Characterization of two genes involved in Neotyphodium lolii growth : a thesis presented in partial fulfillment of the requirements for the degree of MAster of Science in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

Neotyphodium lolii is a filamentous fungus that forms symbiotic associations with Lolium perenne, growing in its intercellular spaces. It is a feature of the symbiosis that growth of the fungus and the plant is synchronized. When the grass leaf-blade grows, the fungus grows at the same rate, hence when the blade ceases extension the hyphae do likewise. In addition, in planta there is little hyphal branching, where as in culture hyphae branch at regular intervals. This suggests the existence of a regulatory mechanism in planta that partially dictates hyphal morphology and growth. The criteria for choosing possible candidate genes relied on whether the gene had a function relating to hyphal branching and/or regulation of hyphal extension in several organisms. Three candidate genes were selected. Protein elongation factor 2 (EF-2; an elongation factor associated with the ribosome) was targeted to add more direct evidence to the high metabolic rate observed in planta using the GUS reporter gene by Tan et al (2001). Cell division control protein 12 (CDC-12); a septin which is involved in the construction of the 10 nm ring structure associated with cell division and whose mutation is lethal in yeast was chosen to help distinguish the growth mode of N.lolii in planta. A Stretch-activated Calcium Channel (SACC) which allows exogenous calcium into the cell upon application of lateral pressure on the membrane was targeted to help distinguish the possible recognition signal the hyphae make to elucidate when the host tissue is growing. This project was then divided into four parts, one part per gene and a final part looking at the in vitro and in vivo expression of these genes. For the first three parts degenerate PCR was performed and appropriate-sized fragments cloned, sequenced and restriction mapped for EF-2 and CDC-12 (2066 bp and 514 bp respectively). Database searches were used to identify the sequences as potentially being the target genes. Degenerate PCR was unsuccessful for the SACC. Southern blots were used to identify restriction enzymes for Inverse PCR; and this was used to obtain the remaining 5' and 3' regions of each target gene. Gene prediction software was used to predict gene structure; 5' and 3' RACE to confirm the length, introns and start/stop points of EF-2 and CDC-12 full gene transcripts (2,900 and 1,612 bp respectively). Internet-based sequence analysis tools subsequently were used to identify sequence features. For the second part, expression of EF-2 and CDC-12 are investigated during various states of hyphal growth. Growth curves were constructed and in vitro expression analysis was achieved by Northern blot. The expression patterns of EF-2 and CDC-12 followed the growth state of N.lolii. RT PCR was used to confirm in planta expression of both genes and validate their uses for future studies.