Project description:To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoots cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Our data suggest that current approaches to correlate host gene expression changes under viral infection conditions to specific infection processes or disease symptom development, based on the interpretation of individual gene functions, have severe limitations. Integrative approaches that can take into account plant development stages, gene threshold levels as well as compensatory, synergistic and antagonistic effects may be necessary to develop a sound systems understanding of the biological significance of host gene expression changes during infection. Compare the transcript profiling of ASGV-infected asymptomatic apple planlets (AP-Vinfect) and virus-free apple plantlets (AP-Vfree) by deep sequencing using Illumina RNA-Seq to check whether lots of genes were modulated by ASGV infection.
Project description:miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance.
Project description:To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoots cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Our data suggest that current approaches to correlate host gene expression changes under viral infection conditions to specific infection processes or disease symptom development, based on the interpretation of individual gene functions, have severe limitations. Integrative approaches that can take into account plant development stages, gene threshold levels as well as compensatory, synergistic and antagonistic effects may be necessary to develop a sound systems understanding of the biological significance of host gene expression changes during infection.
Project description:Virus elimination is indispensable for the maintenance of stone fruit plantations, because these pathogens can cause serious crop damage and crop losses. Currently we do not possess efficient plant protection methods against viruses therefore prevention has a prominent role. In order to prevent infections, pathogen-free propagation material production and application of effective diagnostic methods have essential role. Our examined peach samples derived from isolator houses and stock nurseries of Fruitculture Research Institute of NARIC. With the help of highly sensitive metagenomic diagnostic methods: such as next generation sequencing of small RNAs, we are able to detect all of the presenting pathogens within our samples. During preparation steps, total RNA was isolated from leaf samples, RNA pools were made from the varieties, and then small RNA libraries were prepared. Sequencing was performed on Illumina platform and we used CLC Genomics Workbench for the bioinformatics evaluation. Results were verified by RT-PCR and Northern-blot. PCR products were cloned into pJET vector and Sanger sequenced. As a result, we detected nectarine stem pitting-associated virus (NSPaV), peach associated luteovirus (PaLV) and also peach latent mosaic viroid (PLMVd) which presence has to be checked regularly. Moreover we proved the incidence of PLMVd and PaLV first time in Hungary. We suspect, that the source of the viral infection might be the propagation material, which was used as a base for the variety collection in this isolator house.
Project description:miRNAs are key players in multiple biological processes, therefore analysis and characterization of these small regulatory RNAs is a critical step towards better understanding of animal and plant biology. In apple (Malus domestica) two hundred microRNAs are known, which most probably represents only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of twelve small RNA libraries obtained for fire blight resistant and fire blight sensitive trees. In the sequencing results we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop qPCR and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. In this study we considerably expand the apple miRNAome by identifying and characterizing dozens of novel microRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance. Actively-growing shoot tip tissue samples were collected from twelve apple trees, which includes three biological replicates of each following scion-rootstock combinations: B.9, G.30, M.111 and M.27.