Mary-Lou Edwards

Plant virus detection using a new form of indirect ELISA

A novel form of indirect enzyme-linked immunosorbent assay (ELISA) has been devised for the detection of viruses in plants. The method uses protein A in two applications to sandwich antibody-antigen-antibody layers. The first applied layer of protein A prepares the plate for the coating antibody layer. The second layer of protein A is conjugated to the enzyme and detects the second antibody layer. The orientation of the IgG induced in the coating layer of antibody prevents later unwanted reaction with the conjugated protein A. Using seven antisera, protein A sandwich ELISA (PAS-ELISA) detected homologous virus isolates in standard dilutions of infected plant homogenates at A405 values which were at least one absorbance unit greater than those of healthy controls. The PAS-ELISA method was more sensitive than the direct double antibody sandwich form of ELISA (DAS-ELISA), e.g. not only were A405 values for homologous reactions greater in PAS-ELISA but also an antiserum to a birch isolate of cherry leaf roll virus detected four related isolates with the new method against only one with DAS-ELISA. However, dilution end points for the homologous virus were about the same in both methods. In a practical application, PAS-ELISA detected prune dwarf virus in 18-36% of tested Prunus avium seeds.

Proof of concept pilot study: prevalence of grass virus infection and the potential for effects on the allergenic potency of pollen.

BackgroundWild plants harbour a variety of viruses and these have the potential to alter the composition of pollen. The potential consequences of virus infection of grasses on pollen-induced allergic disease are not known.MethodsWe have collected pollen from Dactylis glomerata (cocksfoot; a grass species implicated as a trigger of allergic rhino-conjunctivitis) from Wytham Wood, Oxfordshire UK. Extracts were prepared from pollen from uninfected grass, and from grass naturally infected by the Cocksfoot streak potyvirus (CSV). Preparations of pollen from virus-infected and non-infected grasses were employed in skin testing 15 grass pollen-allergic subjects with hayfever. Allergen profiles of extracts were investigated by Western blotting for IgE with sera from allergic subjects.ResultsThe prevalence of CSV infection in cocksfoot grasses sampled from the study site varied significantly over an eight-year period, but infection rates of up to 70% were detected. Virus infection was associated with small alterations in the quantities of pollen proteins detected by polyacrylamide gel electrophoresis, and in the patterns of allergens identified by Western blotting with IgE from grass pollen allergic subjects. For individual subjects there were differences in potencies of standardised extracts of pollen from virus-free and virus-infected plants as assessed by skin testing, though a consistent pattern was not established for the group of 15 subjects.ConclusionInfection rates for CSV in cocksfoot grass can be high, though variable. Virus-induced alterations in components of grass pollen have the potential to alter the allergenic potency.

Evidence for GC preference by monocot Dicer-like proteins

Dicot Dicer-like (DCL) enzymes operate preferably on GC rich regions when producing small interfering (si)RNA and micro (mi)RNA. This GC bias, however, is not generic in monocot miRNA productions. From wild Dactylis glomerata naturally infected by Cocksfoot streak potyvirus (CSV), CSV-siRNAs had a greater GC% than the virus genome, indicating that GC rich regions were also preferred by the grass DCLs. This supports the notion that GC preference is an ancient feature for plant DCLs, and suggests that monocot miRNA genes might have evolved to a high GC% resulting in GC bias being not detectable during mature miRNA production.

Predicting the ecological impacts of transgenes for insect and virus resistance in natural and feral populations of Brassica species

Oilseed rape is now widely grown in the UK. Nearly 400 000 ha were planted in 1990, compared with only 50 000 ha in 1978 [1]. There is concern that transgenes may be able to escape from cultivated oilseed rape, either by the formation of feral populations that arise from spilled seed [2, 3], or by gene flow into related wild species (including B. oleracea) [4, 5]. This paper describes research to help predict whether transgenes for insect or virus resistance (two of the commonest genetically modified stress-tolerance traits [6]) will increase the weediness of feral and wild Brassica populations in the UK. We also consider resistance to herbivory by molluscs (although we know of no genetic modifications for mollusc resistance) because slugs and snails are common herbivores of Brassica species.

The conservation of genetic diversity: gene flow from agriculture

One of the most frequently raised concerns about the introduction of genetically modified crops is that transgenes may be transferred to wild relatives, resulting either in the disruption of natural patterns of genetic diversity by “pollution” of species gene pools or in the addition of traits which may cause wild plants to become weedy or invasive. This paper examines these ideas from the viewpoint of conservation genetics. We look first at what is known about genetic diversity in natural populations and the forces that shape it, and then consider some of the issues which arise from hybridisations between crops and their wild relatives. The problems of predicting which traits may confer weediness and the importance of taking a case-by-case approach are illustrated by some of our work on virus-resistance in wild Brassica species.

Seminested PCR for the detection and analysis of flue-cured tobacco ( Nicotiana tabacum ) expressing a chitinase transgene

Standard PCR was ineffective in detecting a baculovirus-derived chitinase transgene in the T1 generation of chitinase-expressingNicotiana tabacum cv. CF80 after leaves were flue-cured at high temperatures. Consequently, a seminested PCR method was developed using fresh leaves from T2 generation plants also expressing the chitinase protein. Seminested PCR was highly effective in detecting the chitinase transgene in fresh leaves ofN. tabacum cvs. Xanthi-nc and K326 and in both fresh and flue-cured leaves ofN. tabacum cv. CF80.