Generation and characterization of single chain antibody fragments binding to the coat protein of grapevine leafroll associated virus 2 and 3

Grapevine (Vitis species) is the main cultivated fruit crop in the world and the most important in economic terms. Increased production efficiency and improved fruit quality has traditionally been based on the modification of management and growing conditions of specific genotypes which have generally been kept constant by vegetative multiplication. Today the limitations of this approach are obvious with increased input costs and practices, some of which are not sustainable, and include the use of chemicals with the potential negative secondary effects on human health and the environment. Grapevine Leafroll-associated virus 2 and 3 (GLRaV-2 and 3) are one of the most widespread graft-transmissible diseases-affecting grapevines. The degeneration of the vascular system of the plants produces a reduction in the berry yields, delayed maturity and causes a poor pigmentation of the berries. All these have negative influence on the vine produced. The feasibility of antibody-based resistance has been demonstrated for plant viruses. It has been shown that plants that express antibodies binding to viral coat proteins gain a degree of protection against the virus in question. The rationale of this work was to generate scFvs by recombinant DNA technology that specifically recognize and inactivate the coat protein of the GLRaV-2 and 3 and therefore potentially inactivate virus disassembly and virus spreading. The constitutive expression of these scFvs may create virus resistant grapevine improving the farming system and reducing the use of environmental unfriendly chemical controls. In the present work, a specific GLRaV-2 coat protein scFv-fragment was generated from the mAb R19. The scFvLR2cp-35 was expressed in E. coli and its reactivity to GLRaV-2 viral particles was confirmed. Furthermore, the functionality of the scFv in the plant cell cytosol was tested by ELISA. In addition, three specific scFv fragments that specifically bind to GLRaV-3 coat protein were isolated from a phage display library derived from immunized mice. One of these specific scFv, scFvLR3cp-1 was further characterized for its reactivity and functionality to GLRaV-3 coat protein by ELISA, EMSA, surface plasmon resonance analysis, electron microscopy and epitope mapping. The scFvLR3cp-1 also recognized with high specificity at least four different members of the Closteroviridae family. The in vivo stability of this scFv was evaluated by transient expression in N. tabacum and stable transformed N. benthamiana plants. The data presented in this study proved that specifics scFvs against GLRaV-2 and 3 coat proteins have been successfully generated and functionally expressed not only in bacteria but also in the cytosol of tobacco plants. The simultaneous expression of these specifics scFv-fragments could be a very powerful strategy to engineered ‘pyramided’ resistance to different GLRaVs in grapevine transgenic plants.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2007

Author dc:creator

• Orecchia, Martin

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Pflanzenkrankheit
• Abwehrreaktion
• Antikörperbibliothek
• Biowissenschaften, Biologie
• Pflanzenvirologie
• Biotechnologie
• molekulare Pflanzenbiotechnologie
• plant virology
• molecular plant biotechnology

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123914%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:62343