Using Rnaseq to Identify Candidate Effectors in the Septoria Canker-Poplar Pathosystem

Sphaerulina musiva, the cause of Septoria leaf spot and stem canker, is one of the most important pathogens of Populus spp. in North America. Although biomass losses due to this disease can be as high as 63% of total yield, no information on the mechanism(s) of virulence is available. To this end, six trees of the susceptible species, Populus trichocarpa, were point inoculated with a virulent isolate of S. musiva. Inoculations were conducted by placing a 5 mm plug of sporulating mycelium over a lenticel, 15 cm from the soil surface. Approximately 100 mg of symptomatic tissue from three trees were harvested at two time points: 2-weeks post-inoculation and 3-weeks post-inoculation. Three fungal cultures grown in liquid V-8 media were used as a comparison (control). The mRNA from S. musiva inoculated stems and the fungal cultures were extracted and pooled to generate RNAseq libraries for the Ion Torrent PGM^™ platform. The S. musiva genome, available online, was used as a reference for mapping reads and differentially expressed genes were estimated using the reads per kilobase per million (RPKM) calculation with the CLCBio software. The RNAseq analyses identified 20 differentially expressed genes representing candidate necrotrophic effectors that were significantly up-regulated in planta in comparison to the controls. The gene exhibiting the greatest differential expression with a predicted secretion signal was transformed into the pCAL-n-EK expression vector. The expressed protein was purified and infiltrated into susceptible P. trichocarpa leaves to validate the role of the protein as a putative necrotrophic effector.