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A low concentration (OD600<0.1) of bacterial suspension resulted in poor transformation thus leading to lower silencing levels, while high concentration (OD600>1.0) occasionally produced necrotic spots around the infiltration zones.
Silencing levels of 30% were obtained (Figure S4) which are similar to that obtained with the GFP tagged transcript, confirming that the sequence specificity of shR-P16 was independent of the reporter tag used (Figure 3).
This structure is consistent with silencing levels at truncated ends which diminish with distance from the telomeric repeats [ 16].
To conclude, these data indicate a direct link between deposition of DNA methylation and gene silencing, with methylation and silencing levels comparable to previous results [ 20].
The deletion of SIR1 produced no discernable differences to the chromatin structure upstream of the URA3 gene at XIL (the left panel in Figure 2B) in accord with the minor effect on the silencing levels seen in this strain.
Also, CaFAD2-C1 showed various silencing levels in both T2 families with strong silencing in three WG4-4 T2 -plants (3 out of 7; 43%) and five WG19-6 T2-plants (5 out of 15; 33%).
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Tyrosinase activity in the individual lines that showed most silencing was decreased when compared to the control (Fig 4), while individual lines that did not present SpTyr-silencing or were at a low silencing level maintained a tyrosinase activity that was similar to the control lines.
This hypothesis was sustained by subsequent results coming from experiments using MDAH 2774 cells where the Skp2 silencing level was optimal.
These subclones exhibited little deviation from the silencing level measured in the cell pool (Additional file 1: Figure S5).
For one experiment silencing level and fungal growth were quantified by qRT-PCR, using RNA from five plants for TRV-EV and 10 plants for TRV-U196237.
The location of the analysed live cells on the cover slips were recorded that allowed confirmation of the TUBB3 silencing level in the same cells after fixation and immunofluorescence with an anti-TUBB3 antibody.
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