Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis

In plants, silencing of mRNA can be transmitted from cell to cell and also over longer distances from roots to shoots. To investigate the long-distance mechanism, WT and mutant shoots were grafted onto roots silenced for an mRNA. We show that three genes involved in a chromatin silencing pathway, NRPD1a encoding RNA polymerase IVa, RNA-dependent RNA polymerase 2 (RDR2), and DICER-like 3 (DCL3), are required for reception of long-distance mRNA silencing in the shoot. A mutant representing a fourth gene in the pathway, argonaute4 (ago4), was also partially compromised in the reception of silencing. This pathway produces 24-nt siRNAs and resulted in decapped RNA, a known substrate for amplification of dsRNA by RDR6. Activation of silencing in grafted shoots depended on RDR6, but no 24-nt siRNAs were detected in mutant rdr6 shoots, indicating that RDR6 also plays a role in initial signal perception. After amplification of decapped transcripts, DCL4 and DCL2 act hierarchically as they do in antiviral resistance to produce 21- and 22-nt siRNAs, respectively, and these guide mRNA degradation. Several dcl genotypes were also tested for their capacity to transmit the mobile silencing signal from the rootstock. dcl1-8 and a dcl2 dcl3 dcl4 triple mutant are compromised in micro-RNA and siRNA biogenesis, respectively, but were unaffected in signal transmission. © 2007 by The National Academy of Sciences of the USA.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Viruses face a double defense by plant small RNAs

Plants fight viral infections with enzymes that digest viral RNA, but viruses retaliate with proteins that suppress these enzymes. To boost their antiviral response plants deploy enzymes with redundant functions.

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Plant and animal microRNAs : similarities and differences

Plant and animal microRNAs (miRNAs) are evolutionarily ancient small RNAs, ∼19-24 nucleotides in length, that are generated by cleavage from larger highly structured precursor molecules. In both plants and animals, miRNAs posttranscriptionally regulate gene expression through interactions with their target mRNAs, and these targets are often genes involved with regulating key developmental events. Despite these similarities, plant and animal miRNAs exert their control in fundamentally different ways. Generally, animal miRNAs repress gene expression by mediating translational attenuation through (multiple) miRNA-binding sites located within the 3′ untranslated region of the target gene. In contrast, almost all plant miRNAs regulate their targets by directing mRNA cleavage at single sites in the coding regions. These and other differences suggest that the two systems may have originated independently, possibly as a prerequisite to the development of complex body plans. © Springer-Verlag 2005.

Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a homolog of dicer-1 from Drosophila

Posttranscriptional silencing (PTGS) in plants, nematodes, Drosophila, and perhaps all eukaryotes operates by sequence-specific degradation or translational inhibition of the target mRNA. These processes are mediated by duplexed RNA. In Drosophila and nematodes, double-stranded (ds)RNA or self-complementary RNA is processed into fragments of approximately 21 nt by Dicer-1 [1, 2]. These small interfering RNAs (siRNAs) serve as guides to target degradation of homologous single-stranded (ss)RNA [1, 3]. In some cases, the approximately 21 nt guide fragments derived from endogenous, imperfectly self-complementary RNAs cause translational inhibition of their target mRNAs, with which they have substantial, but not perfect sequence complementarity [4-6]. These small temporal RNAs (stRNAs) belong to a class of noncoding microRNAs (miRNAs), 20-24 nt in length, that are found in flies, plants, nematodes, and mammals [4, 6-12]. In nematodes, the Dicer-1 enzyme catalyzes the production of both siRNA and stRNA [2, 13-15]. Mutation of the Arabidopsis Dicer-1 homolog, CARPEL FACTORY (CAF), blocks miRNA production [1, 4, 16-18]. Here, we report that the same caf mutant does not block either PTGS or siRNA production induced by self-complementary hairpin RNA. This suggests either that this mutation only impairs miRNA formation or, more interestingly, that plants have two distinct dicer-like enzymes, one for miRNA and another for siRNAi production.

