Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts.

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide.

The product of maize streak virus ORF V1 is associated with secondary plasmodesmata and is first detected with the onset of viral lesions.

We have used a polyclonal antiserum derived from a bacterially expressed viral fusion protein to investigate the expression and subcellular localisation of the maize streak virus V1 product (PV1). Western blot analysis of agroinfected tissue showed that PV1 was detectable from 10 days postinoculation, coinciding with the first appearance of chlorotic viral lesions. The viral protein was only detectable in cell wall fractions of plant protein extracts. PV1 migrated with an apparent size of 14 kDa on SDS-PAGE, larger than the 10.9 kDa predicted from the amino acid sequence and therefore suggestive of posttranslational modification. Immunogold labelling located PV1 to the cell walls within lesion tissue and demonstrated a close association between the viral protein and secondary plasmodesmata. These results are consistent with the V1 product of MSV playing a role in the cell-to-cell movement of the virus in infected plants.

Coordinate regulation of replication and virion sense gene expression in wheat dwarf virus.

We have investigated the relationship between viral DNA replication and virion sense gene expression in wheat dwarf virus (WDV), a member of the geminivirus group, by testing a series of deletion mutants in transfected Triticum monococcum (einkorn) protoplasts. Mutants contained a transcription fusion of the chloramphenicol acetyltransferase coding sequence to the virion sense promoter that replaced the viral coat protein coding sequence. The deletion analysis revealed that WDV replication and virion sense transcription can proceed independently and are controlled in part by nonoverlapping elements in the large intergenic region. These data and those from a C2 open reading frame (ORF) frameshift mutant also showed that the product of the C2 ORF (C1-C2 protein) is independently involved in both DNA replication and activation of the virion sense promoter. The amino acid sequences encoded by C2, which are highly conserved in the geminivirus group, show some homology to the DNA binding domain of the myb-related class of plant transcription factors. The possible involvement of the host in controlling the function of the C1-C2 protein and the implication of these data for the development of WDV-based gene vectors are discussed.

Expression of cauliflower mosaic virus ORF II in a baculovirus system.

The cauliflower mosaic virus ORF II encoding the aphid transmission factor (ATF) was mutagenized to introduce a BamHI restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda (SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antiserum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (P18). Subcellular fractionation of cells infected with the recombinant AcMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified P18 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauliflower mosaic virus (CaMV)-infected turnip tissue, this antiserum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing P18) and not other plant cellular components.

Transcript mapping reveals different expression strategies for the bicistronic RNAs of the geminivirus wheat dwarf virus.

We have characterised the major transcripts of the Czech isolate of wheat dwarf virus (WDV-CJI) which show that WDV uses two different mechanisms for expressing overlapping open reading frames (ORFs). Mapping of the virion sense RNAs identified a single polyadenylated transcript of 1.1kb spanning the overlapping ORFs V1 and V2 which encode cell-cell spread functions and the coat protein respectively. This finding distinguishes WDV from other monocot-infecting geminiviruses studied so far which were shown to encode two 3' co-terminal transcripts capable of expressing either the V1 or V2 ORF. A survey of codon usage at the junction between the V1 and V2 ORF has led us to propose that translational frame shifting analogous to that in the yeast Ty element may occur. Analysis of polymerase chain reaction (PCR) amplified complementary sense cDNA clones has revealed the presence of mature spliced and unspliced RNAs which could encode products of an intron mediated C1:C2 ORF fusion or the C1 ORF product alone. Mapping of the 5' and 3' extremities of the major WDV encoded transcripts has allowed us to identify putative transcription regulatory sequences and the presence of multiple overlapping transcripts may suggest temporal regulation of transcription.

Replication of wheat dwarf virus DNA in protoplasts and analysis of coat protein mutants in protoplasts and plants.

The replication of wheat dwarf virus (WDV) in protoplasts derived from a Triticum monococcum suspension cell system was investigated. The production of circular viral double-stranded DNA (dsDNA) forms consistent with the replication of the viral genome was observed. In comparison to whole plants, the production of viral single-stranded DNA (ssDNA) was reduced, possibly due to only low levels of viral coat protein being produced in the protoplasts. Mutations introduced into the viral coat protein open reading frame (ORF) did not affect the ability of the viral DNA to replicate, and a deletion of ca. 400 bp was tolerated. However, these mutations abolished the infectivity of the viral genome when agroinoculated onto wheat plants, providing evidence that, contrary to the case for the bipartite geminiviruses, the coat protein is essential for infection by WDV.

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA.

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined.

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA.

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.

Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome.

Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.