Potato (S. tuberosum ssp. tuberosum; 2n=4x=48) cv. Rywal plants were obtained from the Institute of Plant Breeding and Acclimatisation—National Research Institute, Młochów (Poland). Plants expressing the NahG transgene (NahG-Rywal) were generated using the binary plasmid pCIB containing salicylate hydroxylase (NahG), which was generously provided by Syngenta Biotechnology. The plasmid was introduced into Agrobacterium tumefaciens LBA4404 and used for potato leaf disc transformation (Chen et al., 1994 (link)). Transgenic potato plants were regenerated as described by Mac et al. (2004) (link). Plants of both genotypes were grown for 4 weeks in soil under controlled environmental conditions (16/8 light/dark cycle, 20 °C) as described previously (Szajko et al., 2008 (link)). PVY strain N-Wilga (PVYN–Wi; accession no. EF558545) was derived from potato cv. Wilga and was multiplied and maintained in tobacco plants, Nicotiana tabacum cv. Samsun.
PVY inoculation was performed on 4-week-old potato plants. Three bottom leaves were dusted with carborundum powder and rubbed with cheesecloth dipped in a sap prepared from the leaves of the PVY-infected tobacco plants. After 10min, the leaves were washed liberally with tap water. In mock inoculations, water was used instead of the sap.
Viral amplification was monitored by semiquantitative reverse-transcription PCR as described before (Szajko et al., 2008 (link)), but by running only 22 cycles. Separate PCR reactions of potato elongation factor 1-α (EF-1) expression were performed as loading control (Varet et al., 2002 (link)).
PVY inoculation was performed on 4-week-old potato plants. Three bottom leaves were dusted with carborundum powder and rubbed with cheesecloth dipped in a sap prepared from the leaves of the PVY-infected tobacco plants. After 10min, the leaves were washed liberally with tap water. In mock inoculations, water was used instead of the sap.
Viral amplification was monitored by semiquantitative reverse-transcription PCR as described before (Szajko et al., 2008 (link)), but by running only 22 cycles. Separate PCR reactions of potato elongation factor 1-α (EF-1) expression were performed as loading control (Varet et al., 2002 (link)).
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