RNA silencing in plants: Flash report!

Earlier this year plant scientists met in Santa Fe, New Mexico at the Keystone Symposium "RNA Silencing Mechanisms in Plants". Sessions included small RNA biogenesis and signalling, development and stress responses, small RNA-directed DNA methylation, and interaction with pathogens. This report highlights some of the prominent and recurring themes at the meeting and emerging arenas of future research.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing

Like other eukaryotes, plants use DICER-LIKE (DCL) proteins as the central enzymes of RNA silencing, which regulates gene expression and mediates defense against viruses. But why do plants like Arabidopsis express four DCLs, a diversity unmatched by other kingdoms? Here we show that two nuclear DNA viruses (geminivirus CaLCuV and pararetrovirus CaMV) and a cytoplasmic RNA tobamovirus ORMV are differentially targeted by subsets of DCLs. DNA virus-derived small interfering RNAs (siRNAs) of specific size classes (21, 22 and 24 nt) are produced by all four DCLs, including DCL1, known to process microRNA precursors. Specifically, DCL1 generates 21 nt siRNAs from the CaMV leader region. In contrast, RNA virus infection is mainly affected by DCL4. While the four DCLs are partially redundant for CaLCuV-induced mRNA degradation, DCL4 in conjunction with RDR6 and HEN1 specifically facilitates extensive virus-induced silencing in new growth. Additionally, we show that CaMV infection impairs processing of endogenous RDR6-derived double-stranded RNA, while ORMV prevents HEN1-mediated methylation of small RNA duplexes, suggesting two novel viral strategies of silencing suppression. Our work highlights the complexity of virus interaction with host silencing pathways and suggests that DCL multiplicity helps mediate plant responses to diverse viral infections

The CaMV transactivator/viroplasmin interferes with RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects

Biochemical Requirements for Two Dicer-Like Activities from Wheat Germ

RNA silencing pathways were first discovered in plants. Through genetic analysis, it has been established that the key silencing components called Dicer-like (DCL) genes have been shown to cooperatively process RNA substrates of multiple origin into distinct 21, 22 and 24 nt small RNAs. However, only few detailed biochemical analysis of the corresponding complexes has been carried out in plants, mainly due to the large unstable complexes that are hard to obtain or reconstitute in heterologous systems. Reconstitution of activity needs thorough understanding of all protein partners in the complex, something that is still an ongoing process in plant systems. Here, we use biochemical analysis to uncover properties of two previously identified native dicer-like activities from wheat germ. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs of 21 and 24 nt in size. The 21 nt dicing activity, likely an siRNA producing complex known as DCL4, is 950 kDa-1.2 mDa in size and is highly unstable during purification processes but has a rather vast range for activity. On the contrary, the 24 nt dicing complex, likely the DCL3 activity, is relatively stable and comparatively smaller in size, but has stricter conditions for effective processing of dsRNA substrates. While both activities could process completely complementary dsRNA albeit with varying abilities, we show that DCL3-like 24 nt producing activity is equally good in processing incompletely complementary RNAs

pH and temperature requirements.

<p><b>a</b>) Activity of DCL complexes across temperature range using S23 and individual DCL complexes. Range of temperature used is mentioned. Please refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116736#pone.0116736.g001" target="_blank">Fig. 1</a> legend for other details. <b>b</b>) Activity of DCL complexes across a pH range using DCL activities.</p

Substrate processing abilities of DCL activities.

<p>Perfectly complementary (P) or imperfectly complementary (IMP; having a mismatch every 4^th base) substrates of the mentioned length were incubated with DCL activities to analyze their processing abilities. Note that 700 bp length substrate is perfectly complementary (Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116736#sec002" target="_blank">methods</a>).</p

Requirements of salt for DCL activities.

<p><b>a</b>) NaCl dependency of DCL activities. S23 extract was used to calculate optimal NaCl concentration required for DCL activities by providing labelled substrates. The extracts were first desalted by passing two times through sephadex G25 columns with buffers having 5 mM of the salt. Range of concentrations of NaCl used is shown. <b>b</b>) KAc dependency of DCL activities. Further details are similar to <b>a</b>.</p