Transcription profiling by high throughput sequencing of grapevine plants inoculated with Neofusicoccum parvum

In the vineyard, symptoms of the grapevine trunk disease Botryosphaeria dieback do not appear until 1 or 2 years after the causal fungus Neofusicoccum parvum establishes a necrotic canker in the permanent woody structure of the vine. There are preventative management practices, but growers tend to wait until symptoms are widespread, at which point prevention has limited efficacy. Toward development of an early detection tool, we examined the leaves of inoculated vs. non-inoculated plants for differential gene expression via RNASeq. Stems were examined to monitor spread of the infection, and its spatial and temporal relationship to anatomical changes via light microscopy and high resolution computed tomography (HRCT). The early stage of infection occurred prior to 2 months post-inoculation (MPI), at which point spread of the pathogen beyond the inoculation site was greatest. This incubation period was also characterized by the largest stem lesions, the highest levels of fungal colonization and xylem vessels fully-occluded by gels, and the lowest starch content of xylem fibers and rays. Prior to 2 MPI, RNASeq and validative qPCR analysis identified eight candidate markers, which are transcriptionally activated by infection, but not by wounding alone. Our best marker genes, a dehydrin, a BURP domain containing protein and an abscissic acid-induced wheat plasma membrane polypeptide 19 (AWPM-19), identified the pathogen’s presence with high specificity. Screening of genome-wide expression data revealed that this signature is not affected by many abiotic and biotic stresses. Asymptomatic leaf mRNA profiles of Neofusicoccum parvum infected wounded plants (IW) and non-infected wounded plants (NIW) at two different time points post inoculation (0 MPI, 0.5-1.5 MPI) were generated by deep sequencing in biological triplicates using Illumina HiSeq2500 technology.