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. 2009 Jan 19;83(7):3276–3287. doi: 10.1128/JVI.02313-08

FIG. 5.

FIG. 5.

Knockdown of HSP70 mRNA levels by VIGS reduces the accumulation of TBSV RNA in N. benthamiana. (A) Total-RNA samples obtained from N. benthamiana leaves silenced as shown were analyzed by gel electrophoresis. Two different segments of HSP70 (pYL619 and pYL622) were used in the TRV silencing vectors. The control experiments included PDS in the TRV vector and the empty TRV vector, as well as infiltration with water. We chose the 6th day after VIGS to inoculate the upper, systemically silenced leaves with TBSV virions. Samples for RNA extraction were taken from the inoculated leaves 3 days postinoculation. (B) (Top) Northern blotting shows the accumulation of TBSV gRNA and subgenomic RNAs (sgRNAs). (Bottom) The ethidium bromide-stained gel shows rRNA and TBSV gRNA levels. These results shown here and in panel A are from different experiments. (C) VIGS reduced NbHSP70 mRNA levels in treated leaves. Semiquantitative RT-PCR was performed with primers that allow the amplification of a region of HSP70 not present in the pYL619 and pYL622 VIGS constructs. (D) Western blotting of silenced N. benthamiana leaves with an anti-Hsp70 antibody 6 days postinoculation. (E) Phenotypes of N. benthamiana plants in which Hsp70 was silenced, shown 6 days after agroinfiltration with pYL619 or pYL622. The pictures were taken just before the inoculation of the upper extended leaves with a TBSV virion preparation. (F) Phenotypes of N. benthamiana plants in which Hsp70 was silenced, shown 9 days after agroinfiltration with pYL619 or pYL622 and 3 days after inoculation with TBSV. Note that no symptoms of TBSV infection are visible at this early time point. The pictures were taken after the samples were collected for TBSV RNA analysis. (G) Symptoms of TBSV-infected N. benthamiana plants in which Hsp70 or PDS was silenced, shown 12 days after agroinfiltration with pYL619 or pPDS. The white/yellow coloring of the leaves is due to the silencing of Hsp70 or PDS after agroinfiltration with pYL619 or pPDS, respectively. The PDS-silenced plants died as quickly as the control plants treated with the empty TRV vector (data not shown).