%0 Journal Article
%~ PubMed
%A Eamens, Andrew L
%A Kim, Ki Wook
%A Curtin, Shaun J
%A Waterhouse, Peter M
%T DRB2 is required for microRNA biogenesis in Arabidopsis thaliana.
%B PloS One
%D 2012
%C United States
%I Public Library of Science
%V 7
%N 4
%P e35933
%@ 1932-6203
%X The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants).
%Z FOR Codes: 60403


%0 Journal Article
%~ PubMed
%A Eamens, Andrew L
%A Wook Kim, Ki
%A Waterhouse, Peter M
%T DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana.
%B Plant Signaling & Behavior
%D 2012
%C United States
%I Landes Bioscience
%V 7
%N 10
%P 1224-1229
%@ 1559-2324
%X DOUBLE-STRANDED RNA BINDING (DRB) proteins have been functionally characterized in viruses, prokaryotes and eukaryotes and are involved in all aspects of RNA biology. Arabidopsis thaliana (Arabidopsis) encodes five closely related DRB proteins, DRB1 to DRB5. DRB1 and DRB4 are required by DICER-LIKE (DCL) proteins DCL1 and DCL4 to accurately and efficiently process structurally distinct double-stranded RNA (dsRNA) precursor substrates in the microRNA (miRNA) and trans-acting small-interfering RNA (tasiRNA) biogenesis pathways respectively. We recently reported that DRB2 is also involved in the biogenesis of specific miRNA subsets. ( 1) Furthermore, the severity of the developmental phenotype displayed by the drb235 triple mutant plant, compared with those expressed by either drb2, drb3 and drb5 single mutants, or double mutant combinations thereof, indicates that DRB3 and DRB5 function in the same non-canonical miRNA pathway as DRB2. Through the use of our artificial miRNA (amiRNA) plant expression vector, pBlueGreen ( 2) (,) ( 3) we demonstrate here that unlike DRB2, DRB3 and DRB5 are not involved in the dsRNA processing stages of the miRNA biogenesis pathway, but are required to mediate RNA silencing of target genes of DRB2-associated miRNAs.
%Z FOR Codes: 60199 604


%0 Journal Article
%~ PubMed
%A Liang, Dacheng
%A White, Rosemary G
%A Waterhouse, Peter M
%T Gene Silencing in Arabidopsis Spreads from the Root to the Shoot, through a Gating Barrier, by Template-Dependent, Nonvascular, Cell-to-Cell Movement.
%B Plant Physiology
%D 2012
%C United States
%I American Society of Plant Biologists
%V 159
%N 3
%P 984-1000
%@ 1532-2548
%X Upward long-distance mobile silencing has been shown to be phloem mediated in several different solanaceous species. We show that the Arabidopsis (Arabidopsis thaliana) seedling grafting system and a counterpart inducible system generate upwardly spreading long-distance silencing that travels not in the phloem but by template-dependent reiterated short-distance cell-to-cell spread through the cells of the central stele. Examining the movement of the silencing front revealed a largely unrecognized zone of tissue, below the apical meristem, that is resistant to the silencing signal and that may provide a gating or protective barrier against small RNA signals. Using a range of auxin and actin transport inhibitors revealed that, in this zone, alteration of vesicular transport together with cytoskeleton dynamics prevented or retarded the spread of the silencing signal. This suggests that small RNAs are transported from cell to cell via plasmodesmata rather than diffusing from their source in the phloem.
%Z FOR Codes: 60699


%0 Book Section
%A Anderssen, Robert S.
%A Waterhouse, Peter
%T Modeling antiviral resistance in plants
%B Methods in Microbiology: Antiviral Resistance in Plants
%D 2012
%C United States
%I Academic Press
%V 
%N 
%P 139-154
%@ 9781617798818
%X 
%Z FOR Codes: 60506


%0 Journal Article
%~ PubMed
%A Fusaro, Adriana F
%A Correa, Regis L
%A Nakasugi, Kenlee
%A Jackson, Craig
%A Kawchuk, Lawrence
%A Vaslin, Maite F S
%A Waterhouse, Peter M
%T The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation.
%B Virology
%D 2012
%C United States
%I Academic Press
%V 426
%N 2
%P 178-187
%@ 0042-6822
%X The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant''s RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0(PE), in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0(PE) has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0(PE) destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.
%Z FOR Codes: 60506


