Abstract
Fusarium head blight (FHB) caused by Fusarium graminearum infection is a devastating disease of wheat, maize, and other cereals. A previously isolated chicken single-chain Fv antibody (scFv), CWP2, that conferred durable resistance in planta was subjected to directed evolution by error-prone PCR and DNA shuffling, generating a mutated library. Panning of the mutated library against cell wall–bound proteins (CWPs) from F. graminearum by phage display enriched phage clones that were used for a further round of DNA shuffling to construct a combinatorial library comprising 3 × 106 variants. Screening of this library by phage display for variants reactive against the CWPs led to the identification of a number of clones. Comparative enzyme-linked immunosorbent assay analyses revealed eight clones exhibiting a higher reactivity than the parent, CWP2, and containing four different single-chain antibody sequences. Surface plasmon resonance measurements confirmed that three mutated scFvs, CWPa, CWPb, and CWPd, displayed 15-fold, 11-fold, and 7-fold higher affinities, respectively, compared with CWP2. Three-dimension modeling of CWPa illustrates a conformational change bringing all six complementary domain regions on the antibody surface in one direction. These results provide promising unique resistance molecules for effective control of FHB and its associated mycotoxins in food/feed chains.
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References
Windels, C. E. (2000). Economic and social impacts of Fusarium head blight: Changing farms and rural communities in the northern great plains. Phytopathology, 90, 17–21.
Chen, L. F., Bai, G. H., & Desjardins, A. E. (2000). Recent advances in wheat head scab research in China. National Agricultural Library Internet Publication, US Department of Agriculture, Beltsville, MD, published online.
Tavladoraki, P., Benvenuto, E., Trinca, S., de Martinis, D., Cattaneo, A., & Galeffi, P. (1993). Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature, 366, 469–472.
Engelen, F. A., Schouten, A., Molthoff, J. W., Roosien, J., Salinas, J., Dirkse, W. G., et al. (1994). Coordinate expression of antibody subunit genes yields high levels of functional antibodies in roots of transgenic tobacco. Plant Molecular Biology, 26, 1701–1710.
Voss, A., Niersbach, M., Hain, R., Hirsch, H. J., Liao, Y. C., Kreuzaler, F., et al. (1995). Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody. Molecular Breeding, 1, 39–50.
Schillberg, S., Zimmermann, S., Zhang, M. Y., & Fischer, R. (2001). Antibody-based resistance to plant pathogens. Transgenic Research, 10, 1–12.
Nölke, G., Cobanov, P., Uhde-Holzem, K., Reustle, G., Fischer, R., & Schillberg, S. (2009). Grapevine fanleaf virus (GFLV)-specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana. Molecular Plant Pathology, 10, 41–49.
Zimmermann, S., Schillberg, S., Liao, Y. C., & Fisher, R. (1998). Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum. Molecular Breeding, 4, 369–379.
Boonrod, K., Galetzka, D., Nagy, P. D., Conrad, U., & Krczal, G. (2004). Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance. Nature Biotechnology, 22, 856–862.
Peschen, D., Li, H. P., Fischer, R., Kreuzaler, F., & Liao, Y. C. (2004). Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen. Nature Biotechnology, 22, 732–738.
Nickel, H., Kawchuk, L., Twyman, R. M., Zimmermann, S., Junghans, H., Winter, S., et al. (2008). Plantibody-mediated inhibition of the Potato leafroll virus P1 protein reduces virus accumulation. Virus Research, 136, 140–145.
Safarnejad, M., Fischer, R., & Commandeur, U. (2009). Recombinant-antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana. Archives of Virology, 154, 457–467.
Cervera, M., Esteban, O., Gil, M., Gorris, M., Martínez, M., Peña, L., et al. (2010). Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance. Transgenic Research, 19, 1001–1015.
Yajima, W., Verma, S. S., Shah, S., Rahman, M. H., Liang, Y., & Kav, N. N. (2010). Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot. New Biotechnology, 27, 816–821.
Fromant, M., Blanquet, S., & Plateau, P. (1995). Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Analytical Biochemistry, 224, 347–353.
Stemmer, W. P. C. (1994). Rapid evolution of a protein in vitro by DNA shuffling. Nature, 370, 389–391.
Stemmer, W. P. C. (1994). DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proceedings of the National Academy of Sciences of the United States of America, 91, 10747–10751.
van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R. C., Hoogenboom, H. R., et al. (2003). Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Letters, 546, 288–294.
Luginbühl, B., Kanyo, Z., Jones, R. M., Fletterick, R. J., Prusiner, S. B., Cohen, F. E., et al. (2006). Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation. Journal of Molecular Biology, 363, 75–97.
Fukuda, I., Kojoh, K., Tabata, N., Doi, N., Takashima, H., Miyamoto-Sato, E., et al. (2006). In vitro evolution of single-chain antibodies using mRNA display. Nucleic Acids Research, 34, e127.
