Supplementary Figure 2 HSVd cannot spread in Cucurbita maxima plants.

1. A B C 1 2 3 4 5 1 2 3 4 5 6 7 1/1 1/3 D 1/1 1/3 E cucumber pumpkin Supplementary Figure 2 HSVd cannot spread in Cucurbita maxima plants. A expression cassette containing a dimeric HSVd cDNA flanked by the 35S promoter and the T-Nos terminator sequences, was cloned in the T-DNA binary vector pMOG 800. The generated vector was used to transform Agrobacterium tumefaciens strain C58C1 carrying the virulence helper plasmid pCH32, via electroporation. Transformed Agrobacterium was cultivated in liquid medium and used to infiltrate cotyledons of pumpkin and cucumber plants (1). The generation of circular HSVd molecules and their putative translocation to distal parts of the plant was analyzed by Northern blot of total RNAs enriched for circular RNAs extracted from cotyledons and apical leaves from HSVd-agroinoculated pumpkin and cucumber plants. A) Total RNAs were precipitated with LiCl, the circular RNAs were fractionated by adsorption to CF11 and first electrophoresed in polyacrylamide gel 5% in buffer TAE and stained with Ethidium Bromide. B) The gel area corresponding to circular HSVd (indicated in the panel A with a frame), was cut, electrophoresed in 8 M Urea denaturing polyacrylamide gel in TBE and transferred to nylon membrane. The membrane was hybridized with HSVd dig-riboprobe and revealed as previously described (2) to detect the circular HSVd. Lanes 1 and 2) cucumber cotyledons at 36 and 72 h post infiltration, 3) cucumber apical leaves 7 days after inoculation, 4 and 5) pumpkin cotyledons at 36 and 72 h post infiltration, 6) pumpkin apical leaves 7 days after infiltration, 7) circular HSVd. It can be observed that the circular HSVd is detected in agroinoculated cotyledons of pumpkin and cucumber (lanes 1-2 and 4-5) but only detected in apical leaves from agroinoculated cucumber plants (compare lane 3 and 6), indicating that the HSVd circular RNA cannot spread in pumpkin plants. Dot-blot assays of total RNA extracted from apical parts from 3 pumpkin varieties at 6 (C) and 13 (D) days after injection in stem tissue with dimeric HSVd-RNA transcripts. The HSVd RNA was no detected in none of the analyzed pumpkin plants (lanes 2 to 4). When the HSVd was injected in cucumber plants (lane 1), the RNA was detected in the apical region. No injected pumpkin (lane 5). E) Cucumber and pumpkin plants, agroinoculated in cotyledons (7 days after germination) with dimeric HSVd, were photographed at 10 days post infiltration. It can be observed that agroinoculated cucumber plants were clearly affected by the systemic infection of HSVd. 1- Bendahmane, A., Querci, M., Kanyuka, K. and Baulcombe, D. 2000. Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. The Plant Journal21, 73-81. 2- Pallás, V., Más, P. & Sánchez-Navarro, J.A. 1998. In: Plant Virus Protocols, from Virus Isolation to Transgenic Resistance. (G. Foster, S. Taylor, eds). Humana Press, Totowa, NY, USA, 461-468.