Investigating the molecular mechanisms underlying avocado sunblotch disease

Abstract

Avocado sunblotch disease is recognised as a major global concern for avocado production, and has a significant impact on the South African avocado industry. The disease is marked by the appearance of distinct chlorotic symptoms on fruit, stems and/or leaves of affected trees. These symptoms are triggered after infection by avocado sunblotch viroid (ASBVd) - a small, single-stranded molecule of RNA which does not encode any proteins. Importantly, infection by ASBVd may remain asymptomatic for long periods of time, though avocado trees without chlorotic symptoms have been known to display altered growth habits and decreased fruit yield. As yet, there have been no studies into the molecular mechanisms underlying the subtle phenotypic changes observed in asymptomatic ASBVd-infected avocado, and the process by which the viroid elicits chlorosis in symptomatic interactions remains unknown. Investigations into other viroid-host interactions have explored how the host transcriptome changes upon viroid infection, and how cleavage of viroids by host RNA silencing machinery may have inadvertent downstream effects on the accumulation of host transcripts, which can lead to symptom development. The aim of this work was therefore to elucidate processes involved in ASBVd pathogenesis using next-generation sequencing (NGS) to explore the host transcriptome in asymptomatic ASBVd-infected avocado, as well as to investigate whether viroid-derived small RNAs (vd-sRNAs) produced from ASBVd can be implicated in the triggering of leaf chlorosis in symptomatic infections. Analysis of the host transcriptome in six asymptomatic ASBVd-infected avocado nursery trees revealed notable changes in host gene expression in symptomless carrier trees. Asymptomatic infection by ASBVd resulted in the differential expression of over 600 avocado genes with roles in several important plant processes. Significantly affected host pathways included plant immune responses, hormone signalling networks, transcription regulation, production of secondary metabolites, transport of cellular compounds and processing of proteins. Observations of transcriptional changes in ASBVd-infected trees without chlorosis indicated that latent viroid infection influences molecular pathways in avocados, even when sunblotch symptoms are absent. This research was the first to examine the impact of ASBVd infection on global gene expression in avocado, highlighting important pathways in this plant-pathogen interaction which can be explored in depth in future investigations. To elucidate molecular mechanisms responsible for manifestation of sunblotch symptoms, we examined the viroid sequence variants and vd-sRNAs present in bleached and asymptomatic leaf tissues from symptomatic avocado trees. The mutation within the ASBVd genome associated with chlorotic symptoms was confirmed to be the addition of a uracil residue within positions 115-118 of the genome. ASBVd variants carrying this pathogenic determinant accumulated to different levels in green and yellow leaf tissues of symptomatic trees. The elevated viroid load in bleached tissues resulted in the production of copious ASBVd-derived small RNAs (ASBVd-sRNAs), which were significantly more abundant in yellow tissues when compared to green tissues. ASBVd-sRNAs containing the pathogenic determinant associated with chlorosis were limited almost entirely to yellow tissues – these were predicted to target many avocado genes for silencing. Twenty-five of the putative targets were shown by transcriptome analysis to be downregulated in yellow tissues relative to green tissues. One of the suppressed genes had strong evidence for RNA silencing guided by ASBVd-sRNAs, and its corresponding protein is predicted to function in avocado chloroplast pathways. The silencing of this gene in yellow tissues suggested that its downregulation might have a role in triggering the bleaching of leaves in ASBVd-infected avocado. This study was the first to employ NGS to examine the ASBVd-sRNAs associated with bleached leaves of sunblotch-affected trees, and represents the earliest exploration of putative avocado genes whose silencing by ASBVd-sRNAs may be involved in the elicitation of bleaching symptoms in avocado sunblotch disease. The findings presented in this thesis signify the first recent exploration of the ASBVd-avocado interaction. Collectively, the results of our investigation start to shed light on the molecular mechanisms involved in ASBVd pathogenesis. This work can be built on in future studies to present an even more comprehensive understanding of how ASBVd infection affects the avocado host and triggers avocado sunblotch disease.