A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus

Barley yellow dwarf virus-PAV (BYDV-PAV) is the most serious and widespread virus of cereals worldwide. Natural resistance genes against this luteovirus give inadequate control, and previous attempts to introduce synthetic resistance into cereals have produced variable results. In an attempt to generate barley with protection against BYDV-PAV, plants were transformed with a transgene designed to produce hairpin (hp)RNA containing BYDV-PAV sequences. From 25 independent barley lines transformed with the BYDV-PAV hpRNA construct, nine lines showed extreme resistance to the virus and the majority of these contained a single transgene. In the progeny of two independent transgenic lines, inheritance of a single transgene consistently correlated with protection against BYDV-PAV. This protection was rated as immunity because the virus could not be detected in the challenged plants by ELISA nor recovered by aphid feeding experiments. In the field, BYDV-PAV is sometimes associated with the related luteovirus Cereal yellow dwarf virus-RPV (CYDV-RPV). When the transgenic plants were challenged with BYDV-PAV and CYDV-RPV together, the plants were susceptible to CYDV-RPV but immune to BYDV-PAV. This shows that the immunity is virus-specific and not broken down by the presence of CYDV. It suggests that CYDV-RPV does not encode a silencing-suppressor gene or that its product does not protect BYDV-PAV against the plant's RNAi-like defence mechanism. Either way, our results indicate that the BYDV-PAV immunity will be robust in the field and is potentially useful in minimizing losses in cereal production worldwide.

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barley yellow dwarf virus transmitted by Rhopalosiphum padi

Barley yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot.

Nucleotide sequence of coat protein gene for GPV isolate of barley yellow dwarf virus and construction of expression plasmid for plant

GPV is a Chinese serotype isolate of barley yellow dwarf virus (BYDV) that has no reaction with antiserum of MAV, PAV, SGV, RPV and RMV The sequence of the coat protein (CP) of GPV isolate of BYDV was identified and its amino acid sequence was deduced. The coding region for the putative GPV CP is 603 bases nucleotides and encodes a Mr 22 218 (22 ku) protein. The same as MAV, PAV and RPV, GPV contained a second ORF within the coat protein coding region. This protein of 17 024 Mr (17 ku) is thought to correspond to the Virion protein genome linked (Vpg). Sequence comparisons of the CP coding region between the GPV isolate of BYDV and other isolates of BYDV have been done. The nucleotide and amino acid sequence homology of GPV has a greater identity to the sequence of RPV than those of PAV and MAV. The GPV CP sequence stored 83.7% of nucleotide similarity and 77.5% of deduced amino acid similarity, whereas that of the PAV and MAV shared 56.9%, 53.2% and 44.1%, 43.8% respectively. According to BYDV-GPV CP sequence, two primers were designed. The cDNA of CP was produced by RT-PCR. Full-length cDNA of CP was inserted into plasmid to construct expression plasmids named pPPI1, pPPI2 and pPPI5 based on different promoters. The recombinant plasmids were identified by using α-32P-dATP labelled CP probe, α-32P-ATP labelled GPV RNA probe and sequencing to confirm real GPV CP gene cDNA in plasmids.

Soybean dwarf luteovirus contains the third variant genome type in the luteovirus group

Complementary DNAs covering the entire RNA genome of soybean dwarf luteovirus (SDV) were cloned and sequenced. Computer analysis of the 5861 nucleotide sequence revealed five major open reading frames (ORFs) possessing conservation of sequence and organisation with known luteovirus sequences. Comparative analyses of the genome structure show that SDV shares sequence homology and features of gene organisation with barley yellow dwarf virus (PAV isolate) in the 5' half of the genome, yet is more closely related to potato leafroll virus in its 3' coding regions. In addition, SDV differs from other known luteoviruses in possessing an exceptionally long 3' terminal sequence with no apparent coding capacity. We conclude from these data that the SDV genome represents a third variant genome type in the luteovirus group.

Characterisation of the subgenomic RNAs of an Australian isolate of barley yellow dwarf luteovirus

We have characterised the subgenomic RNAs of an Australian isolate of BYDV-PAV. Northern blot analyses of infected plants and protoplasts have shown that this isolate synthesises three subgenomic RNAs. Precise mapping of the transcription start sites of all three subgenomic RNAs and translational analyses of subgenomic RNA 2 and 3 have revealed a number of features. First, the transcription start site of subgenomic RNA 1 in this isolate differs markedly from the start site determined for an Illinois isolate of BYDV-PAV. Second, the start sites of subgenomic RNA 1 and 2 occur at a sequence that closely resembles the 5' end sequence of the genomic RNA (5'AGUGAAGA). Third, subgenomic RNA 2 appears to express ORF 6 of BYDV-PAV but the gene product is truncated due to the appearance of a new stop codon in the sequence. Last, subgenomic RNA 3, which is abundantly transcribed and encapsidated by the virus particle, appears to have no coding ability. We postulate that this novel subgenomic RNA has a regulatory function.

Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus

A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter. RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts. Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles. Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants. Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I mutant remained infectious. Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.

A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain

An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotides long, single-stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus " hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudoknot. © 1991.

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene. © 1988.

Testing second order cyclostationarity in the squared envelope spectrum of non-white vibration signals

Cyclostationary models for the diagnostic signals measured on faulty rotating machineries have proved to be successful in many laboratory tests and industrial applications. The squared envelope spectrum has been pointed out as the most efficient indicator for the assessment of second order cyclostationary symptoms of damages, which are typical, for instance, of rolling element bearing faults. In an attempt to foster the spread of rotating machinery diagnostics, the current trend in the field is to reach higher levels of automation of the condition monitoring systems. For this purpose, statistical tests for the presence of cyclostationarity have been proposed during the last years. The statistical thresholds proposed in the past for the identification of cyclostationary components have been obtained under the hypothesis of having a white noise signal when the component is healthy. This need, coupled with the non-white nature of the real signals implies the necessity of pre-whitening or filtering the signal in optimal narrow-bands, increasing the complexity of the algorithm and the risk of losing diagnostic information or introducing biases on the result. In this paper, the authors introduce an original analytical derivation of the statistical tests for cyclostationarity in the squared envelope spectrum, dropping the hypothesis of white noise from the beginning. The effect of first order and second order cyclostationary components on the distribution of the squared envelope spectrum will be quantified and the effectiveness of the newly proposed threshold verified, providing a sound theoretical basis and a practical starting point for efficient automated diagnostics of machine components such as rolling element bearings. The analytical results will be verified by means of numerical simulations and by using experimental vibration data of rolling element bearings.

Managing creativity on the margins : a comparative study of Beijing’s Songzhuang Art District and Hangzhou’s White Horse Lake Creative Eco-City

In the past decade the ‘creative cluster’ has become a driver of urban renewal in China. Many cluster developments attract human capital and investment to post-industrial spaces. This paper looks at two developments which are more post-agricultural than post-industrial: the first is Songzhuang, a large scale contemporary art community situated on the eastern fringe of Beijing, the second is Hangzhou’s White Horse Lake Creative Eco-City, a ‘mixed variety’ cluster model which integrates elements of art, fashion, design and animation. The common factor in both cases is how they came into existence. In both districts urban creative workers moved into a rural environment. Drawing on interviews with planners, officials, and residents we investigate the challenges of sustaining such fringe clusters.

CIB White Paper on IDDS Integrated Design & Delivery Solutions

CIB is developing a priority theme, now termed Improving Construction and Use through Integrated Design & Delivery Solutions (IDDS). The IDDS working group for this theme adopted the following definition: Integrated Design and Delivery Solutions use collaborative work processes and enhanced skills, with integrated data, information, and knowledge management to minimize structural and process inefficiencies and to enhance the value delivered during design, build, and operation, and across projects. The design, construction, and commissioning sectors have been repeatedly analysed as inefficient and may or may not be quite as bad as portrayed; however, there is unquestionably significant scope for IDDS to improve the delivery of value to clients, stakeholders (including occupants), and society in general, simultaneously driving down cost and time to deliver operational constructed facilities. Although various initiatives developed from computer‐aided design and manufacturing technologies, lean construction, modularization, prefabrication and integrated project delivery are currently being adopted by some sectors and specialisations in construction; IDDS provides the vision for a more holistic future transformation. Successful use of IDDS requires improvements in work processes, technology, and people’s capabilities to span the entire construction lifecycle from conception through design, construction, commissioning, operation, refurbishment/ retrofit and recycling, and considering the building’s interaction with its environment. This vision extends beyond new buildings to encompass modifications and upgrades, particularly those aimed at improved local and area sustainability goals. IDDS will facilitate greater flexibility of design options, work packaging strategies and collaboration with suppliers and trades, which will be essential to meet evolving sustainability targets. As knowledge capture and reuse become prevalent, IDDS best practice should become the norm, rather than the exception.