Fruggiero, Carmine (2025) Dual RNA-Sequencing assessment and implementation to study transcriptome alteration of Solanum lycopersicum during the interaction with Orobanchaceae spp. [Tesi di dottorato]

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Abstract

Transcriptomics aims to achieve a comprehensive understanding of each RNA molecule in a given cell (the transcriptome), under specific conditions. The primary method for transcriptome analysis is RNA-sequencing where, typically, each processed sample originates from a single organism. In the case of host-parasite interactions, the close biological interdependence complicates this approach, as the parasite relies on the host for nutrients. To study gene expression changes in both host and parasite simultaneously, dual RNA-seq has emerged as the method of choice. This technique enables the concurrent analysis of gene expression from both interacting organisms, even when their physically separation is difficult. A key challenge in dual RNA-seq lies in the accurate in silico discrimination of reads. The existence of shared homologous sequences between organisms can lead to cross-mapping, a phenomenon where reads from one organism map to the other or to both. Read discrimination becomes most reliable when the host and parasite belong to different kingdoms. In this study, host-parasite interactions were simulated to evaluate the feasibility of dual RNA-seq when both organisms belong to the Plantae kingdom. The simulations demonstrated that the method is reliable and highlighted the possibility of executing it using two approaches (Chapter 2). Dual RNA-seq execution requires programming skills, which may not be accessible to all researchers. In contrast, graphical user interfaces (GUIs) have been developed for the classical bulk RNA- seq, making the analysis process more accessible. To bridge this gap, inDAGO was developed as an open-access software, that enables users to conduct dual RNA-seq analysis without the need for programming. inDAGO provides the option to choose between two approaches and includes an extension for bulk RNA-seq analysis. The software was tested using data from the previous simulations (Chapter 3). The parasitic plant Phelipanche ramosa, a member of the Orobanchaceae family, and Solanum species provide an example of host-parasite interaction to apply dual RNA-seq pipeline using inDAGO. The two tomato species Solanum pennellii and Solanum lycopersicum differ in their response to Phelipanche ramosa infection, with the former showing tolerance and the latter being susceptible. An analysis was carried out to explore the genetic basis of S. pennellii tolerance mechanisms. Genes involved in the strigolactone pathway - known to stimulate Orobanchaceae germination - were identified in S. lycopersicum. Their orthologous in S. pennellii were inferred. Two of these genes exhibited polymorphisms that may explain the resistance mechanism related to parasite seed germination (Chapter 4). This thesis demonstrated three findings: (i) dual RNA-seq is reliable even for taxonomically close organisms; (ii) a GUI was developed to make the analysis accessible to biologists without programming skills; and (iii) the tolerance mechanisms of S. pennellii were preliminarily explored. These pave the way for future transcriptomic investigations of S. pennellii introgression lines during interactions with P. ramosa using the inDAGO software in a dual RNA-seq experiment.

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