Supplemental Methods
Methods S1 TRV-mediated VIGS in N. benthamiana.
Fragments of 5’ NbSACPD-A/B, 5’ NbSACPD-C, and the conserved region of
NbSACPD-A/B and NbSACPD-C cDNAs were amplified by PCR using gene-specific
primers (Table S2). These fragments were then cloned into pTVR2 (Liu et al., 2002) via
EcoR I and Xho I sites to construct pTRV2-AB, pTRV2-C, and pTRV2-ABC,
respectively. 4-week-old N. benthamiana plants were infiltrated with Agrobacteria
cultures carrying different combinations of plasmids by using syringes as described
previously (Liu et al., 2002). Agrobacterium cultures were grown at 28°C in medium
with kanamycin (50 mg l-1), 10 mM MES (morpholine ethanesulfonic acid, pH 5.9), and
50 μM acetosynringone. These were subcultured once and harvested when the OD600
reached 0.8-1.0. Agrobacterium cultures were brought to OD600 = 1.0 in the infiltration
solution (10 mM MgCl2, 10 mM MES, pH 5.9, and 150 μM acetosynringone) and
incubated at room temperature for at least 3 hours. The cultures containing pTRV1 were
mixed with cultures containing pTRV2-GFP, pTRV2-AB, -B or -ABC in a 1:1 ratio (Liu
et al., 2002). Plants co-infiltrated with pTRV1 and pTRV2-GFP were used as controls.
Reference: Liu, Y., Schiff, M., Marathe, R. and Dinesh Kumar, S.P. (2002) Tobacco
Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to
tobacco mosaic virus. Plant J., 30, 415–429.