Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae, the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae. A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae-resistant accessions of the wild sea beet (Beta vulgaris ssp. maritima), which might be used to restrict the transmission of BNYVV, were identifiedPeer reviewe

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

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 rootsPeer reviewe

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes