Carborundum

Dried turnip tissue infected with wild-type CaMV strain W260 was obtained from James E. Schoelz at the University of Missouri. To prepare inoculum (virus sap) for our plants, dried CaMV-infected turnip tissue was ground in two volumes of 20 mM phosphate buffer (pH 7.2). One-week-old canola and watercress plants were mechanically inoculated with the virus sap and used for experiments at 3 weeks post infection. Two leaves per plant were dusted with carborundum (Sigma-Aldrich, St Louis, MO) to facilitate penetration and rub-inoculated with the virus sap using a cotton-stick, as previously described [31 (link), 32 (link)]. Three weeks post infection, two plants from each variety were combined together and their DNA was extracted.

TuMV/GFP was propagated from the infectious clone p35:TuMV/GFP53 (link) by agroinfiltration as described in the ‘Protein expression in Nicotiana spp' section below. PVY-O was obtained from Stewart Gray at Cornell University, Ithaca, NY57 (link) and propagated by rub-inoculation. To prepare inoculum, fully infected N. benthamiana leaves were collected 3 weeks after inoculation and weighed. Leaves were then ground in two volumes 20 mM phosphate buffer (pH 7.2). For experiments, two leaves per plant were dusted with carborundum (Sigma-Aldrich, St Louis, MO) and rub-inoculated with the virus sap using a cotton-stick applicator. A corresponding set of control plants were dusted with carborundum and mock-inoculated with a cotton-stick applicator that was soaked in uninfected N. benthamiana sap in 20 mM phosphate buffer.

N. benthamiana plants were syringe inoculated with A. tumefaciens strain GV2260 carrying pBMV derivatives as described previously^{47 (link)}. The inoculated plants were maintained under greenhouse conditions at 21 ± 2 °C for effective viral infection. A week after inoculation, sap was extracted from inoculated leaves by homogenizing in 0.1 M phosphate buffer (pH 6.0). The BMV titre of 10,000 relative expression units was used for maize inoculation. Six-day-old maize seedlings growing on pots filled with equal amount of uniformly mixed and completely dried potting mix were dusted with carborundum (Sigma-Aldrich, St. Louis, MO, USA) and leaf surface was gently rubbed with 50 μl of N. benthamiana sap. BMV-mediated systemic silencing in maize and the leaves used for VIGS experiments are described in Supplementary Fig. S6.