The Efficacy of RNAi in the Study of the Plant Cytoskeleton

Klink, Vincent P.; Wolniak, Stephen M.

doi:10.1007/s003440000043

Cited by 28 publications

(14 citation statements)

References 112 publications

(79 reference statements)

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“…Additionally, in plants, RNAi transgenes can be inherited in subsequent generations (Chuang and Meyerowitz, 2000), creating a population of transgenic plants with reduced expression of the gene of interest. In ferns, RNAi in regenerating spores provides a simple single-cell system to study gene function (Klink and Wolniak, 2000;Stout et al, 2003). Here, we show that P. patens also uses RNAi for gene silencing.…”

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confidence: 76%

RNA Interference in the Moss Physcomitrella patens

2003

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The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):␤-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUSRNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.

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confidence: 76%

RNA Interference in the Moss Physcomitrella patens

2003

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“…Ultrastructural analyses reveal that, as a discrete spherical particle in the cytosol, the blepharoplast is the precursor for basal body assembly (Mizukami and Gall, 1966;Hepler, 1976;Doonan et al, 1986;Vaughn et al, 1993), and the elongated "eyelash" is actually a cytoskeletal array now known as a multilayered structure, or MLS (Carothers, 1975;Myles and Hepler, 1977;Duckett, 1973; Gunning, 1986, 1988 1995; Wolniak et al, 2000;Klink and Wolniak, 2000), which is common to all plant spermatozoids. The uppermost stratum of the MLS is a ribbon of cross-linked microtubules that appears to control patterns of cell and nuclear elongation Hepler, 1977, 1982) in the developing spermatid and is attached to the basal bodies of the motile apparatus (Carothers, 1975;Hoffman and Vaughn, 1995).…”

Section: Introductionmentioning

confidence: 99%

Centrin Is Necessary for the Formation of the Motile Apparatus in Spermatids ofMarsilea

Klink

Wolniak

2001

MBoC

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126

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During spermiogenesis in the water fern, Marsilea vestita, basal bodies are synthesized de novo in cells that lack preexisting centrioles, in a particle known as a blepharoplast. We have focused on basal body assembly in this organism, asking what components are required for blepharoplast formation. Spermiogenesis is a rapid process that is activated by placing dry microspores into water. Dry microspores contain large quantities of stored protein and stored mRNA, and inhibitors reveal that certain proteins are translated from stored transcripts at specific times during development. Centrin translation accompanies blepharoplast appearance, while ␤-tubulin translation occurs later, during axonemal formation. In asking whether centrin is an essential component of the blepharoplast, we used antisense, sense, and double-stranded RNA probes made from the Marsilea centrin cDNA, MvCen1, to block centrin translation. We employed a novel method to introduce these RNAs directly into the cells. Antisense and sense both arrest spermiogenesis when blepharoplasts should appear, and dsRNA made from the same cDNA is an effective inhibitor at concentrations at least 10 times lower than either of the single-stranded RNA used in these experiments. Blepharoplasts are undetectable and basal bodies fail to form. Antisense, sense, and dsRNA probes made from Marsilea ␤-tubulin permitted normal development until axonemes form. In controls, antisense, sense, and dsRNA, made from a segment of HIV, had no effect on spermiogenesis. Immunoblots suggest that translational blocks induced by centrin-based RNA are gene specific and concentration dependent, since neither ␤-tubulin-nor HIV-derived RNAs affects centrin translation. The disruption of centrin translation affects microtubule distributions in spermatids, since centrin appears to control formation of the cytoskeleton and motile apparatus. These results show that centrin plays an essential role in the formation of a motile apparatus during spermiogenesis of M. vestita. INTRODUCTIONThroughout most of the eukaryotic realm, the formation of male gametes involves the synthesis and assembly of a motile apparatus. The motile apparatus comprises flagellar or ciliary axonemes, whose rapid beat frequency and precise beat shape are the consequence of mechanochemical interactions between scores of proteins that are arranged in a remarkably complex cylindrical array (Dutcher, 1995). A controlling template for axonemal formation is the basal body (Mizukami and Gall, 1966), which resides in the cortical cytoplasm at the base of the axoneme (Ringo, 1967;Fulton, 1971; Dibbayawan et al., 1995). Basal bodies closely resemble centrioles (Gould, 1975;Kuriyama and Borisy, 1981;Kochanski and Borisy, 1990), and they are usually formed from or in close association with centrioles (Dirksen, 1991;Marshall and Rosenbaum, 2000), (however, see Fulton [1971]; Fulton and Dingle [1971]; Gould [1975]). The ubiquity of this association has led to the perception that all basal bodies arise from these cytoplasmic organell...

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“…When introduced into cells, dsRNA can activate mechanisms that target the degradation of cognate cytoplasmic mRNAs Tuschl et al, 1999;Klink and Wolniak, 2000) and thus can effectively silence full gene expression at the posttranscriptional level. This genetic interference effect (termed RNA interference, RNAi) is temporary and the effect is dosage dependent.…”

Section: Introductionmentioning

confidence: 99%

Inducible Systemic RNA Silencing inCaenorhabditis elegans

Timmons

Tabara

Mello

et al. 2003

MBoC

135

109

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Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

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The Efficacy of RNAi in the Study of the Plant Cytoskeleton

Cited by 28 publications

References 112 publications

RNA Interference in the Moss Physcomitrella patens

RNA Interference in the Moss Physcomitrella patens

Centrin Is Necessary for the Formation of the Motile Apparatus in Spermatids ofMarsilea

Inducible Systemic RNA Silencing inCaenorhabditis elegans

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