New dimeric supramolecular structure of mixed (phthalocyaninato)(porphyrinato)-europium(III) sandwiches : preparation and spectroscopic characteristics

The mixed double-decker Eu\[Pc(15C5)4](TPP) (1) was obtained by base-catalysed tetramerisation of 4,5-dicyanobenzo-15-crown-5 using the half-sandwich complex Eu(TPP)(acac) (acac = acetylacetonate), generated in situ, as the template. For comparative studies, the mixed triple-decker complexes Eu2\[Pc(15C5)4](TPP)2 (2) and Eu2\[Pc(15C5)4]2(TPP) (3) were also synthesised by the raise-by-one-story method. These mixed ring sandwich complexes were characterised by various spectroscopic methods. Up to four one-electron oxidations and two one-electron reductions were revealed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). As shown by electronic absorption and infrared spectroscopy, supramolecular dimers (SM1 and SM3) were formed from the corresponding double-decker 1 and triple-decker 3 in the presence of potassium ions in MeOH/CHCl3.

Electrochemistry of Heteroleptic Tris(phthalocyaninato) Rare Earth(III) Complexes

The electrochemical characteristics of a series of heteroleptic tris(phthalocyaninato) complexes with identical rare earths or mixed rare earths (Pc)M(OOPc)M(OOPc) [M = Eu...Lu, Y; H2Pc = unsubstituted phthalocyanine, H2(OOPc) = 3,4,12,13,21,22,30,31-octakis(octyloxy)phthalocyanine] and (Pc)Eu(OOPc)Er(OOPc) have been recorded and studied comparatively by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in CH2Cl2 containing 0.1 M tetrabutylammonium perchlorate (TBAP). Up to five quasi-reversible one-electron oxidations and four one-electron reductions have been revealed. The half-wave potentials of the first, second and fifth oxidations depend on the size of the metal center, but the fifth changes in the opposite direction to that of the first two. Moreover, the difference in redox potentials of the first oxidation and first reduction for (Pc)M(OOPc)M(OOPc), 0.85−0.98 V, also decreases linearly along with decreasing rare earth ion radius, clearly showing the rare earth ion size effect and indicating enhanced π−π interactions in the triple-deckers connected by smaller lanthanides. This order follows the red-shift seen in the lowest energy band of triple-decker compounds. The electronic differences between the lanthanides and yttrium are more apparent for triple-decker sandwich complexes than for the analogous double-deckers. By comparing triple-decker, double-decker and mononuclear [ZnII] complexes containing the OOPc ligand, the HOMO−LUMO gap has been shown to contract approximately linearly with the number of stacked phthalocyanine ligands.

The ageing of alumina hydrolysates synthesized from sec-butoxyaluminium(III)

A combination of X-ray diffraction, thermal analysis and Raman spectroscopy was employed to characterise the ageing of alumina hydrolysates synthesised from the hydrolysis of anhydrous tri-sec-butoxyaluminium(III). X-Ray diffraction showed that the alumino-oxy(hydroxy) hydrolysates were pseudoboehmite. For boehmite the lamellar spacings are in the b direction and multiple d(020) peaks are observed for the un-aged hydrolysate. After 4 h of ageing, a single d(020) peak is observed at 6.53 Å. Thermal analysis showed five endotherms at 70, 140, 238, 351 and 445°C. These endotherms are attributed to the dehydration and dehydroxylation of the boehmite-like hydrolysate. Raman spectroscopy shows the presence of bands for the washed hydrolysates at 333, 355, 414, 455, 475, 495, 530 and 675 cm–1. These bands are attributed to pseudoboehmite. Ageing of the hydrolysates results in an increase in the crystallite size of the pseudoboehmite.

Comparative Electrochemical Study of Unsubstituted and Substituted Bis(phthalocyaninato) Rare Earth(III) Complexes

