Analysis of the apple rubbery wood disease by next generation sequencing of total RNA

Apple rubbery wood (ARW) is a disease causing abnormally flexible (rubber-like) branches in susceptible apple varieties. Despite previous efforts, the cause of the ARW remains unclear. Using Illumina next generation sequencing (NGS) of total RNA (RNA-Seq), an ARW infected apple and a healthy control were sequenced. In order to select NGS reads specific for the ARW sample, a transcriptome of the healthy apple was de novo assembled and served as a reference sequence. Reads from the ARW sample which did not align to the reference were used to search nucleotide and protein sequence databases for a possible ARW-causing pathogen. In this way, the search for a potential ARW causal agent was narrowed down to several sequences homologous to the retroviral proteins, proteins interacting with Geminivirus, Tobamovirus as well as sequences homologous to the genus Phlebovirus. Expression analysis of the genes involved in the synthesis of lignin showed decrease in expression of three out of four genes analyzed in the ARW sample.