Identification of transporter genes from the fungal endophyte Neotyphodium lolii : this thesis is presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Plant Biology at Massey University, Palmerston North, New Zealand /

Abstract

Neotyphodium lolii is an endophytic fungus that lives in the pasture grass, Lolium perenne. They share a mutualistic symbiotic relationship. N. lolii lives out its life cycle within the plant and produces secondary metabolites, including alkaloids peramine, ergovaline and lolitrem which protect the grass from insect and animal herbivory. In fungi the biosynthetic genes of secondary metabolites are often located in gene clusters. These clusters frequently contain one or more genes that code for transporter proteins responsible for the removal of toxic products from the fungal cells. Plants produce defence compounds, including antifungals to protect themselves from colonising fungi. However endophytes are able to neutralise these host toxins, one mechanism for this is possibly by efflux through transporter channels. The goal of this study was to identify ABC and MFS genes from N. lolii. These two families are the largest and most diverse of transporter families, which transport a variety of substrates, including peptides, toxins, ions and sugars across membranes. Using degenerate PCR primers designed from fungal multi-drug transporter sequences, four unique ABC gene fragments were amplified from N. lolii. A further two ABC sequences and two MFS gene fragments were identified in a database of N. lolii EST sequences. RT-PCR was used to compare expression of isolated ABC and MFS genes in N. lolii, growing in culture and in infected plants. Up-regulation of transporter transcripts in planta could suggest a role in symbiosis. Some genes were seen to have a visibly different expression pattern from others, although all genes were strongly expressed in cultured mycelia. Gene expression in the plant host was most evident in tissues more heavily infected with endophyte. To discover possible roles for the isolated transporter genes in transporting endophyte secondary metabolites a strain distribution study was completed. Five of the putative ABC and MFS genes were compared against 12 Epichloë and Neotyphodium endophytes. Amplified PCR products in the genotypes screened produced a unique pattern of gene occurrence for each of the five transporters. This added to the characterisation of the transporter genes and showed that one gene, gABC 4e, was the most diverse in its distribution, while another ABC gene gABC 4g was present across all genotypes. One ABC gene (gABC 4e) plus flanking DNA was sequenced in full. Bioinformatic analyses suggested that gABC 4c may be a half sized ABC transporter gene of 2 kb with four exons. An orotate phosphoribosyltransferase was identified 2 kb upstream of the ABC transporter. Further work will be needed to confirm that the start and stop codons of this ABC transporter have been accurately predicted, as well as to verify the putative intron/ exon boundaries identified by gene prediction programmes. The role of N. lolii ABC transporter gABC 4c has not been determined, however future research could focus on the nature of the substrate(s) transported, the sub-cellular location of the channel, and the effects of gene knockout or over-expression on the symbiosis between N. lolii and perennial ryegrass