Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. A high frequency change, which is both inducible and reversible, results in altered colony morphology of a fungal symbiont (Neotyphodium lolii) and dwarfing of its grass host (Lolium perenne) This thesis is presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Microbiology at Massey University, Palmerston North New Zealand Wayne Roydon Simpson 2009 ii Abstract Fungal endophytes of the genus Neotyphodium form stable symbiotic associations, with grasses, that are symptomless and generally considered to be mutualistic. The benefits that these fungi confer to their grass hosts are exploited in pastoral agriculture systems. The production of a range of secondary metabolites, specifically alkaloids including peramine and ergovaline can give their host plants an ecological advantage in certain environments. Neotyphodium endophytes are asexual and have lost the ability to transfer horizontally between hosts making seed transmission a vital feature of the association. This thesis reports the occurrence of phenotypically different perennial ryegrass plants (Lolium perenne) in a population infected with Neotyphodium lolii. Here we show that the change in the plants is directly attributable to a variant endophyte that they host. Isolation of the variant endophyte reveals a change in colony growth compared to the wild-type resident endophyte in the population, which has a white and cottony phenotype. Colonies of the variant endophyte are smaller than wild-type colonies and mucoid, with hyphal filaments forming aggregates. Evidence shows that the switch between colony morphologies occurs at a very high frequency, is reversible, and appears to be environmentally induced. This suggests that the switching phenomenon involves gene regulation rather than mutation. When endophyte-free plants are infected, with either white and cottony (wild-type) or mucoid (variant) fungal colonies, they assume a morphology consistent with the state of the fungus at the time of inoculation, that is normal or dwarfed, respectively. In addition, re-isolation of endophyte from either normal or dwarfed plants always yields white and cottony or mucoid colonies, respectively, suggesting that the host environment stabilizes the state of the fungus. Proteomic profiling revealed differences in protein expression between plants infected with either the wild-type or mucoid fungus. Furthermore, host plants containing the mucoid fungus have never flowered or produced seed. Thus, if this change in the fungal symbiont occurs in a competitive natural environment the mucoid fungus and its host plant may not persist beyond the first generation. This thesis provides insights into the plastic nature of fungal endophyte/grass symbiota and discusses possible mechanisms for the observed morphological switching in culture and host dwarfing. iii Acknowledgments I would like to acknowledge my supervisors Jan Schmid and Richard Johnson, this project has been conducted over a considerable period and I appreciate the perseverance and commitment demonstrated and the invaluable assistance given. I acknowledge the support of Syd Easton and Dave Hume, both explicit and implicit, for the undertaking of this project and my colleagues Stuart Card, Anouck de Bonth and Bob Skipp for tolerating my ongoing preoccupation with it. This project would not have been possible without the generous assistance of many people at AgResearch Grasslands, Palmerston North. I would particularly like to thank Mike Christensen who has been supportive of this project from its outset and a great mentor to me. Brain Tapper and Liz Davies for their assistance with chemistry along with Wade Mace who most recently has spent a great deal of time in this regard. Marty Faville for invaluable help with SSRs. Jen Pratt for help with RAPD work. Ningxin Zhang (IMBS, Massey University) and Shalome Bassett for teaching me the tricks of the trade required for the protein work. Igor Kardailsky for meristem dissections, Stefan Clerens (AgResearch, Lincoln) for protein spot analysis and John Koolaard for help with statistics. Thanks to Ann Truter and Cynthia Cresswell (IMBS) for their