungus and its sexual relative Epichloë festucae, for which a genome sequence is also available. The genomic complement of Argonaute, RNA dependent RNA polymerase, and Dicer proteins has been shown to vary in fungi (Nakayashi and Nguyen 2008). In Neurospora crassa an RNAi pathway leading to silencing of unpaired DNA in meiosis (MSUD) has been shown to operate during the sexual phase of the lifecycle, while the RNAi dependent silencing of viruses and transposons, known as quelling,  occurs during the vegetative phase of its lifecycle. Comparisons of the RNAi gene complement of Epichloë festucae and Neotyphodium lolii in relation to the genes of other sequenced fungi will be presented. This knowledge will enable further studies of the mechanisms of RNAi in Neotyphodium/Epichloë endophytes. Nakayashi H. and Nguyen Q.B. RNA interference: roles in fungal biology. Current Opinion in Microbiology 2008, 11:494-502.