Dissecting molecular mechanisms of disease in the wheat pathogen, Parastagonospora nodorum Oliver L. Mead, March 2019 A thesis submitted for the degree of Doctor of Philosophy of The Australian National University © Copyright by Oliver Lachlan Mead (2019) All Rights Reserved I, Oliver L. Mead, declare that the following work is entirely the product of my own efforts, except as where otherwise indicated. ……………………………………………………………………. 2 Acknowledgements Peter, thank you for your support, direction and mateship over the last seven years. Within and outside of your you have provided an environment that combines a drive for excellent science and the thrill of enquiry with a casual and supportive gregariousness. I appreciate the time you made for me- especially for my habit of leaning in your door way to ask “Hey uh… quick question”, often chaotic home life and wandering attention. I have enjoyed my time in your lab very much and couldn’t have asked for more. Thank you for taking on that idealistic undergraduate. Megan, Simon, Susan, Eli, Erin and Jordi, thank you for being my second family. Your companionship (and couches) made some of my hardest moments a pleasure. Mum and Dad, thank you for listening to my abundant outbursts (both angry and overly enthusiastic) about primers, fungi and the scientific accuracy of TV programs. Please forgive my absence, both physically and mentally, I’ve been preoccupied having fun. This project was supported by an Australian Government Research Training Program (RTP) Scholarship and a Grain Research and Development Corporation (GRDC) scholarship top-up. 3 “Don’t Panic” -Douglas Adams “Panic!” -Every single neuron in my possession Dashing scientists: “Stago! Give up your secrets!” Spores drift on the breeze 4 Abstract The wheat pathogen Parastagonospora nodorum (Syn. Phaeosphaeria nodorum) belongs to the pathogen-enriched class of fungi known as the Dothideomycetes. P. nodorum is a host specific pathogen that causes annual losses to the Australian wheat industry in excess of $100 million AUD. These losses are due to the pathogens’ mechanism of infection and reproduction. P. nodorum, like other Dothideomycetes, is a polycyclic necrotroph. Typical infection begins with either wind-dispersed ascospores or splash-dispersed pycnidiospores landing on a leaf, followed by invasive hyphal growth. The fungus uses effector proteins in a gene-for-gene manner with the host to kill the plant tissue, generating lesions of necrotic tissue surrounding the infection site. The decaying plant tissue is then assimilated and over the course of several days pycnidia develop, continuing the disease cycle. By decreasing the photosynthetic area of the Flag and Flag-1 leaves, the fungus inhibits grain-filling leading to yield losses. Sporulation and virulence are the two crucial aspects for disease development in the P. nodorum-wheat pathosystem and form the basis of this project. A forward genetics approach was employed to discover novel mechanisms by which P. nodorum facilitates infection on wheat. A library of random insertion mutants of P. nodorum was generated utilising Agrobacterium-mediated transformation. Subsequently, the library was screened for loss of virulence phenotypes on a susceptible cultivar of wheat. This was complemented by a second screen identifying gain of virulence phenotypes on a non-susceptible cultivar. From a library of 950 transformants seven displayed a consistent avirulent phenotype on the susceptible wheat cultivar, and one displayed a partial gain of virulence on the non-susceptible cultivar. The genomes of the seven strains of avirulent P. nodorum were then sequenced via the Illumina 5 MiSeq short-read platform. De novo genome assembly identified two disrupted loci in a single avirulent strain. These loci were identified through BLAST homology as a putative Copper-dependent amine oxidase, and a Catechol-1,2-dioxygenase. A previous study identified a Catechol-1,2-dioxygenase as a key virulence factor in Fusarium oxysporum. However, an independent gene disruption concluded that this gene is not required for virulence in P. nodorum. Complementary to the forward genetics approach to identifying novel mechanisms of virulence, a combined transcriptomics and metabolomics approach was employed to decipher sporulation in this pathogen. This is of particular interest as the canonical sporulation pathways in Aspergillus or Neurospora have been previously shown to be not applicable in P. nodorum. This aspect of my project involved a differential gene analysis of fungal material collected at three key developmental time points. Sporulation was initiated in vitro by the non-proteinaceous amino acid, gamma- aminobutyric acid (GABA). I have previously published a reported describing sporulation induction in P. nodorum and related species by application of exogenous GABA. By comparing gene expression in the fungus just prior to pycnidia formation, sporulation and subsequent to sporulation, I was able to identify several key genes which are involved in initiating a sporulation cascade. Further, I was able to elucidate a polyamine pathway intrinsically linked to P. nodorum sporulation. This study garners genetic and biochemical insight into the two aspects of the P. nodorum life cycle that are crucial for disease development. 6 Table of Contents Dissecting molecular mechanisms of disease in the wheat pathogen, Parastagonospora nodorum ..................................................................................... 1 Acknowledgements .................................................................................................. 3 Abstract .................................................................................................................... 5 Table of Contents ..................................................................................................... 7 List of Figures ......................................................................................................... 12 List of Tables ........................................................................................................... 15 List of Abbreviations ............................................................................................... 16 Chapter 1: General introduction to Parastagonospora nodorum and forward genetics ............................................................................................................................... 19 1.1 Parastagonospora nodorum ........................................................................ 19 1.1.1 Parastagonospora nodorum is a threat to food production ................................ 19 1.1.2 A polycyclic infection cycle is key to disease development.................................. 19 1.1.3 Proteinaceous effectors are the driving cause of SNB ......................................... 21 1.1.3.1 Sn.ToxA .......................................................................................................................... 24 1.1.3.2 Sn.Tox1 .......................................................................................................................... 26 1.1.3.3 Sn.Tox3 .......................................................................................................................... 26 1.1.4 Effector driven virulence is only one component of SNB ..................................... 27 1.1.4.1 A short-chain dehydrogenase is required for differentiation of the pycnidium sub- parietal layer, leading to sporulation ............................................................................................... 29 1.1.5 Sporulation is a vital component for P. nodorum to cause disease ...................... 30 1.2 Forward genetics ......................................................................................... 34 1.2.1 Definition, types and history ............................................................................. 34 1.2.2 Random Insertional Mutagenesis ...................................................................... 36 1.2.3 Random gene disruption via Agrobacterium-mediated transformation .............. 38 1.3 Aims of this project ..................................................................................... 39 1.4 References for Chapter 1 ............................................................................. 41 Chapter 2: Construction and characterisation of a Parastagonospora nodorum random mutation library ........................................................................................ 51 2.1 Introduction ................................................................................................ 52 2.2 Materials and Methods ............................................................................... 53 7 2.2.1 (Table 2.01: Primers used in this chapter) ........................................................... 56 2.2.2 (Table 2.02 Media used in this chapter) ............................................................. 56 2.2.3 (Table 2.03: Buffers and solutions used in this chapter) ...................................... 56 2.2.4 (Table 2.04: Organisms used in this chapter) ...................................................... 58 2.2.5 Protocols .......................................................................................................... 59 2.2.5.1 Culturing of Agrobacterium tumefaciens ...................................................................... 60 2.2.5.2 Culturing of Parastagonospora nodorum ....................................................................