Comparative RNA-seq for the Investigation of Resistance to Phytophthora Root Rot in the Red Raspberry 'Latham'

Mechanisms of resistance to Phytophthora root rot (PRR) (caused by Phytophthora rubi) in the red raspberry (Rubus idaeus) 'Latham' were investigated with a genome-wide expression study. Root-tissues of 'Latham' (resistant to PRR) and 'Titan' (susceptible to PRR) were sampled from controls at 48 h, 5 days, and 20 days post inoculation. Total mRNA was extracted and sequencing libraries were prepared. Over 70,600,000 quality filtered 86 nt reads were sequenced with an Illumina Genome Analyzer IIx. RNA-seq was performed with these data using a freely available bioinformatics pipeline (Bowtie-TopHat and Cufflinks). The recently completed draft genome sequence of the woodland strawberry (Fragaria vesca) was used as the reference for alignment. Of 16,956 unique and putatively expressed genes detected there were 12,823 that we considered significantly expressed because the lower bound of the 95% confidence interval did not include a FPKM of zero. Furthermore, only genes that were significantly differentially expressed after Benjamini Hochberg False Discovery Rate correction for multiple testing were considered. Between the resistant control ('Latham' un-inoculated) and resistant treatment ('Latham' inoculated) 702 genes were significantly differentially expressed with a log fold change greater than 0.5 or less than -0.5 and an FPKM >3 in either the control or treatment. Further analysis was targeted toward genes known to play a role in plant defense. Significant results include: 1) increased expression of WRKY transcription factors, 2) PR genes, and 3) key genes in the citric acid cycle and the lignin biosynthetic pathways. These results are being confirmed with quantitative RT-PCR. This study not only reveals putative mechanisms of durable resistance in 'Latham', but it demonstrates how the newly released draft genome of Fragaria vesca can be used as a resource for genome-wide expression analysis in Rubus species.