NextSeq 550 sequencing; Pepper leaf transcriptome profiling during an incompatible and a compatible pepper-tobamovirus interaction
Pepper leaf transcriptome profiling during an incompatible and a compatible pepper-tobamovirus interaction
Leaf samples were taken from ObPV- and PMMoV-inoculated as well as from mock-treated leaves at 4, 8, 24 and 48 hours post-inoculation (hpi) for total RNA extraction.  Pepper plants were grown under normal greenhouse conditions (18-23 °C; 16 hrs daylight with 160 µmol m-2 s-1 supplemental light for 8 hrs per day; 75-80% relative humidity) and 55–60-day-old plants were used for the experiments.  ObPV and PMMoV inoculations as well as mock-treatments were carried out as described earlier (Rys et al., 2014). The ObPV strain was isolated in Hungary (formerly used synonym: Ob strain of Tomato mosaic virus), whereas the L3-resistance-breaking strain of PMMoV was isolated in Louisiana, USA (formerly used synonym: Samsun latent strain of Tobacco mosaic virus) (Tóbiás et al., 1989; Rys et al., 2014). Virus-inoculated and mock-treated plants were kept at 22 °C in a growth chamber with 16/8 h light/dark cycles.  Total RNA was extracted from pepper leaves by using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany).  The RNA integrity numbers and RNA concentrations were determined by RNA ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and RNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. For mRNA library construction, QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) for Illumina (Lexogen, Wien, Austria) was applied according to the manufacturer's protocol. The quality and quantity of the library QC was performed by using High Sensitivity DNA1000 ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and dsDNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.