ABSTRACT
We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.
Subject(s)
Glutathione Peroxidase/genetics , Solanum tuberosum/parasitology , Tylenchoidea/genetics , Amino Acid Sequence , Animals , Blotting, Western , Cell Line , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Glutathione Peroxidase/metabolism , In Situ Hybridization , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tylenchoidea/enzymology , Tylenchoidea/growth & developmentABSTRACT
A monofungal culture of Spongospora subterranea was unable to acquire and transmit the T isolate of potato mop-top pomovirus (PMTV-T), which has been maintained by manual transmission in the laboratory for 30 years. A recently obtained field isolate (PMTV-S) was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the readthrough (RT) protein-coding region of PMTV-S showed the presence of an additional 543 nt in the 3' half of the coding region relative to that of PMTV-T. These additional nucleotides preserved the reading frame of the RT protein and inserted 181 amino acids into the RT protein. This was confirmed by a comparison by immunoblotting of the sizes of the RT protein of PMTV-T and other recent isolates of PMTV.
Subject(s)
Capsid/chemistry , Myxomycetes/virology , Plant Viruses/chemistry , Solanum tuberosum/virology , Amino Acid Sequence , Animals , Molecular Sequence DataABSTRACT
Potato mop-top furovirus (PMTV) RNA 3 encodes the 20 kDa coat protein and a larger readthrough protein of 67 kDa. The readthrough protein is expressed by suppression of the amber stop codon which terminates the coat protein gene. A 21 kDa C-terminal fragment of the readthrough protein was doned, fused to glutathione S-transferase and expressed in E. coli. An antiserum prepared against purified fusion protein was used in ELISA to detect the readthrough protein in extracts of PMTV-infected leaves. Immunogold labelling studies showed that the readthrough protein was located near one extremity of some of the virus particles.
Subject(s)
Capsid/analysis , Plant Viruses/chemistry , RNA Viruses/chemistry , Animals , Antibodies, Viral , Capsid/genetics , Plant Viruses/genetics , Plant Viruses/ultrastructure , RNA Viruses/genetics , RNA Viruses/ultrastructure , Rabbits , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Solanum tuberosum/virology , Virion/chemistryABSTRACT
DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.
Subject(s)
Capsid/genetics , Luteovirus/genetics , Virus Assembly , Amino Acid Sequence , Animals , Base Sequence , Capsid/metabolism , Cell Line , DNA, Viral , Gene Expression , Genetic Vectors/genetics , Histidine , Luteovirus/physiology , Luteovirus/ultrastructure , Molecular Sequence Data , Nucleopolyhedroviruses/genetics , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Solanum tuberosum/virology , Spodoptera/cytology , Virion/metabolism , Virion/ultrastructureABSTRACT
Particles of isolate T of potato mop-top furovirus (PMTV) contain three RNA species (6.5, 3.0 and 2.5 kb). Hybridization tests with cloned cDNA probes showed that none of these species was derived from another. RNA 2 (2962 nt), which was sequenced, has non-coding regions of 368 nt and 285 nt at the 5' end and 3' end, respectively. Near the 5' terminus, nucleotides 46 to 110 are able to form a stem-loop structure, the stem of which has 23 bp with only one mismatch and one unpaired nucleotide. From the 5' end, the four open reading frames encode proteins of 51K, 13K, 21K and 8K. The first three of these have sequence similarity to the triple-gene-block proteins of other viruses, particularly barley stripe mosaic hordeivirus. The 51K protein contains a putative NTP-binding motif and the 13K and 21K proteins each contain two hydrophobic regions separated by a hydrophilic region. The 8K protein is rich in cysteine. PMTV differs from other furoviruses in having a tripartite genome. Its RNA 2 differs in gene content from the RNA 2 of soil-borne wheat mosaic virus, which lacks a triple gene block, and from that of beet necrotic yellow vein virus, which has a coat protein gene and read-through domain to the 5' side of its triple gene block. The gene arrangement in PMTV is therefore novel for a furovirus.
Subject(s)
Genome, Viral , Plant Viruses/genetics , RNA, Viral/genetics , Solanum tuberosum/virology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Complementary , Gene Library , Molecular Sequence Data , Nucleic Acid Conformation , Open Reading Frames/genetics , RNA, Viral/chemistry , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Viral Proteins/geneticsABSTRACT
Four potato clones with host gene-mediated resistance to potato leafroll virus (PLRV) multiplication were transformed with the PLRV coat protein (CP) gene. Plants of lines expressing high levels of transcript were highly resistant to PLRV multiplication; virus concentration was only 20-40 ng/g of leaf, which is approximately 1% of the concentration reached in susceptible cultivars. The effects of the transgenic and host-derived resistance genes appear to be additive.