Potato virus Y HCPro suppression of antiviral silencing in Nicotiana benthamiana plants correlates with its ability to bind in vivo to small RNAs of 21 and 22 nucleotides in length of viral sequence. Nicotiana benthamiana

We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-tagged Potato virus Y (PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector in Nicotiana benthamiana plants, and purified under non-denaturing conditions. We found that RNAs in purified preparations were differentially enriched in sRNAs of 21 and to a much lesser extent of 22 nucleotides (nt) in length and of viral sequence (vsRNAs) when compared to those found in a control plant protein background bound to the nickel resin in the absence of HCPro, or in a purified alanine substitution HCPro mutant (HCPro mutB) control that lacked suppressor of silencing activity. In both controls, sRNAs were composed almost entirely by molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs, and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21 and 22 nt vsRNAs. HCPro constituted at least 54 % of the total protein content in purified preparations and we were able to calculate its contribution to the 21 and the 22 nt pool of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21 nt vsRNAs of the HCPro preparation 5´ terminal adenines were overrepresented relative to the controls, but not in vsRNAs of other sizes or of plant sequence. Overall design: To analyze sRNA populations, three separate deep sequencing events were performed: The first one analysed the sRNAs present in the plants infected with either PVX or PVX-P1-6x-HCPro used as input for the purification. For that, total RNAs were extracted from ~25 g of infected tissue in both cases. 30 µg of total RNA were sent straight for sequencing of sRNAs (18 to 30 nt in legth). Total RNAs were isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the instruction of the manufacturers. The second one analyzed the short RNAs of 18 to over 120 nt that were present in the purified control and HCPro samples of the first purification experiment. For that, 1.5 ml aliquots from the purified elution samples obtained from tissues infected with either PVX or PVX-P1-6x-HCPro were used to obtain 9.61 and 13.38 ng of gel-eluted RNAs from the control and HCPro samples, respectively: total RNAs from the purified samples were fractioned by electrophoresis in 10%-PAGE containing 8M urea. The gel region resolving ~12 nt to ~500 nt in size was sliced with a razor and RNAs were eluted from the gel with 0.3 M NaCl, and resulting precipitated with 1 µl of glycogen (20 µg/µl) (Roche, Basel, Switzerland) and 1 volume of isopropanol. The third massive sequencing analyzed the sRNAs of 18 to 30 nt present in the Ni2+-NTA column-purified HCPro mutB and HCPro samples of the second purification experiment. For that, 1.5 ml aliquots from the purified elution samples obtained from tissues infected with either PVX-P1-6x-HCPro mutB or PVX-P1-6x-HCPro were used to obtain 597 and 339 ng of total RNAs for sequencing, respectively.