We next used two-step anchored qPCR27 to quantify the relative accumulation of viral ssDNA (viral strand, VS) and the dsDNA intermediate (as complementary strand, CS) (Fig. 3a) in local infection assays with TYLCV-WT or a C3 null mutant in N. benthamiana plants in which POLA2, POLD2, or POLE2 have been silenced by VIGS (Fig. 3b–e). Here, C3 is linked to infection.