Kobayashi, N., Oyama, H., Kato, Y., Goto, J., Söderlind, E., & Borrebaeck, C. A. K. (2010). Two-step in vitro antibody affinity maturation enables estradiol-17β assays with more than 10-fold higher sensitivity. Analytical Chemistry, 82, 1027–1038.
Willats, W. G. T. (2002). Phage display: Practicalities and prospects. Plant Molecular Biology, 50, 837–854.
Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., & Becerril, B. (2005). A strategy for the generation of specific human antibodies by directed evolution and phage display. FEBS Journal, 272, 2591–2601.
Li, H. P., Zhang, J. B., Shi, R. P., Huang, T., Fischer, R., & Liao, Y. C. (2008). Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusarium-specific antibody and an antifungal peptide. Molecular Plant–Microbe Interactions, 21, 1242–1248.
Zhang, J. B., Li, H. P., Dang, F. J., Qu, B., Xu, Y. B., Zhao, C. S., et al. (2007). Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China. Mycological Research, 111, 967–975.
Qu, B., Li, H. P., Zhang, J. B., Xu, Y. B., Huang, T., Wu, A. B., et al. (2008). Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout China. Plant Pathology, 57, 15–24.
Schoffelmeer, E. A. M., Klis, F. M., Sietsma, J. H., & Cornelissen, B. J. C. (1999). The cell wall of Fusarium oxysporum. Fungal Genetics and Biology, 27, 275–282.
Gough, K. C., Li, Y., Vaughan, T. J., Williams, A. J., Cockburn, W., & Whitelam, G. C. (1999). Selection of phage antibodies to surface epitopes of Phytophthora infestans. Journal of Immunological Methods, 228, 97–108.
O’Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., & Hensley, P. (1993). Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: Use of nonlinear least squares analysis methods. Analytical Biochemistry, 212, 457–468.
Gershon, P. D., & Khilko, S. (1995). Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector. Journal of Immunological Methods, 183, 65–76.
Wanyera, R., Kinyua, M. G., Jin, Y., & Singh, R. P. (2006). The spread of stem rust caused by Puccinia graminis f. sp. tritici, with virulence on Sr31 in wheat in Eastern Africa. Plant Disease, 90, 113.
Chen, X. M. (2007). Challenges and solutions for stripe rust control in the United States. Australian Journal of Agricultural Research, 58, 648–655.
Stokstad, E. (2007). Deadly wheat fungus threatens world’s breadbaskets. Science, 315, 1786–1787.
Daugherty, P. S., Chen, G., Iverson, B. L., & Georgiou, G. (2000). Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies. Proceedings of the National Academy of Sciences of the United States of America, 97, 2029–2034.
Christians, F. C., & Loeb, L. A. (1996). Novel human DNA alkyltransferases obtained by random substitution and genetic selection in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 93, 6124–6128.
Martinez, M. A., Pezo, V., Marliere, P., & Wain-Hobson, S. (1996). Exploring the functional robustness of an enzyme by in vitro evolution. EMBO Journal, 15, 1203–1210.
Zaccolo, M., & Gherardi, E. (1999). The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1β-lactamase. Journal of Molecular Biology, 285, 775–783.
Juárez-González, V. R., Riaño-Umbarila, L., Quintero-Hernández, V., Olamendi-Portugal, T., Ortiz-León, M., Ortíz, E., et al. (2005). Directed evolution, phage display and combination of evolved mutants: A strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2. Journal of Molecular Biology, 346, 1287–1297.
Saviranta, P., Pajunen, M., Jauria, P., Karp, M., Pettersson, K., Mäntsälä, P., et al. (1998). Engineering the steroid-specificity of an anti-17β-estradiol Fab by random mutagenesis and competitive phage panning. Protein Engineering, 11, 143–152.
Graff, C. P., Chester, K., Begent, R., & Wittrup, K. D. (2004). Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37°C. Protein Engineering, Design and Selection, 17, 293–304.
Zahnd, C., Spinelli, S., Luginbühl, B., Amstutz, P., Cambillau, C., & Plückthun, A. (2004). Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. Journal of Biological Chemistry, 279, 18870–18877.
Jensen, K. B., Larsen, M., Pedersen, J. S., Christensen, P. A., Álvarez-Vallina, L., Goletz, S., et al. (2002). Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochemical and Biophysical Research Communications, 298, 566–573.
Acknowledgments
We acknowledge financial support from the National Basic Research Program of China (2009CB118806), National Natural Science Foundation of China (30530510, 30571160, 30771337), the Ministry of Agriculture of China (2008ZX08002-001, 2009ZX08002-001B) and the Ministry of Science and Technology of China (2009DFA32330).
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Liu, JL., Hu, ZQ., Xing, S. et al. Attainment of 15-Fold Higher Affinity of a Fusarium-specific Single-Chain Antibody by Directed Molecular Evolution Coupled to Phage Display. Mol Biotechnol 52, 111–122 (2012). https://doi.org/10.1007/s12033-011-9478-3
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DOI: https://doi.org/10.1007/s12033-011-9478-3