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Assembly-hub function of ER-localized SNARE proteins in biogenesis of tombusvirus replication compartment

Fig 6

Knockdown of SYP81 gene inhibits TBSV RNA replication in N. benthamiana plants.

(A) Top panel: Reduced accumulation of TBSV RNA in N. benthamiana leaves expressing Syp81ΔTM, the dominant-negative mutant of Syp81 syntaxin, the ortholog of the yeast Ufe1 protein. Expression of Syp81ΔTM from the 35S promoter was done after agro-infiltration into N. benthamiana leaves. Second panel: Accumulation of the TBSV genomic (g)RNA in the systemically-infected N. benthamiana plants transiently expressing Syp81ΔTM. (B) Top panel: Reduced accumulation of CIRV RNAs in N. benthamiana leaves expressing Syp81ΔTM. Expression of Syp81ΔTM from the 35S promoter was done after agro-infiltration into N. benthamiana leaves. (C) Accumulation of the TBSV genomic (g)RNA in Syp81 knockdown N. benthamiana plants 3 days post-inoculation, based on Northern blot analysis. Inoculation with TBSV gRNA was done 9 days after silencing of Syp81 expression by sap inoculation. VIGS was performed via agroinfiltration of tobacco rattle virus (TRV) vectors carrying NtSyp81 sequence or the TRV-cGFP vector (as a control). Second panel: Ribosomal RNA is shown as a loading control. Note that the TBSV genomic RNA is also visible in the gel. Third panel: RT-PCR analysis of NbSyp81 mRNA level in the silenced and control plants. Fourth panel: RT-PCR analysis of TUBULIN mRNA level in the silenced and control plants. Each experiment was repeated. (D) Accumulation of the unrelated TMV genomic RNA in Syp81 knockdown N. benthamiana plants 3 days post-inoculation, based on Northern blot analysis. See further details in panel C.

Fig 6

doi: https://doi.org/10.1371/journal.ppat.1007028.g006