Incidence of pea seedborne mosaic virus pathotypes in the US National Pisum germplasm collection

One hundred and eighty-nine isolates of pea seedborne mosaic virus (PSbMV) were obtained from seedlings of 435 pea (Pisum sativum) germplasm introductions originally acquired from India, Turkey, Latin America and Europe. Fifty-eight per cent of the isolates were identified as belonging to pathotype P-1, 22% to pathotype P-4, and 7% to the mild form of the lentil pathotype L-1. Some isolates could not be classified into any known pathotype and need further study. No isolate appeared to present a threat to peas beyond that already known to exist with previously described forms of the virus.     

A new pathotype of Pea seedborne mosaic virus explained by properties of the P3-6k1- and viral genome-linked protein (VPg)-coding regions

A fourth pathotype of Pea seedborne mosaic virus, a member of the genus Potyvirus, was identified by analysis of the infection profile on a panel of Pisum sativum lines. The new pathotype, designated P-3, was able to overcome resistance specified by the sbm-1 resistance gene but could not overcome resistance specified by the sbm-2 resistance gene. This infection profile distinguished P-3 from previously described pathotypes, P-1, P-2, and P-4. Analysis of chimeric viruses demonstrated that properties of the P3-6k1- and viral genome-linked protein (VPg)-coding regions accounted for the infection profile of the new pathotype.     

Multiple coding sequences for the genome-linked virus protein (VPg) in dicistroviruses

N-terminal Edman sequencing of the genome-linked viral protein (VPg) of Plautia stali intestine virus (PSIV, Dicistroviridae) detected heterologus residues. The VPg sequence determined was found to be triplicated in the nonstructural protein precursor. Multiple VPg-like sequences were also found in 10 of the 12 dicistroviruses with a maximum of six copies in Solenopsis invicta virus-1. We postulate that redundant VPg coding sequences facilitate multiplication of dicistroviruses, because fewer cycle of translation of the nonstructural protein precursor produces larger amounts of VPg proteins in parallel with the increased production of capsid proteins by the intergenic internal ribosome entry site mediated translation.     

Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum

The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

Transient protein expression in three Pisum sativum (green pea) varieties

The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 ( 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.     

Loss of genetic diversity in pea germplasm by the elimination of individuals infected by pea seedborne mosaic virus

Significant reduction of genetic diversity was observed in seed coat colour and electrosphoretic isozyme genotypes when infected plants were eliminated in genetically heterogeneous pea populations. Some accessions where genetic homogeneity was expected were apparently mixtures. Detection of latent infections by pea seedborne mosaic virus by the ELISA method was more effective on single-plant samples than on bulked samples of four to five plants, and from older plants rather than young seedlings. The implication of these findings in relation to a virus eradication programme in plant germplasm collections are discussed.     

Different mutations in the genome-linked protein VPg of potato virus Y confer virulence on the pvr2(3) resistance in pepper

Five different amino acid substitutions in the VPg of Potato virus Y were shown to be independently responsible for virulence toward pvr2(3) resistance gene of pepper. A consequence of these multiple mutations toward virulence involving single nucleotide substitutions is a particularly high frequency of resistance breaking (37% of inoculated plants from the first inoculation) and suggests a potentially low durability of pvr2(3) resistance. These five mutants were observed with significantly different frequencies, one of them being overrepresented. Genetic drift alone could not explain the observed distribution of virulent mutants. More plausible scenarios were obtained by taking into account either the relative substitution rates, the relative fitness of the mutants in pvr2(3) pepper plants, or both.     

Mutational analysis of the genome-linked protein VPg of poliovirus.

Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal.     

Characterization of a lectin-binding storage protein from pea (Pisum sativum)

From the storage proteins of the pea (Pisum sativum), the fraction which interacts with the pea lectin by the sugar-binding site was studied. By electrophoretical subunit patterns and other criteria, this fraction resembles the group of the 7S storage proteins (vicilins). The fraction was resolved into subunits by micropreparative SDS PAGE. The N-terminal sequences of the individual subunits were determined. Most of these are identical with published vivilin subunit sequences; therefore this lectin-binding fraction belongs to the vicilins. Selected subunits and tryptic fragments were analysed for amino-acid compositions. Though unequivocal assignments to vicilin segments were possible, significant differences could be recognized, in particular in the tryptic fragments.     

Hollow heart of pea (Pisum sativum)

Book reviewed in this article: The Way Ahead in Plant Breeding. Proceedings of the Sixth Congress of Eucarpia. Edited by F. G. H. Lupton , G. Jenkins and R. Johnson . Basic Electron Microscope Techniques. By M. A. Hayat . The Logit Transformation (with special reference to its uses in bioassay). By W. D. Ashton . Families of Frequency Distributions. By J. K. Ord .     

Verticillium wilt of pea (Pisum sativum)

A wilt disease of garden pea (Pisum sativum) caused by Verticillium dahliae is described and the range of pathogenicity of the isolate investigated. It is pathogenic to potato, sweet pea, antirrhinum and broad bean and isolates of V. dahliae from potato, lucerne and sweet pea and V. albo-atrum from lucerne are pathogenic to pea. Since the most common disease symptoms, acropetal progression of chlorosis and necrosis of the leaves followed by premature defoliation are indistinguishable from natural senescence, it is probable that disease and senescence symptoms are confused in the field. The premature defoliation results in marked reduction in green leaf area, leaf dry weight and pod yield.     

Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F

The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (K(a)) and average binding sites (n) for VPg were (8.51 +/- 0.21) x 10(6) M(-1) and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative DeltaH degrees (-29.32 +/- 0.13 kJmol(-1)) and positive DeltaS degrees (36.88 +/- 0.25 Jmol(-1)K(-1)). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m(7)G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 +/- 1.27 and 75.37 +/- 2.95 kJmol(-1) respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.     

The lectin-binding protein from the pea (Pisum sativum); properties and interactions

The lectin-binding protein (lectin binder) from the garden pea (Pisum sativum) was studied. It is a glycoprotein composed of four subunits of about 50 000 Da. Its amino-acid composition and molecular mass differ from those of lectin and of storage proteins. The interaction between lectin and lectin binder is demonstrated and quantified by several different methods and is shown to be specifically sugar-dependent. A biological function of lectin binders and lectins is discussed.     

Occurrence of plantago mottle virus in pea, Pisum sativum, in New York State

Plantago mottle virus (RMV), a member of the tymovirus group, was identified as the causal agent of a disease of pea (Pisum sativum) in New York State. The pea virus isolates were identical in host range and serology to the type strain from Plantago major. In susceptible pea genotypes symptoms were strongly influenced by ambient temperature; high temperature (35^°C) reduced infectivity and suppressed symptoms, whereas low temperature (15 and 25^°C) prolonged the incubation period but favoured the development of conspicuous leaf veinal chlorosis, mottle and necrosis. Resistance to P1MV was found in seventeen of twenty-five domestic pea cultivars and in two of twelve foreign introductions. Many of the P1MV-resistant lines were resistant also to bean yellow mosaic virus. The use of resistant cultivars and the apparent limited conditions for efficient transmission of this virus have minimized its importance to pea crops in New York State.     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA