FIG 1.

Moderately overexpressed p88 stimulates abundant ttsgR accumulation. (a) Schematic representation of the TCV genome with encoded proteins shown as rectangles. The two sgRNAs, namely, sgRNA1 and sgRNA2, are produced during cellular infections and serve as mRNA for p8 and p9 and for p38 (CP), respectively. (b) TCV replicons used in the current study. TCV_sg2R encodes wild-type (wt) p28 and p88 but expresses the red fluorescent mCherry instead of p38 from sgRNA2. Δp88_sg2R contains a 4-nt deletion in the middle of p88 ORF, making it incapable of translating a functional p88. Conversely, RT_sg2R contains a UAC codon in place of the p28 stop codon (UAG), rendering it incapable of translating p28 and yet overexpresses p88. All three replicons use the strong 2X35S promoter of CaMV to drive the transcription of infectious TCV RNA. (c) Constructs that expresses TCV p88 and p28 independent of replication. Three promoters, namely, 2X35S, Core35S, and Micro, were used to drive the expression of p88. Note that p88 expressed from these constructs contain a C-terminal, duplicated hemagglutinin (2HA) tag that permits protein detection with an HA antibody. This tag does not affect the function of p88 (38). In contrast, the p28-expressing construct contained a 2X35S promoter preceding the p28 ORF but no epitope tag at the C terminus. The image beneath the diagrams shows a Western blotting detection of the 2HA-tagged p88 from cells of N. benthamiana receiving the three p88 constructs. (d) Repression and replicational complementation of TCV replicons by p88 are accompanied by preferential overaccumulation of ttsgR and sgRNA2. The Northern blotting autoradiograph shows the accumulation levels of TCV gRNA (g), sgRNAs (sg1 and 2), and ttsgR in the total RNA samples extracted from N. benthamiana leaves that were treated with various constructs or combinations thereof. Note that a TBSV p19-expressing construct was always included to mitigate RNA silencing-mediated mRNA degradation. EB, ethidium bromide stained agarose gel serving as the loading control. (e) The RdRp activity of p88 is required for ttsgR overaccumulation. (f) p88 overexpression from a defective replicon (RT_sg2R) caused ttsgR overaccumulation upon replicational complementation by trans-supplied p28. (g) wt TCV also responded to p88 overexpression with the overaccumulation of ttsgR (and another lncRNA denoted with an arrowhead).