Introduction

1. ScFv Gene Constructs and Protein Products Bivalent, monospecific TBR TBR TBR VH VL Bivalent, bispecific P L TBRB TBRA VHB VLA P Canker challenge test: 16 days post-inoculation VHA VLB L Monovalent, monospecific P L ScFv VL VH P L Control—(Duncan grapefruit) Disease BlockTM--- (Flame grapefruit) X. citri, 5,000 cfu/ml IPG NSF Phase II Project: citrus canker resistance 4 weeks --- 26% “flame” grapefruit survive; 1/2 of these are transformed Introduction Abstract A potentially food-grade technology for controlling plant pathogens involves cloned single chain variable region antibody fragments (SCFVs) expressed in plants (“plantibodies”). We obtained monoclonal antibodies (MAbs) that had been selected to bind two functionally significant domains of a pathogenicity protein required for citrus canker disease. The pathogenicity protein is literally injected by the pathogen into citrus cells to cause canker. The MAbs were used to clone the corresponding SCFVs in various combinations to form monovalent, bivalent and bispecific SCFVs. The SCFV clones were used to transform grapefruit seedlings. Initial tests on some of the SCFVs indicated that they delayed and suppressed canker symptoms but did not confer immunity. However, Western blot analyses revealed that the translational expression levels were below the threshold limit of detection. Disease BlockTM Grapefruit (J605) 50,000 cfu/ml Control Grapefruit 5,000 cfu/ml At least 2,000X fewer X. citri cells released Citrus production for the major producing countries in the world is estimated at approximately 62 million tons or $5.878 billion. One of the leading diseases affecting this industry is citrus canker, which seriously limits citrus production in Asia and S. America (among other places) and now threatens Florida citrus. In heavily infested areas, 50% or more of the fruit fail to develop and fall from the tree prematurely. Canker causes such losses to grapefruit, sweet orange and lime that these simply cannot be grown in many parts of Asia and the Middle East. There is no cure, and resistance cannot be genetically introgressed by breeding. Disease BlockTM: Genetically Engineered Plants with Disease Resistance Chandrika Ramadugu and Dean W. Gabriel, Integrated Plant Genetics, Inc., 12085 Research Drive, Alachua, FL 32615NSF Phase II Award # 011131 Results and Discussion When challenged by inoculation, several transformants exhibited canker resistance as shown, but the resistance was gradually, if only partially, overcome by the canker pathogen. The resistant plants did not appear to be overcome by fast-growing pathogenic variants, but rather the resistance simply appeared to slow the disease process. Western blot analyses revealed that the level of translation of all ScFv transgenes was below the threshold limit of detection. The expression levels of ScFvs are often below the threshold limit of detection in plants, an issue that has recently been addressed in various ways, including addition of promoter enhancer sequences, targeting or retaining the ScFv to the endoplasmic reticulum, and also use of full-length MAbs in place of the ScFvs (De Jaeger et al., 1999; Vine et al., 2001; Xu et al., 2002). Full-length MAbs have the advantage of a greater avidity of binding than the ScFvs, but they tend to accumulate in the apoplastic space (outside the cell wall) or in the plant cell membrane, neither of which is suitable for the interception of the pathogenicity signal protein, PthA. ScFvs have the advantage of being expressed in the cytosol, where they have the best chance of encountering and blocking PthA. We are currently in the process of performing Biacore analyses to assess the binding avidity of the full length MAbs as compared to the ScFvs. We are also currently engineering new gene constructs to increase translation of the ScFvs in the cytosol. References De Jaeger, G., E. Buys et al., 1999. High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida. European Journal of Biochemistry 259:426-434. Vine, N. D., P. Drake, A. Hiatt, and J. K. C. Ma. 2001. Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence. Plant Molecular Biology 45:159-167. Xu, H., F. U. Montoya, et al.,. 2002. Combined use of regulatory elements within the cDNA to increase the production of a soluble mouse single-chain antibody, scFv, from tobacco cell suspension cultures. Protein Expression and Purification 24:384-394. Material and Methods IPG's Disease Block technology is based on expression of antibody fragments (ScFvs) in transgenic plants. The ScFvs are directed against a protein signal, PthA, that is injected by the bacterial pathogen into the plant cell cytoplasm, and is then translocated into the plant cell nucleus to cause disease. Plants are genetically altered express the ScFvs by use of Agrobacterium tumefaciens to inject a piece of engineered DNA into the citrus genome. Grapefruit cultivar "Flame" was used. Southern, Northern and Western blot analyses were performed to confirm transformation and gene expression. Results Over 50 putative transformed grapefruit seedlings were selected on medium containing kanamycin. Surviving seedlings were then grafted onto non-transgenic rootstock, allowed to grow for at least six months, and then evaluated for transformation by PCR, Southern blots and Northern blots (not shown). Most DNA insertions were single copy events, and most plants with inserts showed good transcriptional levels of the transgenes (~60%). Transformants were then challenged by inoculations with the citrus canker pathogen. A few plants exhibited resistance, as shown: Citrus canker results in premature fruit-drop (left). • The Molecular Basis of Canker Disease: • 1. Contagious canker bacteria (Xanthomonas citri) enter through stomata or wounds. • 2. They contact the plant cell wall and draw tight. • 3. They inject a protein signal, PthA, that causes cell division and some epidermal cell death. • 4. The loss of intercellular space creates a capillary effect to draw water from xylem. • 5. Xanthomonas makes xanthan gum, which absorbs water and swells, allowing egress from the epidermis. • 6. Wind-blown rain, high pressure sprays, insects & people do the rest. If X. citri cannot break the epidermis, it cannot spread.