Transcriptomic analysis of the interaction geminivirus-tomato

Geminiviridae family is one of the main families of plant pathogenic viruses with large relevance as they cause great losses worldwide in commercial crops and crops destined to food production. Geminiviruses present a little single-stranded DNA genome and a capsid composed of two twin icosahedral parts. Tomato Yellow Leaf Curl Virus (TYLCV) belongs to the Begomovirus genus and is transmitted by the whitefly Bemisia tabaci. With only 6 viral proteins, this geminivirus must create a proper environment for viral replication, transcription and propagation. Behind the apparent simplicity of geminiviruses lies a complex network of molecular interactions with their host and even their natural vector, which induces a wide variety of transcriptional, post-transcriptional and chromatinic changes in both the plant and the geminivirus. In order to study these changes and decipher the effects of the transmission vector on the infection, we carried out a global approximation of the TYLCV-tomato interaction to generate integrated single-base resolution maps by NGS (next-generation sequencing) of the transcriptome, smallRNAome and methylome of the pathogen and the host. Tomato plants (Moneymaker) were infected with TYLCV under controlled conditions of light and temperature using Agrobacterium tumefaciens or its natural vector. Apical tissue from these plants was collected at different time points (2, 7, 14 and 21 days after inoculation), and three biological replicas were generated for each treatment and time. Total RNA and DNA was extracted and analysed by RNA-Seq, smallRNA-Seq and Bisulfite-Seq. The transcriptome of the tomato-TYLCV interaction will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Genetic and epigenetic characterization of the geminivirus-host interaction

Fecha de lectura de Tesis Doctoral: 29 de abril de 2019Los geminivirus constituyen una familia de virus de plantas transmitidos por insectos, contienen una o dos moléculas de DNA de cadena sencilla empaquetadas en dos partículas gemelas fusionadas. Es uno de los patógenos de plantas más devastadores en el mundo, causando importantes pérdidas de cosechas en plantas de tomate, maíz, algodón, cassava, etc. Durante la infección, los geminivirus interfieren con la maquinaria celular de la planta. Además deben confrontar mecanismos de defensa tales como el silenciamiento génico. Como primer objetivo, se han generado y analizado de forma integrada los mapas transcriptómicos (mediante RNA-Seq y small RNA-Seq) y epigenético (mediante Bisulfite-Seq) del geminivirus TYLCV durante una infección en tomate. Esto nos ha permitido describir, a resolución de nucleótido, la relación existente entre la expresión génica, el patrón de metilación del genoma y los sRNAs derivados (vsRNAs) de TYLCV. Como segundo objetivo, se ha determinado la relevancia de la maquinaria de metilación del DNA de la planta en una infección por geminivirus en Arabidopsis y N. benthamiana. Nuestros resultados indican que la inactivación de la maquinaria de metilación de mantenimiento no interfiere en la acumulación de TYLCV en Arabidopsis. Además, los niveles de DNA de TYLCV no son significativamente diferentes en hojas o tejido floral de plantas silvestres y mutantes. Sin embargo, los niveles de DNA del geminivirus BCTV están incrementados en plantas mutantes en la maquinaria de metilación, tanto en tejido floral como en hojas apicales. Por otro lado, el silenciamiento de los genes involucrados en metilación del DNA en N. benthamiana no tuvo efecto en la acumulación de TYLCV o TYLCSV. Como tercer objetivo, se determinó la transmisión de CaLCuV y BCTV a través de semilla, concluyéndose que no existe evidencia de dicha transmisión

Global analysis of the geminivirus tomato yellow leaf curl virus (TYLCV) genome expression and DNA methylation during infection in tomato plants

Geminiviruses are the plant largest family of DNA viruses that cause devastating diseases in crops worldwide. Upon the infection, viral genomes and transcripts are targeted by the plant gene silencing machinery, that processes the viral transcripts into viral small interfering RNAs (vsRNAs) and directs viral DNA methylation. To counteract these plant responses, geminiviruses encode proteins that are able to supress both, post-transcriptional and transcriptional gene silencing. To get an insight into geminiviral infection, the transcriptional and DNA methylation landscape of the geminivirus TYLCV (Tomato yellow leaf curl virus) from tomato-infected leaves, was characterized for the first time at high coverage by Illumina high-throughput sequencing during a time course infection. Three week-old tomato plants (Solanum lycopersicum cv. Moneymaker) were infected with TYLCV by agroinoculation or using its natural vector, the whitefly Bemisia tabaci. RNA and DNA were extracted from samples collected at 2, 7, 14 and 21 dpi (days post ingfection) and RNAseq, smallRNAseq and WGBS (Whole Genome Bisulfite Sequencing) was performed. We will present the RNA and vsRNA expression profiles along the infection and the complete DNA methylation landscape of a geminivirus by both inoculation methods, discussing the correlation with viral transcripts, vsRNA and viral DNA accumulation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech