Generation Of 13 k-Gene Sugar Beet Transformants And Evaluation Of Their Resistance To BNYVV Infection
Introduction
Sugar beet (Beta vulgaris L.) is one of the main industrial crops in Greece and in many other European countries. Rhizomania, the most important virus disease of the crop, can lead to severe losses in tap root yield and sugar content. The main symptoms of the disease are abnormal proliferation of fine rootlets from the taproot and lateral roots, partial necrosis of these tissues, necrosis of the vascular tissue and stunting of the tap root (reviewed in [1]. The causative agent of the disease is the fungus-transmitted virus beet necrotic yellow vein virus (BNYVV). The vector of the virus, the soilbome obligate intracellular fungus Polymyxa betae transmits the virus in a persistent fashion. It is difficult to control the disease using environmentally acceptable methods, since the virus can survive in the spores of the fungus several years and soil decontamination is effective only after treatment with methylbromide. Thus, if the disease invades the field, the only alternative for the farmer is the use of resistant cultivars. Generation of resistant varieties using transgenic plant approaches offer an alternative. Several approaches have been developed for pathogen-derived transgenic resistance [2]. Transgenic lines containing and expressing the BNYVV coat protein mRNA have been produced, and were found to contain reduced levels of the virus, although the coat protein was not in detectable amounts [3]. Expression of full length coat protein may have a higher risk because of possible recombination or transencapsidation events. Over-expression of viral sequences in a plant may finally lead to the specific suppression of the gene, in a phenomenon called RNA-mediated resistance [4]. This reaction, which is believed to be the result of a general plant defence mechanism, can lead to virus resistant or even immune plants.
As part of a wider sugar beet breeding program aiming to create new sugar beet cultivars resistant to the major diseases of the crop, we present here the first series of experiments for generation of virus resistant plants. BNYVV is a furovirus whose genome consists of four to five plus-sense RNAs. The genes on RNA 1, and 2 encode functions essential for replication and movement in the plant, whereas the genes on RNAs 3,4, and 5 are implicated in symptom expression or host range and vector-mediated infection of sugar beet (review in [5], [6]).
We introduced the viral gene sequence 13 K under the control of the CaMV 35S promoter in sugar beet plants. The viral gene 13 K, which is located on RNA 2 of the virus in an area known as the triple gene block (TGB) [7] encodes for a membrane protein which has a major role in the transport of the virus. Regenerated transgenic plants were challenged with the virus and examined for resistance.
Section snippets
Cloning of the 13 K gene
The cDNA of the selected gene of beet necrotic yellow vein virus was cloned using the immunocapture PCR technique as described in [8] Infected plants were tested for the presence of BNYVV by western blotting using a commercially available anti BNYVV antibody (not shown). Plants showing positive signal (truly infected) were then used for the isolation of the virus. BNYVV was first immunocaptured from extracts of infected roots and then the viral particles were used for the reverse transcription
Phenotype of 13 K gene transformed sugar beet
The 13 K cDNA was subcloned in vector p ART7/ pART27, a binary plasmid for stable transformation of Agrobacterium. Transformants were regenerated on kanamycin selection medium. The 13 K gene was under the control of CaMV 35S promoter. Transgenic sugarbeet plants were regenerated from leaf petioles on selection growth media. Plantlets were transferred on rooting media 2–3 months after the initial shoot induction. Rooted transgenic plants were then transferred to non-sterile conditions in the
Virus infectivity studies
13 K transformed sugar beet plants were tested for resistance to rhizomania in controlled infections in the greenhouse. Transgenic plants were transferred on infected soil and rootlets were used after several intervals for ELISA tests. In order to avoid differences in local concentration of the virus, samples from three different positions of the root were taken and homogenised together for ELISA assays. We tested in preliminary experiments the efficiency of the infection of a commercially
Discussion
Expression of viral sequences, containing either fully functional or truncated ORFs has been shown that can confer resistance in many virus families and several plant species. This type of transgenic resistance can be divided into two groups, one group in which expression of a viral protein is important for resistance (coat protein or other), and another group where expression of RNA sequences are sufficient to confer resistance. The 13 K gene of the viruses containing a triple gene block is a
Materials and methods
Plasmid constructs. All plasmids were constructed according to published routine procedures [18]. The pART7/ 27 vector system used for Agrobacterium transformations has been published [19] and was kindly provided by Dr. A. Gleave. Plasmids were inserted in Agrobacteria by tri-parental mating [20].
Immunocapture PCR and Cloning of 13 K cDNA. Material from Infected rootlets was extracted in Tris/HCl pH 8.2, 0.25% PVP-360,1% PEG 6000, 140 mM NaCI, 0.05%, Tween 20. A commercially available antibody
Acknowledgements
We thank M. Providaki for technical assistance in subcloning the 13 K gene in pART7/27, Dr. G. Skarakis for donating the initial sugar beet plants and Dr. A. Gleave for kindly providing the pART7/27 vectors. This work was supported by the Greek Ministry of Development, General Secretariat of Research and Technology, Framework IV, programme EPETII 233.
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