Plasmopara viticola sporangia and in vitro germinated spores Transcriptome

Leaves with oil spot symptoms were collected from V. vinifera in the Grape Repository of Northwest A&F University, Yangling, Shaanxi, China. Leaves were washed once in water, then three times in sterile water and placed abaxial surface uppermost in the dark at 100% RH until sporulation occurred. Sporangia were washed off the leaves by gentle shaking in distilled water and the resulting sporangial suspensions were used for this study.To collect sporangia, the suspensions were centrifuged at 4? for 15 min at 2200 g. Sporangia were re-suspended in 1 mL of sterile distilled water and transferred to 2.0 mL EP tubes. Suspensions were centrifuged for 2 min at 12,000 g and the supernatant was removed by aspiration. Tubes were then frozen in liquid nitrogen and stored at -80?.In vitro germinated Pl. viticola zoospores were induced as described by Riemann [61]. The sporangia were suspended to homogeneity in 10 mL of distilled water and transferred to a Petri dish. After 3 h of incubation in the light in an incubator at 22°C, NaCl was added to a final concentration of 10 mM and the suspension was returned to the incubator. After checking for spore germination under a light microscope, spores with germ tubes were collected as above.To prepare the inoculum, after counting with a hemocytometer under a light microscope the sporangial suspensions were adjusted to a concentration of 10-4 sporangia/mL, and used directly.Pl. viticola-infected material for RT-PCR experiments was obtained by inoculating the fourth, fifth, and sixth leaves (counting from the base) of V. vinifera cv. Pinot Noir. The leaves were washed as above and inoculated over their entire abaxial surfaces with numerous 50 µL droplets of a suspension of 5×10-4 sporangia/mL. Inoculated leaves were kept in Petri dishes on wet filter paper and incubated in a growing chamber at 20±2? and 100% RH for 24 h in the dark, then under a 16 h light / 8 h dark photoperiod and 70% RH for four days. At each time, three leaves were frozen in liquid nitrogen and stored at -80?.RNA extractions from sporangia (SP) and in vitro germinated spores (GC) were made using the RNeasy Plant Mini Kit (Qiagen) following manufacturer's instructions. Removal of residual genomic DNA was achieved by DNase treatment of RNAs using the Ambion Turbo-DNAfree kit. Prior to sequencing, RNA quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).