Computational protocol: Solanum venturii, a suitable model system for virus-induced gene silencing studies in potato reveals StMKK6 as an important player in plant immunity

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Protocol publication

[…] Phylogenetic tree of potato and its relatives was prepared with online Interactive phylogeny tool within SolRgene database (http://www.plantbreeding.wur.nl/SolRgenes/Phylogeny/species_select.php). In first step all the species were selected and in the second step the accessions used in our experiments were selected. 11 clones out of 73, which were used in our study for responsiveness to TRV silencing, were not available in the database and are therefore not included in the tree. Additionaly two S. lycopersicum and six S. tuberosum accessions were added to the phylogenetic tree to illustrate the relations of wild potato relatives to tomato and cultivated potato. A dendrogram [average linkage (UPGMA)] was created.To check in silico whether the CaPDS construct (part of the sequence with GenBank accession number X68058.1) would have an effect on Solanum species, we have aligned different PDS sequences and checked the identity in comparison to CaPDS. The parts of sequences of PDS genes that were used were from S. tuberosum (GenBank accession number AY484445.1), S. nigrum (GenBank accession number EU434622.1) and N. benthamiana (GenBank accession number EU165355.1). Alignment was prepared and analysed with AlignX software (a component of Vector NTI Suite 9.0.0). [...] RNA from the samples was isolated with innuPREP Plant RNA Kit and treated with DNAse (Invitrogen, USA; 0.1 U/Dnase per μg RNA) prior to reverse transcription. 1 μg of RNA was reversely transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA).Samples were analyzed in the set-up for quantitative real-time PCR (qPCR) analysis as previously described [], using TaqMan chemistry for determining the relative concentration of PVYNTN RNA [] and cytochrome oxidase (Cox; []) as RNA load control. The transcript accumulation was normalized to that of Cox. The relative content of PVYNTN was calculated as follows: Cq value of Cox was subtracted from Cq value for PVY; the resulting value was used as exponential power on value 2; every value was finally divided by the minimum value. The resulting value was transformed with base 10 logarithm. Results were statistically evaluated using Two Way ANOVA in SigmaPlot 13.0 software. Two independent factors were considered in analysis: Genotype (individual species or clones) and Treatment (only PVY infection or PVY infection with prior agroinfiltration with empty TRV vector). The relative PVY RNA content was the dependent variable. For multiple comparison Tukey’s test was selected. […]

Pipeline specifications

Software tools Vector NTI Advance, SigmaPlot
Applications Miscellaneous, Phylogenetics, Protein sequence analysis
Organisms Solanum tuberosum, Tobacco rattle virus, Potato virus Y