%0 Journal Article
%~ PubMed
%A Eamens, Andrew L
%A Agius, Claire
%A Smith, Neil A
%A Waterhouse, Peter M
%A Wang, Ming-Bo
%T Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana.
%B Molecular Plant
%D 2011
%C United Kingdom
%I Oxford University Press
%V 4
%N 1
%P 157-170
%@ 1752-9867
%X We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted. Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants.
%Z FOR Codes: 60199 60499 100499


%0 Book Section
%A Eamens, Andrew
%A Waterhouse, Peter
%T Vectors and Methods for Hairpin RNA and Artificial microRNA-Mediated Gene Silencing in Plants
%B Methods in Molecular Biology: Plant Chromosome Engineering
%D 2011
%C United States
%I Humana Press
%V 
%N 
%P 179-197
%@ 9781617379567
%E Birchler, James A.
%X 
%Z FOR Codes: 60405


%0 Journal Article
%~ PubMed
%A Duan, Guowen
%A Saint, Robert B
%A Helliwell, Chris A
%A Behm, Carolyn A
%A Waterhouse, Peter M
%A Gordon, Karl H J
%T Expression of Caenorhabditis elegans RNA-directed RNA polymerase in transgenic Drosophila melanogaster does not affect morphological development.
%B Transgenic Research
%D 2010
%C Netherlands
%I Springer Netherlands
%V 19
%N 6
%P 1121-1128
%@ 1573-9368
%X Drosophila melanogaster, along with all insects and the vertebrates, lacks an RdRp gene. We created transgenic strains of Drosophila melanogaster in which the rrf-1 or ego-1 RdRp genes from C. elegans were placed under the control of the yeast GAL4 upstream activation sequence. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. RT-PCR confirmed the successful expression of each RdRp gene. The resulting phenotypes indicated that introduction of the RdRp genes had no effect on D. melanogaster morphological development.
%Z FOR Codes: 60499


%0 Book Section
%A Waterhouse, Peter
%T Gene Silencing I A virus defence pathway and a technology
%B Genes to Galaxies : the lecture series of the 35th Professor Harry Messel International Science School
%D 2009
%C Australia
%I University of Sydney
%V 
%N 
%P 56-65
%@ 9780959947120
%E Selinger, Adam
%E Green, Anne
%X 
%Z FOR Codes: 60407


%0 Book Section
%A Waterhouse, Peter
%T Gene Silencing II Gene regulation
%B Genes to Galaxies : the lecture series of the 35th Professor Harry Messel International Science School
%D 2009
%C Australia
%I University of Sydney
%V 
%N 
%P 106-113
%@ 9780959947120
%E Selinger, Adam
%E Green, Anne
%X 
%Z FOR Codes: 60407


%0 Journal Article
%~ PubMed
%A Eamens, Andrew L
%A Smith, Neil A
%A Curtin, Shaun J
%A Wang, Ming-Bo
%A Waterhouse, Peter M
%T The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes.
%B RNA
%D 2009
%C United States, Unite
%I Cold Spring Harbor Laboratory Press
%V 15
%N 12
%P 2219-2235
%@ 1469-9001
%X In Arabidopsis thaliana (Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we use an artificial miRNA (amiRNA) technology, transiently expressed in Nicotiana benthamiana, to produce a series of amiRNA duplexes with differing intermolecular thermostabilities at the 5'' end of duplex strands. Analyses of amiRNA duplex strand accumulation and target transcript expression revealed that strand selection (amiRNA and amiRNA*) is directed by asymmetric thermostability of the duplex termini. The duplex strand possessing a lower 5'' thermostability was preferentially retained by RISC to guide mRNA cleavage of the corresponding target transgene. In addition, analysis of endogenous miRNA duplex strand accumulation in Arabidopsis drb1 and drb2345 mutant plants revealed that DRB1 dictates strand selection, presumably by directional loading of the miRNA duplex onto RISC for passenger strand degradation. Bioinformatic and Northern blot analyses of DCL4/DRB4-dependent small RNAs (miRNAs and siRNAs) revealed that small RNAs produced by this DCL/dsRBP combination do not conform to the same terminal thermostability rules as those governing DCL1/DRB1-processed miRNAs. This suggests that small RNA processing in the DCL1/DRB1-directed miRNA and DCL4/DRB4-directed sRNA biogenesis pathways operates via different mechanisms.
%Z FOR Codes: 110105