Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach

Abstract

Highly specific rabbit polyclonal antibodies for the obligate sugar-beet root parasite, Polymyxa betae, were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro, and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta, and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar-beet roots and with the closely related Polymyxa graminis in barley roots, and to cross-react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross-reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea. In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar-beet, barley, cabbage and potato roots.

Barley elicits a similar early basal defence response during host and non-host interactions with Polymyxa root parasites

“…Alternatively, the host specificity determinants may be encoded not in the plant but within the Polymyxa genomes. Circumstantial evidence to support this exists from the finding that glutathione S-transferase, a well known defence protein in plants (Jwa et al 2006) and a gene which is induced in Mycosphaerella graminicola when the fungus was under oxidative stress in planta (Keon et al 2007), is highly expressed in P. betae during plant infection (Mutasa-Göttgens et al 2000). Clearly, an important key to improved understanding of the Polymyxa plant parasite and virus vector is the availability of reliable and comprehensive genome sequence data which are still lacking.…”

Breaking of Beet necrotic yellow vein virus resistance in sugar beet is independent of virus and vector inoculum densities

“…Either bait plant bioassays using soil dilutions are applied to estimate the most probable numbers (MPN) of infective propagules (Tuitert 1990) or polymerase chain reaction (PCR) is used to estimate P. betae concentration in plant material and soil (Mutasa et al 1995;Mutasa-Gottgens et al 2000;Kingsnorth et al 2003). In a soil sample from The Netherlands, Tuitert (1990) estimated a proportion of 10-15% of the rootinfecting P. betae population to be viruliferous.…”

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

“…Also, tags at the N-and C-terminus are not expected to have significant immunogenic properties (Kumari et al, 2001;Mutasa-Gottgena et al, 2000). It was found in the present study that affinity-purified fusion GFLV CP as the good antigen to raise specific and efficient polyclonal antiserum.…”

“…Although, there are vectors that possess a cleavage facility to remove the tags from the expressed protein before injecting into animal the tags are not expected to have significant immunogenic properties on the CP expressed in E. coli (Gulati-Sakhuja et al, 2009;Kumari et al, 2001;Mutasa-Gottgena et al, 2000). Also, tags at the N-and C-terminus are not expected to have significant immunogenic properties (Kumari et al, 2001;Mutasa-Gottgena et al, 2000).…”