Potato virus Y HCPro suppression of antiviral silencing in Nicotiana benthamiana plants correlates with its ability to bind in vivo to 21- and 22-nucleotide small RNAs of viral sequence
ABSTRACT[size=12.800000190734863px]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 benthamianaplants, 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 this was not observed in vsRNAs of other sizes, or of plant sequence. [size=12.800000190734863px]IMPORTANCE It was previously shown that HCPro can bind to long and small RNAs (sRNAs) in vitro, and in the case of Turnip mosaic virus HCPro also in vivoin arabidopsis AGO2-deficient plants. Our data show that PVY HCPro does bind in vivo to sRNAs during infection in wild-type Nicotiana benthamiana plants when expressed from a heterologous virus vector. Using a suppression of silencing-deficient mutant HCPro that can accumulate in this host when expressed from a virus vector we also show that sRNA binding correlates with silencing suppression activity. We demonstrate that HCPro binds at least to sRNAs of viral sequence of 21 and to a much lesser extent of 22 nucleotide (nt) in length, which were are also differentially enriched in 5’ -end adenines relative to the purified controls. Together, our results support HCPro physical binding to vsRNAs of 21 and 22 nt as a means to interfere with antiviral silencing.
[size=12.800000190734863px]http://jvi.asm.org/content/early/2017/03/30/JVI.00367-17.abstract?papetoc
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发表于 2017-4-6 17:03:12
