Spectrum rna kit

After growth for 7 d in LD conditions, RNA was extracted from 100mg of seedling tissue using the Spectrum RNA kit (Sigma-Aldrich). A 1–2 μg aliquot of RNA was used to produce cDNA with a Superscript III (Life Technologies) or cDNA synthesis kit (Bioline). Non-quantitative PCR for mutant analysis was performed using Red-Hot Taq (Bioline). Real-time PCR was performed with SYBR-Green, Platinum Taq (Life Technologies), using primers for gene expression shown in Supplementary Table 2 at JXB online on an MJ Research Opticon 2 machine with Opticon Monitor 3 software. Quantification of expression was determined from ≥3 experiments and the values derived using the comparative C_T method (2^–ΔΔCt) (Schmittgen and Livak, 2008 ) with ACTIN7 (At5g09810) as the internal control. Control genes At4g33060 and At3g10040 were selected at random from a data set produced from study of the Arabidopsis hypoxia response (Licausi et al., 2011 (link)). From the microarray data described in Fig. 6, the fold change in log₂ expression compared with Col-0 in At4g33060 is 0.054 (nup160-1) or 0.048 (nup62-2) and in At3g10040 is 0.144 (nup160-1) or 0.104 (nup62-2).

RNA was extracted from leaf tissues using a Spectrum RNA Kit (Sigma-Aldrich, St. Louis, MO, United States) following the manufacturer’s instructions. Extracted RNAs were DNase treated and reverse transcribed into cDNA with a QuantiTectReverse Transcription Kit (Qiagen GmbH) following the manufacturer’s instructions. Portions of the movement protein (MP) and coat protein (CP) regions were amplified with the DetF/DetR primer pair (Morelli et al., 2014 (link)) according to the protocol suggested by Bertazzon et al. (2017) (link). Sequencing was carried out in both directions using automated equipment (Genechron Service, Rome, Italy). The obtained sequences were aligned with reference sequences selected from GenBank (Saldarelli et al., 2015 (link); Bertazzon et al., 2017 (link)) using ClustalW (Thompson et al., 1994 (link)) with the following parameters: gap opening penalty: 10, gap extension penalty: 0.2, 30% divergent cutoff. 460-bp-long portions of the aligned sequences were used for construction of a Neighbor Joining (NJ) tree (Saitou and Nei, 1987 (link)) employing Mega X (Kumar et al., 2018 (link)). The reliability of the analyses was subjected to a bootstrap test with 5000 replicates. 12 plants infected with 12 different GPGV strains were selected and used as reported in “Plant Material” section.