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Together forever: Molecular biology of a mycovirus-fungus symbiosis

Cao, Chi Thi Hien (2021) Together forever: Molecular biology of a mycovirus-fungus symbiosis. PhD thesis, Murdoch University.

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The Orchidaceae is the second-largest family of flowering plants, numbering about 900 genera and almost 30,000 species. All orchids maintain intimate relationships with fungi for some or all of their lives. In turn, mycoviruses widely infect fungi. Three mycoviruses co-infected one isolate of an undescribed mycorrhizal fungus, Ceratobasidium sp., isolated from the roots of an undescribed Pterostylis orchid growing naturally in remnant bushland on the Murdoch University campus. The three mycoviruses were evolutionarily close to one another, comprising large RNA viruses that each represented a new species in genus Endornavirus, viz Ceratobasidium endornavirus B, Ceratobasidium endornavirus C, and Ceratobasidium endornavirus D (CtEVB, CtEVC, and CtEVD, respectively). Endornaviruses encode no movement proteins, no capsid protein(s) and have no vectors; they spread between fungi via anastomosis, from cell-to-cell at cell division, and geographically in wind-borne spores or hyphal fragments. It is hypothesized that endornaviruses are ancient, having co-evolved with their hosts. Our project examined aspects of interactions between three endornaviruses and their Ceratobasidium sp host by first attempting to remove (cure) the viruses from their host, and then by applying metabolomic and small RNA approaches to understand deeper aspects of their biology.

A large and critical part of the project was creating a fungal line that lacked the three viruses, thereby enabling a comparison between virus-infected and virus-free isogenic (genetically identical) fungal lines. Curing treatments were with antibiotics (kanamycin, streptromycin, rifampicin, ampicillin and cyclohexamide), most of which had never been used for this purpose, and with hyphal tipping. Surprisingly, various treatments worked very well at eliminating one or two viruses, but not a third. CbEVC was the most recalcitrant virus under all treatments, while CbEVB was relatively simple to eliminate, and CbEVD had different response to different treatments. I present hypotheses that may be tested to explain why this might be so. These experiments provided clues that the three endornaviruses interacted with their host in profoundly different ways, and will direct future research in exciting new directions. For the current project, a virus-free isogenic line of Ceratobasidium sp. was created.

Various classes of small RNAs play important roles in gene regulation and virus defence in most or all eukaryotic organisms, including fungi. These are generated by a nucleoprotein-based system called RNA-interference (RNAi). I assessed one of these classes, micro-RNAs (miRNA), to investigate how virus infection influences gene regulation in the fungal host. Before I could undertake this experiment, the sequence of the fungal genome was required. The fungal genome was sequenced and partially annotated. Fungus-derived miRNAs were subsequently mapped to the genome sequence from virus-infected and virus-free fungal strains and compared. Analysis of miRNA patterns showed that presence of the viruses stimulated differential regulation of some fungal genes. Notable pathways influenced by virus presence were amino acid synthesis, some steps of the TCA cycle, and the putrescine pathway. These were all were significantly up-regulated in the presence of endornaviruses. Other pathways were down-regulated.

The other class of small RNAs studied was those derived from the three viruses. Analysis of these virus-derived small-interfering RNAs (siRNA) revealed RNAi machinery of the fungal host was actively targeting all three virus genomes. Regions of the viral genomes were targeted differentially in the three viruses. Thus, although endornaviruses are thought to have co-evolved with their hosts over vast time periods, in this case the host was actively targeting the viruses using RNAi. Nevertheless, the three viruses escaped complete destruction by the host’s anti-virus machinery, and I hypothesise that at least two of the viruses (CtEVC and CtEVD) encode (inefficient) suppressors of RNAi.

Although these viruses and fungi may have co-evolved together over vast periods of time, survival of the viruses, and perhaps the fungus, appears to rely on a balance between replication of viral genomes and destruction of them by RNAi, with the balance tipped slightly in favour of the virus. The fungus actively destroys the viruses and its biochemistry is fundamentally influenced by them. It seems certain that the viruses encode factors that actively down-regulates but doesn’t turn off the host anti-viral machinery. An unexpected finding was there was some evidence in our data that CtEVB encodes a factor that up-regulates RNAi-based destruction of the three mycoviruses. I present a hypothesis where the viruses control both up-regulation and down-regulation of the RNAi machinery, and how this hypothesis might be tested. How these interactions affect fungal ecology, and, in turn affect the ecology of orchids, is an area of exciting future research.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): College of Science, Health, Engineering and Education
Western Australian State Agricultural Biotechnology Centre
Supervisor(s): Wylie, Steve
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