Alternative splicing in the fungal plant pathogens Ceratocystis fimbriata and C. eucalypticola

Abstract

Alternative splicing (AS) is a complex and dynamic process that results in the production of multiple mRNA isoforms from a single gene. Through the production of multiple mRNA isoforms from a single gene, AS increases eukaryotic complexity by expanding the transcriptome, diversifying the subsequent proteome, and acting as a source of gene regulation. Alternative splicing (AS) offers an efficient and swift method for generating phenotypic novelty when compared to mutations in protein coding sequences. This is because alternative splicing has the potential to add, remove or drastically alter entire protein domains. In addition to changing protein structure, alternative splicing can also influence subcellular localization and secretion. Alternative splicing in fungi was originally considered to be relatively rare compared to higher eukaryotes but recent studies have shown that this is in fact not the case. Fungal alternative splicing is associated with key biological processes including adaptation and pathogenicity. Alternative splicing has been extensively studied in animals and plants, however, our understanding of alternative splicing in fungi is limited due to the absence of in-depth studies. Intron retention, an AS event in which an intron is retained in the processed mRNA, is the most frequently observed pattern of alternative splicing in fungi. Intron retention is typically thought to be associated with gene regulation via the nonsense-mediated decay pathway, however, there is growing evidence indicating that intron retention can influence protein structure, function and localization. The Ceratocystis fimbriata sensu lato (s.l.) complex is a group of closely related plant pathogenic fungi. Although these pathogens are both genetically and morphologically similar, they have a diverse host range and tend to be host specific. Examples of economically important hosts infected by these pathogens include sweet potato, eucalyptus, mango and acacia. Two members of this complex include C. fimbriata sensu stricto (s.s.) and C. eucalypticola. Ceratocystis fimbriata is one of the most well-known members of this group and is the causal agent of black rot disease on sweet potatoes which has resulted in significant postharvest losses worldwide. Ceratocystis eucalypticola on the other hand is a pathogen of eucalyptus and punica, both members of the Myrtaceae. To bridge the large knowledge gap regarding AS in fungi, the overarching goal of this dissertation is to provide further insight and enhance our understanding of alternative splicing in fungi and their potential roles in adaptation. This goal is pursued by investigating two main research questions. Firstly, what is the genomic landscape of alternative splicing among members of the Ceratocystis fimbriata sensu lato complex under both in-vitro and in-planta conditions? Secondly, does alternative splicing contribute to adaption in these fungi? The primary aim of this study was to identify, characterise, and compare alternative splicing events between two closely related species of the Ceratocystis fimbriata s.l. complex, namely, C. fimbriata and C. eucalypticola, under both in-vitro and in-planta conditions. To achieve this aim, we had the following research objectives: i) generate complete genome assemblies for each of these species, ii) perform long and short-read transcriptome sequencing for each species under both in-vitro and in-planta conditions, iii) identify and compare alternative splicing events between the two species and between conditions. The literature review chapter of this dissertation serves to provide an overview of the intricate process of alternative splicing and its regulation. In addition to covering the topic of alternative splicing in general, this chapter explores the current knowledge regarding alternative splicing in fungi. Furthermore, the chapter provides insight into the methodologies regarding RNA sequencing and alternative splicing discovery. In the second chapter, I generate highly complete genome assemblies and annotations for two closely related plant pathogenic fungi, namely, Ceratocystis fimbriata and C. eucalypticola. Following the generation of the genome assemblies and annotations, I report on the discovery and comparison of alternative splicing events between these two closely related fungi. Furthermore, I investigate alternative splicing patterns across in-vitro and in-planta conditions.