The electrochemistry of homoleptic substituted phthalocyaninato rare earth double-decker complexes M(TBPc)2 and M(OOPc)2 [M = Y, La...Lu except Pm; H2TBPc = 3(4),12(13),21(22),30(31)-tetra-tert-butylphthalocyanine, H2OOPc = 3,4,12,13,21,22,30,31-octakis(octyloxy)phthalocyanine] has been comparatively studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in CH2Cl2 containing 0.1 M tetra-n-butylammonium perchlorate (TBAP). Two quasi-reversible one-electron oxidations and three or four quasi-reversible one-electron reductions have been revealed for these neutral double-deckers of two series of substituted complexes, respectively. For comparison, unsubstituted bis(phthalocyaninato) rare earth analogues M(Pc)2 (M = Y, La...Lu except Pm; H2Pc = phthalocyanine) have also been electrochemically investigated. Two quasi-reversible one-electron oxidations and up to five quasi-reversible one-electron reductions have been revealed for these neutral double-decker compounds. The three bis(phthalocyaninato)cerium compounds display one cerium-centered redox wave between the first ligand-based oxidation and reduction. The half-wave potentials of the first and second oxidations and first reduction for double-deckers of the tervalent rare earths depend on the size of the metal center. The difference between the redox potentials of the second and third reductions for MIII(Pc)2, which represents the potential difference between the first oxidation and first reduction of [MIII(Pc)2]−, lies in the range 1.08−1.37 V and also gradually diminishes along with the lanthanide contraction, indicating enhanced π−π interactions in the double-deckers connected by the smaller, lanthanides. This corresponds well with the red-shift of the lowest energy band observed in the electronic absorption spectra of reduced double-decker [MIII(Pc′)2]− (Pc′ = Pc, TBPc, OOPc).

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: Part 14. The infrared and Raman characteristics of phthalocyanine in “pinwheel-like” homoleptic bis[1,8,15,22-tetrakis(3-pentyloxy)phthalocyaninato] rare earth(III) double-decker complexes

The infrared (IR) spectroscopic data and Raman spectroscopic properties for a series of 13 “pinwheel-like” homoleptic bis(phthalocyaninato) rare earth complexes M[Pc(α-OC5H11)4]2 [M = Y and Pr–Lu except Pm; H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected and comparatively studied. Both the IR and Raman spectra for M[Pc(α-OC5H11)4]2 are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues, namely M(Pc)2 and M[Pc(OC8H17)8]2, but resemble (for IR) or are a bit more complicated (for Raman) than those of heteroleptic counterparts M(Pc)[Pc(α-OC5H11)4], revealing the decreased molecular symmetry of these double-decker compounds, namely S8. Except for the obvious splitting of the isoindole breathing band at 1110–1123 cm−1, the IR spectra of M[Pc(α-OC5H11)4]2 are quite similar to those of corresponding M(Pc)[Pc(α-OC5H11)4] and therefore are similarly assigned. With laser excitation at 633 nm, Raman bands derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. The IR spectra reveal that the frequencies of pyrrole stretching and pyrrole stretching coupled with the symmetrical CH bending of –CH3 groups are sensitive to the rare earth ionic size, while the Raman technique shows that the bands due to the isoindole stretchings and the coupled pyrrole and aza stretchings are similarly affected. Nevertheless, the phthalocyanine monoanion radical Pc′− IR marker band of bis(phthalocyaninato) complexes involving the same rare earth ion is found to shift to lower energy in the order M(Pc)2 > M(Pc)[Pc(α-OC5H11)4] > M[Pc(α-OC5H11)4]2, revealing the weakened π–π interaction between the two phthalocyanine rings in the same order.

Development of a Rep-inducible, BBTV-based expression system in banana

Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV. The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed. An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed. The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication. Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed. Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids. The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.

Tris[4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dionato]aluminium(III)-tris[4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dionato]iron(III) (3/1)

In the title compound, [Al(C8H4F3O2S)3]3[Fe(C8H4F3O2S)3], the metal centre is statistically occupied by AlIII and FeIII cations in a 3:1 ratio. The metal centre is within an octahedral O6 donor set defined by three chelating substituted acetoacetonate anions. The ligands are arranged around the periphery of the molecule with a mer geometry of the S atoms.

To WPPSI or to Binet, that is the question : a comparison of the WPPSI-III and SB5 with typically-developing preschoolers

This study compared two popular measures of cognitive ability for preschool children. The Wechsler Preschool and Primary Scale of Intelligence – Third Edition (WPPSI-III) and the Stanford-Binet Intelligence Scale – Fifth Edition (SB5) were administered in a counterbalanced order to 36 typically developing 4-year-old children. There were significant correlations among all WPPSI-III and SB5 composite scores but a small number of children had notable differences between their scores on the two measures. Children tended to prefer the SB5 over the WPPSI-III. Implications for practitioners who assess preschool-aged children are discussed.