professionalism and engaging good humour and Debbie Hudson (AgR) for proof reading the final draft. I would especially like to thank Suzanne Kuijt for her help with cell measurements, reading and commenting on the manuscript and ongoing personal support. There are many others who have helped along the way, thanks to you all. iv Table of Contents Chapter 1 Introduction 1 1.1 Epichloë/Neotyphodium endophytes (epichloae) 1 1.1.1 Epichloë endophytes 1 1.1.2 Neotyphodium endophytes 2 1.2 Features of Neotyphodium endophytes 3 1.2.1 Symbiosis 3 1.2.2 Alkaloids 3 1.2.3 Host specificity 4 1.2.4 Observing Neotyphodium endophytes 5 1.2.5 Isolation and culture of Neotyphodium endophytes 6 1.2.6 Artificial infection of grasses 6 1.3 Neotyphodium lolii 7 1.3.1 Importance of N. lolii in New Zealand 7 1.3.2 Variation in Neotyphodium endophytes 8 1.4 Phenotypic variation 9 1.4.1 Spontaneous mutation 9 1.4.2 Phenotypic switching 10 1.4.3 Phenotypic switching in Candida albicans 10 1.4.4 Switching and the C. albicans mating-type locus 11 1.4.5 Mass conversion 12 1.5 Plant architecture 12 1.6 Aims 16 Chapter 2 Materials and Methods 17 2.1 Table of biological materials 17 2.2 Fungal isolation and culture 17 2.2.1 Media 17 2.2.1.1 ABPDA 17 2.2.1.2 4% water agar 18 2.2.1.3 Murashige and Skoog media 18 v 2.2.1.4 Broth 18 2.2.2 Isolation of fungus on solid media 18 2.2.3 Liquid culture of Neotyphodium endophyte 19 2.2.4 Macerate culture of Neotyphodium endophyte 19 2.3 Seedling inoculation 19 2.4 Endophyte detection 20 2.4.1 Immuno-detection 20 2.4.1.1 Antigen binding 20 2.4.1.2 Processing 21 2.4.2 Microscopic examination 21 2.4.3 Cell measurement 22 2.5 Endophyte elimination 23 2.6 RAPD analysis 23 2.6.1 DNA extraction 23 2.6.2 PCR reactions 24 2.6.3 Gel electrophoresis 24 2.7 Simple Sequence Repeats (SSRs) 24 2.8 Alkaloid determination 25 2.9 Generation, infection and growth of clonal material 25 2.9.1 Source of clonal plant material 25 2.9.2 Axenic culture of clonal plantlets 26 2.9.3 Inoculation of clonal plantlets 27 2.9.4 Growth of clonal plantlets 27 2.10 Proteomics 27 2.10.1 Harvest and preparation of plant material 27 2.10.2 Protein extraction 28 2.10.3 Determination of protein concentration 29 2.10.4 Protein gel separation/2-D analysis of protein 29 2.10.4.1 Rehydration 29 2.10.4.2 Isoelectric focusing 29 2.10.5 Second dimension 30 2.10.6 Staining and fixing 30 2.10.7 Protein analysis 31 vi Chapter 3 Results 32 3.1 A dwarfism-inducing fungus 32 3.1.1 Dwarf plants observed in a perennial ryegrass population 32 3.1.2 Neotyphodium endophyte fungus isolated from dwarfed plants differs from that isolated from normal phenotype plants 32 3.1.3 Removal of the mucoid P1 fungus restores the plant phenotype from dwarfed to normal 34 3.1.4 Infection of endophyte-free seedlings with the mucoid P1 fungus results in dwarfed plants 34 3.1.5 Plantlet clones infected with P1 are dwarfed 36 3.2 Tillering, flowering and cell size 37 3.2.1 P1-infected plants produced more tillers than endophyte-free clones although initially at a similar rate 37 3.2.2 P1 endophyte-infected plants produced a similar number of tillers to P41 endophyte-infected plants 38 3.2.3 Dwarfed plants infected with P1 do not flower under long day conditions following vernalisation 39 3.2.4 Failure of flowers to emerge from P1-infected plants is due to arrest in development of the inflorescence meristems 40 3.2.5 Gibberellin does not restore the dwarf phenotype to normal phenotype 42 3.2.6 The epidermal cells of dwarfed plants are similar in size to those of normal phenotype plants 42 3.3 DNA, protein and metabolic profiling 45 3.3.1 RAPD analysis shows that P1 has N. lolii profile 45 3.3.2 Simple Sequence Repeat data indicate that P1 is closely related to wild-type N. lolii 45 3.3.3 The level of key alkaloids is reduced in symbiosis between P1 and its host compared to P41 47 3.3.4 Ergovaline HPLC shows extreme difference in peak size at 36.0-36.2min 48 3.3.5 Dwarf P1- infected plants differ from normal P41-infected plants in their protein expression 48 3.3.6 LC-MS/MS 52 3.4 In culture studies 52 3.4.1 Fungal morphology is uniform when sub-cultured from newly emerged mycelium 52 3.4.2 Fungal morphology is not