Beta vulgaris
Protocols: Leaves displaying systemic symptoms were harvested (1 g) and grinded in 4 ml 0.05 M phosphate buffer (1 g of KH2PO4 and 7.2 g of Na2HPO4 in 1 l of DEPC-treated water, pH 7 to 7.4). Infectious full-length clones of BNYVV and BSBMV (Laufer et al, 2018) were used for inoculation of sugar beets. A susceptible (KWS03) and a resistant genotype (Rz1) was provided by KWS Saat SE (Einbeck, Germany). Young sugar beet seedlings were infected using the protocol from Bornemann and Varrelmann (2011) with slight modifications. First, plants of Beta macrocarpa were agroinfected with both virus infectious clones when the first two leaves (BBCH12) were fully developed. One true leaf and both cotyledons were inoculated with Rhizobium radiobacter (syn. Agrobacterium tumefaciens) strain GV2260 according to Voinnet et al. (1998) with an OD600 = 0.5. Plants were kept for symptom development in a climate chamber at day and night temperatures of 24°C and 18°C, respectively, and a 14 h photoperiod. 200 mg of leaf tissue was grinded in 1 ml TRIzol Reagent (Invitrogen) using mortar and pestle. Grinded samples were incubated at room temperature for 5 minutes and centrifuged at 12,100 xg for 10 minutes (4°C) to remove crude plant material. Approximately 700 µl of the supernatant was mixed with an equal volume of absolute ethanol (-20°C). Dual cDNA libraries were prepared, sequenced and analyzed as recently described (Verwaaijen et al., 2017) with smaller modifications. In brief, 2.25 μg of RNA per sample was used for preparation of stranded libraries with the TruSeq mRNA Sample Preparation Kit (Illumina Inc., San Diego, U.S.A.).