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22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.

ABSTRACT: The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.

SUBMITTER: Chen HM

PROVIDER: S-EPMC2930544 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.

Chen Ho-Ming HM Chen Li-Teh LT Patel Kanu K Li Yi-Hang YH Baulcombe David C DC Wu Shu-Hsing SH

Proceedings of the National Academy of Sciences of the United States of America 20100719 34

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediat ...[more]

PMID: 20643946

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Project description: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 nondenaturing conditions. We found that RNAs in purified preparations were differentially enriched in 21-nucleotide (nt) and, to a much lesser extent, 22-nt sRNAs of viral sequences (viral sRNAs [vsRNAs]) compared to those found in a control plant protein background bound to nickel resin in the absence of HCPro or in a purified HCPro alanine substitution mutant (HCPro mutB) control that lacked suppressor-of-silencing activity. In both controls, sRNAs were composed almost entirely of 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 pools 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 sequences.IMPORTANCE It was previously shown that HCPro can bind to long RNAs and small RNAs (sRNAs) in vitro and, in the case of Turnip mosaic virus HCPro, also in vivo in arabidopsis AGO2-deficient plants. Our data show that PVY HCPro binds 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 HCPro mutant 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 with viral sequences of 21 nucleotides (nt) and, to a much lesser extent, of 22 nt, which were are also differentially enriched in 5'-end adenines relative to the purified controls. Together, our results support the physical binding of HCPro to vsRNAs of 21 and 22 nt as a means to interfere with antiviral silencing.

Dataset Information

22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.

Publications

22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.

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