Yu-Cai Liao

Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T1 progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T1 progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T1 plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T1 progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.

Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture.

A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.

Engineering Fusarium Head Blight Resistance in Wheat by Expression of a Fusion Protein Containing a Fusarium-Specific Antibody and an Antifungal Peptide

Fusarium head blight (FHB) or scab of wheat is a devastating disease in warm and humid regions at wheat-flowering periods worldwide. Natural resistance against FHB pathogens is inadequate and the development of FHB-resistant wheat cultivars has been a challenge. Expression of pathogen-specific antibodies in plants has been proposed as a strategy for crop protection. In this study, an antibody fusion protein comprising a Fusarium-specific recombinant antibody derived from chicken and an antifungal peptide from Aspergillus giganteus was expressed in wheat as a method for protecting plants against FHB pathogens. Plants expressing the antibody fusion displayed a very significantly enhanced resistance in T2 and T3 generations upon single-floret inoculation with the macroconidia of Fusarium asiaticum, the predominant species causing FHB in China, indicating a type II resistance. Spraying inoculation further revealed an enhanced type I resistance in the transgenic wheat plants. Remarkably, more grains were produced in the transgenic plants than the nontransgenic controls. Our results demonstrated that the antibody fusion protein may be used as an effective tool for the protection of crops against FHB pathogens.

Accumulation of antibody fusion proteins in the cytoplasm and ER of plant cells

To test whether the accumulation of cytoplasmically targeted recombinant antibodies could be improved by fusion to a cytoplasmic protein, we generated a series of single chain antibody-fusion proteins and assayed the levels of functional protein. Glutathione S-transferase (GST) from Schistosoma japonicum, coat protein (CP) from TMV, thioredoxin from tobacco (TRXt) or thioredoxin from Escherichia coli (TRXe) was fused to the N-terminus of scFv24, a TMV specific single chain antibody. Accumulation of functional fusion proteins in the endoplasmic reticulum (ER) and plant cell cytoplasm was analysed by transient expression in tobacco leaves. ELISA analysis demonstrated that the fusion partners did not prevent the binding of scFv24 to TMV virions. However, accumulation of functional scFv24 was dependent on the fusion partner coupled to it. CP-scFv and GST-scFv fusion protein accumulation amounted to 1 mg and 3 mg:g of leaf material, respectively, whereas the thioredoxin fusion proteins were produced at low levels. Western blot and surface plasmon resonance analysis confirmed the integrity of the ER retained CP and GST fusion proteins. In the cytoplasm, only the CP fusion protein was detectable (1‐5 ng:gram of leaf material) and levels of scFv24 alone or fused to the other three fusion partners were below the ELISA detection limit. Addition of a KDEL sequence to the C-terminus of the cytoplasmic CP fusion resulted in a 3-fold increase in protein accumulation indicating that an N-terminal CP and the C-terminal KDEL sequence are suitable elements to stabilize scFv antibodies in the cytoplasm.

GST fusion proteins cause false positives during selection of viral movement protein specific single chain antibodies.

Glutathione S-transferase (GST) fusion proteins are used frequently for investigating protein-protein and protein-DNA interactions. The present study demonstrates that the use of GST fusion proteins caused false positives during selection of phage-displayed single-chain antibody fragments (scFvs) specific for three domains of the movement protein (NS(M)) of tomato spotted wilt virus (TSWV). To identify and exclude the false positives when using GST as a fusion partner linked to the antigen of interest, indirect phage enzyme-linked immunosorbent assay (ELISA) was compared with capture phage ELISA. Of 210 enriched phage clones, indirect phage ELISA identified 106 clones specific for binding to GST-domain fusions but not to GST. In contrast, using capture phage ELISA, all 106 selected clones were identified as false positives, reacting with the GST fusion proteins and GST. This was confirmed by characterization of soluble scFv antibodies. The data indicate that GST fusion proteins seem unsuitable for screening of phage-displayed antibody fragments and it is essential to use capture phage ELISA, instead of the indirect phage ELISA used commonly to exclude false positives in characterization of selected clones with GST fusion proteins.