uniform when sub-cultured from mature P41 colonies 54 3.4.3 Mucoid phenotype colonies can arise spontaneously in cultures of P41 and induce dwarfing of host plants 54 vii 3.4.4 Mass conversions of cottony to mucoid phenotype can be induced in P41 cultures by density 55 3.4.5 Mass conversions of cottony to mucoid phenotype can be induced in P41 cultures by age 55 3.4.6 Evidence that the in culture cottony to mucoid transition is induced by a transmissible substance 57 3.4.7 Conversion from cottony to mucoid phenotype and vice versa can be induced by sub-culturing 57 3.4.7.1 Macerate culture subbing 57 3.4.7.2 Non-macerate subbing 58 3.4.8 Cottony and mucoid fungus grow differently in liquid culture 59 3.4.9 Infection of perennial ryegrass seedlings using cottony P41 fungus and mucoid P41 mycelium produced in culture 60 3.4.9.1 Ryegrass plants artificially infected with cottony P41 fungus display a normal phenotype 60 3.4.9.2 Ryegrass plants artificially infected with mucoid P41 mycelium that develops in culture have a low infection rate and display a dwarf phenotype 60 3.4.9.3 Ryegrass plants artificially infected with cottony P41 fungus that develops from a mucoid colony display a normal phenotype and infection rate 61 Chapter 4 Discussion 62 4.1 Likely mechanisms leading to the observed morphological switching in culture 64 4.2 The dwarf plant phenotype 66 4.3 Implications for the symbiosis 70 Chapter 5 Conclusion 72 Bibliography 73 Appendices Appendix I SSR traces 88 Appendix II Mass spectrometry data 90 viii List of figures Fig.1.1 Hyphal filaments growing from surface sterilised host tissue on PDA 6 Fig.1.2 Simplified model of plant organ size control 15 Fig.2.1 Inoculation of perennial ryegrass seedlings 20 Fig.2.2 Immunoblot 22 Fig.2.3 Aniline blue stained hyphae of Neotyphodium endophyte 23 Fig.2.4 A tiller prepared for surface sterilisation for the axenic production of clonal plantlets 26 Fig.2.5 Harvesting tissue for protein extraction 28 Fig.3.1 Phenotype of dwarf plants 33 Fig.3.2 Colonies of Neotyphodium lolii isolated from perennial ryegrass 33 Fig.3.3 Fungicide treated clone of dwarfed plant 35 Fig.3.4 Phenotypic comparison between uninfected and P1-infected plants 35 Fig.3.5 A single genotype clone of diploid perennial ryegrass Nui D; uninfected and infected 36 Fig.3.6 Tiller numbers of P1-infected and endophyte-free perennial ryegrass 37 Fig.3.7 Tiller numbers of perennial ryegrass clone Nui D infected with P1 and P41 endophyte 39 Fig.3.8 Fully vernalised plants of a dwarfed P1-infected plant and normal phenotype P41 endophyte-infected plant 40 Fig.3.9 Excised tiller meristems of P41-infected and P1-infected perennial ryegrass following vernalisation and exposure to long days 41 Fig.3.10 Tiller lengths of endophyte-infected and endophyte-free clones treated with gibberellin 43 Fig.3.11 Gibberellin treated plants 43 Fig.3.12 Compound light microscope images of leaf tissue imprints 44 Fig.3.13 RAPD PCR products of endophyte DNA 46 Fig.3.14 HPLC trace of endophyte-infected diploid perennial ryegrass 49 Fig.3.15 HPLC chromatograms of P41-infected and P1-infected perennial ryegrass clone Nui D 50 Fig.3.16 2-Dimensional electrophoresis of total protein extracted from P41 and P1 endophyte-infected perennial ryegrass 51 ix Fig.3.17 Isolation of endophyte fungus on ABPDA 53 Fig.3.18 Mature colony of P41 and macerate colonies derived from it 53 Fig.3.19 P41 macerate cultures that developed both cottony and mucoid colony types 54 Fig.3.20 Macerated P41 fungus plated at high and low density 55 Fig.3.21 Macerated P41 fungus plated on solid media that became mucoid with age 56 Fig.3.22 Macerated P41 fungus plated to ABPDA at low, high and split densities 56 Fig.3.23 Colonies on ABPDA from macerated mycelium derived from mucoid and cottony P41 cultures 58 Fig.3.24 Sub-cultured fungus from a macerate culture of P41 59 Fig.3.25 Flask cultures of P41 and P1 in PDB 60 Fig.3.26 Schematic overview of fungal and plant phenotypes following isolation, infection and sub-culturing using both mucoid and cottony N. lolii 61 x List of Tables Table 2.1 List of biological materials used in this study 17 Table 3.1 SSR peaks of P41, AR5 and P1 N. lolii 46