ARTICLES 732 VOLUME 22 NUMBER 6 JUNE 2004 NATURE BIOTECHNOLOGY Fusarium is an important genus of fungal pathogens, responsible for devastating diseases such as cereal scab, which has reached epidemic lev- els 1,2 . There are 17 known species of Fusarium infecting all members of the Gramineae and most genera of other cultivated plants 3 . These fungi cause huge crop losses and produce mycotoxins that are detrimental to human and animal health 2–5 . The best control strategy is the preven- tion of infection in the field and during storage. However, natural resistance against Fusarium is inadequate 2,3 . Current protective meas- ures rely on chemical control, producing undesirable environmental consequences. Alternatives must be found to protect plants from fun- gal pathogens and to reduce or eliminate mycotoxin production. AFPs have been expressed in plants to confer disease resistance 6–9 . In most cases, the expression of individual AFPs only delays the appear- ance of disease symptoms and does not provide effective control of the disease. Monoclonal and recombinant antibodies have been produced in various expression systems including plants 10–13 . In many cases, plant-derived antibodies have been developed for therapeutic applica- tions, but they have also been used for immunomodulation 13 and the protection of plants against pathogens 14–17 . For example, cytoplasmic expression of a single-chain (scFv) antibody against artichoke mottled crinkle virus in transgenic tobacco was shown to reduce viral infection and delay the progression of disease symptoms 15 . However, antibody- mediated resistance against fungal pathogens in transgenic plants has not been demonstrated. To evaluate the potential of antibody-mediated fungal resistance in plants, we generated specific antibodies against Fusarium gramin- earum. No germ plasm exists that provides effective innate resistance to this pathogen under high disease pressure 2,3,18 . Our strategy was based on the use of chicken antibodies that target AFPs to the site of infection by binding to surface components of the invading fungus, thus interfering with fungal growth and development. Recombinant proteins were produced by fusing a Fusarium-specific scFv antibody to AFPs, which specifically inactivate fungal pathogens. The fusion pro- teins showed strong inhibitory effects on the growth of Fusarium spp. in vitro, whereas transgenic A. thaliana plants expressing the fusions showed substantially enhanced resistance to the pathogen. Our results demonstrate that engineered antibody-fusion proteins expressed in transgenic plants can control Fusarium infection, providing a new strategy for the development of environment-friendly pathogen resist- ance management practices. RESULTS Production and characterization of Fusarium specific scFvs Three phage display libraries were created with mRNA from chickens immunized with three different F. graminearum antigens: cell wall– bound proteins (CWPs), mycelium surface proteins and germinated spores. The libraries were pooled to increase the likelihood that the best binders would be selected, and were screened against CWPs. After three rounds of panning, 100 monoclonal phages reactive to CWPs were randomly selected and analyzed using an enzyme-linked immunosorbent assay (ELISA). Fingerprinting of the ten most reac- tive clones identified one scFv (which we named CWP2) that was 1 Institut für Biologie VII (Molekulare Biotechnologie), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany. 2 Fraunhofer Institut für Molekularbiologie und Angewandte Oekologie (IME), Worringerweg 1, D-52074 Aachen, Germany. 3 College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China. 4 Institut für Biologie I (Molekulare Genetik), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany. 5 Correspondence should be addressed to Y.-C.L. (e-mail: yucailiao@mail.hzau.edu.cn or liao@molbiotech.rwth-aachen.de). Published online 16 May 2004; doi:10.1038/nbt970 Fusion proteins comprising a Fusarium -specific antibody linked to antifungal peptides protect plants against a fungal pathogen Dieter Peschen 1 , He-Ping Li 2,3 , Rainer Fischer 1,2 , Fritz Kreuzaler 4 & Yu-Cai Liao 1,3 In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain anti- body specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthi- olae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection. © 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology